Proteomic profile of Toxoplasma gondii stress granules by high-resolution mass spectrometry.

Ribonucleoprotein granules are bio-condensates that form a diverse group of dynamic membrane-less organelles implicated in several cellular functions, including stress response and cellular survival. In Toxoplasma gondii, a type of bio-condensates referred to as stress granules (SGs) are formed prior to the parasites' egress from the host cell and are implicated in the survival and invasion competency of extracellular tachyzoites. We used paraformaldehyde to fix and cross-link SG proteins to allow purification by centrifugation and analysis by mass spectrometry. We profiled protein components of SGs at 10 and 30 min post-egress when parasite's invasion ability is significantly diminished. Thirty-three proteins were identified from 10 min SGs, and additional 43 proteins were identified from 30 min SGs. Notably, common SG components such as proteins with intrinsically disordered domains were not identified. Gene ontology analysis of both 10 and 30 min SGs shows that overall molecular functions of SGs' proteins are ATP-binding, GTP-binding, and GTPase activity. Discernable differences between 10 and 30 min SGs are in the proportions of translation and microtubule-related proteins. Ten-minute SGs have a higher proportion of microtubule-related proteins and a lower proportion of ribosome-related proteins, while a reverse correlation was identified for those of 30 min. It remains to be investigated whether this reverse correlation contributes to the ability of extracellular tachyzoites to reinvade host cells.

Src-mediated phosphorylation of the ribosome biogenesis factor hYVH1 affects its localization, promoting partitioning to the 60S ribosomal subunit.

Yeast VH1-related phosphatase (YVH1) (also known as DUSP12) is a member of the atypical dual-specificity phosphatase subfamily. Although no direct substrate has been firmly established, human YVH1 (hYVH1) has been shown to protect cells from cellular stressors, regulate the cell cycle, disassemble stress granules, and act as a 60S ribosome biogenesis factor. Despite knowledge of hYVH1 function, further research is needed to uncover mechanisms of its regulation. In this study, we investigate cellular effects of a Src-mediated phosphorylation site at Tyr179 on hYVH1. We observed that this phosphorylation event attenuates localization of hYVH1 to stress granules, enhances shuttling of hYVH1 to the nucleus, and promotes hYVH1 partitioning to the 60S ribosomal subunit. Quantitative proteomics reveal that Src coexpression with hYVH1 reduces formation of ribosomal species that represent stalled intermediates through the alteration of associating factors that mediate translational repression. Collectively, these results implicate hYVH1 as a novel Src substrate and provide the first demonstrated role of tyrosine phosphorylation regulating the activity of a YVH1 ortholog. Moreover, the ribosome proteome alterations point to a collaborative function of hYVH1 and Src in maintaining translational fitness.

Label-free quantitative proteomic analysis of extracellular vesicles released from fibroblasts derived from patients with spinal muscular atrophy.

Spinal muscular atrophy (SMA) is an autosomal recessive disorder that represents a significant cause of infant mortality. SMA is characterized by reduced levels of the Survival Motor Neuron protein leading to the loss of alpha motor neurons in the spinal cord and brain stem as well as defects in peripheral tissues such as skeletal muscle and liver. With progress in promising therapies such as antisense oligonucleotide and gene replacement, there remains a need to better understand disease subtypes and develop biomarkers for improved diagnostics and therapeutic monitoring. In this study, we have examined the utility of extracellular vesicles as a source of biomarker discovery in patient-derived fibroblast cells. Proteome examination utilizing data-independent acquisition and ion mobility mass spectrometry identified 684 protein groups present in all biological replicates tested. Label-free quantitative analysis identified 116 statistically significant protein alterations compared to control cells, including several known SMA biomarkers. Protein level differences were also observed in regulators of Wnt signaling and Cajal bodies. Finally, levels of insulin growth factor binding protein-3 were validated as being significantly higher in extracellular vesicles isolated from SMA cells. We conclude that extracellular vesicles represent a promising source for SMA biomarker discovery as well as a relevant constituent for advancing our understanding of SMA pathophysiology.

Characterization of Phosphopeptide Positional Isomers on the Transcriptional Co-activator TAZ.

The transcriptional co-activator with the PDZ binding motif (TAZ) is a critical regulator of numerous cellular processes such as cell differentiation, development, proliferation, and cell growth. Aberrant expression and activity of TAZ are also featured in many human malignancies. A hallmark of TAZ biology is its cytoplasmic retention mediated by 14-3-3 isoforms in response to phosphorylation of Ser89 by members of the LATS family of kinases. Following the observation that TAZ is a highly phosphorylated protein even when Ser89 is mutated, high-resolution mass spectrometry employing data-independent acquisition and ion mobility separation was conducted to elucidate additional TAZ phosphorylation sites that may play a role in regulating this critical transcriptional rheostat. Numerous phosphorylation sites on TAZ were identified, including several novel modifications. Of notable interest was the identification of positional phosphoisomers on a phosphopeptide containing Ser89. Optimized use of a so-called wideband enhancement acquisition technique yielded higher-quality fragmentation data that confirmed the detection of Ser93 as the positional phosphoisomer partner of Ser89 and identified diagnostic fragment ions for the phosphorylation events. Functional analysis indicated that Ser93 phosphorylation reduces the level of 14-3-3 association and increases the level of nuclear translocation, indicating this phosphorylation event attenuates the 14-3-3-mediated TAZ cytoplasmic retention mechanism. These findings suggest that the biological activities of TAZ are likely dynamically regulated by multisite phosphorylation.

Pathologic Alterations in the Proteome of Synaptosomes from a Mouse Model of Spinal Muscular Atrophy.

Spinal muscular atrophy (SMA) is a human genetic disorder characterized by muscle weakness, muscle atrophy, and death of motor neurons. SMA is caused by mutations or deletions in a gene called survival motor neuron 1 (SMN1). SMN1 is a housekeeping gene, but the most prominent pathologies in SMA are atrophy of myofibers and death of motor neurons. Further, degeneration of neuromuscular junctions, of synapses, and of axonal regions are features of SMA disease. Here, we have investigated the proteome dynamics of central synapses in P14 Smn2B/- mice, a model of SMA. Label-free quantitative proteomics on isolated synaptosomes from spinal cords of these animals identified 2030 protein groups. Statistical data analysis revealed 65 specific alterations in the proteome of the central synapses at the early onset stage of disease. Functional analysis of the dysregulated proteins indicated a significant enrichment of proteins associated with mitochondrial dynamics, cholesterol biogenesis, and protein clearance. These pathways represent potential targets for therapy development with the goal of providing stability to the central synapses, thereby preserving neuronal integrity in the context of SMA disease. Data are available via ProteomeXchange with identifier PXD012850.

Proteomic Profile of Daphnia pulex using Data-Independent Acquisition Mass Spectrometry and Ion Mobility Separation.

Daphnia pulex is a keystone species for aquatic habitats and an ecological/evolution model organism. Although significant progress has been made on characterizing its genome, the D. pulex proteome remains largely uncharacterized partially due to abnormally high protein degradation during homogenization and emphasis on genomic analysis. In this study, various sample preparation and mass spectrometry acquisition methods are performed for the purpose of improving D. pulex proteome exploration. Benefits for employing both in-gel and in-solution methods of trypsin digestion are observed. Furthermore, acquisition methods employing ion mobility separation greatly increase peptide identification and more than doubled the proteome coverage. Bioinformatic analysis suggests that mitochondrial and hydrolytic activities are enriched in D. pulex compared to closely related invertebrates or Homo sapiens. Also, novel D. pulex proteins possessing putative genome modifying functional domains are identified. Data are available via ProteomeXchange with identifier PXD008455.

Proteomic characterization of seminal plasma from alternative reproductive tactics of Chinook salmon (Oncorhynchus tswatchysha).

Chinook salmon (Oncorhynchus tshawytscha) are external fertilizers that display sneak-guard alternative reproductive tactics. The larger hooknose males dominate mating positions, while the smaller jack males utilize sneak tactics to achieve fertilization. Although poorly understood, previous studies have suggested that differences in spermatozoa quality may play a critical role in sperm competition. Considering that the seminal plasma strongly regulates spermatozoa quality and other processes critical for fertilization success, we employed label free quantitative mass spectrometry utilizing ion mobility separation coupled to cross-species bioinformatics to examine the seminal plasma proteome of Chinook salmon. A total of 345 proteins were identified in all biological replicates analyzed, including many established seminal plasma proteins that may serve as future biomarkers for Chinook salmon fertility and sperm competition. Moreover, we elucidated statistically significant protein abundance differences between hooknose and jack male tactics. Proteins involved in membrane remodeling, proteolysis, hormonal transport, redox regulation, immunomodulation, and ATP metabolism were among the proteins reproducibly identified at different levels and represent putative factors influencing sperm competition between jack and hooknose males. This study represents the largest seminal plasma proteome from teleost fish and the first reported for Chinook salmon. SIGNIFICANCE: Chinook salmon (Oncorhynchus tshawytscha) males represent an example of male alternative reproductive tactics where diverse reproductive strategies are thought to increase sexual selection. While seminal plasma has been shown to play an important regulatory role in sperm competition in many species, little is known about the protein composition of the seminal plasma of salmon. Therefore, seminal plasma isolated from the two alternative reproductive tactics of Chinook salmon (small sneaky jacks and large dominant hooknoses) were analyzed by label free quantitative mass spectrometry employing data independent acquisition and ion mobility separation. This yielded the largest proteome data set of the seminal plasma from salmon and the first to examine protein abundance differences between male alternative reproductive tactics. The quantitative proteomic data provides insight into possible unique mechanistic aspects of Chinook salmon alternative reproductive tactics utilized for sperm competition and fertilization success.

The Atypical Dual Specificity Phosphatase hYVH1 Associates with Multiple Ribonucleoprotein Particles.

Human YVH1 (hYVH1), also known as dual specificity phosphatase 12 (DUSP12), is a poorly characterized atypical dual specificity phosphatase widely conserved throughout evolution. Recent findings have demonstrated that hYVH1 expression affects cellular DNA content and is a novel cell survival phosphatase preventing both thermal and oxidative stress-induced cell death, whereas studies in yeast have established YVH1 as a novel 60S ribosome biogenesis factor. In this study, we have isolated novel hYVH1-associating proteins from human U2OS osteosarcoma cells using affinity chromatography coupled to mass spectrometry employing ion mobility separation. Numerous ribosomal proteins were identified, confirming the work done in yeast. Furthermore, proteins known to be present on additional RNP particles were identified, including Y box-binding protein 1 (YB-1) and fragile X mental retardation protein, proteins that function in translational repression and stress granule regulation. Follow-up studies demonstrated that hYVH1 co-localizes with YB-1 and fragile X mental retardation protein on stress granules in response to arsenic treatment. Interestingly, hYVH1-positive stress granules were significantly smaller, whereas knocking down hYVH1 expression attenuated stress granule breakdown during recovery from arsenite stress, indicating a possible role for hYVH1 in stress granule disassembly. These results propagate a role for dual specificity phosphatases at RNP particles and suggest that hYVH1 may affect a variety of fundamental cellular processes by regulating messenger ribonucleoprotein (mRNP) dynamics.

Receptor-mediated Endocytosis 8 Utilizes an N-terminal Phosphoinositide-binding Motif to Regulate Endosomal Clathrin Dynamics.

Receptor-mediated endocytosis 8 (RME-8) is a DnaJ domain containing protein implicated in translocation of Hsc70 to early endosomes for clathrin removal during retrograde transport. Previously, we have demonstrated that RME-8 associates with early endosomes in a phosphatidylinositol 3-phosphate (PI(3)P)-dependent fashion. In this study, we have now identified amino acid determinants required for PI(3)P binding within a region predicted to adopt a pleckstrin homology-like fold in the N terminus of RME-8. The ability of RME-8 to associate with PI(3)P and early endosomes is largely abolished when residues Lys(17), Trp(20), Tyr(24), or Arg(26) are mutated resulting in diffuse cytoplasmic localization of RME-8 while maintaining the ability to interact with Hsc70. We also provide evidence that RME-8 PI(3)P binding regulates early endosomal clathrin dynamics and alters the steady state localization of the cation-independent mannose 6-phosphate receptor. Interestingly, RME-8 endosomal association is also regulated by the PI(3)P-binding protein SNX1, a member of the retromer complex. Wild type SNX1 restores endosomal localization of RME-8 W20A, whereas a SNX1 variant deficient in PI(3)P binding disrupts endosomal localization of wild type RME-8. These results further highlight the critical role for PI(3)P in the RME-8-mediated organizational control of various endosomal activities, including retrograde transport.

Investigating redox regulation of protein tyrosine phosphatases using low pH thiol labeling and enrichment strategies coupled to MALDI-TOF mass spectrometry.

A central feature of the protein tyrosine phosphatase (PTP) catalytic mechanism is an attack of the substrate's phosphate moiety by a thiolate ion in the signature CX5R motif. In addition to being an effective nucleophile in this form, the thiolate ion is also susceptible to reversible redox regulation. This attribute permits temporal inhibition of PTP activities, which affects numerous cellular processes utilizing kinase-mediated signal propagation. Accumulating evidence has revealed diverse mechanisms adopted by PTPs to avoid irreversible thiol oxidation of the active site Cys residue, often involving structurally proximal thiols within the active site region. Therefore, there has been a significant effort made to develop thiol labeling strategies coupled to mass spectrometry to identify and characterize redox sensitive thiols within PTPs as a necessary step in understanding how a particular PTP is regulated by redox signaling. A common drawback to many current methods is the use of neutral pH labeling techniques, requiring special attention with regards to non-specific thiol oxidation during sample preparation. This study describes the use of rapid, low pH thiol labeling methods to overcome this issue. Mercury immobilized metal affinity chromatography (Hg-IMAC) demonstrated high selectivity and specificity while enriching for thiol-containing peptides from the atypical dual specificity phosphatase hYVH1 (also known as DUSP12). This approach revealed several reversibly oxidized thiols within the catalytic domain of hYVH1. Subsequently, use of another low pH labeling reagent, 4,4-dithiopyridine (4-DTP) helped identify novel disulfide linkages providing evidence that hYVH1 utilizes a disulfide exchange mechanism to prevent irreversible oxidation of the catalytic Cys residue in the active site.

Differential phosphorylation of the phosphoinositide 3-phosphatase MTMR2 regulates its association with early endosomal subtypes.

Myotubularin-related 2 (MTMR2) is a 3-phosphoinositide lipid phosphatase with specificity towards the D-3 position of phosphoinositol 3-phosphate [PI(3)P] and phosphoinositol 3,5-bisphosphate lipids enriched on endosomal structures. Recently, we have shown that phosphorylation of MTMR2 on Ser58 is responsible for its cytoplasmic sequestration and that a phosphorylation-deficient variant (S58A) targets MTMR2 to Rab5-positive endosomes resulting in PI(3)P depletion and an increase in endosomal signaling, including a significant increase in ERK1/2 activation. Using in vitro kinase assays, cellular MAPK inhibitors, siRNA knockdown and a phosphospecific-Ser58 antibody, we now provide evidence that ERK1/2 is the kinase responsible for phosphorylating MTMR2 at position Ser58, which suggests that the endosomal targeting of MTMR2 is regulated through an ERK1/2 negative feedback mechanism. Surprisingly, treatment with multiple MAPK inhibitors resulted in a MTMR2 localization shift from Rab5-positive endosomes to the more proximal APPL1-positive endosomes. This MTMR2 localization shift was recapitulated when a double phosphorylation-deficient mutant (MTMR2 S58A/S631A) was characterized. Moreover, expression of this double phosphorylation-deficient MTMR2 variant led to a more sustained and pronounced increase in ERK1/2 activation compared with MTMR2 S58A. Further analysis of combinatorial phospho-mimetic mutants demonstrated that it is the phosphorylation status of Ser58 that regulates general endosomal binding and that the phosphorylation status of Ser631 mediates the endosomal shuttling between Rab5 and APPL1 subtypes. Taken together, these results reveal that MTMR2 compartmentalization and potential subsequent effects on endosome maturation and endosome signaling are dynamically regulated through MAPK-mediated differential phosphorylation events.

The dual-specificity phosphatase hYVH1 (DUSP12) is a novel modulator of cellular DNA content.

The dual-specificity phosphatase hYVH1 (DUSP12) is an evolutionary conserved phosphatase that also contains a unique zinc-binding domain. Recent evidence suggests that this enzyme plays a role in cell survival and ribosome biogenesis. Here, we report that hYVH1 expression also affects cell cycle progression. Overexpression of hYVH1 caused a significant increase in polyploidy and in the G 2/M cell population, with a subsequent decrease in the G 0/G 1 population. Phosphatase activity is dispensable, while the zinc-binding domain is necessary and sufficient for hYVH1-mediated cell cycle changes. In agreement with this, siRNA-mediated silencing of hYVH1 expression resulted in a dramatic increase in the G 0/G 1 population and susceptibility to cellular senescence. Additionally, mass spectrometry-based methods identified novel hYVH1 phosphorylation sites, including a C-terminal modification at position Ser ( 335) in the zinc-binding domain. Interestingly, phosphorylation at Ser335 regulates subcellular targeting of hYVH1 and augments the hYVH1 G 2/M phenotype. Collectively we demonstrate that hYVH1 is a novel modulator of cell cycle progression; a function mainly mediated by its C-terminal zinc-binding domain.

Receptor mediated endocytosis 8 is a novel PI(3)P binding protein regulated by myotubularin-related 2.

Myotubularin related protein 2 (MTMR2) is a member of the myotubularin family of phosphoinositide lipid phosphatases. Although MTMR2 dephosphorylates the phosphoinositides PI(3)P and PI(3,5)P2, the phosphoinositide binding proteins that are regulated by MTMR2 are poorly characterized. In this study, phosphoinositide affinity chromatography coupled to mass spectrometry identified receptor mediated endocytosis 8 (RME-8) as a novel PI(3)P binding protein. RME-8 co-localized with the PI(3)P marker DsRed-FYVE, while the N-terminal region of RME-8 is required for PI(3)P and PI(3,5)P(2) binding in vitro. Depletion of PI(3)P by MTMR2 S58A or wortmannin treatment attenuated RME-8 endosomal localization and co-localization with EGFR on early endosomes. Our results suggest a model in which the localization of RME-8 to endosomal compartments is spatially mediated by PI(3)P binding and temporally regulated by MTMR2 activity.

Endosomal targeting of the phosphoinositide 3-phosphatase MTMR2 is regulated by an N-terminal phosphorylation site.

MTMR2 is a member of the myotubularin family of inositol lipid phosphatases, a large protein-tyrosine phosphatase subgroup that is conserved from yeast to humans. Furthermore, the peripheral neuromuscular disease Charcot-Marie Tooth disease type 4B has been attributed to mutations in the mtmr2 gene. Because the molecular mechanisms regulating MTMR2 have been poorly defined, we investigated whether reversible phosphorylation might regulate MTMR2 function. We used mass spectrometry-based methods to identify a high stoichiometry phosphorylation site on serine 58 of MTMR2. Phosphorylation at Ser(58), or a phosphomimetic S58E mutation, markedly decreased MTMR2 localization to endocytic vesicular structures. In contrast, a phosphorylation-deficient MTMR2 mutant (S58A) displayed constitutive localization to early endocytic structures. This localization pattern was accompanied by displacement of a PI(3)P-specific sensor protein and an increase in signal transduction pathways. Thus, MTMR2 phosphorylation is likely to be a critical mechanism by which MTMR2 access to its lipid substrate(s) is temporally and spatially regulated, thereby contributing to the control of downstream endosome maturation events.

Evidence for multiple group 1 late embryogenesis abundant proteins in encysted embryos of Artemia and their organelles.

The presence of late embryogenesis abundant (LEA) proteins in plants and animals has been linked to their ability to tolerate a variety of environmental stresses. Among animals, encysted embryos of the brine shrimp Artemia franciscana are among the most stress resistant eukaryotes, and for that reason it is considered to be an extremophile. The study presented here demonstrates that these embryos contain multiple group 1 LEA proteins with masses of 21, 19, 15.5 and 13 kDa. The LEA proteins first appear in diapause-destined embryos, beginning at ∼4 days post-fertilization, but not in nauplii-destined embryos. After resumption of embryonic development, the LEA proteins decline slowly in the desiccation resistant encysted stages, then disappear rapidly as the embryo emerges from its shell. LEA proteins are absent in fully emerged embryos, larvae and adults. They are abundant in mitochondria of encysted embryos, but barely detectable in nuclei and absent from yolk platelets. LEA proteins were also detected in dormant embryos of six other species of Artemia from hypersaline environments around the world. This study enhances our knowledge of the group 1 LEA proteins in stress tolerant crustacean embryos.

Gold nanoparticle enrichment method for identifying S-nitrosylation and S-glutathionylation sites in proteins.

We present a simple method by which gold nanoparticles (AuNPs) are used to simultaneously isolate and enrich for free or modified thiol-containing peptides, thus facilitating the identification of protein S-modification sites. Here, protein disulfide isomerase (PDI) and dual specificity phosphatase 12 (DUSP12 or hYVH1) were S-nitrosylated or S-glutathionylated, their free thiols differentially alkylated, and subjected to proteolysis. AuNPs were added to the digests, and the AuNP-bound peptides were isolated by centrifugation and released by thiol exchange. These AuNP-bound peptides were analyzed by MALDI-TOF mass spectrometry revealing that AuNPs result in a significant enrichment of free thiol-containing as well as S-nitrosylated, S-glutathionylated, and S-alkylated peptides, leading to the unequivocal assignment of thiols susceptible to modification.

Redox regulation of the human dual specificity phosphatase YVH1 through disulfide bond formation.

YVH1 was one of the first eukaryotic dual specificity phosphatases cloned, and orthologues poses a unique C-terminal zinc-coordinating domain in addition to a cysteine-based phosphatase domain. Our recent results revealed that human YVH1 (hYVH1) protects cells from oxidative stress. This function requires phosphatase activity and the zinc binding domain. This current study provides evidence that the thiol-rich zinc-coordinating domain may act as a redox sensor to impede the active site cysteine from inactivating oxidation. Furthermore, using differential thiol labeling and mass spectrometry, it was determined that hYVH1 forms intramolecular disulfide bonds at the catalytic cleft as well as within the zinc binding domain to avoid irreversible inactivation during severe oxidative stress. Importantly, zinc ejection is readily reversible and required for hYVH1 activity upon returning to favorable conditions. This inimitable mechanism provides a means for hYVH1 to remain functionally responsive for protecting cells during oxidative stimuli.

Characterization of a group 1 late embryogenesis abundant protein in encysted embryos of the brine shrimp Artemia franciscana.

Late embryogenesis abundant (LEA) proteins are hydrophilic molecules that are believed to function in desiccation and low-temperature tolerance in some plants and plant propagules, certain prokaryotes, and several animal species. The brine shrimp Artemia franciscana can produce encysted embryos (cysts) that enter diapause and are resistant to severe desiccation. This ability is based on biochemical adaptations, one of which appears to be the accumulation of the LEA protein that is the focus of this study. The studies described herein characterize a 21 kDa protein in encysted Artemia embryos as a group 1 LEA protein. The amino acid sequence of this protein and its gene have been determined and entered into the NCBI database (no. EF656614). The LEA protein consists of 182 amino acids and it is extremely hydrophilic, with glycine (23%), glutamine (17%), and glutamic acid (12.6%) being the most abundant amino acids. This protein also consists of 8 tandem repeats of a 20 amino acid sequence, which is characteristic of group 1 LEA proteins from non-animal species. The LEA protein and its gene are expressed only in encysted embryos and not in larvae or adults. Evidence is presented to show that the LEA protein functions in the prevention of drying-induced protein aggregation, which supports its functional role in desiccation tolerance. This report describes, for the first time, the purification and characterization of a group 1 LEA protein from an animal species.

The dual-specificity phosphatase hYVH1 interacts with Hsp70 and prevents heat-shock-induced cell death.

hYVH1 [human orthologue of YVH1 (yeast VH1-related phosphatase)] is an atypical dual-specificity phosphatase that is widely conserved throughout evolution. Deletion studies in yeast have suggested a role for this phosphatase in regulating cell growth. However, the role of the human orthologue is unknown. The present study used MS to identify Hsp70 (heat-shock protein 70) as a novel hYVH1-binding partner. The interaction was confirmed using endogenous co-immunoprecipitation experiments and direct binding of purified proteins. Endogenous Hsp70 and hYVH1 proteins were also found to co-localize specifically to the perinuclear region in response to heat stress. Domain deletion studies revealed that the ATPase effector domain of Hsp70 and the zinc-binding domain of hYVH1 are required for the interaction, indicating that this association is not simply a chaperone-substrate complex. Thermal phosphatase assays revealed hYVH1 activity to be unaffected by heat and only marginally affected by non-reducing conditions, in contrast with the archetypical dual-specificity phosphatase VHR (VH1-related protein). In addition, Hsp70 is capable of increasing the phosphatase activity of hYVH1 towards an exogenous substrate under non-reducing conditions. Furthermore, the expression of hYVH1 repressed cell death induced by heat shock, H2O2 and Fas receptor activation but not cisplatin. Co-expression of hYVH1 with Hsp70 further enhanced cell survival. Meanwhile, expression of a catalytically inactive hYVH1 or a hYVH1 variant that is unable to interact with Hsp70 failed to protect cells from the various stress conditions. The results suggest that hYVH1 is a novel cell survival phosphatase that co-operates with Hsp70 to positively affect cell viability in response to cellular insults.

Identification of redox sensitive thiols of protein disulfide isomerase using isotope coded affinity technology and mass spectrometry.

Regulation of the redox state of protein disulfide isomerase (PDI) is critical for its various catalytic functions. Here we describe a procedure utilizing isotope-coded affinity tag (ICAT) technology and mass spectrometry that quantitates relative changes in the dynamic thiol and disulfide states of human PDI. Human PDI contains six cysteine residues, four present in two active sites within the a and a' domains, and two present in the b' domain. ICAT labeling of human PDI indicates a difference between the redox state of the two active sites. Furthermore, under auto-oxidation conditions an approximately 80% decrease in available thiols within the a domain was detected. Surprisingly, the redox state of one of the two cysteines, Cys-295, within the b' domain was altered between the fully reduced and the auto-oxidized state of PDI while the other b' domain cysteine remained fully reduced. An interesting mono- and dioxidation modification of an invariable tryptophan residue, Trp-35, within the active site was also mapped by tandem mass spectrometry. Our findings indicate that ICAT methodology in conjunction with mass spectrometry represents a powerful tool to monitor changes in the redox state of individual cysteine residues within PDI under various conditions.