Ashraf S. Raza

Formation of a stabilized cysteine sulfinic acid is critical for the mitochondrial function of the parkinsonism protein DJ-1.

The formation of cysteine-sulfinic acid has recently become appreciated as a modification that links protein function to cellular oxidative status. Human DJ-1, a protein associated with inherited parkinsonism, readily forms cysteine-sulfinic acid at a conserved cysteine residue (Cys106 in human DJ-1). Mutation of Cys106 causes the protein to lose its normal protective function in cell culture and model organisms. However, it is unknown whether the loss of DJ-1 protective function in these mutants is due to the absence of Cys106 oxidation or the absence of the cysteine residue itself. To address this question, we designed a series of substitutions at a proximal glutamic acid residue (Glu18) in human DJ-1 that alter the oxidative propensity of Cys106 through changes in hydrogen bonding. We show that two mutations, E18N and E18Q, allow Cys106 to be oxidized to Cys106-sulfinic acid under mild conditions. In contrast, the E18D mutation stabilizes a cysteine-sulfenic acid that is readily reduced to the thiol in solution and in vivo. We show that E18N and E18Q can both partially substitute for wild-type DJ-1 using mitochondrial fission and cell viability assays. In contrast, the oxidatively impaired E18D mutant behaves as an inactive C106A mutant and fails to protect cells. We therefore conclude that formation of Cys106-sulfinic acid is a key modification that regulates the protective function of DJ-1.

Characterization of a Novel Dithiocarbamate Glutathione Reductase Inhibitor and Its Use as a Tool to Modulate Intracellular Glutathione

Thiol redox state (TRS) is an important parameter to reflect intracellular oxidative stress and is associated with various normal and abnormal biochemical processes. Agents that can be used to increase intracellular TRS will be valuable tools in TRS-related research. Glutathione reductase (GR) is a critical enzyme in the homeostasis of TRS. The enzyme catalyzes the reduction of GSSG to GSH to maintain a high GSH:GSSG ratio. Inhibition of the enzyme can be used to increase TRS. Despite the reports of various GR inhibitors, N,N-bis(2-chloroethyl)-N-nitrosourea, an anticancer drug with IC50 = 647 μm against yeast GR, remains the most commonly used GR inhibitor in the literature. However, the toxicity caused by nonspecific interactions, as well as inhibition of DNA synthesis, complicates the use of N,N-bis(2-chloroethyl)-N-nitrosourea as a GR inhibitor. We report 2-acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanylthiocarbonylamino)phenylthiocarbamoylsulfanyl]propionic acid (2-AAPA) as a novel irreversible GR inhibitor. 2-AAPA was prepared by one-step synthesis from commercially available reagents. The Ki and kinact of 2-AAPA against yeast GR were determined to be 56 μm and 0.1 min–1, respectively. At the concentration that produced >80% yeast GR inhibition, 2-AAPA showed no inhibition against glutamylcysteine synthetase, glutathione synthetase, catalase, and superoxide dismutase, but minimal inhibition against glutathione S-transferase and glutathione peroxidase. In CV-1 cells, 2-AAPA (0.1 mm) produced 97% GR inhibition, 25% GSH reduction, and a 5-fold increase in GSSG in 20 min. The compound can be a useful tool in TRS-related research.

Analysis of methionine/selenomethionine oxidation and methionine sulfoxide reductase function using methionine-rich proteins and antibodies against their oxidized forms.

Methionine (Met) residues are present in most proteins. However, this sulfur-containing amino acid is highly susceptible to oxidation. In cells, the resulting Met sulfoxides are reduced back to Met by stereospecific reductases MsrA and MsrB. Reversible Met oxidation occurs even in the absence of stress, is elevated during aging and disease, but is notoriously difficult to monitor. In this work, we computationally identified natural Met-rich proteins (MRPs) and characterized three such proteins containing 21-33% Met residues. Oxidation of multiple Met residues in MRPs with H(2)O(2) and reduction of Met sulfoxides with MsrA/MsrB dramatically influenced the mobility of these proteins on polyacrylamide gels and could be monitored by simple SDS-PAGE. We further prepared antibodies enriched for reduced and Met sulfoxide forms of these proteins and used them to monitor Met oxidation and reduction by immunoblot assays. We describe applications of these reagents for the analysis of MsrA and MsrB functions, as well as the development of the assay for high-throughput analysis of their activities. We also show that all Met sulfoxide residues in an MRP can be reduced by MsrA and MsrB. Furthermore, we prepared a selenomethionine form of an MRP and found that selenomethionine selenoxide residues can be efficiently reduced nonenzymatically by glutathione and other thiol compounds. Selenomethionine selenoxide residues were not recognized by antibodies specific for the Met sulfoxide form of an MRP. These findings, reagents, assays, and approaches should facilitate research and applications in the area of Met sulfoxide reduction, oxidative stress, and aging.

Transcriptional Activation of Dehalorespiration IDENTIFICATION OF REDOX-ACTIVE CYSTEINES REGULATING DIMERIZATION AND DNA BINDING

Desulfitobacterium dehalogenans can use chlorinated aromatics including polychlorinated biphenyls as electron acceptors in a process called dehalorespiration. Expression of the cpr gene cluster involved in this process is regulated by CprK, which is a member of the CRP/FNR (cAMP-binding protein/fumarate nitrate reduction regulatory protein) family of helix-turn-helix transcriptional regulators. High affinity interaction of the chlorinated aromatic compound with the effector domain of CprK triggers binding of CprK to an upstream target DNA sequence, which leads to transcriptional activation of the cpr gene cluster. When incubated with oxygen or diamide, CprK undergoes inactivation; subsequent treatment with dithiothreitol restores activity. Using mass spectrometry, this study identifies two classes of redox-active thiol groups that form disulfide bonds upon oxidation. Under oxidative conditions, Cys105, which is conserved in FNR and most other CprK homologs, forms an intramolecular disulfide bond with Cys111, whereas an intermolecular disulfide bond is formed between Cys11 and Cys200. SDS-PAGE and site-directed mutagenesis experiments indicate that the Cys11/Cys200 disulfide bond links two CprK subunits in an inactive dimer. Isothermal calorimetry and intrinsic fluorescence quenching studies show that oxidation does not change the affinity of CprK for the effector. Therefore, reversible redox inactivation is manifested at the level of DNA binding. Our studies reveal a strategy for limiting expression of a redox-sensitive pathway by using a thiol-based redox switch in the transcription factor.

Androgen-Stimulated UDP-Glucose Dehydrogenase Expression Limits Prostate Androgen Availability without Impacting Hyaluronan Levels

UDP-glucose dehydrogenase (UGDH) oxidizes UDP-glucose to UDP-glucuronate, an essential precursor for production of hyaluronan (HA), proteoglycans, and xenobiotic glucuronides. High levels of HA turnover in prostate cancer are correlated with aggressive progression. UGDH expression is high in the normal prostate, although HA accumulation is virtually undetectable. Thus, its normal role in the prostate may be to provide precursors for glucuronosyltransferase enzymes, which inactivate and solubilize androgens by glucuronidation. In this report, we quantified androgen dependence of UGDH, glucuronosyltransferase, and HA synthase expression. Androgen-dependent and androgen-independent human prostate cancer cell lines were used to test the effects of UGDH manipulation on tumor cell growth, HA production, and androgen glucuronidation. Dihydrotestosterone (DHT) increased UGDH expression approximately 2.5-fold in androgen-dependent cells. However, up-regulation of UGDH did not affect HA synthase expression or enhance HA production. Mass spectrometric analysis showed that DHT was converted to a glucuronide, DHT-G, at a 6-fold higher level in androgen-dependent cells relative to androgen-independent cells. The increased solubilization and elimination of DHT corresponded to slower cellular growth kinetics, which could be reversed in androgen-dependent cells by treatment with a UDP-glucuronate scavenger. Collectively, these results suggest that dysregulated expression of UGDH could promote the development of androgen-independent tumor cell growth by increasing available levels of intracellular androgen.

Impeded Electron Transfer From a Pathogenic FMN Domain Mutant of Methionine Synthase Reductase and Its Responsiveness to Flavin Supplementation

Methionine synthase reductase (MSR) is a diflavin oxidoreductase that transfers electrons from NADPH to oxidized cobalamin and plays a vital role in repairing inactive cobalamin-dependent methionine synthase. MSR deficiency is a recessive genetic disorder affecting folate and methionine metabolism and is characterized by elevated levels of plasma homocysteine. In this study, we have examined the molecular basis of MSR dysfunction associated with a patient mutation, A129T, which is housed in the FMN binding domain and is adjacent to a cluster of conserved acidic residues found in diflavin oxidoreductases. We show that the substitution of alanine with threonine destabilizes FMN binding without affecting the NADPH coenzyme specificity or affinity, indicating that the mutations effects may be confined to the FMN module. The A129T MSR mutant transfers electrons to ferricyanide as efficiently as wild type MSR but the rate of cytochrome c, 2,6-dichloroindophenol, and menadione reduction is decreased 10-15 fold. The mutant is depleted in FMN and reactivates methionine synthase with 8% of the efficiency of wild type MSR. Reconstitution of A129T MSR with FMN partially restores its ability to reduce cytochrome c and to reactivate methionine synthase. Hydrogen-deuterium exchange mass spectrometric studies localize changes in backbone amide exchange rates to peptides in the FMN-binding domain. Together, our results reveal that the primary biochemical penalty associated with the A129T MSR mutant is its lower FMN content, provide insights into the distinct roles of the FAD and FMN centers in human MSR for delivering electrons to various electron acceptors, and suggest that patients harboring the A129T mutation may be responsive to riboflavin therapy.

Identification of non‐covalent structure in apocytochrome c by hydrogen exchange and mass spectrometry

Apocytochrome c, the in vivo precursor to active cytochrome c, was analyzed by amide hydrogen exchange and mass spectrometry to search for fixed, non-covalent structure. The protein was incubated in H(2)O at pH 3.3 or 6.7 for various times, then exposed to D(2)O to initiate isotope labeling of unfolded regions. Following acid quenching of hydrogen exchange, the labeled apocytochrome c was digested with pepsin into fragments that were analyzed by directly coupled high-performance liquid chromatography/electrospray ionization mass spectrometry. The intermolecular distribution of deuterium and the deuterium levels in structurally distinctive populations were determined from the mass spectra of the peptic fragments. Spectra of peptic fragments derived from apocytochrome c incubated at pH 3.3 had single envelopes of isotope peaks with masses indicating that all of the amide hydrogens had been replaced with deuterium. These results showed that apocytochrome c at pH 3.3 offered little resistance to hydrogen exchange, indicating that it was unfolded with little fixed structure. However, mass spectra of peptic fragments including residues 81-94 of apocytochrome c incubated at pH 6.7 had two envelopes of isotope peaks, indicating that one population was unfolded and the other population was highly structured in this region. Mass spectra of peptic fragments including residues N-terminal to residue 81 indicated that this region of the protein remained unfolded with little fixed structure at pH 6.7.

Optimization of conditions for studies of protein unfolding by hydrogen exchange/mass spectrometry

Understanding the forces driving protein folding and aggregation is an essential step in developing means for controlling these important processes. Amide hydrogen exchange, coupled with mass spectrometry, has become an important method for studying protein unfolding and refolding. To extend procedures developed to study unfolding of relatively soluble proteins to less soluble, aggregation-prone proteins requires special considerations. This publication describes a general strategy developed using yeast transaldolase, which aggregates easily under conditions required to study its unfolding. Results presented here show that reducing the protein concentration to the nanomolar range is essential for managing aggregation of transaldolase. In addition, the present results point to use of relatively high concentrations of denaturants and short incubation times to minimize aggregation. These results also show how amide hydrogen exchange, coupled with mass spectrometry, can be used to study soluble aggregates.