Gotowa bibliografia na temat „ThiJ/DJ-1/PfpI Family Member Protein”

The DJ-1/ThiJ/PfpI superfamily is a large protein group over diverse organisms, under this superfamily, there are multi-types of proteins such as protease, chaperones, heat shock protein, human parkinson's disease protein. The conserved protein from Staphylococcus aureus SAV1875 is a member of DJ-1 superfamily, but its function is unknown. We have determined the crystal structure of SAV1875 to a resolution of 1.8Å . As expected, the overall fold of the core domain of SAV1875 is similar to that of DJ-1. SAV1875 appears to be a dimer both in solution and the crystal, displaying an oligomerization interface similar to that observed for DJ-1. SAV 1875 contains a possible catalytic triad (Cys105-Glu17-His106) analogous to PfpI, YhbO, and DR1199. The cysteine in this triad (Cys-105) is oxidized in this crystal structure, similar to modifications seen in the cysteine of the DJ-1. This Cys-sulfenic acid is stabilized by hydrogen bonding with Glu17, Gly72, His106. We also have determined the crystal structure of mutated form of reactive Cys, SAV1875 C105D to a resolution of 2.1 Å. Aspartate mutation mimics the the Cys-sulfinic acid, more oxidized form. The aspartate stabilization by hydrogen bonding with neighboring residues are maintained. On the basis of these results, we suggest that SAV1875 might work as a general stress protein involved in the detoxification of the cell from oxygen reactive species.

ABSTRACT Hypermutator Pseudomonas aeruginosa strains, characterized by an increased spontaneous-mutation rate, are found at high frequencies in chronic lung infections. Hypermutability is associated with the loss of antimutator genes related to DNA repair or damage avoidance systems. Only a few antimutator genes have been described in P. aeruginosa, although there is some evidence that additional genes may be involved in naturally occurring hypermutability. In order to find new P. aeruginosa antimutator genes, we constructed and screened a library of random insertions in the PA14 strain. Some previously described P. aeruginosa and/or Escherichia coli antimutator genes, such as mutS, mutL, uvrD, mutT, ung, and mutY, were detected, indicating a good coverage of our insertional library. One additional mutant contained an insertion in the P. aeruginosa PA14-04650 (pfpI) gene, putatively encoding a member of the DJ-1/ThiJ/PfpI superfamily, which includes chaperones, peptidases, and the Parkinson's disease protein DJ-1a. The pfpI-defective mutants in both PAO1 and PA14 showed higher spontaneous mutation rates than the wild-type strains, suggesting that PfpI plays a key role in DNA protection under nonstress conditions. Moreover, the inactivation of pfpI resulted in a dramatic increase in the H2O2-induced mutant frequency. Global transcription studies showed the induction of bacteriophage Pf1 genes and the repression of genes related to iron metabolism, suggesting that the increased spontaneous-mutant frequency may be due to reduced protection against the basal level of reactive oxygen species. Finally, pfpI mutants are more sensitive to different types of stress and are affected in biofilm formation.

A-factor (2-isocapryloyl-3R-hydroxymethyl-γ-butyrolactone) is a chemical signalling molecule, or microbial hormone, that triggers aerial mycelium formation and secondary metabolism in Streptomyces griseus. A-factor pro- duced in a growth-dependent manner switches on the transcription of adpA, encoding a transcriptional activator, by binding to ArpA, the A-factor receptor protein, which has bound to the adpA promoter, and dissociating the bound ArpA from the DNA. AdpA then activates a number of genes of various functions required for morphological development and secondary metabolism, forming an AdpA regulon. ArpA, which belongs to the TetR family, contains a helix–turn–helix DNA-binding motif in its N-terminal portion and an A-factor-binding pocket (5 Å (0.5 nm) diameter and 20 Å (2 nm) long) in its C-terminal portion, as implied by X-ray crystallography of CprB, an ArpA homologue. The ligand pocket, which can accommodate an entire A-factor-type molecule of γ-butyrolactone, is completely embedded in the C-terminal portion. Upon binding A-factor, a long helix connecting the A-factor-binding and ligand-binding domains is relocated, as a result of which the DNA-binding helix moves outside, resulting in dissociation from DNA. AdpA, which belongs to the AraC/XylS family, contains a ThiJ/PfpI/DJ-1-like dimerization domain in its N-terminal portion and an AraC/XylS-type DNA-binding domain in its C-terminal portion. For transcriptional activation, AdpA can bind to various positions with respect to the transcriptional start points of the target genes and sometimes to multiple sites. We show here how A-factor triggers secondary metabolism and morphological development in S. griseus, with emphasis on the two key transcriptional factors, ArpA and AdpA, in the A-factor regulatory cascade.

The DJ-1/ThiJ/PfpI superfamily of proteins is highly conserved across all biological kingdoms showing divergent multifunctions, such as chaperone, catalase, protease, and kinase. The common theme of these functions is responding to and managing various cellular stresses. DJ-1/ThiJ/PfpI superfamily members are classified into three subfamilies according to their quaternary structure (DJ-1-, YhbO-, and Hsp-types). The Hsp-type subfamily includes Hsp31, a chaperone and glyoxalase III. SAV0551, an Hsp-type subfamily member from Staphylococcus aureus, is a hypothetical protein that is predicted as Hsp31. Thus, to reveal the function and reaction mechanism of SAV0551, the crystal structure of SAV0551 was determined. The overall folds in SAV0551 are similar to other members of the Hsp-type subfamily. We have shown that SAV0551 functions as a chaperone and that the surface structure is crucial for holding unfolded substrates. As many DJ-1/ThiJ/PfpI superfamily proteins have been characterized as glyoxalase III, our study also demonstrates SAV0551 as a glyoxalase III that is independent of any cofactors. The reaction mechanism was evaluated via a glyoxylate-bound structure that mimics the hemithioacetal reaction intermediate. We have confirmed that the components required for reaction are present in the structure, including a catalytic triad for a catalytic action, His78 as a base, and a water molecule for hydrolysis. Our functional studies based on the crystal structures of native and glyoxylate-bound SAV0551 will provide a better understanding of the reaction mechanism of a chaperone and glyoxalase III.

In a healthy cell, the ROS levels are stringently regulated by the action of various enzymatic or non-enzymatic antioxidant systems. Imbalance in the ROS homeostasis generates oxidative stress resulting in damage to cellular macromolecules. Besides, pro-oxidants, glyoxals which are normally generated as an intermediate compound in the glycolytic pathway and other metabolic activity are known to cause oxidative stress in the cell. Elevated oxidative stress is one of the prominent cellular aetiologies associated with premature aging, cardiovascular and retinal disorders, atherosclerosis, and several neurological disorders. Parkinson disease (PD) is one of well-known neurodegenerative diseases whose pathogenicity is correlated to mitochondrial dysfunction due to elevated oxidative stress in the neuronal cells. Several proteins which are associated with development of familial form of PD, DJ-1, a member of ThiJ/DJ-1/PfpI super family, is known to act as an oxidative sensor in humans. Interestingly, heat shock protein (Hsp)31 from S. cerevisiae which belongs to DJ-1 family was shown to provide a similar oxidative stress resistance in yeast. However, the mechanistic aspects how these family members functions as an oxidative stress sensor are not clearly defined. The main focus of my investigation is to understand the involvement of these DJ-1 proteins in regulation of redox homeostasis and mitochondrial health, which are major hallmarks in the pathogenesis of PD. My major findings demonstrate the importance of Hsp31 family proteins in protecting cells against oxidative stress, which is induced by methylglyoxal (MG). The deletion of Hsp31 leads to a compromised growth phenotype in yeast upon MG induced stress. Moreover, Hsp31 exhibited robust GSH-independent glyoxalase activity both in vivo and in vitro. Besides, the glyoxalase activity is critical for glyoxal detoxification as well as suppression of ROS levels in cells. On the other hand, in agreement with the observed growth phenotypes, Hsp34 protein possesses a very mild glyoxalase activity as compared to Hsp31. Furthermore, active site mutational analysis reveals that methylglyoxalase activity of Hsp31 protein is critical for providing protection against oxidative stress in yeast. Importantly, endogenous expression of human DJ-1 could complement the growth of yeast under oxidative and glyoxal stress conditions signifying its functional conservation across species. Mechanistically, my findings highlight that Hsp31 regulates cellular GSH and NADPH homeostasis thereby protecting cells against oxidative stress. In addition, cellular localization experiment reveals that though Hsp31 is a cytosolic protein, it predominantly localizes into mitochondria under oxidative stress conditions and protects the organelle from severe oxidative damages. Lastly, my findings uncover the role of Hsp31 paralogs in the maintenance of mitochondrial health integrity and other stress related pathways. To test their role in the mitochondrial health, I have analysed several parameters such as mass, dynamics and functionality. Interestingly, though the single deletions of these paralogs do not have significant effects over the mitochondrial phenotypes, the deletion of DJ-1 homologs in combination of hsp31 and hsp34 in yeast led to enhanced total as well as functional mitochondrial mass in cells. To address how mitochondrial mass enhancement occurs in the cells, the organelle turnover (mitophagy) was assessed. The microscopic and western analysis indicates, there was no alteration in mitophagy among the ∆hsp31∆hsp34 compared to WT. On the contrary, an enhancement in the basal levels of ROS stimulated increased biogenesis of mitochondria in ∆hsp31∆hsp34 cells was observed. Strikingly, ∆hsp31∆hsp34 cells also exhibit upregulation of mitochondrial fusion proteins resulting hyperfusion of mitochondria. Additionally, our results demonstrates that ∆hsp31∆hsp34 cells exhibited a long-term G2/M cell cycle arrest, which was rescued upon overexpression of mitochondrial fission protein, Dnm1. Lastly, absence of these paralogs in yeast, resulted in induction of apoptotic-like features in the cells and decreased lifespan in Saccharomyces cerevisiae. Altogether, my studies highlight the importance of DJ-1 class of proteins in maintaining the cellular redox status, mitochondrial integrity and cellular health in yeast. In conclusion, overall my studies highlight that Hsp31 is a robust methylglyoxalase and regulates cellular NADPH and GSH pool thereby helps in the maintenance of redox homeostasis. Hsp31 predominantly translocate into mitochondria upon oxidative stress to protect the organelle from oxidative damages. Furthermore, my findings provide the first evidence over the involvement of DJ-1 family proteins in the regulation of mitochondrial health and dynamics, cell-cycle arres and reduced lifespan in yeast.