Five novel cytokines (IL-19, IL-20, IL-22 (IL-TIF), IL-24 (human MDA-7, mouse FISP, rat C49A/Mob-5), and IL-26 (AK155)) demonstrating limited primary sequence identity and probable structural homology to IL-10 have been identified. These cellular cytokines, as well as several cytokines encoded in viral genomes (viral cytokines), form a family of IL-10-related cytokines or the IL-10 family. These cytokines share not only homology but also receptor subunits and perhaps activities. Receptors for these cytokines belong to the class II cytokine receptor family. The receptors are IL-10R2 (CRF2-4), IL-22R1 (CRF2-9), IL-22BP (CRF2-10), IL-20R1 (CRF2-8) and IL-20R2 (CRF2-11). Biological activities of these cytokines, receptor utilization and signaling, as well as expression patterns for cytokines and their receptors are summarized. Although data indicate that these cytokines are involved in regulation of inflammatory and immune responses, their major functions remain to be discovered.

To determine the active site cysteine residue in aldehyde dehydrogenase, we mutated amino acid residues 49, 162, and 302 of recombinantly expressed rat liver mitochondrial (class 2) aldehyde dehydrogenase. The C49A and C162A mutants were fully active tetrameric enzymes, although the C162A mutant was found to be highly unstable. The C302A mutant was also a tetramer and bound coenzyme, but lacked both dehydrogenase and esterase activities. To test for the role of cysteine 302 as a nucleophile, the residue was mutated to a serine, a poor nucleophile. this C302S mutant was active but was a much poorer catalyst, with a kcat/Km value 7 x 10(5) times lower than that of the recombinant native enzyme. Unlike with native enzyme where deacylation is rate limiting, formation of the serine hemiacetal intermediate appeared to be the rate-limiting step. Cysteine 302 is the only strictly conserved cysteine residue among all the available sequences of the aldehyde dehydrogenase superfamily, supporting the role of this residue as the active site nucleophile of aldehyde dehydrogenase.

The melanoma differentiation-associated gene-7 (mda-7) was cloned by subtraction hybridization as a molecule whose expression is elevated in terminally differentiated human melanoma cells. Current information based on structural and sequence homology, has led to the recognition of MDA-7 as an IL-10 family cytokine member and its renaming as IL-24. Northern blot analysis revealed mda-7/IL-24 expression in human tissues associated with the immune system such as spleen, thymus, peripheral blood leukocytes and normal melanocytes. The MDA-7/IL-24 mouse counterpart, FISP, appears to be a Th2-specific protein and the rat counterpart, C49A/MOB-5, is associated with wound healing and is also induced as a consequence of ras-transformation. A notable property of MDA-7/IL-24 is its ability to induce apoptosis in a large spectrum of human cancer derived cell lines, in mouse xenografts and upon intratumoral injection in human tumors (phase I clinical trials). Various aspects of this intriguing molecule including its cytokine and anti-tumoral effects are described and discussed.

Plant R2R3 MYB domain proteins comprise one of the largest known families of transcription factors. Discrete evolutionary steps have shaped the plant-specific R2R3 MYB family from the broadly distributed R1R2R3 MYB proteins. R1R2R3 MYB domains have a single Cys residue (Cys-130) that needs to be reduced for DNA binding and transcriptional activity. In contrast, most R2R3 MYB domains contain two cysteines, Cys-49 and Cys-53, with Cys-53 at the equivalent position as Cys-130 in R1R2R3 MYB. Using the maize P1 regulator of flavonoid biosynthesis as a typical R2R3 MYB-domain protein, we investigated here the in vitro REDOX requirement for DNA binding by P1. We show that the C53S mutation requires reducing conditions for DNA-binding, whereas C53A binds DNA under oxidizing and reducing conditions. Neither mutation impairs the in vivo regulatory activity of P1. The C49S and C49A mutants bind DNA in vitro irrespective of the REDOX conditions. A C49I mutant, which simulates the MYB domain of c-MYB, binds DNA only under reducing conditions, and its binding is significantly affected by the C53S replacement. It is interesting that under non-reducing conditions, Cys-49 and Cys-53 form a disulfide bond that prevents the R2R3 MYB domain from binding DNA. Together, our results suggest that the evolutionary origin of Cys-49 within the plants has provided R2R3 MYB domains with a regulatory feature not present in animal MYB domains, highlighting fundamental structural and functional differences between similar DNA-binding domains from plants and animals.

We have developed a new nonoverlapping infectious viral genome (NO-SV40) in order to facilitate structure-based analysis of the simian virus 40 (SV40) life cycle. We first tested the role of cysteine residues in the formation of infectious virions by individually mutating the seven cysteines in the major capsid protein, Vp1. All seven cysteine mutants-C9A, C49A, C87A, C104A, C207S, C254A, and C267L-retained viability. In the crystal structure of SV40, disulfide bridges are formed between certain Cys104 residues on neighboring pentamers. However, our results show that none of these disulfide bonds are required for virion infectivity in culture. We also introduced five different mutations into Cys254, the most strictly conserved cysteine across the polyomavirus family. We found that C254L, C254S, C254G, C254Q, and C254R mutants all showed greatly reduced (around 100,000-fold) plaque-forming ability. These mutants had no apparent defect in viral DNA replication. Mutant Vp1's, as well as wild-type Vp2/3, were mostly localized in the nucleus. Further analysis of the C254L mutant revealed that the mutant Vp1 was able to form pentamers in vitro. DNase I-resistant virion-like particles were present in NO-SV40-C254L-transfected cell lysate, but at about 1/18 the amount in wild-type-transfected lysate. An examination of the three-dimensional structure reveals that Cys254 is buried near the surface of Vp1, so that it cannot form disulfide bonds, and is not involved in intrapentamer interactions, consistent with the normal pentamer formation by the C254L mutant. It is, however, located at a critical junction between three pentamers, on a conserved loop (G2H) that packs against the dual interpentamer Ca(2+)-binding sites and the invading C-terminal helix of an adjacent pentamer. The substitution by the larger side chains is predicted to cause a localized shift in the G2H loop, which may disrupt Ca(2+) ion coordination and the packing of the invading helix, consistent with the defect in virion assembly. Our experimental system thus allows dissection of structure-function relationships during the distinct steps of the SV40 life cycle.

Sco is a mononuclear red copper protein involved in the assembly of cytochrome c oxidase. It is spectroscopically similar to red copper nitrosocyanin, but unlike the latter, which has one copper cysteine thiolate, the former has two. In addition to the two cysteine ligands (C45 and C49), the wild-type (WT) protein from Bacillus subtilis (hereafter named BSco) has a histidine (H135) and an unknown endogenous protein oxygen ligand in a distorted tetragonal array. We have compared the properties of the WT protein to variants in which each of the two coordinating Cys residues has been individually mutated to Ala, using UV/visible, Cu and S K-edge X-ray absorption, electron paramagnetic resonance, and resonance Raman spectroscopies. Unlike the Cu(II) form of native Sco, the Cu(II) complexes of the Cys variants are unstable. The copper center of C49A undergoes autoreduction to the Cu(I) form, which is shown by extended X-ray absorption fine structure to be composed of a novel two-coordinate center with one Cys and one His ligand. C45A rearranges to a new stable Cu(II) species coordinated by C49, H135 and a second His ligand recruited from a previously uncoordinated protein side chain. The different chemistry exhibited by the Cys variants can be rationalized by whether a stable Cu(I) species can be formed by autoredox chemistry. For C49A, the remaining Cys and His residues are trans, which facilitates the formation of the highly stable two-coordinate Cu(I) species, while for C45A such a configuration cannot be attained. Resonance Raman spectroscopy of the WT protein indicates a net weak Cu-S bond strength at approximately 2.24 A corresponding to the two thiolate-copper bonds, whereas the single variant C45A shows a moderately strong Cu-S bond at approximately 2.16 A. S K-edge data give a total covalency of 28% for both Cu-S bonds in the WT protein. These data suggest an average covalency per Cu-S bond lower than that observed for nitrosocyanin and close to that expected for type-2 Cu(II)-thiolate systems. The data are discussed relative to the unique Cu-S characteristics of cupredoxins, from which it is concluded that Sco does not contain highly covalent Cu-S bonds of the type expected for long-range electron-transfer reactivity.

Proper folding of newly synthesized viral proteins in the cytoplasm is a prerequisite for the formation of infectious virions. The major capsid protein Vp1 of simian virus 40 forms a series of disulfide-linked intermediates during folding and capsid formation. In addition, we report here that Vp1 is associated with cellular chaperones (HSP70) and a cochaperone (Hsp40) which can be coimmunoprecipitated with Vp1. Studies in vitro demonstrated the ATP-dependent interaction of Vp1 and cellular chaperones. Interestingly, viral cochaperones LT and ST were essential for stable interaction of HSP70 with the core Vp1 pentamer Vp1 (22-303). LT and ST also coimmunoprecipitated with Vp1 in vivo. In addition to these identified (co)chaperones, stable, covalently modified forms of Vp1 were identified for a folding-defective double mutant, C49A-C87A, and may represent a "trapped" assembly intermediate. By a truncation of the carboxyl arm of Vp1 to prevent the Vp1 folding from proceeding beyond pentamers, we detected several apparently modified Vp1 species, some of which were absent in cells transfected with the folding-defective mutant DNA. These results suggest that transient covalent interactions with known or unknown cellular and viral proteins are important in the assembly process.

Mouse organic anion transporter 1 (mOAT1) belongs to a family of organic anion transporters, which play critical roles in the body disposition of clinically important drugs, including anti-HIV therapeutics, anti-tumour drugs, antibiotics, anti-hypertensives and anti-inflammatories. mOAT1-mediated transport of organic anion PAH ( p -aminohippurate) in HeLa cells was inhibited by the cysteine-modifying reagent PCMBS (p-chloromercuribenzenesulphonate). Therefore the role of cysteine residues in the function of mOAT1 was examined by site-directed mutagenesis. All 13 cysteine residues in mOAT1 were replaced by alanine, singly or in combination. Single replacement of these residues had no significant effect on mOAT1-mediated PAH transport, indicating that no individual cysteine residue is necessary for function. Multiple replacements at a C-terminal region (C335/379/427/434A; Cys(335/379/427/434)->>Ala) resulted in a substantial decrease in transport activity. A simultaneous replacement of all 13 cysteine residues (C-less) led to a complete loss of transport function. The decreased or lack of transport activity of the mutants C335/379/427/434A and C-less was due to the impaired trafficking of the mutant transporters to the cell surface. These results suggest that although cysteine residues are not required for function in mOAT1, their presence appears to be important for the targeting of the transporter to the plasma membrane. We also showed that, although all cysteine mutants of mOAT1 were sensitive to the inhibition by PCMBS, C49A was less sensitive than the wild-type mOAT1, suggesting that the modification of Cys49 may play a role in the inhibition of mOAT1 by PCMBS.

We have used DD-PCR (differential display-polymerase chain reaction) to identify new genes that are over- or underexpressed during wound repair. DD-PCR performed on excisional wounds identified the expression of rat c49a. Cloning and sequence analysis of the rat c49a gene revealed high homology to a novel human melanoma differentiation associated gene, mda-7. The human mda-7gene isolated from melanoma cell lines, has been linked with human melanoma differentiation, and growth suppression. Moreover, transfection of human mda-7 constructs into human tumor cells suppresses the growth and colony formation of tumor cells from diverse origins. To confirm and relatively quantitate expression of rat c49a gene during repair, specific primer, reduced cycle RT-PCR (reverse transcription-PCR) was performed. RT-PCR showed an approximately 9 to 12-fold elevation of rat c49a mRNA at 12 h to 5 days above nonwounded controls that gradually decreased to approximately 1.5 to 3-fold by day 14. Cloning and sequence analysis of the entire 1200 base pair c49a gene product showed 78% nucleotide homology to human mda-7. Immunohistochemistry studies localized rat C49A expression primarily to fibroblast-like cells at the wound edge and base. The marked up-regulation of rat c49a transcripts during the inflammatory and early granulation tissue phases of wound repair where cellular processes such as re-epithelialization, angiogenesis, and fibroplasia predominate--suggest that c49a is associated with proliferation of fibroblasts in wound healing.

MYB proteins are a family of transcription factors that play an important role in plant development and regulatory defense processes. Arabidopsis thaliana MYB30 (AtMYB30), a member of this protein family, is involved in cell death processes during the hypersensitive response (HR) of plants. HR is characterized by a vast production of reactive oxygen species (ROS) and nitric oxide (NO). NO may thus influence the binding of AtMYB30 to DNA. In this work we evaluated the effect of NO on AtMYB30 DNA binding activity, and also in the protein structural properties. A fully active minimal DNA-binding domain (DBD) of AtMYB30 (residues 11-116) containing two cysteine residues (C49 and C53) was overexpressed and purified. Site-directed mutagenesis was used to obtain AtMYB30 DBD mutants C49A and C53A. The DNA binding activity of AtMYB30 DBD, and Cys single mutants is clearly inhibited upon incubation with a NO donor, and S-nitrosylation was confirmed by the biotin switch assay. Finally, in order to understand the mechanism of NO effect on AtMYB30 DNA binding activity we performed circular dichroism analysis, to correlate the observed protein function inhibition and a potential structural impairment on AtMYB30 DBD. Indeed, NO modification of C49 and C53 residues promotes a subtle modification on the secondary structure of this transcription factor. We thus demonstrated, using various techniques, the in vitro effect of NO on AtMYB30 DBD, and thus the potential consequences of NO activity on plant metabolism influenced by this transcription factor.

The acylphosphatase from Escherichia coli (EcoAcP) is the first AcP so far studied with a disulfide bond. A mutational variant of the enzyme lacking the disulfide bond has been produced by substituting the two cysteine residues with alanine (EcoAcP mutational variant C5A/C49A, mutEcoAcP). The native states of the two protein variants are similar, as shown by far-UV and near-UV circular dichroism and dynamic light-scattering measurements. From unfolding experiments at equilibrium using intrinsic fluorescence and far-UV circular dichroism as probes, EcoAcP shows an increased conformational stability as compared with mutEcoAcP. The wild-type protein folds according to a two-state model with a very fast rate constant (k(F)(H2O)=72,600 s(-1)), while mutEcoAcP folds ca 1500-fold slower, via the accumulation of a partially folded species. The correlation between the hydrophobicity of the polypeptide chain and the folding rate, found previously in the AcP-like structural family, is maintained only when considering the mutant but not the wild-type protein, which folds much faster than expected from this correlation. Similarly, the correlation between the relative contact order and the folding rate holds only for mutEcoAcP. The correlation also holds for EcoAcP, provided the relative contact order value is recalculated by considering the disulfide bridge as an alternate path for the backbone to determine the shortest sequence separation between contacting residues. These results indicate that the presence of a disulfide bond in a protein is an important determinant of the folding rate and allows its contribution to be determined in quantitative terms.

The Fenna-Matthews-Olson (FMO) pigment-protein complex in green sulfur bacteria transfers excitation energy from the chlorosome antenna complex to the reaction center. In understanding energy transfer in the FMO protein, the individual contributions of the bacteriochlorophyll pigments to the FMO complex's absorption spectrum could provide detailed information with which molecular and energetic models can be constructed. The absorption properties of the pigments, however, are such that their spectra overlap significantly. To overcome this, we used site-directed mutagenesis to construct a series of mutant FMO complexes in the model green sulfur bacterium Chlorobaculum tepidum (formerly Chlorobium tepidum). Two cysteines at positions 49 and 353 in the C. tepidum FMO complex, which reside near hydrogen bonds between BChls 2 and 3, and their amino acid binding partner serine 73 and tyrosine 15, respectively, were changed to alanine residues. The resulting C49A, C353A, and C49A C353A double mutants were analyzed with a combination of optical absorption and circular dichroism (CD) spectroscopies. Our results revealed changes in the absorption properties of several underlying spectral components in the FMO complex, as well as the redox behavior of the complex in response to the reductant sodium dithionite. A high-resolution X-ray structure of the C49A C353A double mutant reveals that these spectral changes appear to be independent of any major structural rearrangements in the FMO mutants. Our findings provide important tests for theoretical calculations of the C. tepidum FMO absorption spectrum, and additionally highlight a possible role for cysteine residues in the redox activity of the pigment-protein complex.

Mechanisms of disulfide bond formation in the human pathogen Streptococcus pyogenes is currently unknown. To date, no disulfide bond forming thiol-disulfide oxidoreductase (TDOR) has been described and at least one disulfide bonded protein is known in S. pyogenes. This protein is the superantigen SpeA, which contains 3 cysteine residues (Cys 87, Cys90, and Cys98), and has a disulfide bond is formed between Cys87 and Cys98. In this study, candidate TDORs were identified from the genome seuence of S. pyogenes MGAS8232. Using mutational and biochemical approaches, one of the candidate proteins, SpyM18_2037 (named here SdbA), was shown to be the catalyst that introduces the disulfide bond in SpeA. SpeA in the culture supernatant remained reduced when sdbA was inactivated and restored to the oxidized state when a functional copy of sdbA was returned to the sdbA-knockout mutant. SdbA has a typical C₄₆XXC₄₉ active site motif commonly found in TDORs. Site-directed mutagenesis experiments showed that the cysteines in the CXXC motif were required the disulfide bond in SpeA to form. Interactions between SdbA and SpeA were examined using cysteine variant proteins. The results showed that SdbA_C49A formed a mixed disulfide with SpeA_C87A, suggesting that the N-terminal Cys46 of SdbA and the C-terminal Cys98 of SpeA participated in the initial reaction. SpeA oxidized by SdbA displayed biological activities suggesting that SpeA was properly folded following oxidation by SdbA. In conclusion, formation of the disulfide bond in SpeA is catalyzed by SdbA and the findings represent the first report of disulfide bond formation in S. pyogenes. IMPORTANCE Here, we reported the first example of disulfide bond formation in Streptococcus pyogenes. The results showed that a thiol-disulfide oxidoreductase, named SdbA, is responsible for introducing the disulfide bond in the superantigen SpeA. The cysteine residues in the CXXC motif of SdbA are needed for catalyzing the disulfide bond in SpeA. The disulfide bond in SpeA and neighboring amino acids form a disulfide loop that is conserved among many superantigens, including those from Staphylococcus aureus. SpeA and staphylococcal enterotoxins lacking the disulfide bond are biologically inactive. Thus, the discovery of the enzyme that catalyzes the disulfide bond in SpeA is important to understanding the biochemistry of SpeA production and presents a target for mitigating the virulence of S. pyogenes.