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.     

Glutathione Participates in the Regulation of Mitophagy in Yeast

The antioxidant N-acetyl-l-cysteine prevented the autophagy-dependent delivery of mitochondria to the vacuoles, as examined by fluorescence microscopy of mitochondria-targeted green fluorescent protein, transmission electron microscopy, and Western blot analysis of mitochondrial proteins. The effect of N-acetyl-l-cysteine was specific to mitochondrial autophagy (mitophagy). Indeed, autophagy-dependent activation of alkaline phosphatase and the presence of hallmarks of non-selective microautophagy were not altered by N-acetyl-l-cysteine. The effect of N-acetyl-l-cysteine was not related to its scavenging properties, but rather to its fueling effect of the glutathione pool. As a matter of fact, the decrease of the glutathione pool induced by chemical or genetical manipulation did stimulate mitophagy but not general autophagy. Conversely, the addition of a cell-permeable form of glutathione inhibited mitophagy. Inhibition of glutathione synthesis had no effect in the strain Δuth1, which is deficient in selective mitochondrial degradation. These data show that mitophagy can be regulated independently of general autophagy, and that its implementation may depend on the cellular redox status.Autophagy is a major pathway for the lysosomal/vacuolar delivery of long-lived proteins and organelles, where they are degraded and recycled. Autophagy plays a crucial role in differentiation and cellular response to stress and is conserved in eukaryotic cells from yeast to mammals (1, 2). The main form of autophagy, macroautophagy, involves the non-selective sequestration of large portions of the cytoplasm into double-membrane structures termed autophagosomes, and their delivery to the vacuole/lysosome for degradation. Another process, microautophagy, involves the direct sequestration of parts of the cytoplasm by vacuole/lysosomes. The two processes coexist in yeast cells but their extent may depend on different factors including metabolic state: for example, we have observed that nitrogen-starved lactate-grown yeast cells develop microautophagy, whereas nitrogen-starved glucose-grown cells preferentially develop macroautophagy (3).Both macroautophagy and microautophagy are essentially non-selective, in the way that autophagosomes and vacuole invaginations do not appear to discriminate the sequestered material. However, selective forms of autophagy have been observed (4) that target namely peroxisomes (5, 6), chromatin (7, 8), endoplasmic reticulum (9), ribosomes (10), and mitochondria (3, 11–13). Although non-selective autophagy plays an essential role in survival by nitrogen starvation, by providing amino acids to the cell, selective autophagy is more likely to have a function in the maintenance of cellular structures, both under normal conditions as a “housecleaning” process, and under stress conditions by eliminating altered organelles and macromolecular structures (14–16). Selective autophagy targeting mitochondria, termed mitophagy, may be particularly relevant to stress conditions. The mitochondrial respiratory chain is both the main site and target of ROS⁴ production (17). Consequently, the maintenance of a pool of healthy mitochondria is a crucial challenge for the cells. The progressive accumulation of altered mitochondria (18) caused by the loss of efficiency of the maintenance process (degradation/biogenesis de novo) is often considered as a major cause of cellular aging (19–23). In mammalian cells, autophagic removal of mitochondria has been shown to be triggered following induction/blockade of apoptosis (23), suggesting that autophagy of mitochondria was required for cell survival following mitochondria injury (14). Consistent with this idea, a direct alteration of mitochondrial permeability properties has been shown to induce mitochondrial autophagy (13, 24, 25). Furthermore, inactivation of catalase induced the autophagic elimination of altered mitochondria (26). In the yeast Saccharomyces cerevisiae, the alteration of F₀F₁-ATPase biogenesis in a conditional mutant has been shown to trigger autophagy (27). Alterations of mitochondrial ion homeostasis caused by the inactivation of the K⁺/H⁺ exchanger was shown to cause both autophagy and mitophagy (28). We have reported that treatment of cells with rapamycin induced early ROS production and mitochondrial lipid oxidation that could be inhibited by the hydrophobic antioxidant resveratrol (29). Furthermore, resveratrol treatment impaired autophagic degradation of both cytosolic and mitochondrial proteins and delayed rapamycin-induced cell death, suggesting that mitochondrial oxidation events may play a crucial role in the regulation of autophagy. This existence of regulation of autophagy by ROS has received molecular support in HeLa cells (30): these authors showed that starvation stimulated ROS production, namely H₂O₂, which was essential for autophagy. Furthermore, they identified the cysteine protease hsAtg4 as a direct target for oxidation by H₂O₂. This provided a possible connection between the mitochondrial status and regulation of autophagy.Investigations of mitochondrial autophagy in nitrogen-starved lactate-grown yeast cells have established the existence of two distinct processes: the first one occurring very early, is selective for mitochondria and is dependent on the presence of the mitochondrial protein Uth1p; the second one occurring later, is not selective for mitochondria, is not dependent on Uth1p, and is a form of bulk microautophagy (3). The absence of the selective process in the Δuth1 mutant strongly delays and decreases mitochondrial protein degradation (3, 12). The putative protein phosphatase Aup1p has been also shown to be essential in inducing mitophagy (31). Additionally several Atg proteins were shown to be involved in vacuolar sequestration of mitochondrial GFP (3, 12, 32, 33). Recently, the protein Atg11p, which had been already identified as an essential protein for selective autophagy has also been reported as being essential for mitophagy (33).The question remains as to identify of the signals that trigger selective mitophagy. It is particularly intriguing that selective mitophagy is activated very early after the shift to a nitrogen-deprived medium (3). Furthermore, selective mitophagy is very active on lactate-grown cells (with fully differentiated mitochondria) but is nearly absent in glucose-grown cells (3). In the present paper, we investigated the relationships between the redox status of the cells and selective mitophagy, namely by manipulating glutathione. Our results support the view that redox imbalance is a trigger for the selective elimination of mitochondria.     

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.     

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.     

Loss of PINK1 Function Promotes Mitophagy through Effects on Oxidative Stress and Mitochondrial Fission

Mitochondrial dysregulation is strongly implicated in Parkinson disease. Mutations in PTEN-induced kinase 1 (PINK1) are associated with familial parkinsonism and neuropsychiatric disorders. Although overexpressed PINK1 is neuroprotective, less is known about neuronal responses to loss of PINK1 function. We found that stable knockdown of PINK1 induced mitochondrial fragmentation and autophagy in SH-SY5Y cells, which was reversed by the reintroduction of an RNA interference (RNAi)-resistant plasmid for PINK1. Moreover, stable or transient overexpression of wild-type PINK1 increased mitochondrial interconnectivity and suppressed toxin-induced autophagy/mitophagy. Mitochondrial oxidant production played an essential role in triggering mitochondrial fragmentation and autophagy in PINK1 shRNA lines. Autophagy/mitophagy served a protective role in limiting cell death, and overexpressing Parkin further enhanced this protective mitophagic response. The dominant negative Drp1 mutant inhibited both fission and mitophagy in PINK1-deficient cells. Interestingly, RNAi knockdown of autophagy proteins Atg7 and LC3/Atg8 also decreased mitochondrial fragmentation without affecting oxidative stress, suggesting active involvement of autophagy in morphologic remodeling of mitochondria for clearance. To summarize, loss of PINK1 function elicits oxidative stress and mitochondrial turnover coordinated by the autophagic and fission/fusion machineries. Furthermore, PINK1 and Parkin may cooperate through different mechanisms to maintain mitochondrial homeostasis.Parkinson disease is an age-related neurodegenerative disease that affects ∼1% of the population worldwide. The causes of sporadic cases are unknown, although mitochondrial or oxidative toxins such as 1-methyl-4-phenylpyridinium, 6-hydroxydopamine (6-OHDA),³ and rotenone reproduce features of the disease in animal and cell culture models (1). Abnormalities in mitochondrial respiration and increased oxidative stress are observed in cells and tissues from parkinsonian patients (2, 3), which also exhibit increased mitochondrial autophagy (4). Furthermore, mutations in parkinsonian genes affect oxidative stress response pathways and mitochondrial homeostasis (5). Thus, disruption of mitochondrial homeostasis represents a major factor implicated in the pathogenesis of sporadic and inherited parkinsonian disorders (PD).The PARK6 locus involved in autosomal recessive and early-onset PD encodes for PTEN-induced kinase 1 (PINK1) (6, 7). PINK1 is a cytosolic and mitochondrially localized 581-amino acid serine/threonine kinase that possesses an N-terminal mitochondrial targeting sequence (6, 8). The primary sequence also includes a putative transmembrane domain important for orientation of the PINK1 domain (8), a conserved kinase domain homologous to calcium calmodulin kinases, and a C-terminal domain that regulates autophosphorylation activity (9, 10). Overexpression of wild-type PINK1, but not its PD-associated mutants, protects against several toxic insults in neuronal cells (6, 11, 12). Mitochondrial targeting is necessary for some (13) but not all of the neuroprotective effects of PINK1 (14), implicating involvement of cytoplasmic targets that modulate mitochondrial pathobiology (8). PINK1 catalytic activity is necessary for its neuroprotective role, because a kinase-deficient K219M substitution in the ATP binding pocket of PINK1 abrogates its ability to protect neurons (14). Although PINK1 mutations do not seem to impair mitochondrial targeting, PD-associated mutations differentially destabilize the protein, resulting in loss of neuroprotective activities (13, 15).Recent studies indicate that PINK1 and Parkin interact genetically (3, 16-18) to prevent oxidative stress (19, 20) and regulate mitochondrial morphology (21). Primary cells derived from PINK1 mutant patients exhibit mitochondrial fragmentation with disorganized cristae, recapitulated by RNA interference studies in HeLa cells (3).Mitochondria are degraded by macroautophagy, a process involving sequestration of cytoplasmic cargo into membranous autophagic vacuoles (AVs) for delivery to lysosomes (22, 23). Interestingly, mitochondrial fission accompanies autophagic neurodegeneration elicited by the PD neurotoxin 6-OHDA (24, 25). Moreover, mitochondrial fragmentation and increased autophagy are observed in neurodegenerative diseases including Alzheimer and Parkinson diseases (4, 26-28). Although inclusion of mitochondria in autophagosomes was once believed to be a random process, as observed during starvation, studies involving hypoxia, mitochondrial damage, apoptotic stimuli, or limiting amounts of aerobic substrates in facultative anaerobes support the concept of selective mitochondrial autophagy (mitophagy) (29, 30). In particular, mitochondrially localized kinases may play an important role in models involving oxidative mitochondrial injury (25, 31, 32).Autophagy is involved in the clearance of protein aggregates (33-35) and normal regulation of axonal-synaptic morphology (36). Chronic disruption of lysosomal function results in accumulation of subtly impaired mitochondria with decreased calcium buffering capacity (37), implicating an important role for autophagy in mitochondrial homeostasis (37, 38). Recently, Parkin, which complements the effects of PINK1 deficiency on mitochondrial morphology (3), was found to promote autophagy of depolarized mitochondria (39). Conversely, Beclin 1-independent autophagy/mitophagy contributes to cell death elicited by the PD toxins 1-methyl-4-phenylpyridinium and 6-OHDA (25, 28, 31, 32), causing neurite retraction in cells expressing a PD-linked mutation in leucine-rich repeat kinase 2 (40). Whereas properly regulated autophagy plays a homeostatic and neuroprotective role, excessive or incomplete autophagy creates a condition of “autophagic stress” that can contribute to neurodegeneration (28).As mitochondrial fragmentation (3) and increased mitochondrial autophagy (4) have been described in human cells or tissues of PD patients, we investigated whether or not the engineered loss of PINK1 function could recapitulate these observations in human neuronal cells (SH-SY5Y). Stable knockdown of endogenous PINK1 gave rise to mitochondrial fragmentation and increased autophagy and mitophagy, whereas stable or transient overexpression of PINK1 had the opposite effect. Autophagy/mitophagy was dependent upon increased mitochondrial oxidant production and activation of fission. The data indicate that PINK1 is important for the maintenance of mitochondrial networks, suggesting that coordinated regulation of mitochondrial dynamics and autophagy limits cell death associated with loss of PINK1 function.     

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.     

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.     

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.     

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.     

Mechanism of Sustained Activation of Ribosomal S6 Kinase (RSK) and ERK by Kaposi Sarcoma-associated Herpesvirus ORF45: MULTIPROTEIN COMPLEXES RETAIN ACTIVE PHOSPHORYLATED ERK AND RSK AND PROTECT THEM FROM DEPHOSPHORYLATION*

As obligate intracellular parasites, viruses exploit diverse cellular signaling machineries, including the mitogen-activated protein-kinase pathway, during their infections. We have demonstrated previously that the open reading frame 45 (ORF45) of Kaposi sarcoma-associated herpesvirus interacts with p90 ribosomal S6 kinases (RSKs) and strongly stimulates their kinase activities (Kuang, E., Tang, Q., Maul, G. G., and Zhu, F. (2008) J. Virol. 82 ,1838 -1850). Here, we define the mechanism by which ORF45 activates RSKs. We demonstrated that binding of ORF45 to RSK increases the association of extracellular signal-regulated kinase (ERK) with RSK, such that ORF45, RSK, and ERK formed high molecular mass protein complexes. We further demonstrated that the complexes shielded active pERK and pRSK from dephosphorylation. As a result, the complex-associated RSK and ERK were activated and sustained at high levels. Finally, we provide evidence that this mechanism contributes to the sustained activation of ERK and RSK in Kaposi sarcoma-associated herpesvirus lytic replication.The extracellular signal-regulated kinase (ERK)² mitogen-activated protein kinase (MAPK) signaling pathway has been implicated in diverse cellular physiological processes including proliferation, survival, growth, differentiation, and motility (1-4) and is also exploited by a variety of viruses such as Kaposi sarcoma-associated herpesvirus (KSHV), human cytomegalovirus, human immunodeficiency virus, respiratory syncytial virus, hepatitis B virus, coxsackie, vaccinia, coronavirus, and influenza virus (5-17). The MAPK kinases relay the extracellular signaling through sequential phosphorylation to an array of cytoplasmic and nuclear substrates to elicit specific responses (1, 2, 18). Phosphorylation of MAPK is reversible. The kinetics of deactivation or duration of signaling dictates diverse biological outcomes (19, 20). For example, sustained but not transient activation of ERK signaling induces the differentiation of PC12 cells into sympathetic-like neurons and transformation of NIH3T3 cells (20-22). During viral infection, a unique biphasic ERK activation has been observed for some viruses (an early transient activation triggered by viral binding or entry and a late sustained activation correlated with viral gene expression), but the responsible viral factors and underlying mechanism for the sustained ERK activation remain largely unknown (5, 8, 13, 23).The p90 ribosomal S6 kinases (RSKs) are a family of serine/threonine kinases that lie at the terminus of the ERK pathway (1, 24-26). In mammals, four isoforms are known, RSK1 to RSK4. Each one has two catalytically functional kinase domains, the N-terminal kinase domain (NTKD) and C-terminal kinase domain (CTKD) as well as a linker region between the two. The NTKD is responsible for phosphorylation of exogenous substrates, and the CTKD and linker region regulate RSK activation (1, 24, 25). In quiescent cells ERK binds to the docking site in the C terminus of RSK (27-29). Upon mitogen stimulation, ERK is activated by its upstream MAPK/ERK kinase (MEK). The active ERK phosphorylates Thr-359/Ser-363 of RSK in the linker region (amino acid numbers refer to human RSK1) and Thr-573 in the CTKD activation loop. The activated CTKD then phosphorylates Ser-380 in the linker region, creating a docking site for 3-phosphoinositide-dependent protein kinase-1. The 3-phosphoinositide-dependent protein kinase-1 phosphorylates Ser-221 of RSK in the activation loop and activates the NTKD. The activated NTKD autophosphorylates the serine residue near the ERK docking site, causing a transient dissociation of active ERK from RSK (25, 26, 28). The stimulation of quiescent cells by a mitogen such as epidermal growth factor or a phorbol ester such as 12-O-tetradecanoylphorbol-13-acetate (TPA) usually results in a transient RSK activation that lasts less than 30 min. RSKs have been implicated in regulating cell survival, growth, and proliferation. Mutation or aberrant expression of RSK has been implicated in several human diseases including Coffin-Lowry syndrome and prostate and breast cancers (1, 24, 25, 30-32).KSHV is a human DNA tumor virus etiologically linked to Kaposi sarcoma, primary effusion lymphoma, and a subset of multicentric Castleman disease (33, 34). Infection and reactivation of KSHV activate multiple MAPK pathways (6, 12, 35). Noticeably, the ERK/RSK activation is sustained late during KSHV primary infection and reactivation from latency (5, 6, 12, 23), but the mechanism of the sustained ERK/RSK activation is unclear. Recently, we demonstrated that ORF45, an immediate early and also virion tegument protein of KSHV, interacts with RSK1 and RSK2 and strongly stimulates their kinase activities (23). We also demonstrated that the activation of RSK plays an essential role in KSHV lytic replication (23). In the present study we determined the mechanism of ORF45-induced sustained ERK/RSK activation. We found that ORF45 increases the association of RSK with ERK and protects them from dephosphorylation, causing sustained activation of both ERK and RSK.     

The Substrate Recognition Domains of the N-end Rule Pathway

The N-end rule pathway is a ubiquitin-dependent system where E3 ligases called N-recognins, including UBR1 and UBR2, recognize type-1 (basic) and type-2 (bulky hydrophobic) N-terminal residues as part of N-degrons. We have recently reported an E3 family (termed UBR1 through UBR7) characterized by the 70-residue UBR box, among which UBR1, UBR2, UBR4, and UBR5 were captured during affinity-based proteomics with synthetic degrons. Here we characterized substrate binding specificity and recognition domains of UBR proteins. Pull-down assays with recombinant UBR proteins suggest that 570-kDa UBR4 and 300-kDa UBR5 bind N-degron, whereas UBR3, UBR6, and UBR7 do not. Binding assays with 24 UBR1 deletion mutants and 31 site-directed UBR1 mutations narrow down the degron-binding activity to a 72-residue UBR box-only fragment that recognizes type-1 but not type-2 residues. A surface plasmon resonance assay shows that the UBR box binds to the type-1 substrate Arg-peptide with K_d of ∼3.4 μm. Downstream from the UBR box, we identify a second substrate recognition domain, termed the N-domain, required for type-2 substrate recognition. The ∼80-residue N-domain shows structural and functional similarity to 106-residue Escherichia coli ClpS, a bacterial N-recognin. We propose a model where the 70-residue UBR box functions as a common structural element essential for binding to all known destabilizing N-terminal residues, whereas specific residues localized in the UBR box (for type 1) or the N-domain (for type 2) provide substrate selectivity through interaction with the side group of an N-terminal amino acid. Our work provides new insights into substrate recognition in the N-end rule pathway.The N-end rule pathway is a ubiquitin (Ub)²-dependent proteolytic system in which N-terminal residues of short-lived proteins function as an essential component of degradation signals (degrons) called N-degrons (Fig. 1A) (1-15). An N-degron can be created from a pre-N-degron through specific N-terminal modifications (12). Specifically, in mammals, N-terminal Asn and Gln are tertiary destabilizing residues that function through their deamidation by N-terminal amidohydrolases into the secondary destabilizing N-terminal residues Asp and Glu, respectively (6, 16) (Fig. 1A). N-terminal Asp and Glu are secondary destabilizing residues that function through their arginylation by ATE1 R-transferase, which creates the primary destabilizing residue Arg at the N terminus (4, 8) (Fig. 1A). N-terminal Cys can also function as a tertiary destabilizing residue through its oxidation in a manner depending on nitric oxide and oxygen (O₂); the oxidized Cys residue is subsequently arginylated by ATE1 (8, 13, 17).Open in a separate windowFIGURE 1.A, the mammalian N-end rule pathway. N-terminal residues are indicated by single-letter abbreviations for amino acids. Yellow ovals denote the rest of a protein substrate. C^* denotes oxidized N-terminal Cys, either Cys-sulfinic acid [CysO₂(H)] or Cys-sulfonic acid [CysO₃(H)]. The Cys oxidation requires nitric oxide and oxygen (O₂) or its derivatives. The oxidized Cys is arginylated by ATE1 Arg-tRNA-protein transferase (R-transferase). N-recognins also recognize internal (non-N-terminal) degrons in other substrates of the N-end rule pathway. B, the X-peptide pull-down assay. Left, a 12-mer peptide bearing N-terminal Arg (type 1), Phe (type 2), Trp (type 2), or Gly (stabilizing control) residue was cross-linked through its C-terminal Cys residue to Ultralink Iodoacetyl beads. Right, the otherwise identical 12-mer peptide, bearing C-terminal biotinylated Lys instead of Cys, was conjugated, via biotin, to the streptavidin-Sepharose beads. C, the X-peptide pull-down assay of endogenous UBR proteins using testes extracts. Extracts from mouse testes were mixed with bead-conjugated X-peptides bearing N-terminal Phe (F), Gly (G), or Arg (R). After centrifugation, captured proteins were separated and subjected to anti-UBR immunoblotting. Mo, a pull-down reaction with mock beads. D, the X-peptide pull-down assays using rat testis extracts were performed in the presence of varying concentrations of NaCl. After incubation and washing, bound proteins were eluted by 10 mm Tyr-Ala for Phe-peptide, 10 mm Arg-Ala for Arg-peptide, and 5 mm Tyr-Ala and 5 mm Arg-Ala for Val-peptide. Eluted proteins were subjected to immunoblotting for UBR1 and UBR5. E, cytoplasmic fractions of wild-type (+/+), Ubr1^-/-, Ubr2^-/-, Ubr1^-/-Ubr2^-/-, and Ubr1^-/-Ubr2^-/-Ubr4^RNAi MEFs were subjected to X-peptide pull-down assay. Precipitated proteins were separated and analyzed by immunoblotting for UBR1 and UBR4.N-terminal Arg together with other primary destabilizing N-terminal residues are directly bound by specific E3 Ub ligases called N-recognins (3, 7, 9). Destabilizing N-terminal residues can be created through the removal of N-terminal Met or the endoproteolytic cleavage of a protein, which exposes a new amino acid at the N terminus (12, 13). N-terminal degradation signals can be divided into type-1 (basic; Arg, Lys, and His) and type-2 (bulky hydrophobic; Phe, Leu, Trp, Tyr, and Ile) destabilizing residues (2, 12). In addition to a destabilizing N-terminal residue, a functional N-degron requires at least one internal Lys residue (the site of a poly-Ub chain formation) and a conformational feature required for optimal ubiquitylation (1, 2, 18). UBR1 and UBR2 are functionally overlapping N-recognins (3, 7, 9). Our proteomic approach using synthetic peptides bearing destabilizing N-terminal residues captured a set of proteins (200-kDa UBR1, 200-kDa UBR2, 570-kDa UBR4, and 300-kDa UBR5/EDD) characterized by a 70-residue zinc finger-like domain termed the UBR box (10-12). UBR5 is a HECT E3 ligase known as EDD (E3 identified by differential display) (19) and a homolog of Drosophila hyperplastic discs (20). The mammalian genome encodes at least seven UBR box-containing proteins, termed UBR1 through UBR7 (10). UBR box proteins are generally heterogeneous in size and sequence but contain, with the exception of UBR4, specific signatures unique to E3s or a substrate recognition subunit of the E3 complex: the RING domain in UBR1, UBR2, and UBR3; the HECT domain in UBR5; the F-box in UBR6 and the plant homeodomain domain in UBR7 (Fig. 2B). The biochemical properties of more recently identified UBR box proteins, such as UBR3 through UBR7, are largely unknown.Open in a separate windowFIGURE 2.The binding properties of the UBR box family members to type-1 and type-2 destabilizing N-terminal residues. A, the X-peptide pull-down assay with overexpressed, full-length UBR proteins: UBR2, UBR3 (in S. cerevisiae cells), UBR4, UBR5 (in COS7 cells), and UBR6 and UBR7 (in the wheat germ lysates). Precipitates were analyzed by immunoblotting (for UBR2, UBR3, UBR4, and UBR5) with tag-specific antibodies as indicated in B or autoradiography (for UBR6 and UBR7). B, the structures of UBR box proteins. Shown are locations of the UBR box, the N-domain, and other E3-related domains. UBR, UBR box; RING, RING finger; UAIN, UBR-specific autoinhibitory domain; CRD, cysteine-rich domain; PHD, plant homeodomain; HECT, HECT domain.Studies using knock-out mice implicated the N-end rule pathway in cardiac development and signaling, angiogenesis (8, 15), meiosis (9), DNA repair (21), neurogenesis (15), pancreatic functions (22), learning and memory (23, 24), female development (9), muscle atrophy (25), and olfaction (11). Mutations in human UBR1 is a cause of Johanson-Blizzard syndrome (22), an autosomal recessive disorder with multiple developmental abnormalities (26). Other functions of the pathway include: (i) a nitric oxide and oxygen (O₂) sensor controlling the proteolysis of RGS4, RGS5, and RGS16 (8, 13, 17), (ii) a heme sensor through hemin-dependent inhibition of ATE1 function (27), (iii) the regulation of short peptide import through the peptide-modulated degradation of the repressor of the import (28, 29), (iv) the control of chromosome segregation through the degradation of a separate produced cohesin fragment (30), (v) the regulation of apoptosis through the degradation of a caspase-processed inhibitor of apoptosis (31, 32), (vi) the control of the human immunodeficiency virus replication cycle through the degradation of human immunodeficiency virus integrase (10, 33), and (vii) the regulation of leaf senescence in plants (34).In the present study we characterized substrate binding specificities and recognition domains of UBR proteins. In our binding assays, UBR1, UBR2, UBR4, and UBR5 were captured by N-terminal degradation determinants, whereas UBR3, UBR6, and UBR7 were not. We also report that in contrast to other E3 systems that usually recognize substrates through protein-protein interface, UBR1 and UBR2 have a general substrate recognition domain termed the UBR box. Remarkably, a 72-residue UBR box-only fragment fully retains its structural integrity and thereby the ability to recognize type-1 N-end rule substrates. We also report that the N-domain, structurally and functionally related with bacterial N-recognins, is required for recognizing type-2 N-end rule substrates. We discuss the evolutionary relationship between eukaryotic and prokaryotic N-recognins.     

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.     

Galectin-8 Induces Apoptosis in Jurkat T Cells by Phosphatidic Acid-mediated ERK1/2 Activation Supported by Protein Kinase A Down-regulation

Galectins have been implicated in T cell homeostasis playing complementary pro-apoptotic roles. Here we show that galectin-8 (Gal-8) is a potent pro-apoptotic agent in Jurkat T cells inducing a complex phospholipase D/phosphatidic acid signaling pathway that has not been reported for any galectin before. Gal-8 increases phosphatidic signaling, which enhances the activity of both ERK1/2 and type 4 phosphodiesterases (PDE4), with a subsequent decrease in basal protein kinase A activity. Strikingly, rolipram inhibition of PDE4 decreases ERK1/2 activity. Thus Gal-8-induced PDE4 activation releases a negative influence of cAMP/protein kinase A on ERK1/2. The resulting strong ERK1/2 activation leads to expression of the death factor Fas ligand and caspase-mediated apoptosis. Several conditions that decrease ERK1/2 activity also decrease apoptosis, such as anti-Fas ligand blocking antibodies. In addition, experiments with freshly isolated human peripheral blood mononuclear cells, previously stimulated with anti-CD3 and anti-CD28, show that Gal-8 is pro-apoptotic on activated T cells, most likely on a subpopulation of them. Anti-Gal-8 autoantibodies from patients with systemic lupus erythematosus block the apoptotic effect of Gal-8. These results implicate Gal-8 as a novel T cell suppressive factor, which can be counterbalanced by function-blocking autoantibodies in autoimmunity.Glycan-binding proteins of the galectin family have been increasingly studied as regulators of the immune response and potential therapeutic agents for autoimmune disorders (1). To date, 15 galectins have been identified and classified according with the structural organization of their distinctive monomeric or dimeric carbohydrate recognition domain for β-galactosides (2, 3). Galectins are secreted by unconventional mechanisms and once outside the cells bind to and cross-link multiple glycoconjugates both at the cell surface and at the extracellular matrix, modulating processes as diverse as cell adhesion, migration, proliferation, differentiation, and apoptosis (4–10). Several galectins have been involved in T cell homeostasis because of their capability to kill thymocytes, activated T cells, and T cell lines (11–16). Pro-apoptotic galectins might contribute to shape the T cell repertoire in the thymus by negative selection, restrict the immune response by eliminating activated T cells at the periphery (1), and help cancer cells to escape the immune system by eliminating cancer-infiltrating T cells (17). They have also a promising therapeutic potential to eliminate abnormally activated T cells and inflammatory cells (1). Studies on the mostly explored galectins, Gal-1, -3, and -9 (14, 15, 18–20), as well as in Gal-2 (13), suggest immunosuppressive complementary roles inducing different pathways to apoptosis. Galectin-8 (Gal-8)⁴ is one of the most widely expressed galectins in human tissues (21, 22) and cancerous cells (23, 24). Depending on the cell context and mode of presentation, either as soluble stimulus or extracellular matrix, Gal-8 can promote cell adhesion, spreading, growth, and apoptosis (6, 7, 9, 10, 22, 25). Its role has been mostly studied in relation to tumor malignancy (23, 24). However, there is some evidence regarding a role for Gal-8 in T cell homeostasis and autoimmune or inflammatory disorders. For instance, the intrathymic expression and pro-apoptotic effect of Gal-8 upon CD4^highCD8^high thymocytes suggest a role for Gal-8 in shaping the T cell repertoire (16). Gal-8 could also modulate the inflammatory function of neutrophils (26), Moreover Gal-8-blocking agents have been detected in chronic autoimmune disorders (10, 27, 28). In rheumatoid arthritis, Gal-8 has an anti-inflammatory action, promoting apoptosis of synovial fluid cells, but can be counteracted by a specific rheumatoid version of CD44 (CD44vRA) (27). In systemic lupus erythematosus (SLE), a prototypic autoimmune disease, we recently described function-blocking autoantibodies against Gal-8 (10, 28). Thus it is important to define the role of Gal-8 and the influence of anti-Gal-8 autoantibodies in immune cells.In Jurkat T cells, we previously reported that Gal-8 interacts with specific integrins, such as α1β1, α3β1, and α5β1 but not α4β1, and as a matrix protein promotes cell adhesion and asymmetric spreading through activation of the extracellular signal-regulated kinases 1 and 2 (ERK1/2) (10). These early effects occur within 5–30 min. However, ERK1/2 signaling supports long term processes such as T cell survival or death, depending on the moment of the immune response. During T cell activation, ERK1/2 contributes to enhance the expression of interleukin-2 (IL-2) required for T cell clonal expansion (29). It also supports T cell survival against pro-apoptotic Fas ligand (FasL) produced by themselves and by other previously activated T cells (30, 31). Later on, ERK1/2 is required for activation-induced cell death, which controls the extension of the immune response by eliminating recently activated and restimulated T cells (32, 33). In activation-induced cell death, ERK1/2 signaling contributes to enhance the expression of FasL and its receptor Fas/CD95 (32, 33), which constitute a preponderant pro-apoptotic system in T cells (34). Here, we ask whether Gal-8 is able to modulate the intensity of ERK1/2 signaling enough to participate in long term processes involved in T cell homeostasis.The functional integration of ERK1/2 and PKA signaling (35) deserves special attention. cAMP/PKA signaling plays an immunosuppressive role in T cells (36) and is altered in SLE (37). Phosphodiesterases (PDEs) that degrade cAMP release the immunosuppressive action of cAMP/PKA during T cell activation (38, 39). PKA has been described to control the activity of ERK1/2 either positively or negatively in different cells and processes (35). A little explored integration among ERK1/2 and PKA occurs via phosphatidic acid (PA) and PDE signaling. Several stimuli activate phospholipase D (PLD) that hydrolyzes phosphatidylcholine into PA and choline. Such PLD-generated PA plays roles in signaling interacting with a variety of targeting proteins that bear PA-binding domains (40). In this way PA recruits Raf-1 to the plasma membrane (41). It is also converted by phosphatidic acid phosphohydrolase (PAP) activity into diacylglycerol (DAG), which among other functions, recruits and activates the GTPase Ras (42). Both Ras and Raf-1 are upstream elements of the ERK1/2 activation pathway (43). In addition, PA binds to and activates PDEs of the type 4 subfamily (PDE4s) leading to decreased cAMP levels and PKA down-regulation (44). The regulation and role of PA-mediated control of ERK1/2 and PKA remain relatively unknown in T cell homeostasis, because it is also unknown whether galectins stimulate the PLD/PA pathway.Here we found that Gal-8 induces apoptosis in Jurkat T cells by triggering cross-talk between PKA and ERK1/2 pathways mediated by PLD-generated PA. Our results for the first time show that a galectin increases the PA levels, down-regulates the cAMP/PKA system by enhancing rolipram-sensitive PDE activity, and induces an ERK1/2-dependent expression of the pro-apoptotic factor FasL. The enhanced PDE activity induced by Gal-8 is required for the activation of ERK1/2 that finally leads to apoptosis. Gal-8 also induces apoptosis in human peripheral blood mononuclear cells (PBMC), especially after activating T cells with anti-CD3/CD28. Therefore, Gal-8 shares with other galectins the property of killing activated T cells contributing to the T cell homeostasis. The pathway involves a particularly integrated signaling context, engaging PLD/PA, cAMP/PKA, and ERK1/2, which so far has not been reported for galectins. The pro-apoptotic function of Gal-8 also seems to be unique in its susceptibility to inhibition by anti-Gal-8 autoantibodies.     

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.