Structure and Function of an NADPH-Cytochrome P450 Oxidoreductase in an Open Conformation Capable of Reducing Cytochrome P450

NADPH-cytochrome P450 oxidoreductase (CYPOR) catalyzes the transfer of electrons to all known microsomal cytochromes P450. A CYPOR variant, with a 4-amino acid deletion in the hinge connecting the FMN domain to the rest of the protein, has been crystallized in three remarkably extended conformations. The variant donates an electron to cytochrome P450 at the same rate as the wild-type, when provided with sufficient electrons. Nevertheless, it is defective in its ability to transfer electrons intramolecularly from FAD to FMN. The three extended CYPOR structures demonstrate that, by pivoting on the C terminus of the hinge, the FMN domain of the enzyme undergoes a structural rearrangement that separates it from FAD and exposes the FMN, allowing it to interact with its redox partners. A similar movement most likely occurs in the wild-type enzyme in the course of transferring electrons from FAD to its physiological partner, cytochrome P450. A model of the complex between an open conformation of CYPOR and cytochrome P450 is presented that satisfies mutagenesis constraints. Neither lengthening the linker nor mutating its sequence influenced the activity of CYPOR. It is likely that the analogous linker in other members of the diflavin family functions in a similar manner.NADPH-cytochrome P450 oxidoreductase (CYPOR)⁴ is a ∼78-kDa, multidomain, microsomal diflavin protein that shuttles electrons from NADPH → FAD → FMN to members of the ubiquitous cytochrome P450 superfamily (1, 2). In humans, the cytochromes P450 (cyt P450) are one of the most important families of proteins involved in the biosynthesis and degradation of a vast number of endogenous compounds and the detoxification and biodegradation of most foreign compounds. CYPOR also donates electrons to heme oxygenase (3), cytochrome b₅ (4), and cytochrome c (5).The FAD receives a hydride anion from the obligate two electron donor NADPH and passes the electrons one at a time to FMN. The FMN then donates electrons to the redox partners of CYPOR, again one electron at a time. Cyt P450 accepts electrons at two different steps in its complex reaction cycle. Ferric cyt P450 is reduced to the ferrous protein, and oxyferrous cyt P450 receives the second of the two electrons to form the peroxo (Fe⁺³OO)^2- cyt P450 intermediate (6). In vivo, CYPOR cycles between the one- and three-electron reduced forms (7, 8). Although the one-electron reduced form is an air-stable, neutral blue semiquinone (FMN_ox/sq, -110 mV), it is the FMN hydroquinone (FMN_sq/hq, -270 mV), not the semiquinone, that donates an electron to its redox partners (8–11). CYPOR is the prototype of the mammalian diflavin-containing enzyme family, which includes nitric-oxide synthase (12), methionine synthase reductase (13, 14), and a novel reductase expressed in the cytoplasm of certain cancer cells (15). CYPOR is also a target for anticancer therapy, because it reductively activates anticancer prodrugs (16).CYPOR consists of an N-terminal single α-helical transmembrane anchor (∼6 kDa) responsible for its localization to the endoplasmic reticulum and the soluble cytosolic portion (∼66 kDa) capable of reducing cytochrome c. Crystal structures of the soluble form of the wild-type and several mutant CYPORs are available (17, 18). The first ∼170 amino acids of the soluble domain are highly homologous to flavodoxin and bind FMN (FMN domain), whereas the C-terminal portion of the soluble protein consists of a FAD- and NADPH-binding domain with sequence and structural similarity to ferredoxin-NADP⁺ oxidoreductase (FAD domain). A connecting domain, possessing a unique sequence and structure, joins the FMN and FAD domains and is partly responsible for the relative orientation of the FMN and FAD domains. In the crystal structure, a convex anionic surface surrounds FMN. In the wild-type crystal structure, the two flavin isoalloxazine rings are in van der Waals contact, poised for efficient interflavin electron transfer (17). Based on the juxtaposition of the two flavins, an extrinsic electron transfer rate of ∼10¹⁰ s^-1 is predicted (19). However, the experimentally observed electron transfer rate between the two flavins is 30–55 s^-1 (20, 21). This modest rate and slowing of electron transfer in a viscous solvent (75% glycerol) suggest that interflavin electron transfer is likely conformationally gated. Moreover, the “closed” crystal structure, in which the flavins are in contact, is difficult to reconcile with mutagenesis studies that indicate the acidic amino acid residues on the surface near FMN are involved in interacting with cyt P450 (22). The first structural insight into how cyt P450 might interact with the FMN domain of CYPOR was provided by the crystal structure of a complex between the heme and FMN-containing domains of cyt P450 BM3 (23). In this complex, the methyl groups of FMN are oriented toward the heme on the proximal surface of cyt P450 BM3. Considered together, these three observations, the slow interflavin electron transfer, the mutagenesis data, and the structure of the complex between the heme and FMN domains of cyt P450 BM3, suggest that CYPOR will undergo a large conformational rearrangement in the course of shuttling electrons from NADPH to cyt P450. In addition, crystal structures of various CYPOR variants indicate that the FMN domain is highly mobile with respect to the rest of the molecule (18).Consideration of how the reductase would undergo a reorientation to interact with its redox partners led us to hypothesize the existence of a structural element in the reductase that would regulate the conformational changes and the relative dynamic motion of the domains. Our attention focused on the hinge region between the FMN and the connecting domain, because it is often disordered and highly flexible in the crystal structure (supplemental Fig. S1). The length and sequence of the hinge have been altered by site-directed mutagenesis, and the effects of the mutations on the catalytic properties of each mutant have been determined. The results demonstrate that lengthening the linker or altering its sequence do not modify the properties of CYPOR. In contrast, deletion of four amino acids markedly disrupts electron transfer from FAD to FMN, whereas the ability of the FMN domain to donate electrons to cyt P450 remains intact. The hinge deletion variant has been crystallized in three “open” conformations capable of interacting with cyt P450.     

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

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

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

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

Selective Inhibition of Carotenoid Cleavage Dioxygenases: PHENOTYPIC EFFECTS ON SHOOT BRANCHING*S?

Members of the carotenoid cleavage dioxygenase family catalyze the oxidative cleavage of carotenoids at various chain positions, leading to the formation of a wide range of apocarotenoid signaling molecules. To explore the functions of this diverse enzyme family, we have used a chemical genetic approach to design selective inhibitors for different classes of carotenoid cleavage dioxygenase. A set of 18 arylalkyl-hydroxamic acids was synthesized in which the distance between an iron-chelating hydroxamic acid and an aromatic ring was varied; these compounds were screened as inhibitors of four different enzyme classes, either in vitro or in vivo. Potent inhibitors were found that selectively inhibited enzymes that cleave carotenoids at the 9,10 position; 50% inhibition was achieved at submicromolar concentrations. Application of certain inhibitors at 100 μm to Arabidopsis node explants or whole plants led to increased shoot branching, consistent with inhibition of 9,10-cleavage.Carotenoids are synthesized in plants and micro-organisms as photoprotective molecules and are key components in animal diets, an example being β-carotene (pro-vitamin A). The oxidative cleavage of carotenoids occurs in plants, animals, and micro-organisms and leads to the release of a range of apocarotenoids that function as signaling molecules with a diverse range of functions (1). The first gene identified as encoding a carotenoid cleavage dioxygenase (CCD)² was the maize Vp14 gene that is required for the formation of abscisic acid (ABA), an important hormone that mediates responses to drought stress and aspects of plant development such as seed and bud dormancy (2). The VP14 enzyme cleaves at the 11,12 position (Fig. 1) of the epoxycarotenoids 9′-cis-neoxanthin and/or 9-cis-violaxanthin and is now classified as a 9-cis-epoxycarotenoid dioxygenase (NCED) (3), a subclass of the larger CCD family.Open in a separate windowFIGURE 1.Reactions catalyzed by the carotenoid cleavage dioxygenases. a, 11,12-oxidative cleavage of 9′-cis-neoxanthin by NCED; b, oxidative cleavage reactions on β-carotene and zeaxanthin.Since the discovery of Vp14, many other CCDs have been shown to be involved in the production of a variety of apocarotenoids (Fig. 1). In insects, the visual pigment retinal is formed by oxidative cleavage of β-carotene by β-carotene-15,15′-dioxygenase (4). Retinal is produced by an orthologous enzyme in vertebrates, where it is also converted to retinoic acid, a regulator of differentiation during embryogenesis (5). A distinct mammalian CCD is believed to cleave carotenoids asymmetrically at the 9,10 position (6) and, although its function is unclear, recent evidence suggests a role in the metabolism of dietary lycopene (7). The plant volatiles β-ionone and geranylacetone are produced from an enzyme that cleaves at the 9,10 position (8) and the pigment α-crocin found in the spice saffron results from an 7,8-cleavage enzyme (9).Other CCDs have been identified where biological function is unknown, for example, in cyanobacteria where a variety of cleavage specificities have been described (10-12). In other cases, there are apocarotenoids with known functions, but the identity or involvement of CCDs have not yet been described: grasshopper ketone is a defensive secretion of the flightless grasshopper Romalea microptera (13), mycorradicin is produced by plant roots during symbiosis with arbuscular mycorrhyza (14), and strigolactones (15) are plant metabolites that act as germination signals to parasitic weeds such as Striga and Orobanche (16).Recently it was discovered that strigolactones also function as a branching hormone in plants (17, 18). The existence of such a branching hormone has been known for some time, but its identity proved elusive. However, it was known that the hormone was derived from the action of at least two CCDs, max3 and max4 (more axillary growth) (19), because deletion of either of these genes in Arabidopsis thaliana, leads to a bushy phenotype (20, 21). In Escherichia coli assays, AtCCD7 (max3) cleaves β-carotene at the 9,10 position and the apocarotenoid product (10-apo-β-carotene) is reported to be further cleaved at 13,14 by AtCCD8 (max4) to produce 13-apo-β-carotene (22). Also recent evidence suggests that AtCCD8 is highly specific, cleaving only 10-apo-β-carotene (23). How the production of 13-apo-β-carotene leads to the synthesis of the complex strigolactone is unknown. The possibility remains that the enzymes may have different specificities and cleavage activities in planta. In addition, a cytochrome P450 enzyme (24) is believed to be involved in strigolactone synthesis and acts in the pathway downstream of the CCD genes. Strigolactone is thought to effect branching by regulating auxin transport (25). Because of the involvement of CCDs in strigolactone synthesis, the possibility arises that plant architecture and interaction with parasitic weeds and mycorrhyza could be controlled by the manipulation of CCD activity.Although considerable success has been obtained using genetic approaches to probe function and substrate specificity of CCDs in their native biological contexts, particularly in plant species with simple genetic systems or that are amenable to transgenesis, there are many systems where genetic approaches are difficult or impossible. Also, when recombinant CCDs are studied either in vitro or in heterologous in vivo assays, such as in E. coli strains engineered to accumulate carotenoids (26), they are often active against a broad range of substrates (5, 21, 27), and in many cases the true in vivo substrate of a particular CCD remains unknown. Therefore additional experimental tools are needed to investigate both apocarotenoid and CCD functions in their native cellular environments.In the reverse chemical genetics approach, small molecules are identified that are active against known target proteins; they are then applied to a biological system to investigate protein function in vivo (28, 29). This approach is complementary to conventional genetics since the small molecules can be applied easily to a broad range of species, their application can be controlled in dose, time, and space to provide detailed studies of biological functions, and individual proteins or whole protein classes may be targeted by varying the specificity of the small molecules. Notably, functions of the plant hormones gibberellin, brassinosteroid, and abscisic acid have been successfully probed using this approach by adapting triazoles to inhibit specific cytochrome P450 monooxygenases involved in the metabolism of these hormones (30).In the case of the CCD family, the tertiary amines abamine (31) and the more active abamineSG (32) were reported as specific inhibitors of NCED, and abamine was used to show new functions of abscisic acid in legume nodulation (33). However, no selective inhibitors for other types of CCD are known. Here we have designed a novel class of CCD inhibitor based on hydroxamic acids, where variable chain length was used to direct inhibition of CCD enzymes that cleave carotenoids at specific positions. We demonstrate the use of such novel inhibitors to control shoot branching in a model plant.     

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

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

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

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

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.     

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.     

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.     

Covalently Bound Substrate at the Regulatory Site of Yeast Pyruvate Decarboxylases Triggers Allosteric Enzyme Activation

The mechanism by which the enzyme pyruvate decarboxylase from two yeast species is activated allosterically has been elucidated. A total of seven three-dimensional structures of the enzyme, of enzyme variants, or of enzyme complexes from two yeast species, three of them reported here for the first time, provide detailed atomic resolution snapshots along the activation coordinate. The prime event is the covalent binding of the substrate pyruvate to the side chain of cysteine 221, thus forming a thiohemiketal. This reaction causes the shift of a neighboring amino acid, which eventually leads to the rigidification of two otherwise flexible loops, one of which provides two histidine residues necessary to complete the enzymatically competent active site architecture. The structural data are complemented and supported by kinetic investigations and binding studies, providing a consistent picture of the structural changes occurring upon enzyme activation.Pyruvate decarboxylases (EC 4.1.1.1) catalyze the non-oxidative decarboxylation of pyruvate, yielding acetaldehyde and carbon dioxide. Together with the enzyme alcohol dehydrogenase (EC 1.1.1.1), which reduces the acetaldehyde to ethanol with the help of the co-substrate NADH, it represents the metabolic pathway of alcoholic fermentation. PDC³ is localized in the cytosol of cells from yeasts, plant seeds, and a few bacteria. The catalytic activity of PDC depends on the presence of the cofactor thiamine diphosphate (ThDP), which is bound mainly via a divalent metal ion (magnesium in most cases) to the protein moiety. Many detailed kinetic studies have been published on yeast PDC wild types (1–9). A number of ScPDC variants were analyzed, too (1–9). Some active site variants (E51A, D28A, E477Q) proved to be almost catalytically inactive. PDCs are multisubunit enzymes. The typical molecular mass of one subunit is 59–61 kDa. The tetramer is the catalytically active state of most PDCs. Higher oligomers (octamers) have been described for PDCs from plant seeds (10, 11) or some fungi (12). However, studies on structure function relationships of yeast PDCs showed that the dimer is the minimum functional unit of the enzyme displaying considerable catalytic activity (13, 14). The two closely related pyruvate decarboxylases from Saccharomyces cerevisiae (ScPDC) and Kluyveromyces lactis (KlPDC) are well characterized ThDP-dependent enzymes, which share 86.3% identical amino acid residues. They have been studied in great detail by means of kinetic investigations and spectroscopic studies. Both enzymes are allosterically regulated as reflected by sigmoid steady state kinetics and lag phases in their progress curves. The substrate PYR activates the initially inactive yeast PDCs in a time-dependent manner. Kinetic studies reveal a slow isomerization as triggered by substrate binding to a separate regulatory site (15). A number of substrate surrogates have been identified, which are able to activate PDC as well. The effects of pyruvamide (PA; for the chemical structure, see Scheme 1) on the activation kinetics have been studied in detail for ScPDC (15) and for KlPDC (16). Phosphonate analogues (among them methyl acetylphosphonate, MAP, Scheme 1) of pyruvate have been applied to elucidate the catalytic cycle (17–21) or to trap reaction intermediates in crystal structures (22–24). Chemical modification of PDCs with group-specific reagents pointed to an important role of cysteine residues (25). Site-directed mutagenesis of cysteine residues to alanine or serine demonstrated that residue Cys-221 might be the decisive one for enzyme activation (1, 4, 26, 27). Consequently, it was postulated that the region around Cys-221 is the regulatory site of PDC, and formation of a thiohemiketal at this side chain was proposed. However, a number of questions remained elusive. (i) How is the activator fixed at the regulatory site? (ii) What are the prime structural properties of the active state as compared with the inactive state? (iii) How is the signal transmitted from the regulatory to the active site? (iv) Which are the decisive features of the active site in the activated state that render efficient catalysis possible? To answer these questions, we present here the crystal structures of KlPDC with the bound substrate surrogate MAP and of the ScPDC variants D28E and E477Q with bound substrate PYR along with kinetic studies on the activating effect of both activators and binding studies using the small angle x-ray solution scattering (SAXS) method.Open in a separate windowSCHEME 1.Chemical structures of the substrate pyruvate, the activators pyruvamide and methyl acetylphosphonate, and the thiohemiketal from pyruvate and cysteine, respectively.     

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.     

Nitrile-specifier Proteins Involved in Glucosinolate Hydrolysis in Arabidopsis thaliana

Glucosinolates are plant secondary metabolites present in Brassicaceae plants such as the model plant Arabidopsis thaliana. Intact glucosinolates are believed to be biologically inactive, whereas degradation products after hydrolysis have multiple roles in growth regulation and defense. The degradation of glucosinolates is catalyzed by thioglucosidases called myrosinases and leads by default to the formation of isothiocyanates. The interaction of a protein called epithiospecifier protein (ESP) with myrosinase diverts the reaction toward the production of epithionitriles or nitriles depending on the glucosinolate structure. Here we report the identification of a new group of nitrile-specifier proteins (AtNSPs) in A. thaliana able to generate nitriles in conjunction with myrosinase and a more detailed characterization of one member (AtNSP2). Recombinant AtNSP2 expressed in Escherichia coli was used to test its impact on the outcome of glucosinolate hydrolysis using a gas chromatography-mass spectrometry approach. AtNSP proteins share 30–45% sequence homology with A. thaliana ESP. Although AtESP and AtNSP proteins can switch myrosinase-catalyzed degradation of 2-propenylglucosinolate from isothiocyanate to nitrile, only AtESP generates the corresponding epithionitrile. Using the aromatic benzylglucosinolate, recombinant AtNSP2 is also able to direct product formation to the nitrile. Analysis of glucosinolate hydrolysis profiles of transgenic A. thaliana plants overexpressing AtNSP2 confirms its nitrile-specifier activity in planta. In silico expression analysis reveals distinctive expression patterns of AtNSPs, which supports a biological role for these proteins. In conclusion, we show that AtNSPs belonging to a new family of A. thaliana proteins structurally related to AtESP divert product formation from myrosinase-catalyzed glucosinolate hydrolysis and, thereby, likely affect the biological consequences of glucosinolate degradation. We discuss similarities and properties of AtNSPs and related proteins and the biological implications.Brassicaceae plants such as oilseed rape (Brassica napus), turnip (Brassica rapa), and white mustard (Sinapis alba) as well as the model plant Arabidopsis thaliana contain a group of secondary metabolites known as glucosinolates (GSLs)² (1, 2). These are β-thioglucoside N-hydroxysulfates with a sulfur-linked β-d-glucopyranose moiety and a variable side chain that is derived from one of eight amino acids or their methylene group-elongated derivatives. Aliphatic GSLs are derived from alanine, leucine, isoleucine, valine, or predominantly methionine. Tyrosine or phenylalanine give aromatic GSLs, and tryptophan-derived GSLs are called indolic GSLs (for review, see Ref. 3). Although more than 120 different GSLs have been identified in total so far, individual plant species usually contain only a few GSLs (2). Quantitative and qualitative differences of GSL profiles are also observed within a species, such as, for example, for different A. thaliana ecotypes (4–6). In addition, GSL composition varies among organs and during the life cycle of plants (7, 8) and is affected by external factors (9).Intact GSLs are mostly considered to be biologically inactive. Most GSL degradation products have toxic effects on insect, fungal, and bacterial pests, serve as attractants for specialist insects, or may have beneficial health effects for humans (10–15). The enzymatic degradation of GSLs (Fig. 1A), which occurs massively upon tissue damage, is catalyzed by plant thioglucosidases called myrosinases (EC 3.2.1.147; glycoside hydrolase family 1). Depending on several factors (e.g. GSL structure, proteins, cofactors, pH) myrosinase-catalyzed hydrolysis of GSLs can lead to a variety of products (Fig. 1B; for review, see Refs. 16 and 17). Of these, isothiocyanates are the most common as their formation only requires myrosinase activity. Thiocyanates on the other hand are only produced from a very limited number of GSLs, and their formation necessitates the presence of a thiocyanate-forming factor in addition to myrosinase (18). A thiocyanate-forming protein (TFP) has recently been identified in Lepidium sativum (19). Alkenyl GSLs, a subgroup of aliphatic GSLs containing a terminal unsaturation in their side chain, can lead to the production of epithionitriles through the cooperative action of myrosinase and a protein called epithiospecifier protein (ESP (20)) in a ferrous ion-dependent way (21–23). Both TFP and ESP contain a series of Kelch repeats (19). Kelch repeats are involved in protein-protein interactions, and Kelch repeat-containing proteins are involved in a number of diverse biological processes (24). In addition to isothiocyanates, nitriles are the major group of GSL hydrolysis products. Although ESP and TFP activities can generate nitriles (19, 21, 25, 26), indications for an ESP-independent nitrile-specifier activity exist. The GSL hydrolysis profile of A. thaliana roots, an organ that does not show ESP expression or activity (27), reveals predominantly the presence of nitriles (28). In addition, leaf tissue of A. thaliana ecotypes supposedly devoid of ESP activity produces a certain amount of nitriles upon autolysis (21). Under acidic buffer conditions, a non-enzymatic production of nitriles from GSLs is observed (Ref. 29 and references therein). Increasing Fe²⁺ concentrations have also been shown to favor nitrile formation over isothiocyanate formation from a number of GSLs in the presence of myrosinase and absence of ESP (21, 22). Therefore, a non-enzymatic origin of this nitrile production cannot be excluded, although the presence of a nitrile-specifier protein is a tempting alternative. Although ESP is able to generate nitriles, it has also been shown that the conversion rates of GSLs to nitriles are lower than those of GSLs to epithionitriles for ESP (21, 22).Open in a separate windowFIGURE 1.Simplified scheme of enzymatic GSL hydrolysis (A) and structures and names of GSLs and their hydrolysis products that are mentioned in the article. (B). A, myrosinase acts on GSLs to form an unstable aglycone intermediate that can rearrange spontaneously to form an isothiocyanate. Hydrolysis can be diverted from this default route under certain conditions (e.g. the presence of NSPs, ferrous ions, or at pH < 5) to give the corresponding nitrile. ESP is responsible for the formation of epithionitriles from alkenyl GSLs in a ferrous ion-dependent mechanism. B, the general structure of GSLs, indicating the variable side chain as R, is given as well as the three major classes of hydrolysis products (i.e. isothiocyanates, nitriles, and epithionitriles). The listed GSLs are the ones mentioned in this article and are arranged according to the class of GSLs they belong to and with an increase in chain length or complexity. The names of the respective hydrolysis products are given for a better understanding of the present article, and not all were encountered during our studies.A nitrile-specifier protein (NSP) that is able to redirect the hydrolysis of GSLs toward nitriles has been cloned from the larvae of the butterfly Pieris rapae (30). This protein does not, however, exhibit sequence similarity to plant ESP, and a corresponding plant nitrile-specifier protein has not yet been identified. We report here the identification of a group of six A. thaliana genes with some sequence similarity to A. thaliana ESP, providing evidence for a new family of nitrile-specifier proteins and a more detailed characterization of one member that possesses nitrile-specifier activity in vitro, when applied exogenously to plant tissue and after ectopic expression in the two A. thaliana ecotypes Col-0 and C24. Despite its sequence homology to A. thaliana epithiospecifier protein (AtESP), it does not possess epithiospecifier activity under similar conditions. Therefore, we propose to designate this protein as A. thaliana nitrile-specifier protein 2 (AtNSP2). Although the biological roles of AtNSP2 and related proteins are not yet known, their specificities and distinctive expression patterns indicate the presence of a fine-tuned mechanism for GSL degradation controlling the outcome of an array of biologically active molecules.     

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

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

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.     

Structural and Functional Dissection of the Heterocyclic Peptide Cytotoxin Streptolysin S

The human pathogen Streptococcus pyogenes secretes a highly cytolytic toxin known as streptolysin S (SLS). SLS is a key virulence determinant and responsible for the β-hemolytic phenotype of these bacteria. Despite over a century of research, the chemical structure of SLS remains unknown. Recent experiments have revealed that SLS is generated from an inactive precursor peptide that undergoes extensive post-translational modification to an active form. In this work, we address outstanding questions regarding the SLS biosynthetic process, elucidating the features of substrate recognition and sites of posttranslational modification to the SLS precursor peptide. Further, we exploit these findings to guide the design of artificial cytolytic toxins that are recognized by the SLS biosynthetic enzymes and others that are intrinsically cytolytic. This new structural information has ramifications for future antimicrobial therapies.Streptolysin S (SLS)⁴ is secreted by the human pathogen Streptococcus pyogenes, the causative agent of diseases ranging from pharyngitis to necrotizing fasciitis (1). SLS is a potent cytolysin that is ribosomally synthesized, extensively posttranslationally modified, and exported to exert its effects on the target cell (2, 3). The expression of SLS promotes virulence in animal models of invasive infection and accounts for the hall-mark zone of β-hemolysis surrounding colonies of these bacteria grown on blood agar (2, 4). An intriguing feature of SLS is its nonimmunogenic nature (5). This characteristic is likely due to its small size and its capacity to lyse cells involved in both innate and adaptive immunity (6, 7). The β-hemolytic phenotype of S. pyogenes has been studied since the early 1900s, but the molecular structure of SLS has remained elusive (8). In the last decade, transposon mutagenesis studies identified the gene encoding the SLS toxin precursor (sagA, for SLS-associated gene) and eight additional genes in an operon required for toxin maturation and export (9). Targeted mutagenesis of the sag operon yields nonhemolytic S. pyogenes mutants with markedly diminished virulence in mice (2). More recently, it was demonstrated that the protein products of sagA–D are sufficient for the in vitro reconstitution of cytolytic activity (3). The first gene product, SagA, serves as a structural template that after a series of tailoring reactions matures into the active SLS metabolite (see Fig. 1A). A trimeric complex of SagBCD catalyzes these tailoring reactions, which results in the conversion of cysteine, serine, and threonine residues to thiazole, oxazole, and methyloxazole heterocycles, respectively (3).Open in a separate windowFIGURE 1.SagBCD substrate recognition is provided by the SagA leader peptide. A, SagA is converted into an active cytolysin, pro-streptolysin-S (pro-SLS), by the actions of SagBCD (a trimeric oxazole/thiazole synthetase). Heterocycles are schematically represented as shaded pentagons. A marginally conserved motif in the SagA leader peptide, FXXXB (where B is a branched chain amino acid), is highlighted in red. Individual reactions catalyzed by SagC (cyclodehydratase) and SagB (FMN-dehydrogenase) are shown. B, representative amino acid sequences and cytolytic activity of SagA-like substrates. Shown in red are leader peptide residues that comprise the FXXXB motif. The putative leader peptide cleavage sites are shown as asterisks, except for McbA, where the site is known (hyphen). In blue are sites of potential heterocycle formation (for McbA, known sites are blue). The percentage of amino acid similarity to full-length SagA (as determined by ClustalW alignment) is given. The cytolytic activity was tested for these substrates in vitro using purified proteins and in vivo using the SLS-deficient strain, S. pyogenes ΔsagA, complemented with the desired substrate. Activity equal to wild type SagA is designated as (+++); activity that is 30–70% of wild type SagA is (++); detectable activity that is less than 30% of SagA is noted as (+); and nondetectable activity is (-). The activity for McbA is not applicable (n.a.) because this secondary metabolite is a DNA gyrase inhibitor, not a cytolysin. C, sequences and lytic activity of mutant substrates. All of the substrates contain the wild type SagA leader peptide, except for the first entry (FXXXB mutant, SagA-FIA). The percentage of amino acid similarity to the protoxin half of SagA is shown. The second and third entries are SagA leader peptides fused to the protoxin of StaphA and ListA. SagX is an artificially designed toxin, whereas the inverse and scrambled substrates manipulate the sequence of SagA between residues 33–50 (underlined).A DNA gyrase inhibitor, microcin B17, is produced by an orthologous biosynthetic cluster (mcb) found in a subset of Escherichia coli strains (10–12). Microcin B17 contains four thiazole and four oxazole heterocycles, which are indispensable for biological activity. By analogy to microcin B17 and the lantibiotics, the heterocycles of SLS are formed on the C terminus of SagA, whereas the N terminus serves as a leader peptide (13–15). The installation of thiazole and (methyl)-oxazole heterocycles restricts backbone conformational flexibility and provides microcin B17 and SLS with rigidified structures. The SLS heterocycles are formed via two distinct steps; SagC, a cyclodehydratase, generates thiazoline and (methyl)-oxazoline heterocycles, whereas SagB, a dehydrogenase, removes two electrons to afford the aromatic thiazole and (methyl)-oxazole (3, 16, 17). SagD is proposed to play a role in trimer formation and regulation (see Fig. 1A). The final genes in the genetic cluster encode a predicted leader peptidase/immunity protein (SagE), a membrane-associated protein of unknown function (SagF), and three ABC transporters (SagGHI).It is now appreciated that many other prokaryotes harbor similar genetic clusters for the synthesis of thiazole and (methyl)-oxazole heterocycles (3, 18, 19). Additional important mammalian pathogens such as Listeria monocytogenes, Staphylococcus aureus, and Clostridium botulinum, contain sag-like gene clusters that produce SLS-like cytolysins. These toxins are expected to promote pathogen survival and host cell injury during infection, but this has only been conclusively shown for S. pyogenes and L. monocytogenes (2, 18). Like E. coli, many other prokaryotes harbor a sag-like genetic cluster but are not known to produce cytolysins. Some examples are the goadsporin-producing organism, Streptomyces sp. TP-A0584 and cyanobactin producers such as Prochloron didemni (20–22). The molecular targets of these secondary metabolites remain to be elucidated, but it is known that goadsporin exhibits antibiotic activity, and the cyanobactin, patellamide D, reverses multiple drug resistance in a human leukemia cell line (23). Because genetic loci containing sagBCD-like genes have been widely disseminated in prokaryotes (3), nature appears to have found a preferred route to synthesizing such secondary metabolites.In this work, we build upon our initial report on the in vitro reconstitution of SLS biosynthesis to uncover the requisite features of substrate selectivity and cytolytic activity. The impetus for defining substrate tolerance arose from earlier results showing that SagBCD accepts alternate substrates in vitro (3), as evidenced by two key experiments. First, SagBCD converted a noncognate substrate, ClosA (C. botulinum), into a cytolytic entity. Second, mass spectrometry revealed heterocycle formation on the McbA (E. coli) peptide after SagBCD treatment (3). Here, we dissect the N-terminal leader peptide and C-terminal protoxin of SagA to define the residues necessary for conversion into SLS.