The 1.8-A resolution crystal structure of YDR533Cp from Saccharomyces cerevisiae: a member of the DJ-1/ThiJ/PfpI superfamily

The yeast gene YDR533C encodes a protein belonging to the DJ-1/ThiJ/PfpI superfamily. This family includes the human protein DJ-1, which is mutated in autosomal recessive early-onset Parkinson's disease. The function of DJ-1 and its yeast homologue YDR533Cp is unknown. We report here the crystal structure of YDR533Cp at 1.8-A resolution. The structure indicates that the closest relative to YDR533Cp is the Escherichia coli heat shock protein Hsp31 (YedU), which has both chaperone and protease activity. As expected, the overall fold of the core domain of YDR533Cp is also similar to that of DJ-1 and the bacterial protease PfpI. YDR533Cp contains a possible catalytic triad analogous to that of Hsp31 and an additional domain that is present in Hsp31 but is not seen in DJ-1 and other members of the family. The cysteine in this triad (Cys-138) is oxidized in this crystal structure, similar to modifications seen in the corresponding cysteine in the crystal structure of DJ-1. YDR533Cp appears to be a dimer both in solution and the crystal, but this dimer is formed by a different interface than that found in Hsp31 or other members of the superfamily.     

Mutational analysis of DJ-1 in Drosophila implicates functional inactivation by oxidative damage and aging

Inherited mutations in PARK7, the gene encoding DJ-1, are associated with loss of protein function and early-onset parkinsonism. Like human DJ-1 (hDJ-1), Drosophila DJ-1b protects against oxidative insult and is modified with oxidation. We demonstrate that hDJ-1 rescues flies mutant for DJ-1b, and that a conserved cysteine residue in the fly protein (C104, analogous to C106 in hDJ-1) is critical for biological antioxidant function in vivo. Targeted mutagenesis suggests that modification of DJ-1b at this residue inactivates the protective activity of the protein against oxidative stress. Further studies show that DJ-1 modification increases dramatically with age in flies, mice, and humans, with aged flies showing strikingly increased susceptibility to oxidative stress and markedly enhanced DJ-1b modification upon oxidative challenge. Overoxidation of DJ-1 with age and exposure to oxidative toxins may lead to inactivation of DJ-1 function, suggesting a role in susceptibility to sporadic Parkinson's disease.     

Crystal structure of an intracellular protease from Pyrococcus horikoshii at 2-A resolution

The intracellular protease from Pyrococcus horikoshii (PH1704) and PfpI from Pyrococcus furiosus are members of a class of intracellular proteases that have no sequence homology to any other known protease family. We report the crystal structure of PH1704 at 2.0-A resolution. The protease is tentatively identified as a cysteine protease based on the presence of cysteine (residue 100) in a nucleophile elbow motif. In the crystal, PH1704 forms a hexameric ring structure, and the active sites are formed at the interfaces between three pairs of monomers.     

The Parkinson's disease protein DJ-1 is neuroprotective due to cysteine-sulfinic acid-driven mitochondrial localization

Loss-of-function DJ-1 mutations can cause early-onset Parkinson's disease. The function of DJ-1 is unknown, but an acidic isoform accumulates after oxidative stress, leading to the suggestion that DJ-1 is protective under these conditions. We addressed whether this represents a posttranslational modification at cysteine residues by systematically mutating cysteine residues in human DJ-1. WT or C53A DJ-1 was readily oxidized in cultured cells, generating a pI 5.8 isoform, but an artificial C106A mutant was not. We observed a cysteine-sulfinic acid at C106 in crystalline DJ-1 but no modification of C53 or C46. Oxidation of DJ-1 was promoted by the crystallization procedure. In addition, oxidation-induced mitochondrial relocalization of DJ-1 and protection against cell death were abrogated in C106A but not C53A or C46A. We suggest that DJ-1 protects against neuronal death, and that this is signaled by acidification of the key cysteine residue, C106.     

Crystal structure of a multifunctional 2-Cys peroxiredoxin heme-binding protein 23 kDa/proliferation-associated gene product

Heme-binding protein 23 kDa (HBP23), a rat isoform of human proliferation-associated gene product (PAG), is a member of the peroxiredoxin family of peroxidases, having two conserved cysteine residues. Recent biochemical studies have shown that HBP23/PAG is an oxidative stress-induced and proliferation-coupled multifunctional protein that exhibits specific bindings to c-Abl protein tyrosine kinase and heme, as well as a peroxidase activity. A 2.6-A resolution crystal structure of rat HBP23 in oxidized form revealed an unusual dimer structure in which the active residue Cys-52 forms a disulfide bond with conserved Cys-173 from another subunit by C-terminal tail swapping. The active site is largely hydrophobic with partially exposed Cys-173, suggesting a reduction mechanism of oxidized HBP23 by thioredoxin. Thus, the unusual cysteine disulfide bond is involved in peroxidation catalysis by using thioredoxin as the source of reducing equivalents. The structure also provides a clue to possible interaction surfaces for c-Abl and heme. Several significant structural differences have been found from a 1-Cys peroxiredoxin, ORF6, which lacks the C-terminal conserved cysteine corresponding to Cys-173 of HBP23.     

Differentiation of multiple domains in the herpes simplex virus 1 protease encoded by the UL26 gene.

Previous studies have shown that the herpes simplex virus 1 gene UL26 encodes a 635-amino acid protease that cleaves approximately 20 amino acids from the carboxyl terminus of itself and of a 329-amino acid product of the UL26.5 gene. The results of studies with a variety of protease inhibitors showed that the UL26 protease was inhibited by serine protease inhibitors but not by inhibitors of cysteine protease, aspartic acid protease, or metalloprotease. Mutations resulting in amino acid substitutions, deletions, or insertion of stop codons in the gene or of 20-amino acid stretches into the protease have delineated the dispensable domains I and IV at the amino and carboxyl domains of the gene product. The essential carboxyl-proximal domain (III) can be separated from the essential amino-proximal domain (II) by at least 20 amino acids. The amino-proximal domain is the most conserved region among varicella-zoster virus and human cytomegalovirus homologues of UL26. Of the conserved aspartic acid, histidine, or serine amino acids in this domain, only histidine-61 and -148 could not be replaced without impairment of the proteolytic activity.     

The 1.6-A crystal structure of the class of chaperones represented by Escherichia coli Hsp31 reveals a putative catalytic triad

Heat shock proteins (Hsps) play essential protective roles under stress conditions by preventing the formation of protein aggregates and degrading misfolded proteins. EcHsp31, the yedU (hchA) gene product, is a representative member of a family of chaperones that alleviates protein misfolding by interacting with early unfolding intermediates. The 1.6-A crystal structure of the EcHsp31 dimer reveals a system of hydrophobic patches, canyons, and grooves, which may stabilize partially unfolded substrate. The presence of a well conserved, yet buried, triad in each two-domain subunit suggests a still unproven hydrolytic function of the protein. A flexible extended linker between the A and P domains may play a role in conformational flexibility and substrate binding. The alpha-beta sandwich of the EcHsp31 monomer shows structural similarity to PhPI, a protease belonging to the DJ-1 superfamily. The structure-guided sequence alignment indicates that Hsp31 homologs can be divided in three classes based on variations in the P domain that dramatically affect both oligomerization and catalytic triad formation.     

The Parkinson's disease gene DJ-1 is also a key regulator of stroke-induced damage

Recent evidence has indicated that common mechanisms play roles among multiple neurological diseases. However, the specifics of these pathways are not completely understood. Stroke is caused by the interruption of blood flow to the brain, and cumulative evidence supports the critical role of oxidative stress in the ensuing neuronal death process. DJ-1 (PARK7) has been identified as the gene linked to early-onset familial Parkinson's disease. Currently, our work also shows that DJ-1 is central to death in both in Vitro and in Vivo models of stroke. Loss of DJ-1 increases the sensitivity to excitotoxicity and ischemia, whereas expression of DJ-1 can reverse this sensitivity and indeed provide further protection. Importantly, DJ-1 expression decreases markers of oxidative stress after stroke insult in Vivo, suggesting that DJ-1 protects through alleviation of oxidative stress. Consistent with this finding, we demonstrate the essential role of the oxidation-sensitive cysteine-106 residue in the neuroprotective activity of DJ-1 after stroke. Our work provides an important example of how a gene seemingly specific for one disease, in this case Parkinson's disease, also appears to be central in other neuropathological conditions such as stroke. It also highlights the important commonalities among differing neuropathologies.     

Crystal structure of the zymogen form of the group A Streptococcus virulence factor SpeB: an integrin-binding cysteine protease

Pathogenic bacteria secrete protein toxins that weaken or disable their host, and thereby act as virulence factors. We have determined the crystal structure of streptococcal pyrogenic exotoxin B (SpeB), a cysteine protease that is a major virulence factor of the human pathogen Streptococcus pyogenes and participates in invasive disease episodes, including necrotizing fasciitis. The structure, determined for the 40-kDa precursor form of SpeB at 1.6-A resolution, reveals that the protein is a distant homologue of the papain superfamily that includes the mammalian cathepsins B, K, L, and S. Despite negligible sequence identity, the protease portion has the canonical papain fold, albeit with major loop insertions and deletions. The catalytic site differs from most other cysteine proteases in that it lacks the Asn residue of the Cys-His-Asn triad. The prosegment has a unique fold and inactivation mechanism that involves displacement of the catalytically essential His residue by a loop inserted into the active site. The structure also reveals the surface location of an integrin-binding Arg-Gly-Asp (RGD) motif that is a feature unique to SpeB among cysteine proteases and is linked to the pathogenesis of the most invasive strains of S. pyogenes.     

融合DJ-1及其活性多肽片段的抗氧化应激作用及机制

目的 探讨融合DJ-1及其活性多肽片段的抗氧化应激作用及其机制。方法在稳定的抗氧化应激实验系统中,分别观察20μmol／LH2O2作用下不同质量浓度His融合DJ-1对钙调神经磷酸酶（CaN）活性的影响;在1．5μmol／LH2O2作用下DJ-1活性多肽片段102—107IAMCA和106—111CAGPTA对乳酸脱氢酶（LDH）活性的影响。结果两种浓度His融合DJ．1均可对抗H20：诱导的氧化应激,恢复CaN活性;DJ-1活性多肽片段102—107IAAICA和106～111CAGPTA均可部分对抗H2O2诱导的氧化应激,提高LDH活性。结论融合DJ-1及其活性多肽片段具有抗氧化应激作用;DJ-1完整蛋白可能通过自身C106残基氧化来对抗氧化应激,此为今后对抗H2O2活性多肽的发展及应用提供了依据。     

Regulation of tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis by DJ-1 in thyroid cancer cells

DJ-1, a cancer-associated protein protects cells from multiple toxic stresses. The expression of DJ-1 and its influence on thyroid cancer cell death has not been investigated so far. We analyzed DJ-1 expression in human thyroid carcinoma cell lines and the effect of DJ-1 on tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. DJ-1 was expressed in human thyroid carcinoma cell lines; small interfering RNA-mediated downregulation of its levels significantly sensitized thyroid carcinoma cells to TRAIL-induced apoptosis, whereas the forced exogenous expression of DJ-1 significantly suppressed cell death induced by TRAIL. We also report here that TRAIL-induced thyroid cancer cell apoptosis is mediated by oxidative stress and that DJ-1, a potent nutritional antioxidant, protects cancer cells from apoptosis at least in part by impeding the elevation of reactive oxygen species levels induced by TRAIL and impairing caspase-8 activation. Subsequently, we investigated DJ-1 expression in 52 normal and 74 primary thyroid carcinomas from patients of China Medical University. The protein was not detectable in the 52 specimens of normal thyroid, while 70 out of 74 analyzed carcinomas (33 out of 33 follicular, 17 out of 19 papillary, 12 out of 13 medullar, and 8 out of 9 anaplastic) were clearly positive for DJ-1 expression. Our data demonstrated that DJ-1 is specifically expressed in thyroid carcinomas and not in the normal thyroid tissue. In addition, the protein modulates the response to TRAIL-mediated apoptosis in human neoplastic thyroid cells, at least partially through its antioxidant property.     

Inactivation of Drosophila DJ-1 leads to impairments of oxidative stress response and phosphatidylinositol 3-kinase/Akt signaling

Parkinson's disease (PD) is the most common movement disorder characterized by dopaminergic dysfunction and degeneration. The cause of most PD cases is unknown, although postmortem studies have implicated the involvement of oxidative stress. The identification of familial PD-associated genes offers the opportunity to study mechanisms of PD pathogenesis in model organisms. Here, we show that DJ-1A, a Drosophila homologue of the familial PD-associated gene DJ-1, plays an essential role in oxidative stress response and neuronal maintenance. Inhibition of DJ-1A function through RNA interference (RNAi) results in cellular accumulation of reactive oxygen species, organismal hypersensitivity to oxidative stress, and dysfunction and degeneration of dopaminergic and photoreceptor neurons. To identify other genes that may interact with DJ-1A in regulating cell survival, we performed genetic interaction studies and identified components of the phosphatidylinositol 3-kinase (PI3K)/Akt-signaling pathway as specific modulators of DJ-1A RNAi-induced neurodegeneration. PI3K signaling suppresses DJ-1A RNAi phenotypes at least in part by reducing cellular reactive oxygen species levels. Consistent with the genetic interaction results, we also found reduced phosphorylation of Akt in DJ-1A RNAi animals, indicating an impairment of PI3K/Akt signaling by DJ-1A down-regulation. Together with recent findings in mammalian systems, these results implicate impairments of PI3K/Akt signaling and oxidative stress response in DJ-1-associated disease pathogenesis. We also observed impairment of PI3K/Akt signaling in the fly parkin model of PD, hinting at a common molecular event in the pathogenesis of PD. Manipulation of PI3K/Akt signaling may therefore offer therapeutic benefits for the treatment of PD.     

RNA binding activity of the recessive parkinsonism protein DJ-1 supports involvement in multiple cellular pathways

Parkinson's disease (PD) is a major neurodegenerative condition with several rare Mendelian forms. Oxidative stress and mitochondrial function have been implicated in the pathogenesis of PD but the molecular mechanisms involved in the degeneration of neurons remain unclear. DJ-1 mutations are one cause of recessive parkinsonism, but this gene is also reported to be involved in cancer by promoting Ras signaling and suppressing PTEN-induced apoptosis. The specific function of DJ-1 is unknown, although it is responsive to oxidative stress and may play a role in the maintenance of mitochondria. Here, we show, using four independent methods, that DJ-1 associates with RNA targets in cells and the brain, including mitochondrial genes, genes involved in glutathione metabolism, and members of the PTEN/PI3K cascade. Pathogenic recessive mutants are deficient in this activity. We show that DJ-1 is sufficient for RNA binding at nanomolar concentrations. Further, we show that DJ-1 binds RNA but dissociates after oxidative stress. These data implicate a single mechanism for the pleiotropic effects of DJ-1 in different model systems, namely that the protein binds multiple RNA targets in an oxidation-dependent manner.     

The cotton fiber zinc-binding domain of cellulose synthase A1 from Gossypium hirsutum displays rapid turnover in vitro and in vivo

Little is known about the assembly and turnover of cellulose synthase complexes commonly called rosettes. Recent work indicates that rosette assembly could involve the dimerization of CesA (cellulose synthase catalytic subunit) proteins regulated by the redox state of the CesA zinc-binding domain (ZnBD). Several studies in the 1980s led to the suggestion that synthase complexes may have very short half-lives in vivo, but no recent work has directly addressed this issue. In the present work, we show that the half-life of cotton fiber GhCesA1 protein is <30 min in vivo, far less than the average membrane protein. We also show that the reduced monomer of GhCesA1 ZnBD is rapidly degraded when exposed to cotton fiber extracts, whereas the oxidized dimer is resistant to degradation. Low rates of degradation activity were detected in vitro by using extracts from fibers harvested during primary cell-wall formation, but activity increased markedly during transition to secondary cell-wall synthesis. In vitro degradation of reduced GhCesA1 ZnBD is inhibited by proteosome inhibitor MG132 and also by E64 and EGTA, suggesting that proteolysis is initiated by cysteine protease activity rather than the proteosome. We used a yeast two-hybrid system to identify a putative cotton fiber metallothionein and to confirm it as a protein that could interact with the GhCesA1 ZnBD. A model is proposed wherein active cellulose synthase complexes contain CesA proteins in dimerized form, and turnover and degradation of the complexes are mediated through reductive zinc insertion by metallothionein and subsequent proteolysis involving a cysteine protease.     

The solution structure of the Josephin domain of ataxin-3: structural determinants for molecular recognition

The Josephin domain plays an important role in the cellular functions of ataxin-3, the protein responsible for the neurodegenerative Machado-Joseph disease. We have determined the solution structure of Josephin and shown that it belongs to the family of papain-like cysteine proteases, sharing the highest degree of structural similarity with bacterial staphopain. A currently unique structural feature of Josephin is a flexible helical hairpin formed by a 32-residue insertion, which could determine substrate specificity. By using the Josephin structure and the availability of NMR chemical shift assignments, we have mapped the enzyme active site by using the typical cysteine protease inhibitors, transepoxysuccinyl-L-eucylamido-4-guanidino-butane (E-64) and [L-3-trans-(propylcarbamyl)oxirane-2-carbonyl]-L-isoleucyl-L-proline (CA-074). We also demonstrate that the specific interaction of Josephin with the ubiquitin-like domain of the ubiquitin- and proteasome-binding factor HHR23B involves complementary exposed hydrophobic surfaces. The structural similarity with other deubiquitinating enzymes suggests a model for the proteolytic enzymatic activity of ataxin-3.     

Structural aspects of activation pathways of aspartic protease zymogens and viral 3C protease precursors.

The three-dimensional structures of the inactive protein precursors (zymogens) of the serine, cysteine, aspartic, and metalloprotease classes of proteolytic enzymes are known. Comparisons of these structures with those of the mature, active proteases reveal that, in general, the preformed, active conformations of the residues involved in catalysis are rendered sterically inaccessible to substrates by the residues of the zymogens' N-terminal extensions or prosegments. The prosegments interact in nonsubstrate-like fashions with the residues of the active sites in most of the cases. The gastric aspartic proteases have a well-characterized zymogen conversion pathway. Structures of human progastricsin, the inactive intermediate 2, and active human pepsin are known and have been used to define the conversion pathway. The structure of the zymogen precursor of plasmepsin II, the malarial aspartic protease, shows a new twist on the mode of inactivation used by the gastric zymogens. The prosegment of proplasmepsin disrupts the active conformation of the two catalytic aspartic acid residues by inducing a major reorientation of the two domains of the mature protease. The picornaviral 2A and 3C proteases have a chymotrypsin-like tertiary structure but with a cysteine nucleophile. These enzymes cleave themselves from the viral polyprotein in cis (intramolecular cleavage) and carry out trans cleavages of other scissile peptides important for the virus life cycle. Although the structure of the precursor viral polyprotein is unknown, it probably resembles the organization of the proenzymes of the bacterial serine proteases, subtilisin, and alpha-lytic protease. Cleavage of the prosegment is known to occur in cis for these precursor molecules.     

DJ-1 protects the nigrostriatal axis from the neurotoxin MPTP by modulation of the AKT pathway

Loss-of-function DJ-1 (PARK7) mutations have been linked with a familial form of early onset Parkinson disease. Numerous studies have supported the role of DJ-1 in neuronal survival and function. Our initial studies using DJ-1-deficient neurons indicated that DJ-1 specifically protects the neurons against the damage induced by oxidative injury in multiple neuronal types and degenerative experimental paradigms, both in vitro and in vivo. However, the manner by which oxidative stress-induced death is ameliorated by DJ-1 is not completely clear. We now present data that show the involvement of DJ-1 in modulation of AKT, a major neuronal prosurvival pathway induced upon oxidative stress. We provide evidence that DJ-1 promotes AKT phosphorylation in response to oxidative stress induced by H₂O₂ in vitro and in vivo following 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment. Moreover, we show that DJ-1 is necessary for normal AKT-mediated protective effects, which can be bypassed by expression of a constitutively active form of AKT. Taken together, these data suggest that DJ-1 is crucial for full activation of AKT upon oxidative injury, which serves as one explanation for the protective effects of DJ-1.     

Escherichia coli ribosomal protein L7/L12 dimers remain fully active after interchain crosslinking of the C-terminal domains in two orientations.

Cysteine site-directed mutagenesis was used to create variants of Escherichia coli ribosomal protein L7/L12 that have single cysteine substitutions, at residues 63 or 89, located in different exposed loops in the structure of the globular C-terminal domain indicated by the crystallographic structure. That structure shows a possible dimer interaction in which the two sites of cysteine substitution appear to be too distant for disulfide bond formation. After mild oxidation in solution both of the overexpressed purified cysteine-substituted proteins formed interchain disulfide crosslinked dimers in high yield. Both crosslinked dimers were fully active in restoring activity in poly(U)-directed polyphenylalanine synthesis to ribosomal core particles depleted of wild-type L7/L12. These results show that the two C-terminal domains have independent mobility. The activity of dimeric L7/L12 does not require the independent movement of the two globular C-terminal domains in an L7/L12 dimer; moreover, it appears independent of their mutual orientation when joined by crosslinking at the two loops. A third variant with a cysteine substitution at residue 33 near the junction between the alpha-helical N-terminal domain and the flexible hinge was prepared and tested. This protein was active in the protein synthesis assay in the reduced state. Oxidation produced the interchain crosslinked dimer in high yield, but this crosslinked dimer was inactive in polyphenylalanine synthesis. The inactivation was due to the inability of the Cys33-Cys33 oxidized dimer to bind to the core particle.