Analysis of a Critical Interaction within the Archaeal Box C/D Small Ribonucleoprotein Complex

In archaea and eukarya, box C/D ribonucleoprotein (RNP) complexes are responsible for 2′-O-methylation of tRNAs and rRNAs. The archaeal box C/D small RNP complex requires a small RNA component (sRNA) possessing Watson-Crick complementarity to the target RNA along with three proteins: L7Ae, Nop5p, and fibrillarin. Transfer of a methyl group from S-adenosylmethionine to the target RNA is performed by fibrillarin, which by itself has no affinity for the sRNA-target duplex. Instead, it is targeted to the site of methylation through association with Nop5p, which in turn binds to the L7Ae-sRNA complex. To understand how Nop5p serves as a bridge between the targeting and catalytic functions of the box C/D small RNP complex, we have employed alanine scanning to evaluate the interaction between the Pyrococcus horikoshii Nop5p domain and an L7Ae box C/D RNA complex. From these data, we were able to construct an isolated RNA-binding domain (Nop-RBD) that folds correctly as demonstrated by x-ray crystallography and binds to the L7Ae box C/D RNA complex with near wild type affinity. These data demonstrate that the Nop-RBD is an autonomously folding and functional module important for protein assembly in a number of complexes centered on the L7Ae-kinkturn RNP.Many biological RNAs require extensive modification to attain full functionality in the cell (1). Currently there are over 100 known RNA modification types ranging from small functional group substitutions to the addition of large multi-cyclic ring structures (2). Transfer RNA, one of many functional RNAs targeted for modification (3-6), possesses the greatest modification type diversity, many of which are important for proper biological function (7). Ribosomal RNA, on the other hand, contains predominantly two types of modified nucleotides: pseudouridine and 2′-O-methylribose (8). The crystal structures of the ribosome suggest that these modifications are important for proper folding (9, 10) and structural stabilization (11) in vivo as evidenced by their strong tendency to localize to regions associated with function (8, 12, 13). These roles have been verified biochemically in a number of cases (14), whereas newly emerging functional modifications are continually being investigated.Box C/D ribonucleoprotein (RNP)³ complexes serve as RNA-guided site-specific 2′-O-methyltransferases in both archaea and eukaryotes (15, 16) where they are referred to as small RNP complexes and small nucleolar RNPs, respectively. Target RNA pairs with the sRNA guide sequence and is methylated at the 2′-hydroxyl group of the nucleotide five bases upstream of either the D or D′ box motif of the sRNA (Fig. 1, star) (17, 18). In archaea, the internal C′ and D′ motifs generally conform to a box C/D consensus sequence (19), and each sRNA contains two guide regions ∼12 nucleotides in length (20). The bipartite architecture of the RNP potentially enables the complex to methylate two distinct RNA targets (21) and has been shown to be essential for site-specific methylation (22).Open in a separate windowFIGURE 1.Organization of the archaeal box C/D complex. The protein components of this RNP are L7Ae, Nop5p, and fibrillarin, which together bind a box C/D sRNA. The regions of the Box C/D sRNA corresponding to the conserved C, D, C′, and D′ boxes are labeled. The target RNA binds the sRNA through Watson-Crick pairing and is methylated by fibrillarin at the fifth nucleotide from the D/D′ boxes (star).In addition to the sRNA, the archaeal box C/D complex requires three proteins for activity (23): the ribosomal protein L7Ae (24, 25), fibrillarin, and the Nop56/Nop58 homolog Nop5p (Fig. 1). L7Ae binds to both box C/D and the C′/D′ motifs (26), which respectively comprise kink-turn (27) or k-loop structures (28), to initiate the assembly of the RNP (29, 30). Fibrillarin performs the methyl group transfer from the cofactor S-adenosylmethionine to the target RNA (31-33). For this to occur, the active site of fibrillarin must be positioned precisely over the specific 2′-hydroxyl group to be methylated. Although fibrillarin methylates this functional group in the context of a Watson-Crick base-paired helix (guide/target), it has little to no binding affinity for double-stranded RNA or for the L7Ae-sRNA complex (22, 26, 33, 34). Nop5p serves as an intermediary protein bringing fibrillarin to the complex through its association with both the L7Ae-sRNA complex and fibrillarin (22). Along with its role as an intermediary between fibrillarin and the L7Ae-sRNA complex, Nop5p possesses other functions not yet fully understood. For example, Nop5p self-dimerizes through a coiled-coil domain (35) that in most archaea and eukaryotic homologs includes a small insertion sequence of unknown function (36, 37). However, dimerization and fibrillarin binding have been shown to be mutually exclusive in Methanocaldococcus jannaschii Nop5p, potentially because of the presence of this insertion sequence (36). Thus, whether Nop5p is a monomer or a dimer in the active RNP is still under debate.In this study, we focus our attention on the Nop5p protein to investigate its interaction with a L7Ae box C/D RNA complex because both the fibrillarin-Nop5p and the L7Ae box C/D RNA interfaces are known from crystal structures (29, 35, 38). Individual residues on the surface of a monomeric form of Nop5p (referred to as mNop5p) (22) were mutated to alanine, and the effect on binding affinity for a L7Ae box C/D motif RNA complex was assessed through the use of electrophoretic mobility shift assays. These data reveal that residues important for binding cluster within the highly conserved NOP domain (39, 40). To demonstrate that this domain is solely responsible for the affinity of Nop5p for the preassembled L7Ae box C/D RNA complex, we expressed and purified it in isolation from the full Nop5p protein. The isolated Nop-RBD domain binds to the L7Ae box C/D RNA complex with nearly wild type affinity, demonstrating that the Nop-RBD is truly an autonomously folding and functional module. Comparison of our data with the crystal structure of the homologous spliceosomal hPrp31-15.5K protein-U4 snRNA complex (41) suggests the adoption of a similar mode of binding, further supporting a crucial role for the NOP domain in RNP complex assembly.     

Identification and Manipulation of the Caprazamycin Gene Cluster Lead to New Simplified Liponucleoside Antibiotics and Give Insights into the Biosynthetic Pathway

Caprazamycins are potent anti-mycobacterial liponucleoside antibiotics isolated from Streptomyces sp. MK730-62F2 and belong to the translocase I inhibitor family. Their complex structure is derived from 5′-(β-O-aminoribosyl)-glycyluridine and comprises a unique N-methyldiazepanone ring. The biosynthetic gene cluster has been identified, cloned, and sequenced, representing the first gene cluster of a translocase I inhibitor. Sequence analysis revealed the presence of 23 open reading frames putatively involved in export, resistance, regulation, and biosynthesis of the caprazamycins. Heterologous expression of the gene cluster in Streptomyces coelicolor M512 led to the production of non-glycosylated bioactive caprazamycin derivatives. A set of gene deletions validated the boundaries of the cluster and inactivation of cpz21 resulted in the accumulation of novel simplified liponucleoside antibiotics that lack the 3-methylglutaryl moiety. Therefore, Cpz21 is assigned to act as an acyltransferase in caprazamycin biosynthesis. In vivo and in silico analysis of the caprazamycin biosynthetic gene cluster allows a first proposal of the biosynthetic pathway and provides insights into the biosynthesis of related uridyl-antibiotics.Caprazamycins (CPZs)² (Fig. 1, 1) are liponucleoside antibiotics isolated from a fermentation broth of Streptomyces sp. MK730-62F2 (1, 2). They show excellent activity in vitro against Gram-positive bacteria, in particular against the genus Mycobacterium including Mycobacterium intracellulare, Mycobacterium avium, and Mycobacterium tuberculosis (3). In a pulmonary mouse model with M. tuberculosis H37Rv, administration of caprazamycin B exhibited a therapeutic effect but no significant toxicity (4). Structural elucidation (2) revealed a complex and unique composition of elements the CPZs share only with the closely related liposidomycins (LPMs, 2) (5). The core skeleton is the (+)-caprazol (5) composed of an N-alkylated 5′-(β-O-aminoribosyl)-glycyluridine, also known from FR-900493 (6) (6) and the muraymycins (7) (7), which is cyclized to form a rare diazepanone ring. Attached to the 3′″-OH are β-hydroxy fatty acids of different chain length resulting in CPZs A–G (1). They differ from the LPMs in the absence of a sulfate group at the 2″-position of the aminoribose and the presence of a permethylated l-rhamnose β-glycosidically linked to the 3-methylglutaryl (3-MG) moiety.Open in a separate windowFIGURE 1.Nucleoside antibiotics of the translocase I inhibitor family.The LPMs have been shown to inhibit biosynthesis of the bacterial cell wall by targeting the formation of lipid I (8). The CPZs are expected to act in the same way and are assigned to the growing number of translocase I inhibitors that include other nucleoside antibiotics, like the tunicamycins and mureidomycins (9). During peptidoglycan formation, translocase I catalyzes the transfer of UDP-MurNAc-pentapeptide to the undecaprenyl phosphate carrier to generate lipid I (10). This reaction is considered an unexploited and promising target for new anti-infective drugs (11).Recent investigations indicate that the 3″-OH group (12), the amino group of the aminoribosyl-glycyluridine, and an intact uracil moiety (13) are essential for the inhibition of the Escherichia coli translocase I MraY. The chemical synthesis of the (+)-caprazol (5) was recently accomplished (14), however, this compound only shows weak antibacterial activity. In contrast, the acylated compounds 3 and 4 exhibit strong growth inhibition of mycobacteria, suggesting a potential role of the fatty acid side chain in penetration of the bacterial cell (15, 16). Apparently, the acyl-caprazols (4) represent the most simplified antibiotically active liponucleosides and a good starting point for further optimization of this class of potential therapeutics.Although chemical synthesis and biological activity of CPZs and LPMs has been studied in some detail, their biosynthesis remains speculative and only few data exists about the formation of other translocase I inhibitors (17, 18). Nevertheless, we assume that the CPZ biosynthetic pathway is partially similar to that of LPMs, FR-90043 (6), and muraymycins (7) and presents a model for the comprehension and manipulation of liponucleoside formation. Considering the unique structural features of the CPZs we also expect some unusual biotransformations to be involved in the formation of, e.g. the (+)-caprazol.Here we report the identification and analysis of the CPZ gene cluster, the first cluster of a translocase I inhibitor. A set of gene disruption experiments provide insights into the biosynthetic origin of the CPZs and moreover, heterologous expression of the gene cluster allows the generation of novel bioactive derivatives by pathway engineering.     

VCP Mutations Causing Frontotemporal Lobar Degeneration Disrupt Localization of TDP-43 and Induce Cell Death

Frontotemporal lobar degeneration (FTLD) with inclusion body myopathy and Paget disease of bone is a rare, autosomal dominant disorder caused by mutations in the VCP (valosin-containing protein) gene. The disease is characterized neuropathologically by frontal and temporal lobar atrophy, neuron loss and gliosis, and ubiquitin-positive inclusions (FTLD-U), which are distinct from those seen in other sporadic and familial FTLD-U entities. The major component of the ubiquitinated inclusions of FTLD with VCP mutation is TDP-43 (TAR DNA-binding protein of 43 kDa). TDP-43 proteinopathy links sporadic amyotrophic lateral sclerosis, sporadic FTLD-U, and most familial forms of FTLD-U. Understanding the relationship between individual gene defects and pathologic TDP-43 will facilitate the characterization of the mechanisms leading to neurodegeneration. Using cell culture models, we have investigated the role of mutant VCP in intracellular trafficking, proteasomal function, and cell death and demonstrate that mutations in the VCP gene 1) alter localization of TDP-43 between the nucleus and cytosol, 2) decrease proteasome activity, 3) induce endoplasmic reticulum stress, 4) increase markers of apoptosis, and 5) impair cell viability. These results suggest that VCP mutation-induced neurodegeneration is mediated by several mechanisms.Frontotemporal lobar degeneration (FTLD)² accounts for 10% of all late onset dementias and is the third most frequent neurodegenerative disease after Alzheimer disease and dementia with Lewy bodies (1). FTLD with ubiquitin-immunoreactive inclusions is genetically, clinically, and neuropathologically heterogeneous (2, 3). FTLD-U comprises several distinct entities, including sporadic forms and familial cases caused by mutations in the genes encoding VCP (valosin-containing protein), GRN (progranulin), CHMP2B (charged multivesicular body protein 2B), TDP-43 (TAR DNA-binding protein of 43 kDa) and an unknown gene linked to chromosome 9 (2, 3). Frontotemporal dementia with inclusion body myopathy and Paget disease of bone is a rare, autosomal dominant disorder caused by mutations in the VCP gene located on chromosome 9p13-p12 (4-10) (Fig. 1). This multisystem disease is characterized by progressive muscle weakness and atrophy, increased osteoclastic bone resorption, and early onset frontotemporal dementia, also called FTLD (9, 11). Mutations in VCP are also associated with dilatative cardiomyopathy with ubiquitin-positive inclusions (12). Neuropathologic features of FTLD with VCP mutation include frontal and temporal lobar atrophy, neuron loss and gliosis, and ubiquitin-positive inclusions (FTLD-U). The majority of aggregates are ubiquitin- and TDP-43-positive neuronal intranuclear inclusions (NIIs); a smaller proportion is made up of TDP-43-immunoreactive dystrophic neurites (DNs) and neuronal cytoplasmic inclusions (NCIs). A small number of inclusions are VCP-immunoreactive (5, 13). Pathologic TDP-43 in inclusions links a spectrum of diseases in which TDP-43 pathology is a primary feature, including FTLD-U, motor neuron disease, including amyotrophic lateral sclerosis, FTLD with motor neuron disease, and inclusion body myopathy and Paget disease of bone, as well as an expanding spectrum of other disorders in which TDP-43 pathology is secondary (14, 15).Open in a separate windowFIGURE 1.Model of pathogenic mutations and domains in valosin-containing protein. CDC48 (magenta), located within the N terminus (residues 22-108), binds the following cofactors: p47, gp78, and Npl4-Ufd1 (23-25, 28). There are two AAA-ATPase domains (AAA; blue) at residues 240-283 and 516-569, which are joined by two linker regions (L1 and L2; red).TDP-43 proteinopathy in FTLD with VCP mutation has a biochemical signature similar to that seen in other sporadic and familial cases of FTLD-U, including sporadic amyotrophic lateral sclerosis, FTLD-motor neuron disease, FTLD with progranulin (GRN) mutation, and FTLD linked to chromosome 9p (3, 16). TDP-43 proteinopathy in these disorders is characterized by hyperphosphorylation of TDP-43, ubiquitination, and cleavage to form C-terminal fragments detected only in insoluble brain extracts from affected brain regions (16). Identification of TDP-43 as the major component of the ubiquitin-immunoreactive inclusions of FTLD with VCP mutation supports the hypothesis that VCP gene mutations cause an alteration of VCP function, leading to TDP-43 proteinopathy.VCP/p97 (valosin-containing protein) is a member of the AAA (ATPase associated with diverse cellular activities) superfamily. The N-terminal domain of VCP has been shown to be involved in cofactor binding (CDC48 (cell division cycle protein 48)) and two AAA-ATPase domains that form a hexameric complex (Fig. 1) (17). Recently, it has been shown that the N-terminal domain of VCP binds phosphoinositides (18, 19). AKT (activated serine-threonine protein kinase) phosphorylates VCP and is required for constitutive VCP function (20, 21). AKT is activated through phospholipid binding and phosphorylation via the phosphoinositide 3-kinase signaling pathway, which is involved in cell survival (22). The lipid binding domain may recruit VCP to the cell membrane where it is phosphorylated by AKT (19).The diversity of VCP functions is modulated, in part, by a variety of intracellular cofactors, including p47, gp78, and Npl4-Ufd1 (23). Cofactor p47 has been shown to play a role in the maintenance and biogenesis of both the endoplasmic reticulum (ER) and Golgi apparatus (24). The structure of p47 contains a ubiquitin regulatory X domain that binds the N-terminus of VCP, and together they act as a chaperone to deliver membrane fusion machinery to the site of adjacent membranes (25). The function of the p47-VCP complex is dependent upon cell division cycle 2 (CDC2) serine-threonine kinase phosphorylation of p47 (26, 27). Also, VCP has been found to interact with the cytosolic tail of gp78, an ER membrane-spanning E3 ubiquitin ligase that exclusively binds VCP and enhances ER-associated degradation (ERAD) (28). The Npl4-Ufd1-VCP complex is involved in nuclear envelope assembly and targeting of proteins through the ubiquitin-proteasome system (29, 30). The cell survival response of this complex has been found to be important in DNA damage repair though activation by phosphorylation and its recruitment to double-stranded breaks (20, 31). The Npl4-Ufd1-VCP cytosolic complex is also recruited to the ER membrane, interacting with Derlin 1, VCP-interacting membrane proteins (VIMP), and other complexes. At the ER membrane, these misfolded proteins are targeted to the proteasome via ERAD (32-34). VCP also targets IKKβ for ubiquitination to the ubiquitin-proteasome system, implicating VCP in the cell survival pathway and neuroprotection (21, 35-37).To investigate the mechanism of neurodegeneration caused by VCP mutations, we first tested the hypothesis that VCP mutations decrease cell viability in vitro using a neuroblastoma SHSY-5Y cell line and then investigated cellular pathways that are known to lead to neurodegeneration, including decrease in proteasome activity, caspase-mediated degeneration, and a change in cellular localization of TDP-43.     

Alternative Splicing in Class V Myosins Determines Association with Rab10

Rab proteins influence vesicle trafficking pathways through the assembly of regulatory protein complexes. Previous investigations have documented that Rab11a and Rab8a can interact with the tail region of myosin Vb and regulate distinct trafficking pathways. We have now determined that a related Rab protein, Rab10, can interact with myosin Va, myosin Vb, and myosin Vc. Rab10 localized to a system of tubules and vesicles that have partially overlapping localization with Rab8a. Both Rab8a and Rab10 were mislocalized by the expression of dominant-negative myosin V tails. Interaction with Rab10 was dependent on the presence of the alternatively spliced exon D in myosin Va and myosin Vb and the homologous region in myosin Vc. Yeast two-hybrid assays and fluorescence resonance energy transfer studies confirmed that Rab10 binding to myosin V tails in vivo required the alternatively spliced exon D. In contrast to our previous work, we found that Rab11a can interact with both myosin Va and myosin Vb tails independent of their splice isoform. These results indicate that Rab GTPases regulate diverse endocytic trafficking pathways through recruitment of multiple myosin V isoforms.Eukaryotic cells are comprised of networks of highly organized membranous structures that require the efficient and timely movement of diverse intracellular proteins for proper function. Molecular motors provide the physical force needed to move these materials along microtubules and actin microfilaments. Unconventional myosin motors, such as those belonging to classes V, VI, and VII, have roles in the trafficking and recycling of membrane-bound structures in eukaryotic cells (1) and are recruited to discrete vesicle populations. Myosin VI is involved in clathrin-mediated endocytosis (2), whereas myosin VIIa participates in the proper development of stereocilia of inner ear hair cells and the transport of pigment granules in retinal pigmented epithelial cells (3, 4). Similarly, the three members of vertebrate class V myosins, myosin Va, myosin Vb, and myosin Vc, are required for the proper transport of a wide array of membrane cargoes, such as the melanosomes of pigment cells, synaptic vesicles in neurons, apical recycling endosomes in polarized epithelial cells, and bulk recycling vesicles in non-polarized cells (5).Members of the Rab family of small GTPases regulate many cellular systems, including membrane trafficking (6, 7). Certain Rab proteins associate with and regulate the function of class V myosins. Rab27a, in a complex with the adaptor protein melanophilin/Slac2-a, is required to localize myosin Va to the surface of melanin-filled pigment granules in vertebrates (8-10), whereas Rab27a and Slac2-c/MyRIP associate with both Myosin Va and myosin VIIa (3, 11). Rab11a, in a complex with its adaptor protein Rab11-FIP2, associates with myosin Vb on recycling endosomes (12-14) where the tripartite complex regulates the recycling of a variety of cargoes (15-19). In addition, Rab8a associates with both myosin Vb (20) and myosin Vc (21) as part of the non-clathrin-mediated tubular recycling system (20). Recently, Rab11a has also been shown to associate with myosin Va in the transport of AMPA receptors in dendritic spines (22), contributing to the model of myosin V regulation by multiple Rab proteins.Previous investigations have documented alternative splicing of myosin Va in a tissue-specific manner (23-28). Alternate splicing occurs in a region lying between the coiled-coil region of the neck of the motor and the globular tail region. Three exons in particular are subject to alternative splicing: exons B, D, and F (23-25). Exon F is critical for association with melanophilin/Slac2 and Rab27a (8, 9, 29, 30). Additionally, exon B is required for the interaction of myosin Va with dynein light chain 2 (DLC2) (27, 28). Currently no function for the alternatively spliced exon D has been reported. Similar to myosin Va, myosin Vb contains exons A, B, C, D, and E, whereas no exon F has yet been identified in myosin Vb (Fig. 1A). In addition, exon B in myosin Vb does not resemble the dynein light chain 2 (DLC2) binding region in myosin Va (27, 28), and therefore, it likely does not interact with DLC2. On the other hand, exon D is highly conserved among Myosin Va, myosin Vb, and myosin Vc, suggesting a common function in these molecular motors.Open in a separate windowFIGURE 1.Tissue distribution of human myosin Va and myosin Vb splice isoforms. A, schematic of the alternative exon organization in the tails of myosin Va and myosin Vb. It is known that exons B, D, and F are subject to alternative splicing in myosin Va, whereas there is only evidence that exon D is alternatively spliced in myosin Vb, which does not contain exon F. B, alignment of exon D sequences from mouse and human myosin V''s. myosin Va and myosin Vb both contain exon D (amino acids 1320-1346 of myosin Va and 1315-1340 of myosin Vb), whereas myosin Vc contains an exon D-like region (amino acids 1124-1147 of human myosin Vc) that is not known to be alternatively spliced. Alignment of the exon D regions from all three motors reveals a high degree of homology, especially in the center of the exon. Asterisks indicate amino acid identities. C, PCR-based analysis of human tissue panels reveals the alternative splicing pattern of exon D in myosin Va and myosin Vb. Primers flanking the region encoding exon D for both motors were used to amplify cDNA from human MTC™ panels (Clontech). cDNA amplified from HeLa cell RNA as well as myosin Va and myosin Vb tail constructs were used as positive controls. Variants expressing exon D (upper bands) and lacking exon D (lower bands) were visible. Per., peripheral; Pos., positive.Here we report that Rab10, a protein related to Rab8a and thought to have similar function (31-35), localizes to a system of tubules and vesicles overlapping in distribution with Rab8a in HeLa cells. Utilizing dominant-negative myosin V tail constructs, we show that Rab8a and Rab10 can interact with Myosin Va, myosin Vb, and myosin Vc in vivo. In addition, we have determined that the alternatively spliced exon D in both myosin Va and myosin Vb is required for interaction with Rab10. In contrast to our previous findings, we demonstrate that Rab11a is able to interact with both myosin Va and myosin Vb tails in an exon independent-manner. These results reveal that multiple Rab proteins potentially regulate all three class V myosin motors.     

Bacterial ApbC Protein Has Two Biochemical Activities That Are Required for in Vivo Function

The ApbC protein has been shown previously to bind and rapidly transfer iron-sulfur ([Fe-S]) clusters to an apoprotein (Boyd, J. M., Pierik, A. J., Netz, D. J., Lill, R., and Downs, D. M. (2008) Biochemistry 47, 8195–8202. This study utilized both in vivo and in vitro assays to examine the function of variant ApbC proteins. The in vivo assays assessed the ability of ApbC proteins to function in pathways with low and high demand for [Fe-S] cluster proteins. Variant ApbC proteins were purified and assayed for the ability to hydrolyze ATP, bind [Fe-S] cluster, and transfer [Fe-S] cluster. This study details the first kinetic analysis of ATP hydrolysis for a member of the ParA subfamily of “deviant” Walker A proteins. Moreover, this study details the first functional analysis of mutant variants of the ever expanding family of ApbC/Nbp35 [Fe-S] cluster biosynthetic proteins. The results herein show that ApbC protein needs ATPase activity and the ability to bind and rapidly transfer [Fe-S] clusters for in vivo function.Proteins containing iron-sulfur ([Fe-S]) clusters are employed in a wide array of metabolic functions (reviewed in Ref. 1). Research addressing the biosynthesis of the iron-molybdenum cofactor of nitrogenase in Azotobacter vinelandii led to the discovery of an operon (iscA^nifnifUSVcysE1) involved in the biosynthesis of [Fe-S] clusters (reviewed in Ref. 2). Subsequent experiments led to the finding of two more systems involved in the de novo biosynthesis of [Fe-S] clusters, the isc and the suf systems (3, 4). Like Escherichia coli, the genome of Salmonella enterica serovar Typhimurium encodes for the isc and suf [Fe-S] cluster biosynthesis machinery.Recent studies have identified a number of additional or non-isc/-suf-encoded proteins that are involved in bacterial [Fe-S] cluster biosynthesis and repair. Examples include the following: CyaY, an iron-binding protein believed to be involved in iron trafficking and iron delivery (5–7); YggX, an Fe²⁺-binding protein that protects the cell from oxidative stress (8, 9); ErpA, an alternate A-type [Fe-S] cluster scaffolding protein (10); NfuA, a proposed intermediate [Fe-S] delivery protein (11–13); YtfE, a protein proposed to be involved in [Fe-S] cluster repair (14, 15); and CsdA-CsdE, an alternative cysteine desulferase (16).Analysis of the metabolic network anchored to thiamine biosynthesis in S. enterica identified lesions in three non-isc or -suf loci that compromise Fe-S metabolism as follows: apbC, apbE, and rseC (17–21). This metabolic system was subsequently used to dissect a role for cyaY and gshA in [Fe-S] cluster metabolism (6, 22, 23). Of these, the apbC (mrp in E. coli) locus was identified as the predominant site of lesions that altered thiamine synthesis by disrupting [Fe-S] cluster metabolism (17, 18).ApbC is a member of the ParA subfamily of proteins that have a wide array of functions, including electron transfer (24), initiation of cell division (25), and DNA segregation (26, 27). Importantly, ATP hydrolysis is required for function of all well characterized members of this subfamily, and all members contain a “deviant” Walker A motif, which contains two lysine residues instead of one (GKXXXGK(S/T)) (28). ApbC has been shown to hydrolyze ATP (17).Recently, five proteins with a high degree of identity to ApbC have been shown to be involved in [Fe-S] cluster metabolism in eukaryotes. The sequence alignments of the central portion of these proteins and bacterial ApbC are shown in Fig. 1. HCF101 was demonstrated to be involved in chloroplast [Fe-S] cluster metabolism (29, 30). The CFD1, Npb35, and huNbp35 (formally Nubp1) proteins were demonstrated to be involved in cytoplasmic [Fe-S] cluster metabolism (31, 32). Ind1 was demonstrated to be involved in the maturation of [Fe-S] clusters in the mitochondrial enzyme NADH:ubiquinone oxidoreductase (33). There is currently no report of any of these proteins hydrolyzing ATP.Open in a separate windowFIGURE 1.Protein sequence alignments of members of the ApbC/Nbp35 subfamily of ParA family of proteins. Protein alignments were assembled using the Clustal_W method in the Lasergene® software and show only the central portion of the proteins, which have the highest sequence conservation. The three boxed areas highlight the Walker A box, conserved Ser residue, and CXXC motif. Proteins listed are as follows: ApbC (S. enterica serovar Typhimurium LT2), CFD1 (S. cerevisiae), Nbp35 (S. cerevisiae), HCF101 (Arabidopsis thaliana), huNpb35 (formally Nubp1) (Homo sapiens), and Ind1 (Candida albicans).Biochemical analysis of ApbC indicated that it could bind and transfer [Fe-S] clusters to Saccharomyces cerevisiae apo-isopropylmalate isomerase (34). Additional genetic studies indicated that ApbC has a degree of functional redundancy with IscU, a known [Fe-S] cluster scaffolding protein (35, 36).In this study we investigate the correlation between the biochemical properties of ApbC (i.e. ATPase activity, [Fe-S] cluster binding, and [Fe-S] cluster transfer rates) and the in vivo function of this protein. This is the first detailed kinetic analysis of ATP hydrolysis for a member of the ParA subfamily of deviant Walker A proteins and the first functional analysis of a member of the ever expanding family of ApbC/Nbp35 proteins. Data presented indicate that noncomplementing variants have distinct biochemical properties that place them in three distinct classes.     

Characterization of Enantiomeric Bile Acid-induced Apoptosis in Colon Cancer Cell Lines

Bile acids are steroid detergents that are toxic to mammalian cells at high concentrations; increased exposure to these steroids is pertinent in the pathogenesis of cholestatic disease and colon cancer. Understanding the mechanisms of bile acid toxicity and apoptosis, which could include nonspecific detergent effects and/or specific receptor activation, has potential therapeutic significance. In this report we investigate the ability of synthetic enantiomers of lithocholic acid (ent-LCA), chenodeoxycholic acid (ent-CDCA), and deoxycholic acid (ent-DCA) to induce toxicity and apoptosis in HT-29 and HCT-116 cells. Natural bile acids were found to induce more apoptotic nuclear morphology, cause increased cellular detachment, and lead to greater capase-3 and -9 cleavage compared with enantiomeric bile acids in both cell lines. In contrast, natural and enantiomeric bile acids showed similar effects on cellular proliferation. These data show that bile acid-induced apoptosis in HT-29 and HCT-116 cells is enantiospecific, hence correlated with the absolute configuration of the bile steroid rather than its detergent properties. The mechanism of LCA- and ent-LCA-induced apoptosis was also investigated in HT-29 and HCT-116 cells. These bile acids differentially activate initiator caspases-2 and -8 and induce cleavage of full-length Bid. LCA and ent-LCA mediated apoptosis was inhibited by both pan-caspase and selective caspase-8 inhibitors, whereas a selective caspase-2 inhibitor provided no protection. LCA also induced increased CD95 localization to the plasma membrane and generated increased reactive oxygen species compared with ent-LCA. This suggests that LCA/ent-LCA induce apoptosis enantioselectively through CD95 activation, likely because of increased reactive oxygen species generation, with resulting procaspase-8 cleavage.Bile acids are physiologic steroids that are necessary for the proper absorption of fats and fat-soluble vitamins. Their ability to aid in these processes is largely due to their amphipathic nature and thus their ability to act as detergents. Despite the beneficial effects, high concentrations of bile acids are toxic to cells (1-11). High fat western diets induce extensive recirculation of the bile acid pool, resulting in increased exposure of the colonic epithelial cells to these toxic steroids (12, 13). A high fat diet is also a risk factor for colon carcinogenesis; increased bile acid exposure is responsible for some of this risk. Bile acids can contribute to both colon cancer formation and progression, and their effects on colonic proliferation and apoptosis aid this process by disrupting the balance between cell growth and cell death, as well as helping to select for bile acid-resistant cells (14, 15).In colonocyte-derived cell lines bile acid-induced apoptosis is thought to proceed through mitochondrial destabilization with resulting mitochondrial permeability transition formation and cytochrome c release as well as generation of oxidative stress (1, 9-11). Bile acid-induced apoptosis has also been extensively explored in hepatocyte derived cell lines with mechanisms including mitochondria dysfunction (16-23), endoplasmic reticulum stress (24), ligand-independent activation of death receptor pathways (18, 25-28), and modulation of cellular apoptotic and anti-apoptotic Bcl-2 family proteins (29).Although ample evidence exists for multiple mechanisms of bile acid-induced apoptosis, the precise interactions responsible for initiating these apoptotic pathways are still unclear. Bile acids have been shown to interact directly with specific receptors (30, 31). These steroids can also initiate cellular signaling through nonspecific membrane perturbations (32), and evidence exists showing that other simple detergents (i.e. Triton X-100) are capable of inducing caspase cleavage nonspecifically with resultant apoptosis (33). Therefore, hydrophobic bile acids may interact nonspecifically with cell membranes to alter their physical properties, bind to receptors specific for these steroids, or utilize a combination of both specific and nonspecific interactions to induce apoptosis.Bile acid enantiomers could be useful tools for elucidating mechanisms of bile acid toxicity and apoptosis. These enantiomers, known as ent-bile acids, are synthetic nonsuperimposable mirror images of natural bile acids with identical physical properties except for optical rotation. Because bile acids are only made in one absolute configuration naturally, ent-bile acids must be constructed using a total synthetic approach. Recently we reported the first synthesis of three enantiomeric bile acids: ent-lithocholic acid (ent-LCA),² ent-chenodeoxycholic acid (ent-CDCA), and ent-deoxycholic acid (ent-DCA) (Fig. 1) (34, 35). Enantiomeric bile acids have unique farnesoid X receptor, vitamin D receptor, pregnane X receptor, and TGR5 receptor activation profiles compared with the corresponding natural bile acids (34). This illustrates that natural and enantiomeric bile acids interact differently within chiral environments because of their distinct three-dimensional configurations (Fig. 1). Despite these differences in chiral interactions, ent-bile acids have physical properties identical to those of their natural counterparts including solubility and critical micelle concentrations (34, 35). With different receptor interaction profiles and identical physical properties compared with natural bile acids, ent-bile acids are ideal compounds to differentiate between the receptor-mediated and the non-receptor-mediated functions of natural bile acids.Open in a separate windowFIGURE 1.Natural and enantiomeric bile acids. Structures and three-dimensional projection views of natural LCA, CDCA, DCA, and their enantiomers (ent-LCA, ent-CDCA, and ent-DCA). The three-dimensional ent-steroid structure is rotated 180° around the long axis for easier comparison with the natural steroid.In this study we explore the enantioselectivity of LCA-, CDCA-, and DCA-mediated toxicity and apoptosis in two human colon adenocarcinoma cell lines, HT-29 and HCT-116. Because the mechanism of natural LCA induced apoptosis has never been characterized, we then examined in more detail LCA- and ent-LCA-mediated apoptosis in colon cancer cells. These studies will not only explore the LCA apoptotic mechanism but will also determine whether ent-LCA signals through similar cellular pathways.     

Serum Opacity Factor Is a Streptococcal Receptor for the Extracellular Matrix Protein Fibulin-1

The adhesion of bacteria to host tissues is often mediated by interactions with extracellular matrices. Herein, we report on the interactions of the group A streptococcus, Streptococcus pyogenes, with the extracellular matrix protein fibulin-1. S. pyogenes bound purified fibulin-1 in a dose-dependent manner. Genetic ablation of serum opacity factor (SOF), a virulence determinant of S. pyogenes, reduced binding by ∼50%, and a recombinant peptide of SOF inhibited binding of fibulin-1 to streptococci by ∼45%. Fibulin-1 bound to purified SOF2 in a dose-dependent manner with high affinity (K_d = 1.6 nm). The fibulin-1-binding domain was localized to amino acid residues 457–806 of SOF2, whereas the fibronectin-binding domain is contained within residues 807–931 of SOF2, indicating that these two domains are separate and distinct. Fibulin-1 bound to recombinant SOF from M types 2, 4, 28, and 75 of S. pyogenes, indicating that the fibulin-1-binding domain is likely conserved among SOF from different serotypes. Mixed binding experiments suggested that gelatin, fibronectin, fibulin-1, and SOF form a quaternary molecular complex that enhanced the binding of fibulin-1. These data indicate that S. pyogenes can interact with fibulin-1 and that SOF is a major streptococcal receptor for fibulin-1 but not the only receptor. Such interactions with fibulin-1 may be involved in the adhesion of S. pyogenes to extracellular matrices of the host.Adhesion of bacteria to host surfaces is the first stage in establishing bacterial infections in the human host, and a variety of molecular mechanisms are utilized to initiate adhesion. A common mechanism for adhesion involves interactions between bacterial adhesins and components of the extracellular matrices of the host. The identification and characterization of microbial surface components recognizing adhesive matrix molecules (MSCRAMM) has led to important advances in vaccines and immunotherapies for preventing and treating bacterial infections (1).The group A streptococcus, Streptococcus pyogenes, is a major human pathogen causing diseases ranging from relative minor infections such as pharyngitis and cellulitis to severe infections with high levels of morbidity and mortality such as necrotizing fasciitis and toxic shock syndrome (2). This pathogen expresses adhesins that interact with various components of the extracellular matrix including laminin, elastin, fibronectin, fibrinogen, and collagen (3–7). The interactions between fibronectin and S. pyogenes have been intensely studied, and these investigations have revealed at least 10 different streptococcal proteins that bind fibronectin (4).Serum opacity factor (SOF)² is a major fibronectin-binding protein that is involved in adhesion to host cells (8–11). SOF is a virulence determinant that is expressed by approximately half of the clinical isolates of S. pyogenes (8). SOF opacifies serum by binding and displacing apoA-I in high density lipoproteins (8, 12–15). SOF is covalently linked to the streptococcal cell wall via an LPSTG sortase recognition site and is also released in a soluble form. SOF has two functionally distinct domains, an N-terminal domain that opacifies serum and a C-terminal domain that binds fibronectin. The role of SOF in adhesion involves both its C-terminal fibronectin-binding domain and an N-terminal region (see Fig. 1 for a schematic of structure) (9, 11). However, the nature of the interactions between the N-terminal region of SOF and host components is not well characterized.Open in a separate windowFIGURE 1.A, a schematic of the structure of SOF and its functional domains is shown. The assignment of functional domains are based on the findings of Rakonjac et al. (33), Kreikemeyer et al. (34), Courtney et al. (8, 13), and results presented in this work. Fn, fibronectin. B, the data for the binding of SOF peptides to fibronectin are from previous publications (8, 13), and the data for fibulin-1 are from the present work.Herein, we report on the interactions between a truncated form of SOF in which its fibronectin-binding domain has been deleted and the extracellular matrix protein fibulin-1. Fibulin-1 is a member of the fibulin family that currently consists of seven glycoproteins. All fibulins contain epidermal growth factor-like repeats and a unique fibulin-type module at its C terminus that define this family (16, 17). Fibulin-1 is found within the extracellular matrices and in human plasma at 30–50 μg/ml (18). It interacts with many of the components of the extracellular matrix including fibronectin, laminin, fibrinogen, nidogen-1, endostatin, aggrecan, and versican (16, 19). Due to its intimate relationship with the extracellular matrix, it is not surprising that the defects in fibulin-1 have a wide-ranging impact. Genetic evidence suggests that fibulin-1 is involved in tissue organization, the maturation and maintenance of blood vessels, and multiple embryonic pathways (16, 20–22).Although it has been established that many of the other components of the extracellular matrix can interact with bacteria, there has been no previous report on the binding of fibulins to bacteria. Our findings indicate that fibulin-1 does bind to streptococci and that SOF is a major streptococcal receptor for fibulin-1.     

Structure and Functional Analysis of RifR, the Type II Thioesterase from the Rifamycin Biosynthetic Pathway

Two thioesterases are commonly found in natural product biosynthetic clusters, a type I thioesterase that is responsible for removing the final product from the biosynthetic complex and a type II thioesterase that is believed to perform housekeeping functions such as removing aberrant units from carrier domains. We present the crystal structure and the kinetic analysis of RifR, a type II thioesterase from the hybrid nonribosomal peptide synthetases/polyketide synthase rifamycin biosynthetic cluster of Amycolatopsis mediterranei. Steady-state kinetics show that RifR has a preference for the hydrolysis of acyl units from the phosphopantetheinyl arm of the acyl carrier domain over the hydrolysis of acyl units from the phosphopantetheinyl arm of acyl-CoAs as well as a modest preference for the decarboxylated substrate mimics acetyl-CoA and propionyl-CoA over malonyl-CoA and methylmalonyl-CoA. Multiple RifR conformations and structural similarities to other thioesterases suggest that movement of a helical lid controls access of substrates to the active site of RifR.Assembly line complexes, which include modular polyketide synthases (PKS)³ and nonribosomal peptide synthetases (NRPS), are multifunctional proteins composed of modules that work in succession to synthesize secondary metabolites, many of which are precursors of potent antibiotics, immunosuppressants, anti-tumor agents, and other bioactive compounds. Rifamycin, the precursor to the anti-tuberculosis drug rifampicin, is produced by the rifamycin assembly line complex, which is an NRPS/PKS hybrid system composed of one NRPS-like and 10 PKS modules (1). Each module in an assembly line complex extends and modifies the intermediate compound before passing it on to the next module in the series (Fig. 1A). The intermediate compounds are covalently attached through a thioester linkage to the phosphopantetheine arm (Ppant) of carrier domains, one associated with each module, until they are released from the synthase, usually by a type I thioesterase (TEI) (2, 3).Open in a separate windowFIGURE 1.Proposed functions of thioesterase II proteins. A, chain elongation by a PKS module. The chain elongation intermediate is transferred from the ACP of the upstream module to the ketosynthase (KS) domain. The acyltransferase (AT) domain transfers an acyl group building block from CoA to the ACP within the module. The KS domain catalyzes condensation of the new building block with the intermediate, releasing CO₂. B, production of a decarboxylated acyl unit by the ketosynthase domain and the subsequent hydrolysis by a TEII. C, mispriming of a PKS by transfer of an acyl-phosphopantetheine arm by a promiscuous phosphopantetheinyl transferase (Pptase) and the subsequent hydrolysis by a TEII. D, hydrolysis of an amino acid derivative by a TEII from an NRPS module comprising an adenylation domain (A) and a peptide carrier protein (PCP) domain.TEIs are usually integrated into the final module of the assembly line complex and remove the final product through macrocyclization or hydrolysis. Occasionally, tandem type I thioesterases are integrated at the C terminus of the final module of NRPS pathways (4).Although TEIs are covalently attached to the terminal module and generally process only the final product of an assembly line complex, type II thioesterases (TEIIs) are discrete proteins that can remove intermediates from any module in the complex. A variety of functions have been attributed to TEIIs, the most prevalent of which is a “housekeeping function,” the removal of aberrant acyl units from carrier domains. These aberrant acyl units may be due to premature decarboxylation by a PKS ketosynthase domain (5) (Fig. 1B) or to mispriming of the carrier domain by a promiscuous phosphopantetheinyl transferase (6–8) (Fig. 1C). Other proposed functions for TEIIs include the removal of intermediates from the synthase as in the case of the mammary gland rat fatty acid synthase (FAS) TEII in lactating rats, which removes medium chain C₈-C₁₂ fatty acids from the ACP domain (9) and the removal of amino acid derivatives from a carrier domain (10–13), allowing these derivatives to be incorporated into the natural product by a later module in the assembly line complex (Fig. 1D).Disruption of the TEI function results in a complete loss of product, whereas disruption of TEII function results in a significant decrease in product yield (30–95%) (4, 14–24). Removal of the TEII from the rifamycin assembly line resulted in a 60% decrease in product yield (25). Neither TEIs nor TEIIs may rescue the disrupted function of the other (6), but a TEII from another pathway may rescue the function of a disrupted TEII (26).Two models have been proposed for the TEII housekeeping function (5). In the high specificity model, the TEII scans the complex and efficiently removes only aberrant acyl units. In the low specificity model, the TEII removes both correct and incorrect acyl units from the Ppant arm at an inefficient rate. Correct acyl units are quickly incorporated into the growing intermediate compound. In contrast, incorrect acyl units stall the assembly line, providing a longer window of opportunity for removal by a TEII. Thus a slow, low specificity enzyme can be effective.TEIIs from different pathways have differing specificities, but general trends include a preference for decarboxylated acyl units over carboxylated acyl units (5, 6, 27), substrates linked to a carrier domain over substrates linked to CoA or the phosphopantetheine mimic N-acetylcysteamine (7, 28), and single amino acids over di- or tri-peptides (6, 7). TEIIs are able to hydrolyze substrates attached to carrier domains from their native pathway as well as other pathways (6, 20, 28).PKS/NRPS/FAS thioesterases belong to the α/β hydrolase family. Structures are reported for seven PKS/NRPS/FAS thioesterases: crystal structures for the TEIs from the pikromycin (PikTE) PKS (29), 6-deoxyerythronolide B (DEBSTE) PKS (30), surfactin NRPS (SrfTE) (31), fengycin NRPS (FenTE) (32), and human fatty acid synthase (hFasTE) (33) systems, and NMR structures for enterobactin TEI (34), and surfactin TEII (35). Like PKS modules, PKS TEIs are dimers. The dimer interface comprises two N-terminal helices that are unique to the PKS TEIs. NRPS TEIs are monomeric, like NRPS modules. The NRPS TEII of surfactin is also monomeric (31). Although the FAS complex is dimeric, the FAS TEI is a monomer (33). All of the TEs have an α-helical insertion after strand β5 that forms a lid over the active site. Additionally, in the PKS TEIs, the N-terminal dimer-forming helices contribute to the lid structure, forming a fixed channel that runs the length of the TE and contains the active site. In contrast, the active site pocket of monomeric NRPS TEIs and TEIIs is flexible; two conformations of the lid and active site pocket were observed in the surfactin TEI (SrfTEI) crystal structure (7), and chemical shift observations suggested greater flexibility for residues of the lid region in the surfactin TEII (SrfTEII) solution structure (35). These movements seem to be of functional importance, because a movement of a linker peptide in SrfTEI determines the shape of the active site pocket and a movement of the first lid helix appears to modulate access to the active site (31).We report the structure and activity of recombinant RifR, the TEII of the rifamycin biosynthetic cluster. Steady-state kinetic analysis of the hydrolytic activity of RifR on a wide range of acyl-CoA and acyl-ACP substrates demonstrates that acyl-ACP substrates are preferred over the acyl-CoAs. Aberrant, decarboxylated acyl units are processed more efficiently than are the natural rifamycin building blocks. We report the crystal structure of RifR, the first for any hybrid PKS/NRPS TEII. The size and shape of the substrate chamber are variable, because one of the elements forming the chamber, an extended linker segment, is highly flexible, and different crystal forms reveal different shapes for the substrate binding site. Access to the active site is severely restricted, and structural comparisons with other thioesterases suggest that a conformational change in the lid and the flexible linker region is required for access to the substrate pocket.     

RamA, a Protein Required for Reductive Activation of Corrinoid-dependent Methylamine Methyltransferase Reactions in Methanogenic Archaea

Archaeal methane formation from methylamines is initiated by distinct methyltransferases with specificity for monomethylamine, dimethylamine, or trimethylamine. Each methylamine methyltransferase methylates a cognate corrinoid protein, which is subsequently demethylated by a second methyltransferase to form methyl-coenzyme M, the direct methane precursor. Methylation of the corrinoid protein requires reduction of the central cobalt to the highly reducing and nucleophilic Co(I) state. RamA, a 60-kDa monomeric iron-sulfur protein, was isolated from Methanosarcina barkeri and is required for in vitro ATP-dependent reductive activation of methylamine:CoM methyl transfer from all three methylamines. In the absence of the methyltransferases, highly purified RamA was shown to mediate the ATP-dependent reductive activation of Co(II) corrinoid to the Co(I) state for the monomethylamine corrinoid protein, MtmC. The ramA gene is located near a cluster of genes required for monomethylamine methyltransferase activity, including MtbA, the methylamine-specific CoM methylase and the pyl operon required for co-translational insertion of pyrrolysine into the active site of methylamine methyltransferases. RamA possesses a C-terminal ferredoxin-like domain capable of binding two tetranuclear iron-sulfur proteins. Mutliple ramA homologs were identified in genomes of methanogenic Archaea, often encoded near methyltrophic methyltransferase genes. RamA homologs are also encoded in a diverse selection of bacterial genomes, often located near genes for corrinoid-dependent methyltransferases. These results suggest that RamA mediates reductive activation of corrinoid proteins and that it is the first functional archetype of COG3894, a family of redox proteins of unknown function.Most methanogenic Archaea are capable of producing methane only from carbon dioxide. The Methanosarcinaceae are a notable exception as representatives are capable of methylotrophic methanogenesis from methylated amines, methylated thiols, or methanol. Methanogenesis from these substrates requires methylation of 2-mercaptoethanesulfonic acid (coenzyme M or CoM) that is subsequently used by methylreductase to generate methane and a mixed disulfide whose reduction leads to energy conservation (1–4).Methylation of CoM with trimethylamine (TMA),⁴ dimethylamine (DMA), or monomethylamine (MMA) is initiated by three distinct methyltransferases that methylate cognate corrinoid-binding proteins (3). MtmB, the MMA methyltransferase, specifically methylates cognate corrinoid protein, MtmC, with MMA (see Fig. 1) (5, 6). The DMA methyltransferase, MtbB, and its cognate corrinoid protein, MtbC, interact specifically to demethylate DMA (7, 8). TMA is demethylated by the TMA methyltransferase (MttB) in conjunction with the TMA corrinoid protein (MttC) (8, 9). Each of the methylated corrinoid proteins is a substrate for a methylcobamide:CoM methyltransferase, MtbA, which produces methyl-CoM (10–12).Open in a separate windowFIGURE 1.MMA:CoM methyl transfer. A schematic of the reactions catalyzed by MtmB, MtmC, and MtbA is shown that emphasizes the key role of MtmC in the catalytic cycle of both methyltransferases. Oxidation to Co(II)-MtmC of the supernucleophilic Co(I)-MtmC catalytic intermediate inactivates methyl transfer from MMA to the thiolate of coenzyme M (HSCoM). In vitro reduction of the Co(II)-MtmC with either methyl viologen reduced to the neutral species or with RamA in an ATP-dependent reaction can regenerate the Co(I) species. In either case in vitro Ti(III)-citrate is the ultimate source of reducing power.CoM methylation with methanol requires the methyltransferase MtaB and the corrinoid protein MtaC, which is then demethylated by another methylcobamide:CoM methyltransferase, MtaA (13–15). The methylation of CoM with methylated thiols such as dimethyl sulfide in Methanosarcina barkeri is catalyzed by a corrinoid protein that is methylated by dimethyl sulfide and demethylated by CoM, but in this case an associated CoM methylase carries out both methylation reactions (16).In bacteria, analogous methyltransferase systems relying on small corrinoid proteins are used to achieve methylation of tetrahydrofolate. In Methylobacterium spp., CmuA, a single methyltransferase with a corrinoid binding domain, along with a separate pterin methylase, effect the methylation of tetrahydrofolate with chloromethane (17, 18). In Acetobacterium dehalogenans and Moorella thermoacetica various three-component systems exist for specific demethylation of different phenylmethyl ethers, such as vanillate (19) and veratrol (20), again for the methylation of tetrahydrofolate. Sequencing of the genes encoding the corrinoid proteins central to the archaeal and bacterial methylotrophic pathways revealed they are close homologs. Furthermore, genes predicted to encode such corrinoid proteins and pterin methyltransferases are widespread in bacterial genomes, often without demonstrated metabolic function. All of these corrinoid proteins are similar to the well characterized cobalamin binding domain of methionine synthase (21, 22).In contrast, the TMA, DMA, MMA, and methanol methyltransferases are not homologous proteins. The methylamine methyltransferases do share the common distinction of having in-frame amber codons (6, 8) within their encoding genes that corresponds to the genetically encoded amino acid pyrrolysine (23–25). Pyrrolysine has been proposed to act in presenting a methylammonium adduct to the central cobalt ion of the corrinoid protein for methyl transfer (3, 23, 26). However, nucleophilic attack on a methyl donor requires the central cobalt ion of a corrinoid cofactor is in the nucleophilic Co(I) state rather than the inactive Co(II) state (27). Subsequent demethylation of the methyl-Co(III) corrinoid cofactor regenerates the nucleophilic Co(I) cofactor. The Co(I)/Co(II) in the cobalamin binding domain of methionine synthase has an E_m value of -525 mV at pH 7.5 (28). It is likely to be similarly low in the homologous methyltrophic corrinoid proteins. These low redox potentials make the corrinoid cofactor subject to adventitious oxidation to the inactive Co(II) state (Fig. 1).During isolation, these corrinoid proteins are usually recovered in a mixture of Co(II) or hydroxy-Co(III) states. For in vitro studies, chemical reduction can maintain the corrinoid protein in the active Co(I) form. The methanol:CoM or the phenylmethyl ether:tetrahydrofolate methyltransferase systems can be activated in vitro by the addition of Ti(III) alone as an artificial reductant (14, 19). In contrast, activation of the methylamine corrinoid proteins further requires the addition of methyl viologen as a redox mediator. Ti(III) reduces methyl viologen to the extremely low potential neutral species. In vitro activation with these agents does not require ATP (5, 7, 9).Cellular mechanisms also exist to achieve the reductive activation of corrinoid cofactors in methyltransferase systems. Activation of human methionine synthase involves reduction of the co(II)balamin by methionine synthase reductase (29), whereas the Escherichia coli enzyme requires flavodoxin (30). The endergonic reduction is coupled with the exergonic methylation of the corrinoid with S-adenosylmethionine (27). An activation system exists in cellular extracts of A. dehalogenans that can activate the veratrol:tetrahydrofolate three-component system and catalyze the direct reduction of the veratrol-specific corrinoid protein to the Co(I) state; however, the activating protein has not been purified (31).For the methanogen methylamine and methanol methyltransferase systems, an activation process is readily detectable in cell extracts that is ATP- and hydrogen-dependent (32, 33). Daas et al. (34, 35) examined the activation of the methanol methyltransferase system in M. barkeri and purified in low yield a methyltransferase activation protein (MAP) which in the presence of a preparation of hydrogenase and uncharacterized proteins was required for ATP-dependent reductive activation of methanol:CoM methyl transfer. MAP was found to be a heterodimeric protein without a UV-visible detectable prosthetic group. Unfortunately, no protein sequence has been reported for MAP, leaving the identity of the gene in question. The same MAP protein was also suggested to activate methylamine:CoM methyl transfer, but this suggestion was based on results with crude protein fractions containing many cellular proteins other than MAP (36).Here we report of the identification and purification to near-homogeneity of RamA (reductive activation of methyltransfer, amines), a protein mediating activation of methylamine:CoM methyl transfer in a highly purified system (Fig. 1). Quite unlike MAP, which was reported to lack prosthetic groups, RamA is an iron-sulfur protein that can catalyze reduction of a corrinoid protein such as MtmC to the Co(I) state in an ATP-dependent reaction (Fig. 1). Peptide mapping of RamA allowed identification of the gene encoding RamA and its homologs in the genomes of Methanosarcina spp. RamA belongs to COG3894, a group of uncharacterized metal-binding proteins found in a number of genomes. RamA, thus, provides a functional example for a family of proteins widespread among bacteria and Archaea whose physiological role had been largely unknown.     

Characterization of the Tautomycin Biosynthetic Gene Cluster from Streptomyces spiroverticillatus Unveiling New Insights into Dialkylmaleic Anhydride and Polyketide Biosynthesis

Tautomycin (TTM) is a highly potent and specific protein phosphatase inhibitor isolated from Streptomyces spiroverticillatus. The biological activity of TTM makes it an important lead for drug discovery, whereas its spiroketal-containing polyketide chain and rare dialkylmaleic anhydride moiety draw attention to novel biosynthetic chemistries responsible for its production. To elucidate the biosynthetic machinery associated with these novel molecular features, the ttm biosynthetic gene cluster from S. spiroverticillatus was isolated and characterized, and its involvement in TTM biosynthesis was confirmed by gene inactivation and complementation experiments. The ttm cluster was localized to a 86-kb DNA region, consisting of 20 open reading frames that encode three modular type I polyketide synthases (TtmHIJ), one type II thioesterase (TtmT), five proteins for methoxymalonyl-S-acyl carrier protein biosynthesis (Ttm-ABCDE), eight proteins for dialkylmaleic anhydride biosynthesis and regulation (TtmKLMNOPRS), as well as two additional regulatory proteins (TtmF and TtmQ) and one tailoring enzyme (TtmG). A model for TTM biosynthesis is proposed based on functional assignments from sequence analysis, which agrees well with previous feeding experiments, and has been further supported by in vivo gene inactivation experiments. These findings set the stage to fully investigate TTM biosynthesis and to biosynthetically engineer new TTM analogs.Tautomycin (TTM)² is a polyketide natural product first isolated in 1987 from Streptomyces spiroverticillatus (1). The structure and stereochemistry of TTM were established on the basis of chemical degradation and spectroscopic evidence (2-4). TTM contains several features not common to polyketide natural products, including a spiroketal group, a methoxymalonate-derived unit, and an acyl chain bearing a dialkylmaleic anhydride moiety. Structurally related to TTM is tautomycetin (TTN), which was first isolated in 1989 from Streptomyces griseochromogenes following the discovery of TTM (5, 6). The structure of TTN was deduced by chemical degradation and spectroscopic analysis (6), and its stereochemistry was established by comparison of spectral data with those of TTN degradation products and synthetic fragments (7). Both TTM and TTN exist as tautomeric mixtures composed of two interconverting anhydride and diacid forms in approximately a 5:4 ratio under neutral conditions (Fig. 1A) (1, 2).Open in a separate windowFIGURE 1.A, structures of TTM and TTN in anhydride or diacid forms, and biosynthetic origin of the dialkylmaleic anhydride by feeding experiments using ¹³C-labeled acetate and propionate. The methoxymalonate-derived unit in TTM is highlighted by the dotted oval. R, polyketide moiety of TTM or TTN. B, selected natural product inhibitors of PP-1 and PP-2A featuring a spiroketal or dialkylmaleric anhydride moiety. C, selected natural products containing a dialkylmaleic anhydride moiety.Early studies of TTM revealed its ability to induce morphological changes in leukemia cells (8). However, it was later realized that TTM is a potent and specific inhibitor of protein phosphatases (PPs) PP-1 and PP-2A (9). PP-1 and PP-2A are two of the four major serine/threonine protein phosphatases that regulate diverse cellular events such as cell division, gene expression, muscle contraction, glycogen metabolism, and neuronal signaling in eukaryotic cells (10-12). Many natural product PP-1 and PP-2A inhibitors are known, including okadaic acid (13), calyculin-A (14), phoslactomycin, spirastrellolide, and cantharidin (15) (Fig. 1B), as well as TTM (16, 17), and TTN (18). They have served as useful tools to study PP-involved intracellular events in vivo and as novel leads for drug discovery (10-12). Among these PP inhibitors, TTM and TTN are unique because of their PP-1 selectivity. Despite their structural similarities, TTM exhibits potent specific inhibition of PP-1 and PP-2A with IC₅₀ values of 22-32 nm and only a slight preference for PP-1 (18). Conversely, TTN shows nearly a 40-fold higher binding affinity to PP-1 (IC₅₀ = 1.6 nm) than to PP-2A (IC₅₀ = 62 nm) (18). Because the major structural differences between TTM and TTN reside in the region distal to the dialkylmaleic anhydride moiety (Fig. 1A), it has been proposed that differences in these moieties might be responsible for the PP-1 selectivity (17-19). Finally, TTN also has an impressive immuno-suppressive activity (20, 21), which is apparently devoid for TTM. Clearly, the structural differences between these two polyketides translate into large, exploitable differences in bio-activities, yet an understanding of the biosynthetic origins of these differences remains elusive.The spiroketal and dialkylmaleic anhydride features of TTM are uncommon for polyketide natural products, as is the methoxymalonate-derived unit (Fig. 1A). Few studies have been carried out for spiroketal biosynthesis, yet it is reasonably common among the phosphatase inhibitors such as calyculin A, okadaic acid, and a few others (Fig. 1B). Less common, but still found in the phosphatase inhibitor cantharidin, as well as TTM and TTN, is the dialkylmaleic anhydride moiety (Fig. 1B); this unit appears in a number of other natural products (Fig. 1C), although the biosynthetic steps leading to this reactive moiety (a protected version of a dicarboxylate) have not been rigorously investigated. Feeding experiments with ¹³C-labeled precursors indicated that the anhydride of TTM and TTN is assembled from a propionate and an as yet undefined C-5 unit (Fig. 1A), which would require novel chemistry for polyketide biosynthesis (22). TTM differentiates itself from all known PP-1 and PP-2A inhibitors by virtue of its unique combination of both the dialkymaleic anhydride and spiroketal functionalities.Multiple total syntheses of TTM and a small number of analogs have been reported, confirming the predicted structure and absolute stereochemistry and facilitating structure-activity relationship studies on PP inhibition and apoptosis induction (19, 23-25). These studies revealed that: (i) the C22-C26 carbon chain and the dialkylmaleic anhydride are the minimum requirements for TTM bioactivity; (ii) the C18-C21 carbon chain and 22-hydroxy group are important for PP inhibition; (iii) the spiroketal moiety determines the affinity to specific protein phosphatases; (iv) the active form is most likely the dicarboxylate; and (v) 3′-epi-TTM exhibits 1,000-fold less activity than TTM. However, taken as a whole, none of the analogs had an improved potency or selectivity for PP-1 inhibition than the natural TTM (19, 22-25). As a result, a more specific inhibitor of PP-1 is urgently awaited to differentiate the physiological roles of PP-1 and PP-2A in vivo and to explore PPs as therapeutic targets for drug discovery.We have undertaken the cloning and characterization of the TTM biosynthetic gene cluster from S. spiroverticillatus as the first step toward engineering TTM biosynthesis for novel analogs (26). We report here: (i) cloning and sequencing of the complete ttm gene cluster, (ii) determination of the ttm gene cluster boundaries, (iii) bioinformatics analysis of the ttm cluster and a proposal for TTM biosynthesis, and (iv) genetic characterization of the TTM pathway to support the proposed pathway. Of particular interest has been the identification of genes possibly related to dialkylmaleic anhydride biosynthesis, the unveiling of the ttm polyketide synthase (PKS) genes predicted to select and incorporate four different starter and extender units for TTM production, and the apparent lack of candidate genes associated with spiroketal formation. These findings now set the stage to engineer TTM analogs for novel PP-1- and PP-2A-specific inhibitors by applying combinatorial biosynthetic methods to the TTM biosynthetic machinery.     

Involvement of All-trans-retinal in Acute Light-induced Retinopathy of Mice

Exposure to bright light can cause visual dysfunction and retinal photoreceptor damage in humans and experimental animals, but the mechanism(s) remain unclear. We investigated whether the retinoid cycle (i.e. the series of biochemical reactions required for vision through continuous generation of 11-cis-retinal and clearance of all-trans-retinal, respectively) might be involved. Previously, we reported that mice lacking two enzymes responsible for clearing all-trans-retinal, namely photoreceptor-specific ABCA4 (ATP-binding cassette transporter 4) and RDH8 (retinol dehydrogenase 8), manifested retinal abnormalities exacerbated by light and associated with accumulation of diretinoid-pyridinium-ethanolamine (A2E), a condensation product of all-trans-retinal and a surrogate marker for toxic retinoids. Now we show that these mice develop an acute, light-induced retinopathy. However, cross-breeding these animals with lecithin:retinol acyltransferase knock-out mice lacking retinoids within the eye produced progeny that did not exhibit such light-induced retinopathy until gavaged with the artificial chromophore, 9-cis-retinal. No significant ocular accumulation of A2E occurred under these conditions. These results indicate that this acute light-induced retinopathy requires the presence of free all-trans-retinal and not, as generally believed, A2E or other retinoid condensation products. Evidence is presented that the mechanism of toxicity may include plasma membrane permeability and mitochondrial poisoning that lead to caspase activation and mitochondria-associated cell death. These findings further understanding of the mechanisms involved in light-induced retinal degeneration.The retinoid cycle is a fundamental metabolic process in the vertebrate retina responsible for continuous generation of 11-cis-retinal from its all-trans-isomer (1-3). Because 11-cis-retinal is the chromophore of rhodopsin and cone visual pigments (4), disabling mutations in genes encoding proteins of the retinoid cycle can cause a spectrum of retinal diseases affecting sight (3). Moreover, the efficiency of the mammalian visual system and health of photoreceptors and retinal pigment epithelium (RPE)² decrease significantly with age. Even in the presence of a functional retinoid cycle, A2E, retinal dimer (RALdi), and other toxic all-trans-retinal condensation products (5-7) can accumulate as a consequence of aging (8). Under experimental conditions, these compounds can produce toxic effects on RPE cells (9-11). Patients affected by age-related macular degeneration, Stargardt disease, or other retinal diseases associated with accumulation of surrogate markers, such as A2E, all develop retinal degeneration (12). Thus, elucidating the fundamental causes of these age-dependent changes is of increasing importance. Encouragingly, our understanding of both retinoid metabolism outside the eye and production of 11-cis-retinal unique to the eye has accelerated recently (Scheme 1) (1-3), and genetic mouse models are readily available to study these processes and their potential aberrations in vivo (13). Thus, a central question can be addressed, namely what initiates the death of photoreceptor cells and the underlining RPE?Open in a separate windowSCHEME 1.Retinoid flow and all-trans-retinal clearance in the visual cycle. After diffusion from the RPE, the visual chromophore, 11-cis-retinal, combines with rhodopsin and then is photoisomerized to all-trans-retinal. Most of the all-trans-retinal dissociates from opsin into the cytoplasm, where it is reduced to all-trans-retinol by RDHs, including RDH8. The fraction of all-trans-retinal that dissociates into the disc lumen is transported by ABCA4 into the cytoplasm (23) before it is reduced. All-trans-retinol then is translocated to the RPE, esterified by LRAT, and recycled back to 11-cis-retinal. Mutations of ABCA4 are associated with human macular degeneration, Stargardt disease, and age-related macular degeneration (55, 56).Several mechanisms associated with retinoid metabolism may contribute to different retinopathies (1). For example, lack of retinoids in LRAT (lecithin:retinol acyltransferase) or chromophore in retinoid isomerase knock-out (Rpe65^-/-) mice leads to rapid degeneration of cone photoreceptors and slowly progressive death of rods (14). Such mice do not produce toxic condensation products from all-trans-retinal. Instead, their retinopathies have been attributed to continuous activation of visual phototransduction (15) due to either the basal activity of opsin (16-18) or disordered vectorial transport of cone visual pigments without bound chromophore (19). Paradoxically, an abnormally high flux of retinoids through the retinoid cycle can also lead to retinopathy in other mouse models (20, 21). Animal models featuring anomalies in the retinoid cycle illustrate the importance of chromophore regeneration and provide an approach to elucidating mechanisms involved in human retinal dysfunction and disease.Recently, we showed that mice carrying a double knock-out of Rdh8 (retinol dehydrogenase 8), one of the main enzymes that reduces all-trans-retinal in rod and cone outer segments (22), and Abca4 (ATP-binding cassette transporter 4), which transports all-trans-retinal from the inside to the outside of disc membranes (23), rapidly accumulate all-trans-retinal condensation products and exhibit accentuated RPE/photoreceptor dystrophy at an early age (24). Although these studies suggest retinoid toxicity, it is still unclear if the elevated levels of retinal and/or its condensation products, such as A2E, are the cause of this retinopathy or merely a nonspecific reflection of impaired retinoid metabolism. Here, we report that spent chromophore, all-trans-retinal, is most likely responsible for photoreceptor degeneration in Rdh8^-/-Abca4^-/- mice. Toxic effects of all-trans-retinal include caspase activation and mitochondria-associated cell death.     

Bortezomib Overcomes Tumor Necrosis Factor-related Apoptosis-inducing Ligand Resistance in Hepatocellular Carcinoma Cells in Part through the Inhibition of the Phosphatidylinositol 3-Kinase/Akt Pathway

Hepatocellular carcinoma (HCC) is one of the most common and aggressive human malignancies. Recombinant tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anti-tumor agent. However, many HCC cells show resistance to TRAIL-induced apoptosis. In this study, we showed that bortezomib, a proteasome inhibitor, overcame TRAIL resistance in HCC cells, including Huh-7, Hep3B, and Sk-Hep1. The combination of bortezomib and TRAIL restored the sensitivity of HCC cells to TRAIL-induced apoptosis. Comparing the molecular change in HCC cells treated with these agents, we found that down-regulation of phospho-Akt (P-Akt) played a key role in mediating TRAIL sensitization of bortezomib. The first evidence was that bortezomib down-regulated P-Akt in a dose- and time-dependent manner in TRAIL-treated HCC cells. Second, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, a PI3K inhibitor, also sensitized resistant HCC cells to TRAIL-induced apoptosis. Third, knocking down Akt1 by small interference RNA also enhanced TRAIL-induced apoptosis in Huh-7 cells. Finally, ectopic expression of mutant Akt (constitutive active) in HCC cells abolished TRAIL sensitization effect of bortezomib. Moreover, okadaic acid, a protein phosphatase 2A (PP2A) inhibitor, reversed down-regulation of P-Akt in bortezomib-treated cells, and PP2A knockdown by small interference RNA also reduced apoptosis induced by the combination of TRAIL and bortezomib, indicating that PP2A may be important in mediating the effect of bortezomib on TRAIL sensitization. Together, bortezomib overcame TRAIL resistance at clinically achievable concentrations in hepatocellular carcinoma cells, and this effect is mediated at least partly via inhibition of the PI3K/Akt pathway.Hepatocellular carcinoma (HCC)² is currently the fifth most common solid tumor worldwide and the fourth leading cause of cancer-related death. To date, surgery is still the only curative treatment but is only feasible in a small portion of patients (1). Drug treatment is the major therapy for patients with advanced stage disease. Unfortunately, the response rate to traditional chemotherapy for HCC patients is unsatisfactory (1). Novel pharmacological therapy is urgently needed for patients with advanced HCC. In this regard, the approval of sorafenib might open a new era of molecularly targeted therapy in the treatment of HCC patients.Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a type II transmembrane protein and a member of the TNF family, is a promising anti-tumor agent under clinical investigation (2). TRAIL functions by engaging its receptors expressed on the surface of target cells. Five receptors specific for TRAIL have been identified, including DR4/TRAIL-R1, DR5/TRAIL-R2, DcR1, DcR2, and osteoprotegerin. Among TRAIL receptors, only DR4 and DR5 contain an effective death domain that is essential to formation of death-inducing signaling complex (DISC), a critical step for TRAIL-induced apoptosis. Notably, the trimerization of the death domains recruits an adaptor molecule, Fas-associated protein with death domain (FADD), which subsequently recruits and activates caspase-8. In type I cells, activation of caspase-8 is sufficient to activate caspase-3 to induce apoptosis; however, in another type of cells (type II), the intrinsic mitochondrial pathway is essential for apoptosis characterized by cleavage of Bid and release of cytochrome c from mitochondria, which subsequently activates caspase-9 and caspase-3 (3).Although TRAIL induces apoptosis in malignant cells but sparing normal cells, some tumor cells are resistant to TRAIL-induced apoptosis. Mechanisms responsible for the resistance include receptors and intracellular resistance. Although the cell surface expression of DR4 or DR5 is absolutely required for TRAIL-induced apoptosis, tumor cells expressing these death receptors are not always sensitive to TRAIL due to intracellular mechanisms. For example, the cellular FLICE-inhibitory protein (c-FLIP), a homologue to caspase-8 but without protease activity, has been linked to TRAIL resistance in several studies (4, 5). In addition, inactivation of Bax, a proapoptotic Bcl-2 family protein, resulted in resistance to TRAIL in MMR-deficient tumors (6, 7), and reintroduction of Bax into Bax-deficient cells restored TRAIL sensitivity (8), indicating that the Bcl-2 family plays a critical role in intracellular mechanisms for resistance of TRAIL.Bortezomib, a proteasome inhibitor approved clinically for multiple myeloma and mantle cell lymphoma, has been investigated intensively for many types of cancer (9). Accumulating studies indicate that the combination of bortezomib and TRAIL overcomes the resistance to TRAIL in various types of cancer, including acute myeloid leukemia (4), lymphoma (10–13), prostate (14–17), colon (15, 18, 19), bladder (14, 16), renal cell carcinoma (20), thyroid (21), ovary (22), non-small cell lung (23, 24), sarcoma (25), and HCC (26, 27). Molecular targets responsible for the sensitizing effect of bortezomib on TRAIL-induced cell death include DR4 (14, 27), DR5 (14, 20, 22–23, 28), c-FLIP (4, 11, 21–23, 29), NF-κB (12, 24, 30), p21 (16, 21, 25), and p27 (25). In addition, Bcl-2 family also plays a role in the combinational effect of bortezomib and TRAIL, including Bcl-2 (10, 21), Bax (13, 22), Bak (27), Bcl-xL (21), Bik (18), and Bim (15).Recently, we have reported that Akt signaling is a major molecular determinant in bortezomib-induced apoptosis in HCC cells (31). In this study, we demonstrated that bortezomib overcame TRAIL resistance in HCC cells through inhibition of the PI3K/Akt pathway.     

Combining RNA Interference Mutants and Comparative Proteomics to Identify Protein Components and Dependences in a Eukaryotic Flagellum

Eukaryotic flagella from organisms such as Trypanosoma brucei can be isolated and their protein components identified by mass spectrometry. Here we used a comparative approach utilizing two-dimensional difference gel electrophoresis and isobaric tags for relative and absolute quantitation to reveal protein components of flagellar structures via ablation by inducible RNA interference mutation. By this approach we identified 20 novel components of the paraflagellar rod (PFR). Using epitope tagging we validated a subset of these as being present within the PFR by immunofluorescence. Bioinformatic analysis of the PFR cohort reveals a likely calcium/calmodulin regulatory/signaling linkage between some components. We extended the RNA interference mutant/comparative proteomic analysis to individual novel components of our PFR proteome, showing that the approach has the power to reveal dependences between subgroups within the cohort.The eukaryotic cilium/flagellum is a multifunctional organelle involved in an array of biological processes ranging from cell motility to cell signaling. Many cells in the human body, across a range of tissues and organs, produce either single or multiple, motile or nonmotile cilia where they perform diverse biological processes essential for maintaining human health. This diversity of function is reflected in an equally diverse range of pathologies and syndromes that result from ciliary/flagellar dysfunction via inherited mutations. This diversity is a reflection of the molecular complexity, both in components and in protein interactions of this organelle (1, 2).The canonical eukaryotic flagellum displays a characteristic “9 + 2” microtubular profile, where nine outer doublet microtubules encircle two singlet central pair microtubules, an arrangement found in organisms as diverse as trypanosomes, green algae, and mammals. Although this 9 + 2 microtubule arrangement has been highly conserved through eukaryotic evolution, there are examples where this standard layout has been modified, including the “9 + 0” layout of primary cilia and the “9 + 9 + 2” of many insect sperm flagella. In addition to this highly conserved 9 + 2 microtubule structure, flagella and cilia show a vast range of discrete substructures, such as the inner and outer dynein arms, nexin links, radial spokes, bipartite bridges, beak-like projections, ponticuli, and other microtubule elaborations that are essential for cilium/flagellum function. Cilia and flagella can also exhibit various extra-axonemal elaborations, and although these are often restricted to specific lineages, there is evidence that some functions, such as metabolic specialization, provided by these diverse structures are conserved (3, 4). Examples of such extraaxonemal elaborations include the fibrous or rod-like structures in the flagellum of the parasite Giardia lamblia (5), kinetoplastid protozoa (6, 7), and mammalian sperm flagella, along with extra sheaths of microtubules in insect sperm flagella (8).Several recent studies have set out to determine the protein composition of the flagellum and demonstrated the existence of both an evolutionarily conserved core of flagellum/cilium proteins and a large number of lineage-restricted components (9–13). Although these approaches provide an invaluable catalogue of the protein components of the flagellum, they provide only limited information on the substructural localization of proteins and do not address either the likely protein-protein interactions or the function of these proteins within the flagellum. To address these issues, the protein composition of some axonemal substructures (radial spoke complexes; for example see Ref. 14) has been determined by direct isolation of these structures, and a number of complexes have been resolved by the use of co-immunoprecipitation of indicator proteins (for example see Refs. 15 and 16). In addition the localization and function of a number of flagellar proteins have been investigated by detailed analysis of mutant cell lines (particularly of Chlamydomonas reinhardtii) that exhibit defined structural defects within the assembled axoneme. Early studies employed two-dimensional PAGE to compare the proteomic profile of purified flagella derived from C. reinhardtii mutants and wild type cells (17–22) that showed numerous proteomic differences in the derived profiles. The available technology did not allow identification of the individual proteins within the profiles. Recent proteomic advances offer the opportunity for this identification. For instance the comparative proteomic technique isotope coded affinity tagging has been used to identify components of the outer dynein arm (23). This technique utilizes stable isotope tagging to quantify the relative concentration of proteins between two samples.Trypanosomatids are important protozoan parasites whose flagellum is a critical organelle for their cell biology and pathogenicity. Their experimental tractability also provides opportunities for generic insights to the eukaryotic flagellum. They are responsible for a number of devastating diseases of humans and other mammals, including commercially important livestock, in some of the poorest areas of the world (24–26). All kinetoplastids build a flagellum that contains an extra-axonemal structure termed the paraflagellar rod (PFR).³ In the case of the African trypanosome Trypanosoma brucei brucei, this consists of a complex subdomain organization of a proximal, intermediate, and distal domain as well as links to specific doublets of the axoneme and a structure known as the flagellum attachment zone (FAZ) by which the flagellum is attached to the cell body for much of its length (6, 7). The PFR is required for cell motility (27, 28) and serves as a scaffold for metabolic and signaling enzymes (3, 29, 30). We have previously shown that the presence of this structure is essential for the survival of the mammalian bloodstream form of the parasite both in vitro (in culture) (12) and in vivo (in mice) (31) as part of a wider requirement for motility in this life cycle stage (12, 32, 33).Two major protein components of the PFR (PFR1 and PFR2) have been identified (34–38) along with several minor PFR protein components (3, 29, 30, 39–43). The availability of RNAi techniques in T. brucei allowed the generation of the inducible mutant cell line snl2 (44), in which RNAi-mediated ablation of the PFR2 protein causes the specific loss of both the distal and intermediate PFR subdomains (see Fig. 1A). After RNAi induction cells become paralyzed but remain viable (44). Our laboratory (3) has previously identified two PFR-specific adenylate kinases by comparing two-dimensional SDS-PAGE gels of purified flagella from induced and noninduced snl2 cells. These proteins cannot be incorporated into the PFR after PFR2 ablation.Open in a separate windowFIGURE 1.A, electron microscopy images (prepared as described previously (12)) of T. brucei snl2 noninduced and RNAi-induced flagellar transverse sections shows the loss of a large part of the PFR structure. Bar, 100 nm. B, frequencies (resolution 0.25) of log₂ protein abundance ratios of noninduced to noninduced samples from quadruplex iTRAQ. C, averaged frequencies (resolution 0.25) of log₂ protein abundance ratios of induced to noninduced samples from quadruplex iTRAQ. D, log₂ protein abundance ratios of induced to noninduced samples from all iTRAQ experiments for all proteins that show at least a 2-fold decrease after RNAi induction of snl2. α- and β-tubulin show a less than 2-fold change as expected. The results of individual sample pairs are graphed separately as per key.The ability to ablate PFR2 and hence disable assembly of a major portion of the PFR affords an opportunity to apply advanced proteomic approaches to identify additional PFR proteins. In this present study we have used two complementary proteomic approaches, two-dimensional fluorescence difference gel electrophoresis (DIGE) (45) and isobaric tags for relative and absolute quantitation (iTRAQ; Applied Biosystems), to investigate PFR+ and PFR–flagella to define 30 components of these two PFR subdomains. We have also conducted a bioinformatic analysis of amino acid motifs present in this protein cohort to gain insights into the possible functions of novel proteins and used epitope tagging approaches to confirm the PFR localization of a test set of identified proteins. We then asked whether it was possible to combine comparative proteomics with further analysis of RNAi mutant trypanosomes to provide detailed information on the individual interactions and assembly dependences within the novel PFR components we had identified. By iterating the subtractive proteomic analysis with novel putative PFR proteins, we were able to reveal the existence of distinct PFR protein dependence relationships and provide intriguing new insight into regulatory processes potentially operating within the trypanosome flagellum. Finally, this study establishes the mutant/proteomic combination as a powerful enabling approach for revealing dependences within subcohorts of the flagellar proteome.     

Functional UDP-xylose Transport across the Endoplasmic Reticulum/Golgi Membrane in a Chinese Hamster Ovary Cell Mutant Defective in UDP-xylose Synthase

In mammals, xylose is found as the first sugar residue of the tetrasaccharide GlcAβ1-3Galβ1-3Galβ1-4Xylβ1-O-Ser, initiating the formation of the glycosaminoglycans heparin/heparan sulfate and chondroitin/dermatan sulfate. It is also found in the trisaccharide Xylα1-3Xylα1-3Glcβ1-O-Ser on epidermal growth factor repeats of proteins, such as Notch. UDP-xylose synthase (UXS), which catalyzes the formation of the UDP-xylose substrate for the different xylosyltransferases through decarboxylation of UDP-glucuronic acid, resides in the endoplasmic reticulum and/or Golgi lumen. Since xylosylation takes place in these organelles, no obvious requirement exists for membrane transport of UDP-xylose. However, UDP-xylose transport across isolated Golgi membranes has been documented, and we recently succeeded with the cloning of a human UDP-xylose transporter (SLC25B4). Here we provide new evidence for a functional role of UDP-xylose transport by characterization of a new Chinese hamster ovary cell mutant, designated pgsI-208, that lacks UXS activity. The mutant fails to initiate glycosaminoglycan synthesis and is not capable of xylosylating Notch. Complementation was achieved by expression of a cytoplasmic variant of UXS, which proves the existence of a functional Golgi UDP-xylose transporter. A ∼200 fold increase of UDP-glucuronic acid occurred in pgsI-208 cells, demonstrating a lack of UDP-xylose-mediated control of the cytoplasmically localized UDP-glucose dehydrogenase in the mutant. The data presented in this study suggest the bidirectional transport of UDP-xylose across endoplasmic reticulum/Golgi membranes and its role in controlling homeostasis of UDP-glucuronic acid and UDP-xylose production.Xylose is only known to occur in two different mammalian glycans. First, xylose is the starting sugar residue of the common tetrasaccharide, GlcAβ1,3Galβ1,3Galβ1,4Xylβ1-O-Ser, attached to proteoglycan core proteins to initiate the biosynthesis of glycosaminoglycans (GAGs)² (1). Second, xylose is found in the trisaccharide Xylα1,3Xylα1,3Glcβ1-O-Ser in epidermal growth factor (EGF)-like repeats of proteins, such as blood coagulation factors VII and IX (2) and Notch (3) (Fig. 1). Two variants of O-xylosyltransferases (XylT1 and XylT2) are responsible for the initiation of glycosaminoglycan biosynthesis, which differ in terms of acceptor specificity and tissue distribution (4-7), and two different enzymatic activities have been identified that catalyze xylosylation of O-glucose residues added to EGF repeats (8-10). On Notch, O-glucose occurs on EGF repeats in a similar fashion as O-fucose, which modifications have been shown to influence ligand-mediated Notch signaling (11-16). Recently, rumi, the gene encoding the Notch O-glucosyltransferase in Drosophila, has been identified, and inactivation of the gene was found to cause a temperature-sensitive Notch phenotype (17). Although this finding clearly demonstrated that O-glucosylation is essential for Notch signaling, the importance of xylosylation for Notch functions remains ambiguous.Open in a separate windowFIGURE 1.UDP-xylose metabolism in mammalian cells. A, UDP-Xyl is synthesized in two steps from UDP-Glc by the enzymes UGDH, forming UDP-GlcA, and UXS, also referred to as UDP-glucuronic acid decarboxylase. UGDH is inhibited by the product of the second enzyme, UDP-Xyl (42). B, in mammals, UDP-Xyl is synthesized within the lumen of the ER/Golgi, where it is substrate for different xylosyltransferases incorporating xylose in the glycosaminoglycan core (XylT1 and XylT2) or in O-glucose-linked glycans. The nucleotide sugar transporter SLC35D1 (52) has been shown to transport UDP-GlcA over the ER membrane and SLC35B4 (29) to transport UDP-Xyl over the Golgi membrane. The function of this latter transporter is unclear.Several different Chinese hamster ovary (CHO) cell lines with defects in GAG biosynthesis have been isolated by screening for reduced incorporation of sulfate (18) and reduced binding of fibroblast growth factor 2 (FGF-2) (19, 20) and by direct selection with FGF-2 conjugated to the plant cytotoxin saporin (21). Isolated cells (called pgs, for proteoglycan synthesis mutants) (21) exhibited defects in various stages of GAG biosynthesis, ranging from the initiating xylosyltransferase to specific sulfation reactions (18, 19, 21-25). Mutants that affect overall GAG biosynthesis were shown to have a defect in the assembly of the common core tetrasaccharide. Interestingly, these latter mutants could be separated into clones in which GAG biosynthesis can be restored by the external addition of xylosides as artificial primers and those that cannot (18). The two mutants belonging to the first group are pgsA-745 and pgsB-761. Although pgs-745 is defective in XylT2 (4-6, 18), pgsB-761 exhibits a defect in galactosyltransferase I (B4GalT7), the enzyme that catalyzes the first step in the elongation of the xylosylated protein (25 (see Fig. 1B). Restoration of GAG biosynthesis in the latter mutant presumably occurs through a second β1-4-galactosyltransferase, able to act on xylosides when provided at high concentration but not on the endogenous protein-linked xylose.Here we describe the isolation of a third CHO cell line (pgsI-208) with the xyloside-correctable phenotype. The mutant is deficient in UDP-xylose synthase (UXS), also known as UDP-glucuronic acid decarboxylase. This enzyme catalyzes the synthesis of UDP-Xyl, the common donor substrate for the different xylosyltransferases, by decarboxylation of UDP-glucuronic acid. Importantly, UXS in the animal cell is localized in the lumen of the ER and/or Golgi (26-28), superseding at first sight the need for the Golgi UDP-xylose transporter, which has been recently cloned and characterized (29). Using this cell variant, experiments were designed that establish the functional significance of UDP-Xyl transport with respect to UDP-glucuronic acid production and xylosylation.     

Specific Activation of mTORC1 by Rheb G-protein in Vitro Involves Enhanced Recruitment of Its Substrate Protein

Rheb G-protein plays critical roles in the TSC/Rheb/mTOR signaling pathway by activating mTORC1. The activation of mTORC1 by Rheb can be faithfully reproduced in vitro by using mTORC1 immunoprecipitated by the use of anti-raptor antibody from mammalian cells starved for nutrients. The low in vitro kinase activity against 4E-BP1 of this mTORC1 preparation is dramatically increased by the addition of recombinant Rheb. On the other hand, the addition of Rheb does not activate mTORC2 immunoprecipitated from mammalian cells by the use of anti-rictor antibody. The activation of mTORC1 is specific to Rheb, because other G-proteins such as KRas, RalA/B, and Cdc42 did not activate mTORC1. Both Rheb1 and Rheb2 activate mTORC1. In addition, the activation is dependent on the presence of bound GTP. We also find that the effector domain of Rheb is required for the mTORC1 activation. FKBP38, a recently proposed mediator of Rheb action, appears not to be involved in the Rheb-dependent activation of mTORC1 in vitro, because the preparation of mTORC1 that is devoid of FKBP38 is still activated by Rheb. The addition of Rheb results in a significant increase of binding of the substrate protein 4E-BP1 to mTORC1. PRAS40, a TOR signaling (TOS) motif-containing protein that competes with the binding of 4EBP1 to mTORC1, inhibits Rheb-induced activation of mTORC1. A preparation of mTORC1 that is devoid of raptor is not activated by Rheb. Rheb does not induce autophosphorylation of mTOR. These results suggest that Rheb induces alteration in the binding of 4E-BP1 with mTORC1 to regulate mTORC1 activation.Rheb defines a unique member of the Ras superfamily G-proteins (1). We have shown that Rheb proteins are conserved and are found from yeast to human (2). Although yeast and fruit fly have one Rheb, mouse and human have two Rheb proteins termed Rheb1 (or simply Rheb) and Rheb2 (RhebL1) (2). Structurally, these proteins contain G1-G5 boxes, short stretches of amino acids that define the function of the Ras superfamily G-proteins including guanine nucleotide binding (1, 3, 4). Rheb proteins have a conserved arginine at residue 15 that corresponds to residue 12 of Ras (1). The effector domain required for the binding with downstream effectors encompasses the G2 box and its adjacent sequences (1, 5). Structural analysis by x-ray crystallography further shows that the effector domain is exposed to solvent, is located close to the phosphates of GTP especially at residues 35–38, and undergoes conformational change during GTP/GDP exchange (6). In addition, all Rheb proteins end with the CAAX (C is cysteine, A is an aliphatic amino acid, and X is the C-terminal amino acid) motif that signals farnesylation. In fact, we as well as others have shown that these proteins are farnesylated (7–9).Rheb plays critical roles in the TSC/Rheb/mTOR signaling, a signaling pathway that plays central roles in regulating protein synthesis and growth in response to nutrient, energy, and growth conditions (10–14). Rheb is down-regulated by a TSC1·TSC2 complex that acts as a GTPase-activating protein for Rheb (15–19). Recent studies established that the GAP domain of TSC2 defines the functional domain for the down-regulation of Rheb (20). Mutations in the Tsc1 or Tsc2 gene lead to tuberous sclerosis whose symptoms include the appearance of benign tumors called hamartomas at different parts of the body as well as neurological symptoms (21, 22). Overexpression of Rheb results in constitutive activation of mTOR even in the absence of nutrients (15, 16). Two mTOR complexes, mTORC1 and mTORC2, have been identified (23, 24). Whereas mTORC1 is involved in protein synthesis activation mediated by S6K and 4EBP1, mTORC2 is involved in the phosphorylation of Akt in response to insulin. It has been suggested that Rheb is involved in the activation of mTORC1 but not mTORC2 (25).Although Rheb is clearly involved in the activation of mTOR, the mechanism of activation has not been established. We as well as others have suggested a model that involves the interaction of Rheb with the TOR complex (26–28). Rheb activation of mTOR kinase activity using immunoprecipitated mTORC1 was reported (29). Rheb has been shown to interact with mTOR (27, 30), and this may involve direct interaction of Rheb with the kinase domain of mTOR (27). However, this Rheb/mTOR interaction is a weak interaction and is not dependent on the presence of GTP bound to Rheb (27, 28). Recently, a different model proposing that FKBP38 (FK506-binding protein 38) mediates the activation of mTORC1 by Rheb was proposed (31, 32). In this model, FKBP38 binds mTOR and negatively regulates mTOR activity, and this negative regulation is blocked by the binding of Rheb to FKBP38. However, recent reports dispute this idea (33).To further characterize Rheb activation of mTOR, we have utilized an in vitro system that reproduces activation of mTORC1 by the addition of recombinant Rheb. We used mTORC1 immunoprecipitated from nutrient-starved cells using anti-raptor antibody and have shown that its kinase activity against 4E-BP1 is dramatically increased by the addition of recombinant Rheb. Importantly, the activation of mTORC1 is specific to Rheb and is dependent on the presence of bound GTP as well as an intact effector domain. FKBP38 is not detected in our preparation and further investigation suggests that FKBP38 is not an essential component for the activation of mTORC1 by Rheb. Our study revealed that Rheb enhances the binding of a substrate 4E-BP1 with mTORC1 rather than increasing the kinase activity of mTOR.     

Structural and Functional Studies of Archaeal Viruses

Viruses populate virtually every ecosystem on the planet, including the extreme acidic, thermal, and saline environments where archaeal organisms can dominate. For example, recent studies have identified crenarchaeal viruses in the hot springs of Yellowstone National Park and other high temperature environments worldwide. These viruses are often morphologically and genetically unique, with genomes that show little similarity to genes of known function, complicating efforts to understand their viral life cycles. Here, we review progress in understanding these fascinating viruses at the molecular level and the evolutionary insights coming from these studies.The last decade has seen resurgent interest in the study of viruses that lie outside traditional agricultural and medical interests. One reason is the growing appreciation of the enormous abundance and impact of viruses on the greater biosphere. For example, the oceans are thought to contain ∼10³¹ viruses, a truly astronomical number (1), making viruses the most abundant biological entities in this ecosystem, where they catalyze turnover of 20% of the oceanic biomass per day (1). Remarkably, the virosphere has now been shown to extend to almost every known environment on earth, including the extreme acidic, thermal, and saline environments where archaeal organisms can be dominant. Thus, because of their abundance and variety, viruses are now thought to represent the greatest reservoir of genetic diversity on the planet (2).A second reason to study archaeal viruses is a growing appreciation for the roles viruses play in evolution. Remarkably with >500 cellular genomes sequenced to date, most show a significant amount of viral or virus-like sequence within their genome, further evidence that viruses play a central role in horizontal gene transfer and help drive the evolution of their hosts. Roles for viruses in cellular evolution are also being considered. Current hypotheses contend that viruses have catalyzed several major evolutionary transitions, including the invention of DNA and DNA replication mechanisms (3), the origin of the eukaryotic nucleus (4), and thus a role in the formation of the three domains of life. In addition, there is also considerable interest in viral genesis and evolution in and of itself. To evaluate these hypotheses and to analyze evolutionary relationships among viruses, knowledge of viruses infecting the archaea is essential, yet these viruses are vastly understudied. Finally, interest in archaeal viruses stems also from the exceptional molecular insight viruses have traditionally provided into host processes; archaeal viruses are certain to provide new insights into the molecular biology of this poorly understood domain of life.Pioneering studies by Wolfram Zillig et al. (5) identified the first archaeal viruses. Although initial studies suggested that viruses infecting the euryarchaea (principally halophiles and methanogens) were similar to head-tail bacteriophage, studies of viruses infecting the hyperthermophilic crenarchaea revealed morphologies suggesting new viral families. Indeed, work by several laboratories has led to the identification of seven new viral families infecting the crenarchaea, the Globuloviridae, Guttaviridae, Fuselloviridae, Bicaudaviridae, Ampullaviridae, Rudiviridae, and Lipothrixviridae (Fig. 1) (6, 7), with STIV³ (8) and STSV1 (9) awaiting assignment. All of these viruses contain double-stranded DNA genomes ranging in size from 13.7 to 75.3 kilobase pairs, encoding 31–74 ORFs. Although many package a circular genome, the filamentous Lipothrixviridae and rod-shaped Rudiviridae are notable exceptions and are the only viruses in any domain known to encapsidate linear double-stranded DNA. Although most crenarchaeal viruses are enveloped, the Rudiviridae are devoid of lipid, and with the exception of the Fuselloviridae, they employ a lytic life cycle, although only STIV and ATV (Bicaudaviridae) are known to cause cell lysis (11).⁴Open in a separate windowFIGURE 1.Morphological diversity in crenarchaeal viruses. A, clockwise, beginning at upper left: STIV (8), a PSV-like virus, Sulfolobus neozealandicus droplet-shaped virus (SNDV) (47), SSV1 (48), STSV1 (9), an ATV-like virus, an SIRV virus, and S. icelandicus filamentous virus (SIFV) (10). Micrographs of SIRV, PSV-like, and ATV-like viruses from Yellowstone National Park are the courtesy of M. J. Y. Other panels are reproduced, with permission, from Refs. 8–10, 47, and 48. B, cryoelectron microscopy reconstruction of the STIV particle (8) showing a cutaway view (20) of the T = 31 icosahedral capsid with turret-like projections that extend from each of the 5-fold vertices. Portions of the protein shell (blue) and inner lipid layer (yellow) have been removed to reveal the interior.The exceptional morphology of these viruses has been reviewed (6, 7) and thus is only summarized here (Fig. 1). For the rod-shaped Rudiviridae, plugs are seen at both ends, from which three short tail fibers emanate, whereas the Lipothrixviridae show mop- or claw-like structures at both ends (6). Similarly, the non-tailed icosahedral viruses, STIV and euryarchaeal SH1, have large turrets or spikes that project from the surface (8, 12). In each case, these structures are thought to facilitate virus-host interactions. In contrast, other crenarchaeal viruses utilize a fusiform or lemon-shaped virion, a morphology unique to archaeal viruses. These fusiform viruses generally contain tail fibers or an extended tail on one end that is also involved in host recognition. For ATV, however, nascent particles are devoid of tails when released from the host (13). Remarkably, extended tails develop at both ends of the virion in an extracellular maturation process. Finally, Acidianus bottle-shaped virus (Ampullaviridae) shows an exceptional morphology that differs in its basic architecture from any known virus.