Haloferax volcanii archaeosortase is required for motility,mating, and C‐terminal processing of the S‐layer glycoprotein

Cell surfaces are decorated by a variety of proteins that facilitate interactions with their environments and support cell stability. These secreted proteins are anchored to the cell by mechanisms that are diverse, and, in archaea, poorly understood. Recently published in silico data suggest that in some species a subset of secreted euryarchaeal proteins, which includes the S‐layer glycoprotein, is processed and covalently linked to the cell membrane by enzymes referred to as archaeosortases. In silico work led to the proposal that an independent, sortase‐like system for proteolysis‐coupled, carboxy‐terminal lipid modification exists in bacteria (exosortase) and archaea (archaeosortase). Here, we provide the first in vivo characterization of an archaeosortase in the haloarchaeal model organism Haloferax volcanii. Deletion of the artA gene (HVO_0915) resulted in multiple biological phenotypes: (a) poor growth, especially under low‐salt conditions, (b) alterations in cell shape and the S‐layer, (c) impaired motility, suppressors of which still exhibit poor growth, and (d) impaired conjugation. We studied one of the ArtA substrates, the S‐layer glycoprotein, using detailed proteomic analysis. While the carboxy‐terminal region of S‐layer glycoproteins, consisting of a putative threonine‐rich O‐glycosylated region followed by a hydrophobic transmembrane helix, has been notoriously resistant to any proteomic peptide identification, we were able to identify two overlapping peptides from the transmembrane domain present in the ΔartA strain but not in the wild‐type strain. This clearly shows that ArtA is involved in carboxy‐terminal post‐translational processing of the S‐layer glycoprotein. As it is known from previous studies that a lipid is covalently attached to the carboxy‐terminal region of the S‐layer glycoprotein, our data strongly support the conclusion that archaeosortase functions analogously to sortase, mediating proteolysis‐coupled, covalent cell surface attachment.     

Two guard cell mitogen‐activated protein kinases,MPK9 and MPK12, function in methyl jasmonate‐induced stomatal closure in Arabidopsis thaliana

Methyl jasmonate (MeJA) and abscisic acid (ABA) signalling cascades share several signalling components in guard cells. We previously showed that two guard cell‐preferential mitogen‐activated protein kinases (MAPKs), MPK9 and MPK12, positively regulate ABA signalling in Arabidopsis thaliana. In this study, we examined whether these two MAP kinases function in MeJA signalling using genetic mutants for MPK9 and MPK12 combined with a pharmacological approach. MeJA induced stomatal closure in mpk9‐1 and mpk12‐1 single mutants as well as wild‐type plants, but not in mpk9‐1 mpk12‐1 double mutants. Consistently, the MAPKK inhibitor PD98059 inhibited the MeJA‐induced stomatal closure in wild‐type plants. MeJA elicited reactive oxygen species (ROS) production and cytosolic alkalisation in guard cells of the mpk9‐1, mpk12‐1 and mpk9‐1 mpk12‐1 mutants, as well in wild‐type plants. Furthermore, MeJA triggered elevation of cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) in the mpk9‐1 mpk12‐1 double mutant as well as wild‐type plants. Activation of S‐type anion channels by MeJA was impaired in mpk9‐1 mpk12‐1. Together, these results indicate that MPK9 and MPK12 function upstream of S‐type anion channel activation and downstream of ROS production, cytosolic alkalisation and [Ca²⁺]_cyt elevation in guard cell MeJA signalling, suggesting that MPK9 and MPK12 are key regulators mediating both ABA and MeJA signalling in guard cells.     

NaTrxh is an essential protein for pollen rejection in Nicotiana by increasing S‐RNase activity

In self‐incompatible Solanaceae, the pistil protein S‐RNase contributes to S‐specific pollen rejection in conspecific crosses, as well as to rejecting pollen from foreign species or whole clades. However, S‐RNase alone is not sufficient for either type of pollen rejection. We describe a thioredoxin (Trx) type h from Nicotiana alata, NaTrxh, which interacts with and reduces S‐RNase in vitro. Here, we show that expressing a redox‐inactive mutant, NaTrxh_SS, suppresses both S‐specific pollen rejection and rejection of pollen from Nicotiana plumbaginifolia. Biochemical experiments provide evidence that NaTrxh specifically reduces the Cys₁₅₅‐Cys₁₈₅ disulphide bond of S_C10‐Rnase, resulting in a significant increase of its ribonuclease activity. This reduction and increase in S‐RNase activity by NaTrxh helps to explain why S‐RNase alone could be insufficient for pollen rejection.     

FLOURY ENDOSPERM11‐2 encodes plastid HSP70‐2 involved with the temperature‐dependent chalkiness of rice (Oryza sativa L.) grains

The frequent occurrence of chalky rice (Oryza sativa L.) grains becomes a serious problem as a result of climate change. The molecular mechanism underlying chalkiness is largely unknown, however. In this study, the temperature‐sensitive floury endosperm11‐2 (flo11‐2) mutant was isolated from ion beam‐irradiated rice of 1116 lines. The flo11‐2 mutant showed significantly higher chalkiness than the wild type grown under a mean temperature of 28°C, but similar levels of chalkiness to the wild type grown under a mean temperature of 24°C. Whole‐exome sequencing of the flo11‐2 mutant showed three causal gene candidates, including Os12g0244100, which encodes the plastid‐localized 70‐kDa heat shock protein 2 (cpHSP70‐2). The cpHSP70‐2 of the flo11‐2 mutant has an amino acid substitution on the 259th aspartic acid with valine (D259V) in the conserved Motif 5 of the ATPase domain. Transgenic flo11‐2 mutants that express the wild‐type cpHSP70‐2 showed significantly lower chalkiness than the flo11‐2 mutant. Moreover, the accumulation level of cpHSP70‐2 was negatively correlated with the chalky ratio, indicating that cpHSP70‐2 is a causal gene for the chalkiness of the flo11‐2 mutant. The intrinsic ATPase activity of recombinant cpHSP70‐2 was lower by 23% at V_max for the flo11‐2 mutant than for the wild type. The growth of DnaK‐defective Escherichia coli cells complemented with DnaK with the D201V mutation (equivalent to the D259V mutation) was severely reduced at 37°C, but not in the wild‐type DnaK. The results indicate that the lowered cpHSP70‐2 function is involved with the chalkiness of the flo11‐2 mutant.     

Expression and characterization of Flavikunin: A Kunitz‐type serine protease inhibitor identified in the venom gland cDNA library of Bungarus flaviceps

Trancriptomic analysis of the venom gland cDNA library of Bungarus flaviceps revealed Kunitz‐type serine protease inhibitor as one of the major venom protein families with three groups A, B, C. One of the group B isoforms named Flavikunin, which lacked an extra cysteine residue involved in disulfide bond formation in β‐bungarotoxin, was synthesized, cloned, and overexpressed in Escherichia coli. To decipher the structure‐function relationship, the P1 residue of Flavikunin, histidine, was mutated to alanine and arginine. Purified wild‐type and mutant Flavikunins were screened against serine proteases‐thrombin, factor Xa, trypsin, chymotrypsin, plasmin, and elastase. The wild‐type and mutant Flavikunin (H?R) inhibited plasmin with an IC ₅₀ of 0.48 and 0.35 µM, respectively. The in‐silico study showed that P1 residue of wild‐type and mutant (H?R) Flavikunin interacted with S1′ and S1 site of plasmin, respectively. Thus, histidine at the P1 position was found to be involved in plasmin inhibition with mild anticoagulant activity.     

RpoS and oxidative stress conditions regulate succinyl‐CoA: 3‐ketoacid‐coenzyme A transferase (SCOT) expression in Burkholderia pseudomallei

Burkholderia pseudomallei, a pathogenic gram‐negative bacterium, causes the severe human disease melioidosis. This organism can survive in eukaryotic host cells by escaping reactive oxygen species via the regulation of stress responsive sigma factors, including RpoS. In B. pseudomallei, RpoS has been reported to play a role in the oxidative stress response through enhanced activity of OxyR and catalase. In this study, the RpoS dependent oxidative stress responsive system was further characterized using comparative proteomic analysis. The proteomic profiles of wild‐type B. pseudomallei following exposure to H₂O₂ and between wild‐type and the rpoS mutant strains were analyzed. Using stringent criteria, 13 oxidative responsive proteins, eight of which are regulated by RpoS, were identified with high confidence. It was observed that ScoA, a subunit of the SCOT enzyme not previously shown to be involved directly in the oxidative stress response, is significantly down‐regulated after hydrogen peroxide treatment. ScoA and ScoB have been predicted to be organized in a single operon using computational methods: in this study it was confirmed by RT‐PCR that these genes are indeed co‐transcribed as a single mRNA. The present study is the first to report a role for RpoS in the down‐regulation of SCOT expression in response to oxidative stress in B. pseudomallei.     

Identification of three potent hydroxyproline O‐galactosyltransferases in Arabidopsis

Arabinogalactan proteins (AGPs) are plant‐specific extracellular glycoproteins implicated in a variety of processes during growth and development. AGP biosynthesis involves O‐galactosylation of hydroxyproline (Hyp) residues followed by a stepwise elongation of the complex sugar chains. However, functionally dominant Hyp O‐galactosyltransferases, such that their disruption produces phenocopies of AGP‐deficient mutants, remain to be identified. Here, we purified and identified three potent Hyp O‐galactosyltransferases, HPGT1, HPGT2 and HPGT3, from Arabidopsis microsomal fractions. Loss‐of‐function analysis indicated that approximately 90% of the endogenous Hyp O‐galactosylation activity is attributable to these three enzymes. AGP14 expressed in the triple mutant migrated much faster on SDS‐PAGE than when expressed in wild‐type, confirming a considerable decrease in levels of glycosylation of AGPs in the mutant. Loss‐of‐function mutant plants exhibited a pleiotropic phenotype of longer lateral roots, longer root hairs, radial expansion of the cells in the root tip, small leaves, shorter inflorescence stems, reduced fertility and shorter siliques. Our findings provide genetic evidence that Hyp‐linked arabinogalactan polysaccharide chains are critical for AGP function and clues to how arabinogalactan moieties of AGPs contribute to cell‐to‐cell communication during plant growth and development.     

A quantitative proteomic analysis of biofilm adaptation by the periodontal pathogen Tannerella forsythia

Tannerella forsythia is a Gram‐negative anaerobe that is one of the most prominent inhabitants of the sub‐gingival plaque biofilm, which is crucial for causing periodontitis. We have used iTRAQ proteomics to identify and quantify alterations in global protein expression of T. forsythia during growth in a biofilm. This is the first proteomic study concentrating on biofilm growth in this key periodontal pathogen, and this study has identified several changes in protein expression. Moreover, we introduce a rigorous statistical method utilising peptide‐level intensities of iTRAQ reporters to determine which proteins are significantly regulated. In total, 348 proteins were identified and quantified with the expression of 44 proteins being significantly altered between biofilm and planktonic cells. We identified proteins from all cell compartments, and highlighted a marked upregulation in the relative abundances of predicted outer membrane proteins in biofilm cells. These included putative transport systems and the T. forsythia S‐layer proteins. These data and our finding that the butyrate production pathway is markedly downregulated in biofilms indicate possible alterations in host interaction capability. We also identified upregulation of putative oxidative stress response proteins, and showed that biofilm cells are 10 to 20 fold more resistant to oxidative stress. This may represent an important adaptation of this organism to prolonged persistence and immune evasion in the oral cavity.     

Sterols of Saccharomyces cerevisiae erg6 Knockout Mutant Expressing the Pneumocystis carinii S‐Adenosylmethionine:Sterol C‐24 Methyltransferase

The AIDS‐associated lung pathogen Pneumocystis is classified as a fungus although Pneumocystis has several distinct features such as the absence of ergosterol, the major sterol of most fungi. The Pneumocystis carinii S‐adenosylmethionine:sterol C24‐methyltransferase (SAM:SMT) enzyme, coded by the erg6 gene, transfers either one or two methyl groups to the C‐24 position of the sterol side chain producing both C₂₈ and C₂₉ 24‐alkylsterols in approximately the same proportions, whereas most fungal SAM:SMT transfer only one methyl group to the side chain. The sterol compositions of wild‐type Sacchromyces cerevisiae, the erg6 knockout mutant (Δerg6), and Δerg6 expressing the P. carinii or the S. cerevisiae erg6 gene were analyzed by a variety of chromatographic and spectroscopic procedures to examine functional complementation in the yeast expression system. Detailed sterol analyses were obtained using high performance liquid chromatography and proton nuclear magnetic resonance spectroscopy (¹H‐NMR). The P. carinii SAM:SMT in the Δerg6 restored its ability to produce the C₂₈ sterol ergosterol as the major sterol, and also resulted in low levels of C₂₉ sterols. This indicates that while the P. carinii SAM:SMT in the yeast Δerg6 cells was able to transfer a second methyl group to the side chain, the action of Δ²⁴⁽²⁸⁾‐sterol reductase (coded by the erg4 gene) in the yeast cells prevented the formation and accumulation of as many C₂₉ sterols as that found in P. carinii.     

Mutation design of a thermophilic Rubisco based on three‐dimensional structure enhances its activity at ambient temperature

Ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco) plays a central role in carbon dioxide fixation on our planet. Rubisco from a hyperthermophilic archaeon Thermococcus kodakarensis (Tk‐Rubisco) shows approximately twenty times the activity of spinach Rubisco at high temperature, but only one‐eighth the activity at ambient temperature. We have tried to improve the activity of Tk‐Rubisco at ambient temperature, and have successfully constructed several mutants which showed higher activities than the wild‐type enzyme both in vitro and in vivo. Here, we designed new Tk‐Rubisco mutants based on its three‐dimensional structure and a sequence comparison of thermophilic and mesophilic plant Rubiscos. Four mutations were introduced to generate new mutants based on this strategy, and one of the four mutants, T289D, showed significantly improved activity compared to that of the wild‐type enzyme. The crystal structure of the Tk‐Rubisco T289D mutant suggested that the increase in activity was due to mechanisms distinct from those involved in the improvement in activity of Tk‐Rubisco SP8, a mutant protein previously reported to show the highest activity at ambient temperature. Combining the mutations of T289D and SP8 successfully generated a mutant protein (SP8‐T289D) with the highest activity to date both in vitro and in vivo. The improvement was particularly pronounced for the in vivo activity of SP8‐T289D when introduced into the mesophilic, photosynthetic bacterium Rhodopseudomonas palustris, which resulted in a strain with nearly two‐fold higher specific growth rates compared to that of a strain harboring the wild‐type enzyme at ambient temperature. Proteins 2016; 84:1339–1346. © 2016 Wiley Periodicals, Inc.     

S‐nitrosylated protein disulfide isomerase contributes to mutant SOD1 aggregates in amyotrophic lateral sclerosis

A major hallmark of mutant superoxide dismutase (SOD1)‐linked familial amyotrophic lateral sclerosis is SOD1‐immunopositive inclusions found within motor neurons. The mechanism by which SOD1 becomes aggregated, however, remains unclear. In this study, we aimed to investigate the role of nitrosative stress and S‐nitrosylation of protein disulfide isomerase (PDI) in the formation of SOD1 aggregates. Our data show that with disease progression inducible nitric oxide synthase (iNOS) was up‐regulated, which generated high levels of nitric oxide (NO) and subsequently induced S‐nitrosylation of PDI in the spinal cord of mutant SOD1 transgenic mice. This was further confirmed by in vitro observation that treating SH‐SY5Y cells with NO donor S‐nitrosocysteine triggered a dose‐dependent formation of S‐nitrosylated PDI. When mutant SOD1 was over‐expressed in SH‐SY5Y cells, the iNOS expression was up‐regulated, and NO generation was consequently increased. Furthermore, both S‐nitrosylation of PDI and the formation of mutant SOD1 aggregates were detected in the cells expressing mutant SOD1^G93A. Blocking NO generation with the NOS inhibitor N‐nitro‐l ‐arginine attenuated the S‐nitrosylation of PDI and inhibited the formation of mutant SOD1 aggregates. We conclude that NO‐mediated S‐nitrosylation of PDI is a contributing factor to the accumulation of mutant SOD1 aggregates in amyotrophic lateral sclerosis.     

A fluorescence‐activated cell sorting‐based strategy for rapid isolation of high‐lipid Chlamydomonas mutants

There is significant interest in farming algae for the direct production of biofuels and valuable lipids. Chlamydomonas reinhardtii is the leading model system for studying lipid metabolism in green algae, but current methods for isolating mutants of this organism with a perturbed lipid content are slow and tedious. Here, we present the Chlamydomonas high‐lipid sorting (CHiLiS) strategy, which enables enrichment of high‐lipid mutants by fluorescence‐activated cell sorting (FACS) of pooled mutants stained with the lipid‐sensitive dye Nile Red. This method only takes 5 weeks from mutagenesis to mutant isolation. We developed a staining protocol that allows quantification of lipid content while preserving cell viability. We improved separation of high‐lipid mutants from the wild type by using each cell's chlorophyll fluorescence as an internal control. We initially demonstrated 20‐fold enrichment of the known high‐lipid mutant sta1 from a mixture of sta1 and wild‐type cells. We then applied CHiLiS to sort thousands of high‐lipid cells from a pool of about 60 000 mutants. Flow cytometry analysis of 24 individual mutants isolated by this approach revealed that about 50% showed a reproducible high‐lipid phenotype. We further characterized nine of the mutants with the highest lipid content by flame ionization detection and mass spectrometry lipidomics. All mutants analyzed had a higher triacylglycerol content and perturbed whole‐cell fatty acid composition. One arbitrarily chosen mutant was evaluated by microscopy, revealing larger lipid droplets than the wild type. The unprecedented throughput of CHiLiS opens the door to a systems‐level understanding of green algal lipid biology by enabling genome‐saturating isolation of mutants in key genes.     

Sinorhizobium meliloti succinylated high‐molecular‐weight succinoglycan and the Medicago truncatula LysM receptor‐like kinase MtLYK10 participate independently in symbiotic infection

The formation of nitrogen‐fixing nodules on legume hosts is a finely tuned process involving many components of both symbiotic partners. Production of the exopolysaccharide succinoglycan by the nitrogen‐fixing bacterium Sinorhizobium meliloti 1021 is needed for an effective symbiosis with Medicago spp., and the succinyl modification to this polysaccharide is critical. However, it is not known when succinoglycan intervenes in the symbiotic process, and it is not known whether the plant lysin‐motif receptor‐like kinase MtLYK10 intervenes in recognition of succinoglycan, as might be inferred from work on the Lotus japonicus MtLYK10 ortholog, LjEPR3. We studied the symbiotic infection phenotypes of S. meliloti mutants deficient in succinoglycan production or producing modified succinoglycan, in wild‐type Medicago truncatula plants and in Mtlyk10 mutant plants. On wild‐type plants, S. meliloti strains producing no succinoglycan or only unsuccinylated succinoglycan still induced nodule primordia and epidermal infections, but further progression of the symbiotic process was blocked. These S. meliloti mutants induced a more severe infection phenotype on Mtlyk10 mutant plants. Nodulation by succinoglycan‐defective strains was achieved by in trans rescue with a Nod factor‐deficient S. meliloti mutant. While the Nod factor‐deficient strain was always more abundant inside nodules, the succinoglycan‐deficient strain was more efficient than the strain producing only unsuccinylated succinoglycan. Together, these data show that succinylated succinoglycan is essential for infection thread formation in M. truncatula, and that MtLYK10 plays an important, but different role in this symbiotic process. These data also suggest that succinoglycan is more important than Nod factors for bacterial survival inside nodules.     

Role of HemF and HemN in the heme biosynthesis of Vibrio vulnificus under S‐adenosylmethionine‐limiting conditions

Vibrio vulnificus contains two coproporphyrinogen III oxidases (CPOs): O₂‐dependent HemF and O₂‐independent HemN. The growth of the hemF mutant HF1 was similar to wild‐type cells at pH 7.5 under 2% O₂ conditions where HemN was active and had a half‐life of 64 min. However, HF1 did not grow when the medium pH decreased to pH 5.0, where oxidative stress affects endogenous S‐adenosylmethionine (SAM) levels. The growth of HF1 was restored not only by elevating the expression of MnSOD but also through the exogenous addition of SAM. For HF1 to grow under these SAM‐limiting conditions, a mutation arose in hemN, encoding HemN_Y74F. Refolding of the denatured enzymes in vitro revealed that the apparent binding affinity of HemN_Y74F for the cofactor SAM1, which coordinates the 4Fe‐4S cluster, was approximately sixfold higher than that of HemN. The K_m of HemN_Y74F for the co‐substrate SAM2, which provides radicals for CPO reactions, was threefold lower than that of HemN. Thus, affinities for both SAM1 and SAM2 were higher with the Y74F mutation. Taken together, when SAM is limiting, HemN is apparently nonfunctional, and heme synthesis is continued by HemF.     

Paradoxical gain‐of‐function mutant of the G‐protein‐coupled receptor PROKR2 promotes early puberty

The human genome encodes ~750 G‐protein‐coupled receptors (GPCRs), including prokineticin receptor 2 (PROKR2) involved in the regulation of sexual maturation. Previously reported pathogenic gain‐of‐function mutations of GPCR genes invariably encoded aberrant receptors with excessive signal transduction activity. Although in vitro assays demonstrated that an artificially created inactive mutant of PROKR2 exerted paradoxical gain‐of‐function effects when co‐transfected with wild‐type proteins, such a phenomenon has not been observed in vivo. Here, we report a heterozygous frameshift mutation of PROKR2 identified in a 3.5‐year‐old girl with central precocious puberty. The mutant mRNA escaped nonsense‐mediated decay and generated a GPCR lacking two transmembrane domains and the carboxyl‐terminal tail. The mutant protein had no in vitro signal transduction activity; however, cells co‐expressing the mutant and wild‐type PROKR2 exhibited markedly exaggerated ligand‐induced Ca²⁺ responses. The results indicate that certain inactive PROKR2 mutants can cause early puberty by enhancing the functional property of coexisting wild‐type proteins. Considering the structural similarity among GPCRs, this paradoxical gain‐of‐function mechanism may underlie various human disorders.     

Improved fruit α‐tocopherol,carotenoid, squalene and phytosterol contents through manipulation of Brassica juncea 3‐HYDROXY‐3‐METHYLGLUTARYL‐COA SYNTHASE1 in transgenic tomato

3‐Hydroxy‐3‐methylglutaryl‐coenzyme A synthase (HMGS) in the mevalonate (MVA) pathway generates isoprenoids including phytosterols. Dietary phytosterols are important because they can lower blood cholesterol levels. Previously, the overexpression of Brassica juncea wild‐type (wt) and mutant (S359A) BjHMGS1 in Arabidopsis up‐regulated several genes in sterol biosynthesis and increased sterol content. Recombinant S359A had earlier displayed a 10‐fold higher in vitro enzyme activity. Furthermore, tobacco HMGS overexpressors (OEs) exhibited improved sterol content, plant growth and seed yield. Increased growth and seed yield in tobacco OE‐S359A over OE‐wtBjHMGS1 coincided with elevations in NtSQS expression and sterol content. Herein, the overexpression of wt and mutant (S359A) BjHMGS1 in a crop plant, tomato (Solanum lycopersicum), caused an accumulation of MVA‐derived squalene and phytosterols, as well as methylerythritol phosphate (MEP)‐derived α‐tocopherol (vitamin E) and carotenoids, which are important to human health as antioxidants. In tomato HMGS‐OE seedlings, genes associated with the biosyntheses of C10, C15 and C20 universal precursors of isoprenoids, phytosterols, brassinosteroids, dolichols, methylerythritol phosphate, carotenoid and vitamin E were up‐regulated. In OE‐S359A tomato fruits, increased squalene and phytosterol contents over OE‐wtBjHMGS1 were attributed to heightened SlHMGR2, SlFPS1, SlSQS and SlCYP710A11 expression. In both tomato OE‐wtBjHMGS1 and OE‐S359A fruits, the up‐regulation of SlGPS and SlGGPPS1 in the MEP pathway that led to α‐tocopherol and carotenoid accumulation indicated cross‐talk between the MVA and MEP pathways. Taken together, the manipulation of BjHMGS1 represents a promising strategy to simultaneously elevate health‐promoting squalene, phytosterols, α‐tocopherol and carotenoids in tomato, an edible fruit.     

The endoplasmic reticulum‐associated protein,OS‐9, behaves as a lectin in targeting the immature calcium‐sensing receptor

The mechanisms responsible for the processing and quality control of the calcium‐sensing receptor (CaSR) in the endoplasmic reticulum (ER) are largely unknown. In a yeast two‐hybrid screen of the CaSR C‐terminal tail (residues 865–1078), we identified osteosarcoma‐9 (OS‐9) protein as a binding partner. OS‐9 is an ER‐resident lectin that targets misfolded glycoproteins to the ER‐associated degradation (ERAD) pathway through recognition of specific N‐glycans by its mannose‐6‐phosphate receptor homology (MRH) domain. We show by confocal microscopy that the CaSR and OS‐9 co‐localize in the ER in COS‐1 cells. In immunoprecipitation studies with co‐expressed OS‐9 and CaSR, OS‐9 specifically bound the immature form of wild‐type CaSR in the ER. OS‐9 also bound the immature forms of a CaSR C‐terminal deletion mutant and a C677A mutant that remains trapped in the ER, although binding to neither mutant was favored over wild‐type receptor. OS‐9 binding to immature CaSR required the MRH domain of OS‐9 indicating that OS‐9 acts as a lectin most likely to target misfolded CaSR to ERAD. Our results also identify two distinct binding interactions between OS‐9 and the CaSR, one involving both C‐terminal domains of the two proteins and the other involving both N‐terminal domains. This suggests the possibility of more than one functional interaction between OS‐9 and the CaSR. When we investigated the functional consequences of altered OS‐9 expression, neither knockdown nor overexpression of OS‐9 was found to have a significant effect on CaSR cell surface expression or CaSR‐mediated ERK1/2 phosphorylation.     

S‐sulfhydration of MEK1 leads to PARP‐1 activation and DNA damage repair

The repair of DNA damage is fundamental to normal cell development and replication. Hydrogen sulfide (H₂S) is a novel gasotransmitter that has been reported to protect cellular aging. Here, we show that H₂S attenuates DNA damage in human endothelial cells and fibroblasts by S‐sulfhydrating MEK1 at cysteine 341, which leads to PARP‐1 activation. H₂S‐induced MEK1 S‐sulfhydration facilitates the translocation of phosphorylated ERK1/2 into nucleus, where it activates PARP‐1 through direct interaction. Mutation of MEK1 cysteine 341 inhibits ERK phosphorylation and PARP‐1 activation. In the presence of H₂S, activated PARP‐1 recruits XRCC1 and DNA ligase III to DNA breaks to mediate DNA damage repair, and cells are protected from senescence.