Identification of the amino acid residues and domains in the cysteine‐rich protein of Chinese wheat mosaic virus that are important for RNA silencing suppression and subcellular localization

Cysteine‐rich proteins (CRPs) encoded by some plant viruses in diverse genera function as RNA silencing suppressors. Within the N‐terminal portion of CRPs encoded by furoviruses, there are six conserved cysteine residues and a Cys–Gly–X–X–His motif (Cys, cysteine; Gly, glycine; His, histidine; X, any amino acid residue) with unknown function. The central domains contain coiled‐coil heptad amino acid repeats that usually mediate protein dimerization. Here, we present evidence that the conserved cysteine residues and Cys–Gly–X–X–His motif in the CRP of Chinese wheat mosaic virus (CWMV) are critical for protein stability and silencing suppression activity. Mutation of a leucine residue in the third coiled‐coil heptad impaired CWMV CRP activity for suppression of local silencing, but not for the promotion of cell‐to‐cell movement of Potato virus X (PVX). In planta and in vitro analysis of wild‐type and mutant proteins indicated that the ability of the CRP to self‐interact was correlated with its suppression activity. Deletion of up to 40 amino acids at the C‐terminus did not abolish suppression activity, but disrupted the association of CRP with endoplasmic reticulum (ER), and reduced its activity in the enhancement of PVX symptom severity. Interestingly, a short region in the C‐terminal domain, predicted to form an amphipathic α‐helical structure, was responsible for the association of CWMV CRP with ER. Overall, our results demonstrate that the N‐terminal and central regions are the functional domains for suppression activity, whereas the C‐terminal region primarily functions to target CWMV CRP to the ER.     

Overexpression of a soybean nuclear localized type–III DnaJ domain‐containing HSP40 reveals its roles in cell death and disease resistance

Heat‐shock proteins such as HSP70 and HSP90 are important molecular chaperones that play critical roles in biotic and abiotic stress responses; however, the involvement of their co‐chaperones in stress biology remains largely uninvestigated. In a screen for candidate genes stimulating cell death in Glycine max (soybean), we transiently overexpressed full‐length cDNAs of soybean genes that are highly induced during soybean rust infection in Nicotiana benthamiana leaves. Overexpression of a type‐III DnaJ domain‐containing HSP40 (GmHSP40.1), a co‐chaperone of HSP70, caused hypersensitive response (HR)‐like cell death. The HR‐like cell death was dependent on MAPKKKα and WIPK, because silencing each of these genes suppressed the HR. Consistent with the presence of a nuclear localization signal (NLS) motif within the GmHSP40.1 coding sequence, GFP‐GmHSP40.1 was exclusively present in nuclear bodies or speckles. Nuclear localization of GmHSP40.1 was necessary for its function, because deletion of the NLS or addition of a nuclear export signal abolished its HR‐inducing ability. GmHSP40.1 co‐localized with HcRed‐SE, a protein involved in pri‐miRNA processing, which has been shown to be co‐localized with SR33‐YFP, a protein involved in pre‐mRNA splicing, suggesting a possible role for GmHSP40.1 in mRNA splicing or miRNA processing, and a link between these processes and cell death. Silencing GmHSP40.1 enhanced the susceptibility of soybean plants to Soybean mosaic virus, confirming its positive role in pathogen defense. Together, the results demonstrate a critical role of a nuclear‐localized DnaJ domain‐containing GmHSP40.1 in cell death and disease resistance in soybean.     

Mass spectrometry unravels disulfide bond formation as the mechanism that activates a molecular chaperone

The heat shock protein Hsp33 is a very potent molecular chaperone with a distinctive mode of functional regulation; its activity is redox-regulated. In its reduced form all six cysteinyl residues of Hsp33 are present as thiols, and Hsp33 displays no folding helper activity. Exposure of Hsp33 to oxidizing conditions like H(2)O(2), however, rapidly converts Hsp33 into an efficient molecular chaperone. Activated Hsp33 binds tightly to refolding intermediates of chemically denatured luciferase and suppresses efficiently their aggregation in vitro. Matrix-assisted laser desorption/ionization-mass spectrometry peptide mapping in combination with in vitro and on target protein chemical modification showed that this activation process of Hsp33 is accompanied by the formation of two intramolecular disulfide bonds within Hsp33: Cys(232)-S-S-Cys(234) and Cys(265)-S-S-Cys(268). Cys(141), although not involved in disulfide bond formation, was found highly reactive toward chemical modifications. In contrast, Cys(239) is readily accessible under reducing conditions but becomes poorly accessible though still reduced when Hsp33 is in its active state. This indicates a significant conformational change during the activation process of Hsp33. Mass spectrometry, thus, unraveled a novel molecular mechanism by which alteration of the disulfide bond structure, as a result of changes in the cellular redox potential, results in the activation of a molecular chaperone.     

Utilization of host‐derived cysteine‐containing peptides overcomes the restricted sulphur metabolism of Campylobacter jejuni

The non‐glycolytic food‐borne pathogen Campylobacter jejuni successfully colonizes the intestine of various hosts in spite of its restricted metabolic properties. While several amino acids are known to be used by C. jejuni as energy sources, none of these have been found to be essential for growth. Here we demonstrated through phenotype microarray analysis that cysteine utilization increases the metabolic activity of C. jejuni. Furthermore, cysteine was crucial for its growth as C. jejuni was unable to synthesize it from sulphate or methionine. Our study showed that C. jejuni compensates this limited anabolic capacity by utilizing sulphide, thiosulphate, glutathione and the dipeptides γGlu–Cys, Cys–Gly and Gly–Cys as sulphur sources and cysteine precursors. A panel of C. jejuni mutants in putative peptidases and peptide transporters were generated and tested for their participation in the catabolism of the cysteine‐containing peptides, and the predicted transporter protein CJJ81176_0236 was discovered to facilitate the growth with the dipeptide Cys–Gly, Ile–Arg and Ile–Trp. It was named Campylobacter peptide transporter A (CptA) and is the first representative of the oligopeptide transporter OPT family demonstrated to participate in the glutathione‐derivative Cys–Gly catabolism in prokaryotes. Our study provides new insights into how host‐ and microbiota‐derived substrates like sulphide, thiosulphate and short peptides are used by C. jejuni to compensate its restricted metabolic capacities.     

Specific protein homeostatic functions of small heat‐shock proteins increase lifespan

During aging, oxidized, misfolded, and aggregated proteins accumulate in cells, while the capacity to deal with protein damage declines severely. To cope with the toxicity of damaged proteins, cells rely on protein quality control networks, in particular proteins belonging to the family of heat‐shock proteins (HSPs). As safeguards of the cellular proteome, HSPs assist in protein folding and prevent accumulation of damaged, misfolded proteins. Here, we compared the capacity of all Drosophila melanogaster small HSP family members for their ability to assist in refolding stress‐denatured substrates and/or to prevent aggregation of disease‐associated misfolded proteins. We identified CG14207 as a novel and potent small HSP member that exclusively assisted in HSP70‐dependent refolding of stress‐denatured proteins. Furthermore, we report that HSP67BC, which has no role in protein refolding, was the most effective small HSP preventing toxic protein aggregation in an HSP70‐independent manner. Importantly, overexpression of both CG14207 and HSP67BC in Drosophila leads to a mild increase in lifespan, demonstrating that increased levels of functionally diverse small HSPs can promote longevity in vivo.     

Activity of the tetrapyrrole regulator CrtJ is controlled by oxidation of a redox active cysteine located in the DNA binding domain

CrtJ from Rhodobacter capsulatus is a regulator of genes involved in the biosynthesis of haem, bacteriochlorophyll, carotenoids as well as structural proteins of the light harvesting‐II complex. Fluorescence anisotropy‐based DNA‐binding analysis demonstrates that oxidized CrtJ exhibits ～ 20‐fold increase in binding affinity over that of reduced CrtJ. Liquid chromatography electrospray tandem ionization mass spectrometric analysis using DAz‐2, a sulfenic acid (–SOH)‐specific probe, demonstrates that exposure of CrtJ to oxygen or to hydrogen peroxide leads to significant accumulation of a sulfenic acid derivative of Cys420 which is located in the helix–turn–helix (HTH) motif. In vivo labelling with 4‐(3‐azidopropyl)cyclohexane‐1,3‐dione (DAz‐2) shows that Cys420 also forms a sulfenic acid modification in vivo when cells are exposed to oxygen. Moreover, a Cys420 to Ala mutation leads to a ～ 60‐fold reduction of DNA binding activity while a Cys to Ser substitution at position 420 that mimics a cysteine sulfenic acid results in a ～ 4‐fold increase in DNA binding activity. These results provide the first example where sulfenic acid oxidation of a cysteine in a HTH‐motif leads to differential effects on gene expression.     

Application of quantitative immunoprecipitation combined with knockdown and cross‐linking to Chlamydomonas reveals the presence of vesicle‐inducing protein in plastids 1 in a common complex with chloroplast HSP90C

Knowledge of the interaction partners of a protein of interest may provide important information on its function. Common to currently available tools for the identification of protein–protein interactions, however, is their high rates of false positives. Only recently an assay was reported that allowed for the unequivocal identification of protein–protein interactions in mammalian cells in a single experiment. This assay, termed quantitative immunoprecipitation combined with knockdown (QUICK), combines RNAi, stable isotope labeling with amino acids in cell culture, immunoprecipitation, and quantitative MS. We are using the unicellular green alga Chlamydomonas reinhardtii to understand the roles of chaperones in chloroplast biogenesis. The goal of this work was to apply QUICK to Chlamydomonas for the identification of novel interaction partners of vesicle‐inducing protein in plastids 1 (VIPP1), a protein required for the biosynthesis/maintenance of thylakoid membranes and known substrate of chloroplast HSP70B. We report here a robust QUICK protocol for Chlamydomonas that has been improved (i) by introducing a cross‐linking step (‐X) to improve protein complex stability and (ii) by including a control for the correction of unequal immunoprecipitation and/or labeling efficiencies. Using QUICK and cross‐linking we could verify that HSP70B and CGE1 form a complex with VIPP1 and could also demonstrate that chloroplast HSP90C is part of this complex. Moreover, we could show that the chaperones interact with VIPP1 also in membrane fractions.     

The small heat shock protein 20 RSI2 interacts with and is required for stability and function of tomato resistance protein I‐2

Race‐specific disease resistance in plants depends on the presence of resistance (R) genes. Most R genes encode NB‐ARC‐LRR proteins that carry a C‐terminal leucine‐rich repeat (LRR). Of the few proteins found to interact with the LRR domain, most have proposed (co)chaperone activity. Here, we report the identification of RSI2 (Required for Stability of I‐2) as a protein that interacts with the LRR domain of the tomato R protein I‐2. RSI2 belongs to the family of small heat shock proteins (sHSPs or HSP20s). HSP20s are ATP‐independent chaperones that form oligomeric complexes with client proteins to prevent unfolding and subsequent aggregation. Silencing of RSI2‐related HSP20s in Nicotiana benthamiana compromised the hypersensitive response that is normally induced by auto‐active variants of I‐2 and Mi‐1, a second tomato R protein. As many HSP20s have chaperone properties, the involvement of RSI2 and other R protein (co)chaperones in I‐2 and Mi‐1 protein stability was examined. RSI2 silencing compromised the accumulation of full‐length I‐2 in planta, but did not affect Mi‐1 levels. Silencing of heat shock protein 90 (HSP90) and SGT1 led to an almost complete loss of full‐length I‐2 accumulation and a reduction in Mi‐1 protein levels. In contrast to SGT1 and HSP90, RSI2 silencing led to accumulation of I‐2 breakdown products. This difference suggests that RSI2 and HSP90/SGT1 chaperone the I‐2 protein using different molecular mechanisms. We conclude that I‐2 protein function requires RSI2, either through direct interaction with, and stabilization of I‐2 protein or by affecting signalling components involved in initiation of the hypersensitive response.     

Structure of the Mycobacterium tuberculosis type VII secretion system chaperone EspG5 in complex with PE25–PPE41 dimer

The growth or virulence of Mycobacterium tuberculosis bacilli depends on homologous type VII secretion systems, ESX‐1, ESX‐3 and ESX‐5, which export a number of protein effectors across membranes to the bacterial surface and environment. PE and PPE proteins represent two large families of highly polymorphic proteins that are secreted by these ESX systems. Recently, it was shown that these proteins require system‐specific cytoplasmic chaperones for secretion. Here, we report the crystal structure of M. tuberculosis ESX‐5‐secreted PE25–PPE41 heterodimer in complex with the cytoplasmic chaperone EspG₅. EspG₅ represents a novel fold that is unrelated to previously characterized secretion chaperones. Functional analysis of the EspG₅‐binding region uncovered a hydrophobic patch on PPE41 that promotes dimer aggregation, and the chaperone effectively abolishes this process. We show that PPE41 contains a characteristic chaperone‐binding sequence, the hh motif, which is highly conserved among ESX‐1‐, ESX‐3‐ and ESX‐5‐specific PPE proteins. Disrupting the interaction between EspG₅ and three different PPE target proteins by introducing different point mutations generally affected protein secretion. We further demonstrate that the EspG₅ chaperone plays an important role in the ESX secretion mechanism by keeping aggregation‐prone PE–PPE proteins in their soluble state.     

Generation of a phage‐display library of single‐domain camelid VHH antibodies directed against Chlamydomonas reinhardtii antigens,and characterization of VHHs binding cell‐surface antigens

Single‐domain antibodies (sdAbs) are powerful tools for the detection, quantification, purification and subcellular localization of proteins of interest in biological research. We have generated camelid (Lama pacos) heavy chain‐only variable V_H domain (V_HH) libraries against antigens in total cell lysates from Chlamydomonas reinhardtii. The sdAbs in the sera from immunized animals and V_HH antibody domains isolated from the library show specificity to C. reinhardtii and lack of reactivity to antigens from four other algae: Chlorella variabilis, Coccomyxa subellipsoidea, Nannochloropsis oceanica and Thalassiosira pseudonana. Antibodies were produced against a diverse representation of antigens as evidenced by sera ELISA and protein‐blot analyses. A phage‐display library consisting of the V_HH region contained at least 10⁶ individual transformants, and thus should represent a wide range of C. reinhardtii antigens. The utility of the phage library was demonstrated by using live C. reinhardtii cells to pan for V_HH clones with specific recognition of cell‐surface epitopes. The lead candidate V_HH clones (designated B11 and H10) bound to C. reinhardtii with EC₅₀ values ≤0.5 nm . Treatment of cells with V_HH B11 fused to the mCherry or green fluorescent proteins allowed brilliant and specific staining of the C. reinhardtii cell wall and analysis of cell‐wall genesis during cell division. Such high‐complexity V_HH antibody libraries for algae will be valuable tools for algal researchers and biotechnologists.     

Cysteine inhibits amyloid fibrillation of lysozyme and directs the formation of small worm‐like aggregates through non‐covalent interactions

In this article, we discuss the effects of amino acids on amyloid aggregation of lysozyme. l ‐cysteine (Cys) dramatically inhibited fibrillation of lysozyme, whereas other amino acids (including l ‐arginine) did not. In the presence of Cys, the aggregation pathway of lysozyme shifted from fibrillation to the formation of the small worm‐like aggregates with unfolding. The interaction between Cys and lysozyme was observed to be non‐covalent, suggesting that the thiophilic interaction between the thiol group on the side chain of Cys and the core sequence of lysozyme significantly contributes to the inhibition of amyloid aggregation. These findings provide a new basis for the design of a biocompatible additive to prevent amyloid fibrillation. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:470–478, 2014     

Exploiting algal NADPH oxidase for biophotovoltaic energy

Photosynthetic microbes exhibit light‐dependent electron export across the cell membrane, which can generate electricity in biological photovoltaic (BPV) devices. How electrons are exported remains to be determined; the identification of mechanisms would help selection or generation of photosynthetic microbes capable of enhanced electrical output. We show that plasma membrane NADPH oxidase activity is a significant component of light‐dependent generation of electricity by the unicellular green alga Chlamydomonas reinhardtii. NADPH oxidases export electrons across the plasma membrane to form superoxide anion from oxygen. The C. reinhardtii mutant lacking the NADPH oxidase encoded by RBO1 is impaired in both extracellular superoxide anion production and current generation in a BPV device. Complementation with the wild‐type gene restores both capacities, demonstrating the role of the enzyme in electron export. Monitoring light‐dependent extracellular superoxide production with a colorimetric assay is shown to be an effective way of screening for electrogenic potential of candidate algal strains. The results show that algal NADPH oxidases are important for superoxide anion production and open avenues for optimizing the biological component of these devices.     

Interaction of ATP with a Small Heat Shock Protein from Mycobacterium leprae: Effect on Its Structure and Function

Adenosine-5’-triphosphate (ATP) is an important phosphate metabolite abundantly found in Mycobacterium leprae bacilli. This pathogen does not derive ATP from its host but has its own mechanism for the generation of ATP. Interestingly, this molecule as well as several antigenic proteins act as bio-markers for the detection of leprosy. One such bio-marker is the 18 kDa antigen. This 18 kDa antigen is a small heat shock protein (HSP18) whose molecular chaperone function is believed to help in the growth and survival of the pathogen. But, no evidences of interaction of ATP with HSP18 and its effect on the structure and chaperone function of HSP18 are available in the literature. Here, we report for the first time evidences of “HSP18-ATP” interaction and its consequences on the structure and chaperone function of HSP18. TNP-ATP binding experiment and surface plasmon resonance measurement showed that HSP18 interacts with ATP with a sub-micromolar binding affinity. Comparative sequence alignment between M. leprae HSP18 and αB-crystallin identified the sequence 49KADSLDIDIE58 of HSP18 as the Walker-B ATP binding motif. Molecular docking studies revealed that β4-β8 groove/strands as an ATP interactive region in M. leprae HSP18. ATP perturbs the tertiary structure of HSP18 mildly and makes it less susceptible towards tryptic cleavage. ATP triggers exposure of additional hydrophobic patches at the surface of HSP18 and induces more stability against chemical and thermal denaturation. In vitro aggregation and thermal inactivation assays clearly revealed that ATP enhances the chaperone function of HSP18. Our studies also revealed that the alteration in the chaperone function of HSP18 is reversible and is independent of ATP hydrolysis. As the availability and binding of ATP to HSP18 regulates its chaperone function, this functional inflection may play an important role in the survival of M. leprae in hosts.     

N‐ACYL HOMOSERINE LACTONe LACTONASE,AiiA, INACTIVATION OF QUORUM‐SENSING AGONISTS PRODUCED BY CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA) AND CHARACTERIZATION OF aiiA TRANSGENIC ALGAE1

Eukaryotes such as plants and the unicellular green alga Chlamydomonas reinhardtii P. A. Dang. produce and secrete compounds that mimic N‐acyl homoserine lactone (AHL) bacterial quorum‐sensing (QS) signals and alter QS‐regulated gene expression in the associated bacteria. Here, we show that the set of C. reinhardtii signal‐mimic compounds that activate the CepR AHL receptor of Burkholderia cepacia are susceptible to inactivation by AiiA, an AHL lactonase enzyme of Bacillus. Inactivation of these algal mimics by AiiA suggests that the CepR‐stimulatory class of mimics produced by C. reinhardtii may have a conserved lactone ring structure in common with AHL QS signals. To examine the role of AHL mimic compounds in the interactions of C. reinhardtii with bacteria, the aiiA gene codon optimized for Chlamydomonas was generated for the expression of AiiA as a chimeric fusion with cyan fluorescent protein (AimC). Culture filtrates of transgenic strains expressing the fusion protein AimC had significantly reduced levels of CepR signal‐mimic activities. When parental and transgenic algae were cultured with a natural pond water bacterial community, a morphologically distinct, AHL‐producing isolate of Aeromonas veronii was observed to colonize the transgenic algal cultures and form biofilms more readily than the parental algal cultures, indicating that secretion of the CepR signal mimics by the alga can significantly affect its interactions with bacteria it encounters in natural environments. The parental alga was also able to sequester and/or destroy AHLs in its growth media to further disrupt or manipulate bacterial QS.     

The role of the cysteine residue in the chaperone and anti‐apoptotic functions of human Hsp27

The small heat shock protein Hsp27 is a molecular chaperone and an anti‐apoptotic protein. Human Hsp27 has one cysteine residue at position 137. We investigated the role of this cysteine residue in the chaperone and anti‐apoptotic functions of Hsp27 by mutating the cysteine residue to an alanine (Hsp27_C137A) and comparing it to wild‐type protein (Hsp27_WT). Both proteins were multi‐subunit oligomers, but subunits of Hsp27_WT were disulfide‐linked unlike those of Hsp27_C137A, which were monomeric. Hsp27_C137A was indistinguishable from Hsp27_WT with regard to its secondary structure, surface hydrophobicity, oligomeric size and chaperone function. S‐thiolation and reductive methylation of the cysteine residue had no apparent effect on the chaperone function of Hsp27_WT. The anti‐apoptotic function of Hsp27_C137A and Hsp27_WT was studied by overexpressing them in CHO cells. No difference in the caspase‐3 or ‐9 activity was observed in staurosporine‐treated cells. The rate of apoptosis between Hsp27_C137A and Hsp27_WT overexpressing cells was similar whether the cells were treated with staurosporine or etoposide. However, the mutant protein was less protective relative to the wild‐type protein in preventing caspase‐3 and caspase‐9 activation and apoptosis induced by 1 mM H₂O₂ in CHO and HeLa cells. These data demonstrate that in human Hsp27, disulfide formation by the lone cysteine does not affect its chaperone function and anti‐apoptotic function against chemical toxicants. However, oxidation of the lone cysteine in Hsp27 might at least partially affect the anti‐apoptotic function against oxidative stress. J. Cell. Biochem. 110: 408–419, 2010. © 2010 Wiley‐Liss, Inc.