Involvement of the Azotobacter vinelandii Rhodanese-Like Protein RhdA in the Glutathione Regeneration Pathway

The phenotypic features of the Azotobacter vinelandii RhdA mutant MV474 (in which the rhdA gene was deleted) indicated that defects in antioxidant systems in this organism were related to the expression of the tandem-domain rhodanese RhdA. In this work, further insights on the effects of the oxidative imbalance generated by the absence of RhdA (e.g. increased levels of lipid hydroperoxides) are provided. Starting from the evidence that glutathione was depleted in MV474, and using both in silico and in vitro approaches, here we studied the interaction of wild-type RhdA and Cys(230) Ala site-directed RhdA mutant with glutathione species. We found that RhdA was able to bind in vitro reduced glutathione (GSH) and that RhdA-Cys(230) residue was mandatory for the complex formation. RhdA catalyzed glutathione-disulfide formation in the presence of a system generating the glutathione thiyl radical (GS(.), an oxidized form of GSH), thereby facilitating GSH regeneration. This reaction was negligible when the Cys230 Ala RhdA mutant was used. The efficiency of RhdA as catalyst in GS(.)-scavenging activity is discussed on the basis of the measured parameters of both interaction with glutathione species and kinetic studies.Vigoni project/0815171Deutscher Akademischer Austausch DienstUniversita` degli Studi di Milano/Fondo interno ricerca scientifica e tecnologic

Arsenic Biotransformations in Microbes and Humans, and Catalytic Properties of Human AS3MT Variants

Arsenic is the most pervasive environmental toxic substance. As a consequence of its ubiquity, nearly every organism has genes for resistance to inorganic arsenic. In one project I examined the role of glutaredoxin 2 (Grx2) in reduction of arsenate to arsenite. I demonstrated that Grx2 has both glutaredoxin thiol transfer activity and glutathione S-transferase (GST) activity. In a second project investigated arsenic resistance in a microbiome organism. I discovered that the human gut microflora B. vulgatus has eight continuous genes in its genome and these genes form an arsenical-inducible transcriptional unit. In two other projects I investigated the properties of two As(III) S-adenosylmethionine (SAM) methyltransferase (ArsM in microbes and AS3MT in animals). In this project we demonstrate that most fungal species have ArsM orthologs with only three conserved cysteine residues, and AfArsM from Aspergillus fumigatus methylates only MAs(III) and not As(III). For human, arsenic methylation process is thought to be protective from acute high-level arsenic exposure. However, with long term low-level exposure, hAS3MT is thought to produce intracellular methylarsenite (MAs(III)) and dimethylarsenite (DMAs(III)), which are considerably more toxic than inorganic As(III) and may contribute to arsenic-related diseases. Several single nucleotide polymorphisms (SNPs) in putative regulatory elements of the hAS3MT gene have been shown to be protective. In contrast, three previously identified exonic SNPs (R173W, M287T and T306I) may be deleterious. I identified five additional intragenic variants in hAS3MT (H51R, C61W, I136T, W203C and R251H). I purified the eight polymorphic hAS3MT proteins and characterized their enzymatic properties. Each enzyme had low methylation activity through decreased affinity for substrate, lower overall rates of catalysis and/or lower stability. I propose that amino acid substitutions in hAS3MT with decreased catalytic activity lead to detrimental responses to environmental arsenic and may increase the risk of arsenic-related diseases

In vivo structure-mediated regulation of ribonucleotide reductase in S. pombe

Sufficient and balanced pools of deoxyribonucleotide triphophates (dNTPs) is crucial for high-fidelity DNA replication as well as correct DNA repair. The enzyme RiboNucleotide Reductase (RNR) catalyses NDP to dNDP and is therefore an essential enzyme by providing the “building blocks” to the cells. dNTPs production needs to be tightly regulated in order to minimize mutation frequencies and prevent genome instability. RNR in S. pombe is composed of two proteins, Cdc22R1 and Suc22R2, and has been described as a heterotetramer with a dimer of each subunit: the big subunit Cdc22R1 and the small subunit Suc22R2. S. pombe also posseses an RNR inhibitor: Spd1, as well as a second RNR regulator Spd2 which has been newly discovered. Spd1 has been demonstrated to inhibit RNR and to regulate its activity throughout the cell cycle. The detailed mechanism of the RNR regulation during the cell cycle or after DNA damage is not entirely clear, as are the means of inhibition by Spd1. In order to shed some light on the RNR complex and its regulation, we used various microscopybased methods to study RNR in vivo as well as in vitro. The data of this thesis suggest there are different forms of active RNR heterocomplexes, found throughout the cell cycle in the cytoplasm as well as in the nucleus. We propose that the precise stoichiometry of subunits in the complexes may vary, or that the complex conformation may be modified in an Spd1-dependent manner. In addition, treatment of the cells with a UV mimetic agent, 4NQO, seems to promote RNR regulation in an Spd1-dependent manner. On the contrary, inhibition of RNR by HydroxyUrea (HU) affects the RNR in a possible structure-related manner, independently of Spd1 or Spd2. The in vivo observations correlate with structural and/or oligomerization modifications of the RNR, representing a novel RNR regulation in S. pombe

Mechanisms of Arsenic Detoxification and Resistance

Arsenic is a ubiquitous environmental toxic substance. As a consequence of continual exposure to arsenic, nearly every organism, from Escherichia coli to humans have evolved arsenic detoxification pathways. One of the pathways is extrusion of arsenic from inside the cells, thereby conferring resistance. The R773 arsRDABC operon in E. coli encodes an ArsAB efflux pump that confers resistance to arsenite. ArsA is the catalytic subunit of the pump, while ArsB forms the oxyanion conducting pathway. ArsD is an arsenite metallochaperone that binds arsenite and transfers it to ArsA. The interaction of ArsA and ArsD allows for resistance to As(III) at environmental concentrations. The interaction between ArsA ATPase and ArsD metallochaperone was examined. A quadruple mutant in the arsD gene encoding a K2A/K37A/K62A/K104A ArsD is unable to interact with ArsA. An error-prone mutagenesis approach was used to generate random mutations in the arsA gene that restored interaction with the quadruple arsD mutant in yeast two-hybrid assays. Three such mutants encoding Q56R, F120I and D137V ArsA were able to restore interaction with the quadruple ArsD mutant. Structural models generated by in silico docking suggest that an electrostatic interface favors reversible interaction between ArsA and ArsD. Mutations in ArsA that propagate changes in hydrogen bonding and salt bridges to the ArsA-ArsD interface also affect their interactions. The second objective was to examine the mechanism of arsenite resistance through methylation and subsequent volatilization. Microbial ArsM (As(III) S-adenosylmethyltransferase) catalyzes the formation of trimethylarsine as the volatile end product. The net result is loss of arsenic from cells. The gene for CrArsM from the eukaryotic green alga Chlamydomonas reinhardtii was chemically synthesized and expressed in E. coli. The purified protein catalyzed the methylation of arsenite into methyl-, dimethyl- and trimethyl products. Synthetic purified CrArsM was crystallized in an unliganded form. Biochemical and biophysical studies conducted on CrArsM sheds new light on the pathways of biomethylation. While in microbes ArsM detoxifies arsenic, the human homolog, hAS3MT, converts inorganic arsenic into more toxic and carcinogenic forms. An understanding of the enzymatic mechanism of ArsM will be critical in deciphering its parallel roles in arsenic detoxification and carcinogenesis

Intricate aspects of the thioredoxin system in redox signaling

Reversible modifications of redox sensitive protein thiols by reactive oxygen and nitrogen species have emerged as a major posttranslational mechanism that affects the function of the respective proteins and therewith all downstream events. These modifications can be reversed by redox catalysts of which the thioredoxin system forms one of the most prominent. It is ubiquitously expressed and consists of thioredoxin reductase (TrxR) that takes electrons from NADPH to reduce thioredoxin (Trx) as well as a myriad of other substrates. Within this thesis we have studied several aspects of cellular signaling pathways modulated by the Trx system. Paper I. The Trx system is overexpressed in many types of cancers and considered to contribute to their survival by countering elevated ROS levels that are typical for these cells. Thus, inhibiting TrxR in order to attenuate the antioxidant capacity of cancer cells might tip the balance in favor of ROS induced cell death pathways as a principle of cancer therapy. TrxR1 is a particularly suitable target in this context due to its highly reactive and accessible selenocysteine (Sec) residue within its C-terminal active site. At physiological pH the Sec is mostly de-protonated and thus easily targeted by electrophilic compounds. In characterizing Au, Pt and Pd based salts we found that all inhibited the Sec-depended activity of the enzyme in a specific manner, with Au and Pd being more potent than Pt in vitro. In context of cellular TrxR1, however, inhibition and cytotoxicity were mainly dependent on the ligand substituents of the compounds and thus their cellular uptake and metabolism. We furthermore discovered cisplatin triggered covalent complex formation of TrxR1 with either Trx1 or TRP14 (thioredoxin like protein of 14 kDa), which potentially contributes to the mechanism of cisplatin mediated cytotoxicity. Paper II. TrxR1 has in addition to its main isoform at least five minor splice variants that are distinguished by their N-terminal extensions. These may directly influence the activity of the TrxR1 core module or mediate subcellular localization via potential translocation signals. One of these variants, named “v3” (carrying a unique glutaredoxin domain), was previously shown to associate with the plasma membrane where it provoked dynamic filopodia. Within this study we found that v3 associates with specific membrane raft microdomains upon N-myristoylation and palmitoylation. These membrane structures were shown to serve as signaling platforms, including redox dependent processes, suggesting that v3 is potentially involved in redox signaling. Paper III. Transcription factors are a specific group of proteins that regulate the rapid transcription of genes. Many are functionally intertwined, activated under redox perturbing conditions and highly controlled by regulatory networks like the thioredoxin and glutathione systems. Signaling pathways leading to their activation are complex and expected to be modulated by numerous factors. In order to simultaneously characterize several transcription factors on single cell level we developed a method that is based on a three-colored fluorescence-based reporter plasmid (pTRAF). We demonstrated the use by quantifying responses of the three medically important transcription factors Nrf2, HIF and NF?B, utilizing HEK293 cells that were subjected to diverse stimulants. In conclusion, we studied TrxR1 targeting by noble metal based compounds and characterized their ability to transform TrxR1 into its pro-oxidant SecTRAP form as a principle of anti-cancer therapy. We also identified the mechanism behind the intracellular localization of “v3” and show that it is targeted to lipid rafts where it is a potentially important regulator of signaling processes. Finally we developed a tool to study the activation of three redox sensitive, intertwined transcription factors

Characterization Of Arsd: An Arsenic Chaperone For The Arsab As(iii)-Translocating Atpase

Arsenic is a metalloid toxicant that is widely distributed throughout the earth\u27s crust and causes a variety of health and environment problems. As an adaptation to arsenic-contaminated environments, organisms have developed resistance systems. In bacteria and archaea various ars operons encode ArsAB ATPases that pump the trivalent metalloids As(III) or Sb(III) out of cells. In these operons, an arsD gene is almost always adjacent to the arsA gene, suggesting a related function. ArsA is the catalytic subunit of the pump that hydrolyzes ATP in the presence of arsenite or antimonite. ArsB is a membrane protein which containing arsenite-conducting pathway. ArsA forms complex with ArsB, therefore ATP hydrolysis is coupled to extrusion of As(III) or Sb(III) through ArsB. Most transition and heavy metal ions do not exist as free ions in the cytosol but are sequestered by a variety of proteins called metal ion chaperones, scaffolds or intracellular carriers. ArsD was recently shown to be a chaperone for transfer of cytosolic As(III) to the 583-residue ArsA ATPase, the catalytic subunit of the efflux pump. ArsD is a 120-residue protein with three conserved cysteine residues, Cys12, Cys13 and Cys18 required for chaperone activity. ArsA exhibits a low, basal rate of ATPase activity in the absence of As(III) or Sb(III) and a higher, activated rate in their presence. ArsA has a high affinity metalloid binding site composed of Cys113 and Cys422 and a third residue, Cys172, which participates in high affinity binding and activation of ATP hydrolysis. By directly transferring As(III) to ArsA, ArsD also increased ArsA ATPase activity at environmental concentrations of arsenic. Therefore, ArsAB pump efficiency is increased and less As(III) will be accumulated in the cells. In analogy with the mechanism of copper transfer from chaperones to copper pumps or enzymes, a step-wise transfer of As(III) from the cysteines of ArsD to the cysteines of ArsA, was proposed. The properties of As(III) binding by ArsD and subsequent transfer to ArsA were examined. X-ray absorption spectroscopy was used to show that As(III) is coordinated with three sulfur atoms, consistent with Cys12, Cys13 and Cys18 forming the As(III) binding site. An assay using intrinsic protein fluorescence was developed as a probe of metalloid binding to ArsD. Two single tryptophan derivatives of ArsD were constructed by changing either Thr15 or Val17 to tryptophan in a tryptophan-free background. Both exhibited quenching of fluorescence upon binding of As(III) or Sb(III), from which the apparent affinity for metalloid could be estimated. Since it is likely that cytosolic As(III) is bound to reduced glutathione (GSH), the effect of GSH on binding to ArsD was examined. GSH greatly increased the rate of binding As(III) to ArsD, suggesting that ArsD accepts metalloid from the As(GS)3 complex. In contrast, GSH did not affect the As(III)-stimulated ArsA ATPase activity, suggesting that As(III) is directly transferred from ArsD to ArsA, as opposed to release from ArsD, binding to GSH and then interaction of ArsA with the As(GS)3 complex. To differentiate between these two possibilities, the effect of the As(III) chelator dimercaptosuccinic acid (DMSA) was examined. The chelator did not affect transfer, indicating channeling of As(III) from ArsD to ArsA. Transfer occurs only under conditions where ArsA hydrolyzes ATP, suggesting that ArsD transfer As(III) to an ArsA conformation transiently formed during catalysis and not simply to the closed conformation that ArsA adopts when As(III) and MgATP are bound. R773 ArsD was shown to be a dimer in crystal structure. Whether the dimerization form is a physiological one existing in the solution, was studied by mutagenesis. Residues, Ser68, Arg87 and Arg96, involved in dimerization were mutated to alanine. ArsD dimerization equilibrium was shifted to the monomer direction by mutating these residues to alanine, but not totally a monomeric form. One mutant ArsDG86E was selected from reverse yeast two-hybrid analysis, showing no dimerization with wild-type ArsD. Gel-filtration chromatography confirmed mutation G86E shifts ArsD dimerization equilibrium to the monomer direction, but not totally change ArsD to a monomeric form. Since Gly86 sits on the dimerization interface in the crystal structure, it is most likely the crystallographic ArsD dimer forms in the solution. All these mutants still retain the ability to stimulate ArsA ATPase activity, suggesting dimerization is not strictly required for ArsD metallochaperone function. ArsA and ArsD crystal structure have been solved individually. But little is known about ArsA-ArsD interaction interface. Yeast two-hybrid and reverse yeast two-hybrid are combined to select for totally 14 ArsD mutants with weaker or stronger interaction with ArsA. Additionally, Lys37 and Lys62 were shown to be important for ArsD function by site-directed mutagenesis. ArsD loses function when Lys37 and Lys62 were mutated to alanine as well as acetylated by Sulfo-NHS acetate. The charge carried by Lys37 and Lys62 was shown to be important since protein is still active when they are mutated to arginine. Yeast two-hybrid confirmed mutating Lys37 and Lys62 to alanine has effect on ArsA-ArsD interaction. Mapping all the mutations on ArsD structure gives us information on ArsA-ArsD interaction interface. Four residues, Ser14, Val17, Thr20 and Val22, are in the loop containing the important metal binding site Cys12-Cys13-Cys18. This suggests the metal binding site may be directly involved in the interaction with ArsA. Seven residues, Gln24, Val27, Asp28, Thr31, Gln34, Lys37 and Gln38 are located on helix Α1. They are aligned at one side of helix Α1 and solvent exposed, suggesting this region might be directly involved in interaction. A structure model of ArsA-ArsD complex was generated by docking. The model suggested an extensive interaction interface at multiple directions, consistent with most of the yeast two-hybrid results

Characterization of cellular stress systems using biological mass spectrometry

In recent years mass spectrometry has become an invaluable tool to address an array of biological questions. The versatility of this experimental approach does not only allow assignment of protein identity and identification of sequence specific modifications, but with the help of particular derivatization techniques facilitates the determination of peptide quantity. Each of these approaches were applied to the following biological projects: The 21 kDa heat stable protein purified from the encysted embryo of Artemia franciscana was characterized by time-of-flight mass spectrometry. De novo sequencing of peptides identified this protein as a group 1 Late embryogenesis abundant (LEA) protein. The amino acid sequence assignment to these peptides allowed amplification of the entire gene sequence from an embryonic cDNA library. This was deposited into the NCBI database (EF656614). The expression of group 1 LEA protein is consistent with and supports a role in desiccation tolerance. In addition, this is a first report describing identification of a group 1 LEA protein in an animal species. A MS-based quantitative analysis was performed in order to analyze relative changes in the dynamic thiol and disulfide states of the redox sensitive protein disulfide isomerase, PDI. PDI cysteine residues were derivatized with an isotope-coded affinity tag (ICAT), thus allowing a direct comparison between the reduced and auto-oxidized forms. Quantitation was based on relative ratios between light and heavy isotopically labeled cysteine containing peptides. The application of the ICAT-labeling approach to PDI related studies, allowed direct assignment of individual cysteine residues and their oxidation status, compared to the previously employed techniques, that only provided information regarding the average number of modified cysteine residues within PDI, not their exact identity. A combination of a phosphopeptide enrichment step and a MS-based approach was utilized to identify three phosphorylation sites on hYVH1, an atypical dual specificity phosphatase that functions as a cell survival factor. With the help of phosphomimetic and non-phosphorylable mutants, we were able to decipher their effect on localization and progression through the cell cycle. Collectively, these studies manifest the power of MS-generated data to influence and guide many different fields ranging from molecular embryology to biochemistry

Proteomics of spindle checkpoint complexes and characterisation of novel interactors

The eukaryotic cell cycle is governed by molecular checkpoints that ensure genomic integrity and the faithful transmission of chromosomes to daughter cells. They inhibit the cycle until conditions prevail that guarantee accurate DNA duplication and chromosome segregation. Two major mechanisms are the ‘spindle assembly checkpoint’ and the ‘DNA damage checkpoint’. During pro-metaphase, the spindle checkpoint monitors the orientation process of chromatid pairs on the bipolar microtubule array nucleated by spindle pole bodies. In the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae, six proteins are at the heart of spindle checkpoint function: Mad1, Mad2, Mad3, Bub1, Bub3 and Mph1/Mps1. The formation of spindle checkpoint complexes signals the presence of incorrect spindle microtubule attachments to kinetochores. These complexes cooperate to suppress the activity of the anaphase promoting complex (APC) and inhibit the onset of anaphase. By isolating these distinct complexes and analysing their composition by mass-spectrometry (MS) this work revealed several intriguing disparities between the two yeast species, and the way in which the Bub and Mad proteins cooperate to achieve inhibition. The ‘mitotic checkpoint complex’, which in S.cerevisiae consists of Mad2, Mad3, Bub3 and the APC activator Cdc20, was found to lack Bub3 in S.pombe. The S.pombe complex was shown to interact with the APC, but no stable interaction was found to be required in S.cerevisiae cells. And whereas Bub1 and Bub3 were found to form a complex with Mad1 in S.cerevisiae, in S.pombe they were shown to associate with Mad3 to form the ‘BUB+ spindle checkpoint complex’. In addition, MS analysis uncovered TAPAS: a novel S.pombe complex that was found to interact with the BUB+ complex and revealed to consist of Tfg3, Abo1 (gene product of SPAC31G5.19), Pob3 and Spt16. TAPAS mutant cells were shown to lose viability as a result of genotoxic stress, a phenotype that was surprisingly shared with bub1Δ and bub1kd ‘kinase dead’ mutants. Sensitivity of cells deficient in TAPAS or Bub1 did not appear to be due to the loss of DNA damage checkpoint or DNA replication checkpoint functions. Further examination revealed that Bub1 functions in the repair of DNA double strand breaks. Taken together, this work demonstrates that even though the molecular components of the spindle checkpoint pathway are conserved, their regulatory connections have to some extent diverged through molecular evolution. This process not only rewired, but entwined two molecular processes that together safeguard the genetic heritage of cells