Cysteines In Domain Research Articles

Molecular cloning and expression profiles of an insulin-like growth factor binding protein IGFBP5 in the pearl oyster, Pinctada fucata

ABSTRACTThe insulin-like growth factor binding protein (IGFBP) family participates in transportation, localization, and biological regulation of insulin-like growth factors (IGFs). In the present study, IGFBP5 gene from the Pinctada fucata (PfIGFBP5) was cloned and characterized. The full-length cDNA sequence (1319 bp) contained an open reading frame of 399 bp encoding a predicted protein of 132 amino acids. The amino acid sequence of PfIGFBP5 included an IGF binding domain and multiple cysteine residues. The genomic sequence of PfIGFBP5 consisted of three exons and two introns. PfIGFBP5 was found to be expressed in all tissues and developmental stages investigated, although the expression level was significantly higher in the pearl sac and in trochophore larvae than in other tissues or developmental stages (P < 0.05). Expression of PfIGFBP5 was induced by notching the oyster shell margin; the gene was mainly expressed in the outer epithelium of the mantle, and to a lesser extent in the whole pearl sac, including connective tissues. These findings suggest that PfIGFBP5 is very likely to be involved in modulating biomineralization in the mantle and pearl sac of P. fucata since they are the main organs for shell or pearl formation.

Systematic Review of Cysteine-Sparing NOTCH3 Missense Mutations in Patients with Clinical Suspicion of CADASIL.

CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy) is caused by mutations in the NOTCH3 gene, affecting the number of cysteines in the extracellular domain of the receptor, causing protein misfolding and receptor aggregation. The pathogenic role of cysteine-sparing NOTCH3 missense mutations in patients with typical clinical CADASIL syndrome is unknown. The aim of this article is to describe these mutations to clarify if any could be potentially pathogenic. Articles on cysteine-sparing NOTCH3 missense mutations in patients with clinical suspicion of CADASIL were reviewed. Mutations were considered potentially pathogenic if patients had: (a) typical clinical CADASIL syndrome; (b) diffuse white matter hyperintensities; (c) the 33 NOTCH3 exons analyzed; (d) mutations that were not polymorphisms; and (e) Granular osmiophilic material (GOM) deposits in the skin biopsy. Twenty-five different mutations were listed. Four fulfill the above criteria: p.R61W; p.R75P; p.D80G; and p.R213K. Patients carrying these mutations had typical clinical CADASIL syndrome and diffuse white matter hyperintensities, mostly without anterior temporal pole involvement. Cysteine-sparing NOTCH3 missense mutations are associated with typical clinical CADASIL syndrome and typical magnetic resonance imaging (MRI) findings, although with less involvement of the anterior temporal lobe. Hence, these mutations should be further studied to confirm their pathological role in CADASIL.

Dimerization of the BASIC PENTACYSTEINE Domain in Plant GAGA-Factors is Mediated by Disulfide Bonds and Required for DNA-Binding.

GAGA-binding proteins in plants are encoded by the BARLEY B-RECOMBINANT / BASIC PENTACYSTEINE (BBR/BPC) family, which can be spilt into several groups on the basis of sequence divergence. The proteins of the different groups share an evolutionary conserved BASIC PENTACYSTEINE (BPC) domain at their very C-terminus that is important for DNA binding. Hallmark of this domain are five Cysteines at defined positions and spacing, which are considered to form a zinc-finger like structure that is involved in GAGA-motif recognition. Here, we report the formation of stabile homodimers between Arabidopsis thaliana group I member BPC1 or between group II member BPC6 in SDS-PAGE. Serial mutations of the highly conserved five Cysteines in the BPC domain of Arabidopsis thaliana BPC1 were tested for their capacity to bind to GAGA-motifs by DPI-ELISA. Our results do not support the idea of a direct involvement of these residues in making physical contact with the DNA, e.g. by formation of a zinc-finger structure. Instead, the data implies an indispensable function for the five Cysteines in homodimerization and stabilization of the protein structure by disulfide bonds. Accordingly, protein folding and structure prediction suggests the formation of a scaffold for dimerization that is supported by three intermolecular and one intramolecular S-S bond. The high degree of conservation between the BPC domains from the different groups and from different species denotes that this role for the five Cysteines might be evolutionary retained.

The structure-function relationship of disulfide bonds in etanercept

Etanercept is a TNFα receptor Fc fusion protein used for the treatment of rheumatic disease and psoriasis. Physicochemical and functional investigation of process fractions during development of the etanercept biosimilar GP2015 (Erelzi®) revealed a correlation between reduced potency and incorrect disulfide bridging between specific cysteines in the receptor domain. This novel structure-function relationship was found to be the molecular basis for reduced potency in recent Enbrel® batches, which exhibit higher levels of incorrect disulfide bridging. Interestingly, incorrect disulfide bridging was found to be reversible under serum-like redox conditions, restoring potency to normal levels. This redox dependent reversibility suggests that these variants are likely not relevant for clinical efficacy once the drug enters the bloodstream. Nonetheless, incorrect disulfide bridging in etanercept represents a new quality attribute that is critical for biopharmaceutical functionality and should thus be carefully monitored and controlled to guarantee patient safety.

Select targeting of intracellular Cysteine in Toll-Interleukin-1 receptor resistance domains for protection against Influenza.

Abstract Regulation of Toll-like receptor (TLR) signaling using small molecule agonists and antagonists is widely sought after for controlling inflammation and disease. Recently, small molecule and TIR-based-decoy-peptide inhibitors targeting intracellular Toll-Interleukin-1 receptor (TIR) resistance domains have been identified and functionally characterized to protect against lethality in animal models of infection, inflammation and disease. We observe multiple instances of targeting, modification or involvement of conserved Cysteine residues within intracellular TIR domains by functionally characterized small molecule and decoy peptide inhibitors. We hypothesize that cysteine interactions among intracellular Toll and Interleukin-1 receptors may possess therapeutic value. We have used molecular modeling, X-ray crystallography and in vitro and in vivo functional characterization to characterize TIR cysteine interactions in molecular detail using previously functionally verified derived small molecule and decoy-peptide inhibitors. As a proof of concept, we have used the small molecule receptor antagonist TAK242, which selectively binds Cys747 located in the intracellular TIR domain of TLR4. We show that intracellular targeting of TLR4 TIR domain cysteine using TAK242 is able block LPS activated TLR4 NF-kB signaling in cells and protects mice against lethal influenza (PR8) infection at a lower dose than the ectodomain TLR4 antagonist Eritoran. Structural analysis shows this Cysteine residue to be highly conserved among other surface receptor TLRs 1,2,4,5,6 and 10. Additionally, surface plasmon resonance binding of cysteine containing TIR-decoy peptides, is sensitive to reduction using 2-Mercaptoethanol.

Novel insights into the interaction of UBA5 with UFM1 via a UFM1-interacting sequence

The modification of proteins by ubiquitin-fold modifier 1 (UFM1) is implicated in many human diseases. Prior to conjugation, UFM1 undergoes activation by its cognate activating enzyme, UBA5. UBA5 is a non-canonical E1 activating enzyme that possesses an adenylation domain but lacks a distinct cysteine domain. Binding of UBA5 to UFM1 is mediated via an amino acid sequence, known as the UFM1-interacting sequence (UIS), located outside the adenylation domain that is required for UFM1 activation. However, the precise boundaries of the UIS are yet not clear and are still under debate. Here we revisit the interaction of UFM1 with UBA5 by determining the crystal structure of UFM1 fused to 13 amino acids of human UBA5. Using binding and activity assays, we found that His 336 of UBA5, previously not reported to be part of the UIS, occupies a negatively charged pocket on UFM1’s surface. This His is involved in UFM1 binding and if mutated perturbs activation of UFM1. Surprisingly, we also found that the interaction between two UFM1 molecules mimics how the UIS binds UFM1. Specifically, UFM1 His 70 resembles UBA5 His336 and enters a negatively charged pocked on the other UFM1 molecule. Our results refine our understanding of UFM1-UBA5 binding.

Selective Chemical Labeling of Type II Cannabinoid Receptor CB2 for 19F-NMR and EPR Studies

Human cannabinoid receptor CB2, a G protein-coupled receptor (GPCR) is implicated in an array of health related systemic body functions including the immune response, inflammation, and pain sensing, and it is an important target for pharmaceutical drug development. Structural and functional studies of GPCR by NMR, EPR and fluorescence techniques require non-intrusive, site-specific labeling of the protein. This can be achieved by chemical labeling of cysteines of the receptor. In this study we explored feasibility of labeling of selected cysteines by minimizing the number of reactive SH groups through systematic replacement of the majority of the 13 naturally-occurring cysteines of CB2. The replacement of the six water-exposed cysteines resulted in a well-expressed, fully functional receptor as evidenced by ligand binding- and G protein activation studies. While the replacement of four additional cysteines in the transmembrane domain of the protein lowered ligand binding affinity several fold, the receptor could still be activated by an excess of a high affinity agonists and performed competently in an in vitro G protein activation assay. The reduced cysteine templates of CB2 were either labeled directly or used to introduce new cysteines in selected positions at the extracellular and intracellular surfaces of the protein. The cysteine mutants of CB2 were chemically labeled with 2,2,2-trifluoroethanethiol (TET) and their spectra recorded by 19F NMR in micelles composed of dodecyl maltoside and CHAPS or in particles formed by the NVoy polymer. The latter was advantageous since it yielded monomeric receptor suitable for recording solution-state 19F NMR spectra with decent resolution. This study is a precursor for exploring the structure-function relationship of CB2 by NMR and EPR.

Fluorescent Labeling Preserving OCP Photoactivity Reveals Its Reorganization during the Photocycle

Orange carotenoid protein (OCP), responsible for the photoprotection of the cyanobacterial photosynthetic apparatus under excessive light conditions, undergoes significant rearrangements upon photoconversion and transits from the stable orange to the signaling red state. This is thought to involve a 12-Å translocation of the carotenoid cofactor and separation of the N- and C-terminal protein domains. Despite clear recent progress, the detailed mechanism of the OCP photoconversion and associated photoprotection remains elusive. Here, we labeled the OCP of Synechocystis with tetramethylrhodamine-maleimide (TMR) and obtained a photoactive OCP-TMR complex, the fluorescence of which was highly sensitive to the protein state, showing unprecedented contrast between the orange and red states and reflecting changes in protein conformation and the distances from TMR to the carotenoid throughout the photocycle. The OCP-TMR complex was sensitive to the light intensity, temperature, and viscosity of the solvent. Based on the observed Förster resonance energy transfer, we determined that upon photoconversion, the distance between TMR (donor) bound to a cysteine in the C-terminal domain and the carotenoid (acceptor) increased by 18 Å, with simultaneous translocation of the carotenoid into the N-terminal domain. Time-resolved fluorescence anisotropy revealed a significant decrease of the OCP rotation rate in the red state, indicating that the light-triggered conversion of the protein is accompanied by an increase of its hydrodynamic radius. Thus, our results support the idea of significant structural rearrangements of OCP, providing, to our knowledge, new insights into the structural rearrangements of OCP throughout the photocycle and a completely novel approach to the study of its photocycle and non-photochemical quenching. We suggest that this approach can be generally applied to other photoactive proteins.

Preclinical evaluation of drugs to block inflammation-driven preterm birth.

Intrauterine inflammation, the major cause of early preterm birth, can have microbial and sterile aetiologies. We assessed in a Transwell model the anti-inflammatory efficacies of five drugs on human extraplacental membranes delivered after preterm spontaneous labour (30-34 wk). Drugs [TPCA1 (IKKβ inhibitor), 5 z-7-oxozeaenol (OxZ, TAK1 inhibitor), inhibitor of NF-κB essential modulator binding domain (iNBD), SB239063 (p38 MAPK inhibitor) and N-acetyl cysteine (free radical scavenger free radicals)] were added after 12 h equilibration to the amniotic compartment. Concentrations of IL-6, TNF-α, MCP-1, IL-1β and PGE2 in the media, and IL6, TNFA and PTGS2 mRNA expression levels in membranes, were determined after 12 h. Data were analysed using mixed models analyses. Thirteen of the 28 membranes had histological chorioamnionitis (HCA+); five were positive for bacterial culture and six for fetal inflammatory reaction. Baseline PGE2 and cytokine production was similar between HCA- and HCA+ membranes. Anti-inflammatory effects were also similar between HCA- and HCA+ membranes. TPCA1 and OxZ were the most effective drugs; each inhibited amniotic secretion of 4/5 pro-inflammatory mediators and mRNA levels of 2/3, regardless of stimulus. We conclude that treatment with TPCA1 or OxZ, in combination with antibiotics, may minimise the adverse effects of intrauterine inflammation in pregnancy.

Heterotrimeric G protein signaling in plant

Heterotrimeric guanine nucleotide-binding protein (G protein) signaling is an evolutionarily conserved mechanism in diverse eukaryotic organisms. In the past half century, five Nobel Prizes are awarded to researchers working in the G protein field, these seminal studies make the G protein pathway become the best understood signaling transmit system in the world. In animals, heterotrimeric G proteins contains α , β and γ subunits, perceives extracellular stimuli through G protein-coupled receptors (GPCR), and transmit signals to ion channels, enzymes and other effectors to affect numerous cellular behaviors. In recent years, much has been learned about the diversity of signal transduction through plant G proteins thanks to the identification and mutation of genes in Arabidopsis and rice. Plant cells have most of the core elements found in animal G signaling, such as α , β and γ subunits. However, plant G proteins transmit signals by atypical mechanisms. The plant G α subunit spontaneously releases GDP and forms a stable GTP bound state in vitro , and thus are self-activating. The self-activating characteristic also means that plant G protein paradigm do not need and therefore do not have typical animal GPCR. Indeed, the current evidence supports that no typical GPCR exists in plant. In most plants, the role of GPCR is performed by GTPase-accelerating protein (GAP), such as Arabidopsis regulator of G protein signaling 1 (AtRGS1). Interestingly, there is no ortholog of AtRGS1 in rice genome. Recently, an important advance has been proved that up-regulation of COLD1 is positively correlated with chilling tolerance in rice. COLD1 has nine-transmembrane domains, and localize to plasma membrane (PM) and endoplasmic reticulum (ER). The COLD1 physically interacts with RGA1 and acts as RGS protein that senses low temperature signals and accelerates G-protein GTPase activity. These results indicate that non-GPCR proteins in plant can topologically and functionally resemble animal GPCRs. In plants, the repertoire of the heterotrimeric G protein is much simpler than that in mammalian, but G γ subunits exhibit an extraordinary level of structural diversity and show significant differences from their animal counterparts. The non-canonical G γ subunits in plant kingdom have a large cysteine domain on C terminal, can be four times larger than the average mammalian G γ subunits size. The non-canonical G γ subunits present an inhibitory effects of N terminal on C terminal. Recent studies have shown that the manipulation of two non-canonical G γ subunits, GS3 (grain size 3) and DEP1 (dense and erect panicle 1), represents new strategy to simultaneously increase grain yield and nitrogen use efficiency in rice breeding. In this review, we focus on the recent insights of G protein network derived from the research about the model plant rice and Arabidopsis , the difference of G protein signal between plants and animals, and the potential of G protein to improve the crops productivity.

A novel missense mutation of CMT2P alters transcription machinery.

Charcot-Marie-Tooth type 2P (CMT2P) has been associated with frameshift mutations in the RING domain of LRSAM1 (an E3 ligase). This study describes families with a novel missense mutation of LRSAM1 gene and explores pathogenic mechanisms of CMT2P. Patients with CMT2P were characterized clinically, electrophysiologically, and genetically. A neuronal model with the LRSAM1 mutation was created using CRISPR/Cas9 technology. The neuronal cell line along with fibroblasts isolated from the patients was used to study RNA-binding proteins. This American family with dominantly inherited axonal polyneuropathy reveals a phenotype similar to those in previously reported non-US families. The affected members in our family cosegregated with a novel missense mutation Cys694Arg that alters a highly conserved cysteine in the RING domain. This mutation leads to axonal degeneration in the in vitro neuronal cell line. Moreover, using protein mass spectrometry, we identified a group of RNA-binding proteins (including FUS, a protein critically involved in motor neuron degeneration) that interacted with LRSAM1. The interactions were disrupted by the Cys694Arg mutation, which resulted in reduction of intranuclear RNA-binding proteins. Our findings suggest that the mutant LRSAM1 may aberrantly affect the formation of transcription machinery. Given that a similar mechanism has been reported in motor neuron degeneration of amyotrophic lateral sclerosis, abnormalities of RNA/RNA-binding protein complex may play a role in the neuronal degeneration of CMT2P. Ann Neurol 2016;80:834-845.

S-Nitrosylation Induces Structural and Dynamical Changes in a Rhodanese Family Protein

S-Nitrosylation is well established as an important post-translational regulator in protein function and signaling. However, relatively little is known about its structural and dynamical consequences. We have investigated the effects of S-nitrosylation on the rhodanese domain of the Escherichia coli integral membrane protein YgaP by NMR, X-ray crystallography, and mass spectrometry. The results show that the active cysteine in the rhodanese domain of YgaP is subjected to two competing modifications: S-nitrosylation and S-sulfhydration, which are naturally occurring in vivo. It has been observed that in addition to inhibition of the sulfur transfer activity, S-nitrosylation of the active site residue Cys63 causes an increase in slow motion and a displacement of helix 5 due to a weakening of the interaction between the active site and the helix dipole. These findings provide an example of how nitrosative stress can exert action at the atomic level.

The ubiquitin signal and autophagy: an orchestrated dance leading to mitochondrial degradation

The quality of mitochondria, essential organelles that produce ATP and regulate numerous metabolic pathways, must be strictly monitored to maintain cell homeostasis. The loss of mitochondrial quality control systems is acknowledged as a determinant for many types of neurodegenerative diseases including Parkinson's disease (PD). The two gene products mutated in the autosomal recessive forms of familial early-onset PD, Parkin and PINK1, have been identified as essential proteins in the clearance of damaged mitochondria via an autophagic pathway termed mitophagy. Recently, significant progress has been made in understanding how the mitochondrial serine/threonine kinase PINK1 and the E3 ligase Parkin work together through a novel stepwise cascade to identify and eliminate damaged mitochondria, a process that relies on the orchestrated crosstalk between ubiquitin/phosphorylation signaling and autophagy. In this review, we highlight our current understanding of the detailed molecular mechanisms governing Parkin-/PINK1-mediated mitophagy and the evidences connecting Parkin/PINK1 function and mitochondrial clearance in neurons.

Investigating the Role of Transmembrane Domain Cysteine Residues in the Organization of TNF Receptors

We recently showed that disulfide bond formation via cysteine residues in the transmembrane (TM) domains of the long isoform of Death Receptor 5 play a significant role in receptor homodimerization and activation. This is significant because the dominant paradigm in TNF Receptor signaling had been that receptor trimers, not dimers, are the signaling competent oligomeric state. Here, we surveyed the full TNF Receptor superfamily and found that potential TM domain disulfide bonds exist in approximately one third of the superfamily members. To investigate the broader importance of these cysteine residues and the potential for disulfide bond formation in the TNF Receptor superfamily, we studied Fas and TNF-Receptor 1 in cells. We also performed computational calculations (potential of mean force) to explore the thermodynamics of conformational changes of disulfide-bonded TM domain dimers. Additionally, we performed a bioinformatics study on transmembrane domains to further investigate the prevalence of TM domain cysteine residues and potential disulfide bonded TM dimers.

Genetic diversity of transmission-blocking vaccine candidate Pvs48/45 in Plasmodium vivax populations in China.

BackgroundThe male gamete fertilization factor P48/45 in malaria parasites is a prime transmission-blocking vaccine (TBV) candidate. Efforts to develop antimalarial vaccines are often thwarted by genetic diversity of the target antigens. Here we evaluated the genetic diversity of Pvs48/45 gene in global Plasmodium vivax populations.MethodsWe determined 200 Pvs48/45 sequences collected from temperate and subtropical parasite populations in China. Population genetic and evolutionary analyses were performed to determine the levels of genetic diversity, potential signature of selection, and population differentiation.ResultsAnalysis of the Pvs48/45 sequences from 200 P. vivax parasites collected in a temperate and a tropical region revealed a low level of genetic diversity (π = 0.0012) with 14 single nucleotide polymorphisms, of which 11 were nonsynonymous. Analysis of 344 Pvs48/45 sequences from nine worldwide P. vivax populations detected a total of 38 haplotypes, of which 13 haplotypes were present only once. Multiple tests for selection confirmed a signature of positive selection on Pvs48/45 with selection skewed to the second cysteine domain. Haplotype network analysis and Wright’s fixation index showed large geographical differentiation with the presence of continent-or region-specific mutations in this gene.ConclusionsPvs48/45 displays low levels of genetic diversity with the presence of region-specific mutations. Some of the mutations may be potential epitope targets based on their positions in the predicted structure, highlighting the need for future evaluation of these mutations in designing Pvs48/45-based TBV.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-015-1232-4) contains supplementary material, which is available to authorized users.

BIOPHYSICAL AND STRUCTURAL ANALYSES OF THE ANTI-ATHEROSCLEROTIC PROPERTIES OF HSP-27

BIOPHYSICAL AND STRUCTURAL ANALYSES OF THE ANTI-ATHEROSCLEROTIC PROPERTIES OF HSP-27

The Human Acid-Sensing Ion Channel ASIC1a: Evidence for a Homotetrameric Assembly State at the Cell Surface.

The chicken acid-sensing ion channel ASIC1 has been crystallized as a homotrimer. We address here the oligomeric state of the functional ASIC1 in situ at the cell surface. The oligomeric states of functional ASIC1a and mutants with additional cysteines introduced in the extracellular pore vestibule were resolved on SDS-PAGE. The functional ASIC1 complexes were stabilized at the cell surface of Xenopus laevis oocytes or CHO cells either using the sulfhydryl crosslinker BMOE, or sodium tetrathionate (NaTT). Under these different crosslinking conditions ASIC1a migrates as four distinct oligomeric states that correspond by mass to multiples of a single ASIC1a subunit. The relative importance of each of the four ASIC1a oligomers was critically dependent on the availability of cysteines in the transmembrane domain for crosslinking, consistent with the presence of ASIC1a homo-oligomers. The expression of ASIC1a monomers, trimeric or tetrameric concatemeric cDNA constructs resulted in functional channels. The resulting ASIC1a complexes are resolved as a predominant tetramer over the other oligomeric forms, after stabilization with BMOE or NaTT and SDS-PAGE/western blot analysis. Our data identify a major ASIC1a homotetramer at the surface membrane of the cell expressing functional ASIC1a channel.

Abstract 1216: Thiol-modification induced reactivation of mutant p53 protein in human cancers using a nontoxic stabilized homoglutathione disulfide

Abstract Loss of functional p53 gene is a frequent and most critical event in the genesis and progression of half of all human cancers. Conversion of the non-DNA binding and misfolded-p53 mutant proteins to their wild-type conformation using small molecules to induce apoptosis is currently an intense area of research. Although a few compounds with this property have been described, their abilities to alter the p53 structure in animal models have not been documented and they appear toxic as well. Human cancers harbor elevated levels of oxidative stress and are vulnerable to even slight redox oscillations. To exploit this susceptibility, we have designed a glutathione mimic, namely, homoglutathione disulfide that has been stabilized with the addition of small amounts of cisplatin (1000:10). This compound perturbs the cellular redox, alters kinase activities and induces protein-glutathionylation. Since the human p53 contains several redox-sensitive cysteines in the DNA-binding domain, we explored the possible reactivation of mutant p53 (R273H) present in HT29 and SW620 colon cancer cells by the stabilized hGSSG.Pt (hGTX). Electrophoretic mobility shift assays demonstrated a concentration and time-dependent restoration of DNA-binding for the mutant p53 in HT29 cells by hGTX. DNA affinity immunoblotting, immunoprecipitation and direct western blotting using specific antibodies against the mutant (Mab240) or wild type p53 (Mab1620) or pan p53 (MabDo1) confirmed a progressive generation of wild type p53 and a gradual decline of mutant p53 levels in HT29 cells. The half-life of the altered p53 remained similar to the endogenous mutant form. Treatment of HT29 nuclear extracts with hGTX also induced the binding of mutant p53 with DNA, indicating that thiolation of specific cysteines mediates the reactivation. RT-PCR and western analyses showed significant upregulation of many p53 target genes (2-5 fold). Furthermore, when nude mice bearing the HT29 xenografts were administered HGTX (100 mg/kg daily for 7 days), marked p53 reactivation with concomitant reduction of mutant p53 was observed in tumor tissues. Treatment of hGTX also modulated redox sensitive DNA repair systems like MGMT and Topoisomerase-I. A highly significant amplification of cytotoxicity was induced by anticancer drugs in association with hGTX. We conclude that the alteration of redox milieu following hGTX modifies the reactive thiols in the p53 protein to restore the native conformation. These findings acquired with a nontoxic endogenous metabolite suggest therapeutic applications in combination with chemotherapy agents. Molecular modeling and mass spectrometry studies are underway to determine the structural changes and residues altered by hGTX treatment (Supported by grants from CPRIT (RP130266), NIH (R03CA125872) to KSS. Citation Format: Debasish Basak, Kalkunte S. Srivenugopal. Thiol-modification induced reactivation of mutant p53 protein in human cancers using a nontoxic stabilized homoglutathione disulfide. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1216. doi:10.1158/1538-7445.AM2015-1216

The N-terminal degenerated metal-binding domain is involved in the heavy metal transport activity of TaHMA2.

We identified key residues of TaHMA2, and the N- and C-terminal regions of the protein have different roles in its transport function when heterologously expressed in yeast. TaHMA2, a P1B-type ATPase from wheat (Triticum aestivum L.), plays an important role in heavy metal homeostasis in plants. A previous study showed that overexpressing TaHMA2 in rice (Oryza sativa L.), Arabidopsis thaliana, or tobacco (Nicotiana tabacum L.) resulted in various responses to heavy metals. Here, we report the heterologous expression of TaHMA2 in the yeast Saccharomyces cerevisiae. TaHMA2 expression increased the yeast's sensitivity to Cd, but not to Zn, Pb or Co, and increased Cd accumulation was concurrently observed. The eGFP-TaHMA2 fusion protein was localized to the plasma membrane and showed a discontinuous pattern. Mutagenesis of the cysteine and glutamate residues in the N-terminal metal-binding domain (N-MBD) impaired the function of TaHMA2. Deletion of most of the C terminus (TaHMA2ΔC, 712-1003) partially abolished the protein's function, whereas deletion of the N terminus (TaHMA2ΔN, 2-699) completely abolished Cd sensitivity. These data suggest that cysteine and glutamate residues are important for the metal-binding/translocation function of TaHMA2. Additional studies are needed to further understand the selectivity of TaHMA2 in planta.

The conservative cysteines in transmembrane domain of AtVKOR/LTO1 are critical for photosynthetic growth and photosystem II activity in Arabidopsis.

Thylakoid protein vitamin K epoxide reductase (AtVKOR/LTO1) is involved in oxidoreduction. The deficiency of this compound causes pleiotropic defects in Arabidopsis thaliana, such as severely stunted growth, smaller sized leaves, and delay of flowering. Transgenic complementation of wild-type AtVKOR (VKORWT) to vkor mutant lines ultimately demonstrates that the phenotype changes are due to this gene. However, whether AtVKOR functions in Arabidopsis through its protein oxidoreduction is unknown. To further study the redox-active sites of AtVKOR in vivo, a series of plasmids containing cysteine-mutant VKORs were constructed and transformed into vkor deficient lines. Compared with transgenic AtVKORWT plants, the size of the transgenic plants with a single conservative cysteine mutation (VKORC109A, VKORC116A, VKORC195A, and VKORC198A) were smaller, and two double-cysteine mutations (VKORC109AC116A and VKORC195AC198A) showed significantly stunted growth, similar with the vkor mutant line. However, mutations of two non-conservative cysteines (VKORC46A and VKORC230A) displayed little obvious changes in the phenotypes of Arabidopsis. Consistently, the maximum and actual efficiency of photosystem II (PSII) in double-cysteine mutation plants decreased significantly to the level similar to that of the vkor mutant line both under normal growth light and high light. A significantly decreased amount of D1 protein and increased accumulation of reactive oxygen species were observed in two double-cysteine mutations under high light. All of the results above indicated that the conservative cysteines in transmembrane domains were the functional sites of AtVKOR in Arabidopsis and that the oxidoreductase activities of AtVKOR were directly related to the autotrophic photosynthetic growth and PSII activity of Arabidopsis thaliana.