Background: Membrane-associated UCH-L1 has been proposed as a target for treating neurodegeneration.Results: UCH-L1 association with neuronal membranes does not involve farnesylation. C-terminal truncation causes protein misfolding and neuronal death.Conclusion: The C terminus of UCH-L1 regulates protein aggregation and stability.Significance: The CTTΔ4 mutant provides a model for studying neurotoxic UCH-L1 aggregation events. C) and a membrane-associated form (UCH-L1M). Alzheimer brains show reduced levels of soluble UCH-L1C correlating with the formation of UCH-L1-immunoreactive tau tangles, whereas UCH-L1M has been implicated in α-synuclein dysfunction. Given these reports of divergent roles, we investigated the properties of UCH-L1 membrane association. Surprisingly, our results indicate that UCH-L1 does not partition to the membrane in the cultured cell lines we tested. Furthermore, in primary cultured neurons, a proportion of UCH-L1M does partition to the membrane, but, contrary to a previous report, this does not require farnesylation. Deletion of the four C-terminal residues caused the loss of protein solubility, abrogation of substrate binding, increased cell death, and an abnormal intracellular distribution, consistent with protein dysfunction and aggregation. These data indicate that UCH-L1 is differently processed in neurons compared with clonal cell lines and that farnesylation does not account for the membrane association in neurons.Ubiquitin C-terminal hydrolase L1 (UCH-L1) is a deubiquitinating enzyme that is highly expressed in neurons. A possible role for UCH-L1 in neurodegeneration has been highlighted because of its presence in Lewy bodies associated with Parkinson disease and neurofibrillary tangles observed in Alzheimer disease. UCH-L1 exists in two forms in neurons, a soluble cytoplasmic form (UCH-L1 Ubiquitination is a dynamic, posttranslational modification that is fundamentally important in controlling multiple aspects of neuronal function, including turnover, trafficking, and synaptic plasticity , 2. Indein vivo remain to be determined.Ubiquitin C-terminal hydrolase L1 (UCH-L1) is a highly abundant neuronal deubiquitinase, a cysteine protease that cleaves small peptide adducts from the C terminus of ubiquitin. Despite the fact that UCH-L1 comprises 1–5% of total neuronal protein , the widin vitro, especially in comparison with the more widely expressed UCH-L3, with which it shares 52% sequence homology (UCH-L1 binds ubiquitin with high affinity but possesses poor hydrolase activity homology . Aside fhomology .C) and membrane (UCH-L1M) fractions. In neurons, it has been estimated that 20–50% is membrane-associated  caused the loss of ubiquitin substrate binding, increased neuronal death, altered UCH-L1 distribution within neurons, and removed soluble, cytoplasmic UCH-L1 with a corresponding increase in Triton X-100-insoluble UCH-L1. A proportion of truncated UCH-L1 was still detected in the membrane fraction, but the levels of soluble cytosolic UCH-L1 were significantly decreased. Circular dichroism analysis of WT and a C-terminal truncation mutant (CTTΔ4) revealed that the removal of the last four residues of UCH-L1 caused a minor disruption to the secondary structure but dramatically increased aggregation.These changes are in line with previous reports on the disruption to the tertiary structure and aggregation of UCH-L1 and are consistent with the distribution and soluble properties of UCH-L1 in Alzheimer brains , 15, sugPrimary hippocampal and cortical neuronal cultures were prepared from embryonic day 18 rats exactly as described previously . The N2aAll SDS-PAGE and Western blotting procedures were carried out according to standard protocols. The following primary antibodies were used: mouse anti-UCH-L1 , rabbit anti-GluA1 , rabbit anti-RhoGDI , mouse anti-H-ras , mouse anti-Rac1 , rabbit anti-HA , goat anti-Lamin B , and rabbit anti-PSD95 . All blots were developed using Li-Cor Odyssey imaging software, except for anti-Lamin B, which was developed on film using HRP-conjugated anti-goat . For Li-Cor Odyssey Western blot imaging, the IRDye secondary antibodies  used were 800CW donkey anti-mouse, 800CW donkey anti-rabbit, 680RD donkey anti-mouse, and 680RD donkey anti-rabbit, all 1:10,000. Western blot signal quantification was performed using Li-Cor Odyssey imaging software.Fractions were obtained as described previously .m sucrose, 20 mm HEPES (pH 7.4), 10 mm KCl, 1.5 mm MgCl2, 1 mm EDTA, 1 mm DTT, and protease inhibitors (Roche)), lysed by passing the cell suspension through a 25-gauge needle 12 times using a 1-ml syringe, and then sonicated briefly three times on low power. Lysates were centrifuged at 100,000 × g in a benchtop ultracentrifuge (Beckman) for 1 h at 4 °C. The supernatant was retained as the cytosolic fraction. The pellet was washed three times in subcellular fractionation buffer and resuspended in a standard lysis buffer containing 1% Triton and 0.1% SDS and solubilized on a rotating wheel at 4 °C for 30 min. The suspension was centrifuged at 10,000 × g in a benchtop centrifuge for 10 min at 4 °C. The supernatant was retained as the membrane fraction. The Triton X-100-insoluble fraction was prepared by resuspending the remaining pellet in lysis buffer containing 1% Triton and 0.1% SDS, rotated at 4 °C for 30 min, passed through a 25-gauge needle 12 times using a 1-ml syringe, and then sonicated three times on low power. Protein concentration was determined using a Bradford assay (Bio-Rad) to determine protein concentration compared with a set of BSA standards. Sarkosyl solubility was determined by resuspending the Triton X-100-insoluble fraction in lysis buffer containing 1% N-lauroylsarcosine sodium salt (Sigma), passed through a needle and sonicated as before, rotated at room temperature for 1 h, and ultracentrifuged at 100,000 × g at 4 °C for 45 min. 20 μl of the supernatant was loaded on the gel as the sarkosyl-soluble fraction. The remaining pellet was resuspended in lysis buffer containing 2% SDS, passed through a needle, resonicated, and then rotated on a wheel at room temperature for a further 30 min. 20 μl was loaded on the gel as the sarkosyl-insoluble fraction.Cells were scraped in detergent-free subcellular fractionation buffer  or the farnesyltransferase inhibitor FTI-276  for 48 h prior to undergoing the subcellular fractionation protocol at day The GFP-UCH-L1 human cDNA plasmid was a gift from Prof. Mike Clague (University of Liverpool). His6-TAT-HA-UCH-L1 was a gift from Dr. Ottavio Arancio. HA-UCH-L1 constructs were generated using standard cloning procedures and PCR-based mutagenesis. Sindbis virus expressing HA-UCH-L1 constructs were created using the pSinRep5 plasmid and Ambion virus production tools.m of UbVMECultured cortical neurons were infected with Sindbis virus-expressing HA-UCH-L1 constructs for 24 h prior to lysis. N2a cells were transfected with HA-UCH-L1 constructs at ∼75% confluency using Lipofectamine LTX Plus (Invitrogen) for 40 h prior to lysis. 20 μg of cell lysates was incubated with 2 μin vitro 18 dissociated hippocampal neurons infected with Sindbis virus expressing HA-UCH-L1 constructs for 24 h. Anti-UCH-L1 and anti-HA antibodies were used at 1:500. Fluorophore-conjugated secondary antibodies used were goat anti-mouse-DyLight 488 and goat anti-rabbit DyLight 649 (Jackson ImmunoResearch Laboratories) and Hoechst nuclear stain . Confocal imaging was performed using a ×63 oil immersion objective lens  on a Zeiss LSM 150 META inverted confocal microscope. Maximum projection confocal z stacks were taken using 0.4-μm steps using a 1024 × 1024 pixel resolution and a pinhole ∼1 airy unit and prepared using ImageJ software .For fixed cell confocal imaging, standard 4% PFA fixation and immunostaining protocols were used on day in vitro toxicology assay kit (Sigma). Briefly, day in vitro 18 dissociated cortical neurons cultured in 3-cm dishes were infected with Sindbis virus expressing HA-UCH-L1 constructs for 24 h. 100 μl of conditioned culture medium was taken from each dish. 30 μl of medium was transferred to each well of a 96-well plate. A lactate dehydrogenase assay mixture containing substrate, cofactor, and dye solutions was prepared according to the instructions of the manufacturer, and 60 μl of the mixture was added to each well of a 96-well plate. Following 25 min of incubation, the reaction was quenched using 10 μl of 1 N HCl, and the absorbance at 492 nm and at reference 620 nm was measured using a Tecan program on a plate reader.lactate dehydrogenase levels in conditioned neuronal culture media were assessed using an m in PBS with 1 mm Tris (2-carboxyethyl)phosphine and 0.01% SDS. CD data were collected on a Jasco J810 spectropolarimeter fitted with a Peltier temperature-controlled sample stage. Samples were placed in a 1-mm path length quartz cuvette (Starna), and the scan rate was 50 nm min−1 with a 1-nm interval, a 1-nm bandwidth, and a 1-s response time.Purified TAT-HA-UCH-L1 and TAT-HA-CTTΔ4 were analyzed by circular dichroism spectroscopy at a concentration of 5 μA). Immunoblotting for the cytosolic marker RhoGDI showed that the presence of UCH-L1 in these fractions was not due to cytosolic contamination.Subcellular fractionation of adult rat brain was used to identify the neuronal compartments that contain UCH-L1. As expected, UCH-L1 was most abundant in the cytosolic fractions, but it was also detected in several of the membrane-enriched fractions A. ImmunoB). Importantly, we also confirmed that this protocol preserves the cytosolic/membrane ratio of proteins that partition into both compartments by analyzing Rac1. Rac1 is a partially prenylated protein that is posttranslationally modified by geranylgeranylation (B). UCH-L1 was detected by Western blot in both the cytosolic and membrane fractions (B) with ∼30% UCH-L1M (C), consistent with values reported previously and, potentially, for a role of farnesylation in regulating membrane partitioning  and membrane (GluA1) protein markers B. Importnylation  leading nylation B. UCH-L1ractions B with ∼3 UCH-L1M C, consisitioning , 11.AAX , with the resulting cleavage of the final three residues  compared with cultured cortical neurons (∼32.2%) (D and E). To test whether the proportion of UCH-L1M in neurons is linked to neurite outgrowth, we induced N2a cell differentiation. 24 h of serum starvation produced neuronal-like processes characteristic of differentiation, but the proportion of UCH-L1M was unchanged (F and H). Taken together, our data strongly suggest that membrane association occurs selectively in neurons but is not dependent on differentiation.Farnesylation involves the irreversible posttranslational addition of a 15-carbon lipid tail to the cysteine at the C-terminal end of proteins containing the sequence Cresidues . The memresidues . However(∼32.2%) , D and Enchanged , F and HM to the farnesyltransferase inhibitor FTI-276. Because membrane-association of H-ras is mediated by farnesylation at the C terminus .Several posttranslational modifications that generate membrane association, such as prenylation (farnesylation and geranylgeranylation) and palmitoylation, rely on covalent attachments to cysteine residues. We therefore mutated three cysteine residues in UCH-L1 (3CS) Cys-90, Cys-152, Cys-220) because these side chains are likely accessible for modification in the cytosol. Although Cys-90 is part of the catalytic triad in the active site, Cys-152 can bind lipid products that mediate the cellular response to oxidative stress and inflammation A.B). These data suggest that mutation of any individual cysteine residue does not disrupt UCH-L1 conformation.Mutation of each of the six cysteine residues in HA-tagged UCH-L1 was tested in N2a cells to assess whether UCH-L1 could still bind covalently to a ubiquitin vinyl methyl ester suicide substrate (Boston Biochem), which forms a bond with Cys-90 in the active site. Notably, the C152S and C220S mutants, individually or combined, showed comparable substrate binding compared with the WT, whereas binding was abolished in the C90S mutant B. These C, HA-UCH-L1 bound to UbVME, and ∼25% partitioned into the membrane, consistent with ∼30% of endogenously expressed UCH-L1 (D and E). Therefore, the HA-tagged virally overexpressed UCH-L1 behaves in a similar manner as endogenous protein. Interestingly, ∼20% of the HA-UCH-L1–3CS mutant was also present in the membrane fraction, suggesting that the cysteine modification at Cys-90, Cys-152, or Cys-220 is not involved in membrane partitioning.We next infected cultured cortical neurons with WT or the triple cysteine mutant HA-UCH-L1 (3CS). As shown in Fig. 3C), although the overexpressed HA-UCH-L1-D30K mutant still partitioned into the membrane fraction (∼22%) . Therefore, together with the data from the 3CS mutant, we conclude that membrane association of UCH-L1 is not dependent on an interaction with the ubiquitin-binding region of UCH-L1.Because UCH-L1 has a high affinity for ubiquitin, we tested whether membrane association of UCH-L1 might arise from binding to a protein containing a domain resembling a ubiquitin fold. The Asp-30 site on UCH-L1 is required for ubiquitin binding . The D30Fig. 3M. Our results indicate that I93M partitions into the membrane and retains sufficient overall structure to bind UbVME to the same extent as WT UCH-L1.The I93M UCH-L1 point mutation has been linked to a familial strain of Parkinson disease . InteresA). UCH-L1 has a complex “knotted” backbone structure, and it has been predicted that even minor N- or C-terminal truncations would denature the protein (B). Furthermore, there was no significant change to the proportion of HA-UCH-L1 in the membrane fraction with either the N-terminal truncation (5–223) or the C-terminal truncation  (C).To investigate the possible roles of signaling sequences at the N and C termini in UCH-L1 membrane association, we removed the first four N-terminal amino acids (MQLK) and the final two residues (AA) A. UCH-L1 protein . Our dat protein B. Furthe, 1–221) C.B), suggesting that the protein conformation had been altered. Consistent with this, there was a significant loss of the soluble cytosolic form of UCH-L1, with the majority of the signal being detected in a 1% Triton X-100-insoluble fraction. There was also an increase in the proportion of UCH-L1M in neurons infected with the CTTΔ4 mutant compared with the WT (C and D).Although the CTTΔ2 truncation behaved the same as the WT, an HA-tagged C-terminal truncation , which deleted the final four residues (CKAA) encompassing the proposed site of farnesylation, reduced cell viability. Initially we noticed that UbVME binding was abolished B, suggesh the WT , C and DE).It has been reported that UCH-L1 is enriched in the insoluble protein aggregates, consistent with several types of neurodegenerative disease . To inveFig. 4A). In each case, the protein is approximately one-third α-helical. The most significant difference between the proteins is the marked increase in aggregation of truncated UCH-L1, as shown by the greatly increased scattering evident in the spectrum, especially at lower wavelengths.To explore the possible reasons for the changes in solubility, we analyzed the secondary structure using circular dichroism. The spectra show that the truncated protein has a shallower minimum at 222 nm, indicating a slightly reduced α-helical content compared with the WT A. In eacA). Furthermore, overexpression of this mutant markedly increased neuronal cell death, as measured by lactate dehydrogenase release (B).Confocal microscopy of hippocampal neurons infected with the different UCH-L1 constructs using Sindbis virus revealed significant differences in the distributions of CTTΔ4 compared with the WT. CTTΔ4 displayed a non-uniform, mostly perinuclear staining and was largely absent from the processes A. Furthe release B.M). A previous report proposed that farnesylation mediates membrane association in cell lines  with which UCH-L1 associates has remained unclear because the very high levels of expression of native UCH-L1 in neurons, combined with its relatively uniform distribution, make it difficult to resolve using imaging techniques such as confocal microscopy. Also, unlike the previous report in clonal cell lines  caused a dramatic increase in neurotoxicity, whereas deletion of the final two residues  had no marked effect . The I93M mutation is associated with Parkinson disease and has been reported to decrease UCH-L1 solubility and hydrolytic activity (D).A common theme that emerges from both point mutation and oxidative modification studies of the misfolding and/or aggregation of UCH-L1 is disruption to the central hydrophobic core , 31. AltFig. 5activity , 31. Theactivity . Howeveractivity . Overallactivity D.B). Analogous to this disruption of the packed core structure by prostaglandins, our results indicate that disruption of the extreme C terminus may also have a similar effect and provide a potential mechanism for how UCH-L1 structure and stability may be regulated by other forms of oxidative stress.Cyclopentenone prostaglandins such as 15d-PGJ2 are produced during an immune response or under oxidative stress. 15d-PGJ2 specifically modifies UCH-L1 at C152 site, leading to a loss of protein stability B. Analog2ETL, Ref. Crystal structures of UCH-L1 suggest that Lys-221 might be integral to the tertiary structure by forming intramolecular bonds to hold the three-dimensional shape of the protein (PDB code We therefore propose that exposure of the hydrophobic core causes a toxic gain of function in which misfolded UCH-L1 aberrantly binds to hydrophobic intracellular membranes, and this leads to cell death. The CTTΔ4 mutant we report here represents an extreme form of unfolded UCH-L1. Importantly, we attribute this to a toxic gain of function rather than a loss of hydrolytic activity because C90S and D30K do not have the same effect. Taken together, our findings suggest that modifications or mutations that alter the folding and/or stability of UCH-L1 are neurotoxic. The CTTΔ4 truncation provides a useful new tool for studying how UCH-L1 misfolding and dysfunction lead to protein instability and cell death, which pathways are involved, and what measures can be taken to reduce or prevent toxicity.In conclusion, our results demonstrate that membrane association of UCH-L1 is differentially regulated in different cell types. We investigated a series of possible mechanisms for the membrane association, and our data indicate that, contrary to a previous report, farnesylation and/or other cysteine posttranslational modifications are not responsible for membrane partitioning. Therefore, further work is required to define how UCH-L1 is recruited and retained at membranes selectively in neurons and to elucidate the functional consequences of this membrane association.


Background: The human multiple aminoacyl-tRNA synthetase complex (MSC) has an elongated and multiarmed structure, which might be sensitive to proteases such as calpain in vivo.Results: Partial calpain hydrolysis of MSC components generated specific fragments.Conclusion: Calpain hydrolysis could dissociate the MSC, thereby affecting its function.Significance: Calpain proteolytically degrades MSC components and might regulate the canonical and non-canonical functions of aaRS. in vivo. A yeast two-hybrid screen for proteins interacting with LeuRS, a representative MSC member, identified calpain 2, a calcium-activated neutral cysteine protease. Calpain 2 and calpain 1 could partially hydrolyze most MSC components to generate specific fragments that resembled those reported previously. The cleavage sites of calpain in ArgRS, GlnRS, and p43 were precisely mapped. After cleavage, their N-terminal regions were removed. Sixty-three amino acid residues were removed from the N terminus of ArgRS to form ArgRSΔN63; GlnRS formed GlnRSΔN198, and p43 formed p43ΔN106. GlnRSΔN198 had a much weaker affinity for its substrates, tRNAGln and glutamine. p43ΔN106 was the same as the previously reported p43-derived apoptosis-released factor. The formation of p43ΔN106 by calpain depended on Ca2+ and could be specifically inhibited by calpeptin and by RNAi of the regulatory subunit of calpain in vivo. These results showed, for the first time, that calpain plays an essential role in dissociating the MSC and might regulate the canonical and non-canonical functions of certain components of the MSC.Nine aminoacyl-tRNA synthetases (aaRSs) and three scaffold proteins form a super multiple aminoacyl-tRNA synthetase complex (MSC) in the human cytoplasm. Domains that have been added progressively to MSC components during evolution are linked by unstructured flexible peptides, producing an elongated and multiarmed MSC structure that is easily attacked by proteases  Specifi of tRNA . In addi of tRNA , 3.In the human cytoplasm, a multiple aminoacyl-tRNA synthetase complex (MSC) has been found that comprises 11 polypeptides and harbors the activities of nine aaRSs, bifunctional glutamylprolyl- (GluPro/EP-), isoleucyl- (Ile), leucyl- (Leu), methionyl- (Met), glutaminyl- (Gln), lysyl- (Lys), arginyl- (Arg), and aspartyl- (Asp) tRNA synthetases, and three non-synthetase components, p18, p38, and p43, which are also named AIMP3, AIMP2, and AIMP1, respectively  requires a lower concentration of Ca2+ for activation   is a Caroteases . The actpastatin . Among tpastatin . Calpaincalpain) . Comparecalpain) . Therefocalpain) , 14.in vivo from the MSC, with LeuRS as the bait, we performed a yeast two-hybrid screening on a human cDNA library. We found that calpain 2 interacts with LeuRS. Moreover, calpain cleaved other MSC components, which affected their aminoacylation activity and released their specific fragments with potential new functions. Our results suggested that the calpain family is involved in dissociation of the MSC and might regulate the functions of MSC components and connect their canonical and non-canonical functions.To identify new factors able to release functional fragments l-glutamine, 3×FLAG peptides, and anti-FLAG M2 affinity gel were from Sigma-Aldrich; and l-[3H]Gln was from PerkinElmer Life Sciences. Lipofectamine 2000 reagent, puromycin, and fetal bovine serum (FBS) were obtained from Life Technologies, and the cell culture medium was from Biowest (France). Human erythrocyte calpain 1 and calpeptin , a cell-permeable specific calpain inhibitor, were purchased from Merck; the protease inhibitor mixture was from Roche Applied Science; and the rProtein A/G-agarose was from Abmart Inc. (China). All cell lines were purchased from the cell resource center of the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.Materials for biochemical experiments were reported previously . The Mat6 tag was from BD Biosciences; and the mouse monoclonal antibodies against Myc and FLAG epitope were from Abmart Inc. Rabbit polyclonal antibodies directed against LeuRS, IleRS, EPRS, LysRS, p43, p38, and p18 were prepared by the antibody research center of the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. Rabbit polyclonal antibodies for ArgRS were prepared by Willget Biotech (China). Human full-length LeuRS, ArgRS, human p43ΔN106, unique domain of human IleRS, mouse full-length p38, p18, LysRS, and WHEP domains (domains present in TrpRS (W), HisRS (H), and GluProRS (EP)) of mouse EPRS were used as antigens for rabbit polyclonal antibody preparation. Mouse antibodies were shown to cross-react with the corresponding human proteins. The mouse monoclonal antibody against human GlnRS  was produced by Abiocode, Inc.Antibodies were obtained from different companies. HRP-labeled anti-mouse and anti-rabbit secondary antibodies were purchased from Sigma-Aldrich; the mouse monoclonal antibody against the peptide representing residues 2–27 at the N terminus of calpain 2 was from Santa Cruz Biotechnology, Inc.; rabbit polyclonal antibodies for calpain 1 and the regulatory small subunit CAPNS1 were from Proteintech Group, Inc. (China); the mouse monoclonal antibody against the HisEscherichia coli (BL21) and those for immunoprecipitation or co-immunoprecipitation experiments, protein overexpression, and immunofluorescence analysis in 293T cells were constructed as shown in The plasmids for protein purification in CCACAGAACTCATGAACATTTCAAGAGAATGTTCATGAGTTCTGTGGTTTTTG; reverse primer, AATTCAAAAACCACAGAACTCATGAACATTTCAAGAGAATGTTCATGAGTTCTGTGG. Random (shcontrol) primer sequences were as follows: forward primer, CCGGCAACAAGATGAAGAGCACCAACTTCAAGAGAGTTGGTGCTCTTCATCTTGTTGTTTTTG; reverse primer, AATTCAAAAACAACAAGATGAAGAGCACCAACTTCAAGAGAGTTGGTGCTCTTCATCTTGTTG.For CAPNS1 knockdown, we inserted its shRNA into vector pLKO.1. The shCAPNS1 primer sequences were as follows (the core shRNA sequences for targeting CAPNS1 coding sequence (from 350 to 368 bp) are underlined): forward primer, CCGGE. coli (BL21) according to the method described by Hata et al. (E. coli (BL21)  with CAPNS1ΔN26  by co-expression of their genes in a et al. . The puri (BL21) . PurificThe gene encoding the C-terminal domain of LeuRS (residues 708–1176) was cloned into pGBKT7 and transformed into yeast Y2HGold strain. The transformed strains were mated with Y187 strains transformed with a simplified human cDNA library. After perfect mating, they were plated on selective plates for positive clone screening. The yeast two-hybrid screening was performed approximately according to the protocol supplied by Takara (Japan).m HEPES, pH 7.5, 10 mm potassium acetate, 1.5 mm MgAc2, and 8 μg/ml recombinant calpain 2 with 5 mm Ca2+ or 24 μg/ml calpain 1 with 0.5 mm Ca2+  was carried out at 37 °C for 30 min in a reaction mixture containing 20 m mm Ca2+ . In someA previous report showed that Jurkat cells have considerable calpain 2, and their cell lysates were used as a calpain 2 source to perform the proteolysis reaction toward substrates . Jurkat 7 cells were washed with phosphate-buffered saline (PBS) solution twice and cultured with RPMI 1640 medium plus 0.2% FBS for 48 h. Starved U937 cells and culture medium were collected by centrifugation at 240 × g for 2 min at 4 °C. After rinsing twice with PBS, cell pellets were lysed with 2× SDS loading buffer. Protein in the culture medium was precipitated according to the method of Park et al.  at their C termini were fixed in 4% paraformaldehyde and stained with 4′, 6-diamidino-2-phenylindole (DAPI). After washing, immunofluorescence was analyzed under an Olympus FV1000 microscope.Gln(CUG) was recombined into pTrc99b under the control of a T7 RNA polymerase promoter. In vitro transcription of tRNAGln(CUG) by T7 RNA polymerase was performed similarly to that described previously (Gln(CUG) was 1410 pmol/A260. 3′-End 32P labeling of tRNAGln(CUG) was performed as described previously with minor changes , 0.5 mm DTT, 15 μm ATP, 50 μm tetrasodium pyrophosphate, 0.333 μm [α-32P]ATP, and 10 μmE. coli CCA-adding enzyme for 10 min. 0.7 unit/μl pyrophosphatase was added to the mixture for another 5 min. Phenol/chloroform extraction of [32P]tRNAGln(CUG) was conducted as described previously , and 0.1 mg/ml BSA at 37 °C. Reactions were initiated by adding 20 nm GlnRS or GlnRSΔN198. At various time intervals, aliquots were quenched and precipitated with 5% trichloroacetic acid as described previously , and 10 or 50 mm Gln for GlnRS or GlnRSΔN198, respectively. Cold tRNAGln(CUG) concentrations varied between 0.1 and 1.7 μm for GlnRS and between 1 and 16 μm for GlnRSΔN198. The reaction was initiated by adding 5 nm GlnRS or 10 nm GlnRSΔN198. The mK value of GlnRSΔN198 for Gln was assayed by a similar experiment with 0.1 μm [32P]tRNAGln(CUG) and 15 μm cold tRNAGln(CUG), and the concentration of Gln varied between 0.05 and 1.6 mm. At specific time intervals, aliquots were removed for quenching in 0.5 m sodium acetate, pH 5.2, and ethanol-precipitated. Sample treatment and data collection were performed as described previously , and 0.1 mg/ml BSA at 37 °C. The concentration of [3H]Gln varied between 2.5 and 25 μm. The reaction was initiated by adding 5 nm GlnRS. Sample treatment and data collection were performed as described previously . We used the supernatant of Jurkat extracts (Jurkat S100) containing calpain 2 in vitro to assay hydrolysis of recombinant LeuRS (N terminus His6-tagged) and found that LeuRS was cleaved under Ca2+ stimulation as shown by Western blotting with the His6 antibody (B). We obtained the purified recombinant calpain 2 containing human calpain 2 (large subunit) and CAPNS1ΔN26  by co-expressing their genes to further confirm the digestion of LeuRS (C). The recombinant calpain 2 could cleave casein under Ca2+ stimulation (D), indicating that it had hydrolysis activity, which could be inhibited by calpeptin or EDTA  treatment. The recombinant calpain 2 could also hydrolyze the recombinant LeuRS with a pattern similar to that by Jurkat S100 (E). Consistently, calpeptin or EDTA completely inhibited the hydrolysis reactions of human LeuRS by recombinant calpain 2 (B and E). The data showed that human LeuRS was hydrolyzed by the recombinant calpain 2 indeed.A yeast two-hybrid screen was performed to identify new partners that could interact with the MSC and have potential regulatory functions. Based on our comprehensive biochemical research on LeuRS, human cytosolic LeuRS was chosen as a representative of MSC members, and its C-terminal domain (residues 708–1176) was used as the bait to screen a human cDNA library. 3.4 × 10d system A. We useantibody B. We obtof LeuRS C. The remulation D, indicakat S100 E. Consisalpain 2 , B and EE. coli, except IleRS, MetRS, and EPRS, which had unfavorable expression or solubility (data not shown). In the presence of Ca2+ ions or Ca2+ ions plus calpeptin inhibitor or EDTA, we analyzed hydrolysis of the seven components by recombinant calpain 2. Except for p18, the other proteins  were partially cleaved by the recombinant calpain 2 in a Ca2+-dependent manner as detected by Coomassie Blue staining (A). The proteolysis reactions were also inhibited by calpeptin (A) or EDTA (data not shown). ArgRS (N terminus His6-tagged) displayed a truncated peptide very similar in size to its short form generated by translation reinitiation reported in our laboratory  . Similarly, recombinant calpain 2 cleaved GlnRS  into two fragments (∼23 and ∼70 kDa) as demonstrated by Coomassie Blue staining . The antibody against His6 tag could detect the ∼70-kDa fragment of GlnRS, and the FLAG antibody could detect the ∼23-kDa fragment, showing that the 23-kDa fragment was located in the N-terminal part and the 70-kDa fragment was located in the C-terminal part (B). Compared with prokaryotes, human GlnRS, with a molecular mass of about 95 kDa, has a ∼200-amino acid-long N-terminal extension containing the calpain cleavage site, probably located in the unstructured coil linking the N-terminal appended domain. AspRS (N terminus His6-tagged) was only cleaved to remove a peptide of about 5 kDa ; however, Western blotting against His6 tag did not reveal any proteolytic fragment, indicating that its N-terminal extension, which is involved in the association with the MSC, was removed (B) (6-tagged) into three main truncated LysRSs with various N termini deleted ; Western blotting against the His6 tag did not show any fragments . These data suggested that ArgRS, GlnRS, AspRS, and LysRS were cleaved in their N-terminal extensions and that their intact catalytic cores were retained.We purified seven other components of human MSC by expressing their genes in staining A. The pralpeptin A or EDTAboratory A, lane 2 lane 2) . Westernxtension B. Similastaining A, lane 5nal part B. Comparut 5 kDa A, lane 8 removed B 21). T. TE. col6-taggged) generated an ∼25-kDa fragment after cleavage by recombinant calpain 2 . Western blot analysis with the His6 antibody suggested that its N-terminal half was removed . Similarly, the p38 (N terminus His6-taggged) was cleaved, and the generated ∼23-kDa hydrolytic fragment was not detected in a Western blot using the His6 antibody , suggesting that the N-terminal part of p38 was also removed by calpain2. As auxiliary factors of the MSC, p43 and p38 have similar secondary structures formed by a leucine zipper motif in their N-terminal regions and an endothelial monocyte-activating polypeptide II or GST domain in the C-terminal region for p43 or p38, respectively . Recombinant calpain 2 did not cleave factor p18 (N terminus His6-taggged) as assayed by either Coomassie Blue staining  or Western blotting . As a negative control, the separate editing domain of LeuRS, LeuRS-CP1 (N terminus His6-taggged), was stable against hydrolysis by recombinant calpain 2 .The auxiliary factor p43 , and ArgRS and LeuRS (data not shown) could be co-immunoprecipitated with FLAG-calpain 2, indicating that calpain 2 could interact with and cleave MSC components in vivo.To check the nRS, p43 C, and ArC), whereas it was not hydrolyzed by recombinant calpain 2.Calpain 1 and calpain 2 share ∼60% amino acid identity and have similar tertiary structures and recognition specificities toward substrates . We usedFig. 32+ to stimulate the hydrolytic activity; therefore, we observed that it could also hydrolyze these components, except p38 and p18, without extra Ca2+ addition. The hydrolytic efficiency without Ca2+ addition was in the following estimated order: p43 > LysRS > AspRS > GlnRS > LeuRS/ArgRS > p18/p38 . This result may be the consequence of the transient interaction of calpain 1 with MSC components because this easily activated protease may be rapidly released from these molecules after cleavage. This highly dynamic type of interaction between calpain 1 and its substrates might also explain why calpain 1 was not selected by the yeast two-hybrid screen.In another assay, calpain 1 expressed in 293T cells was subjected to a co-immunoprecipitation experiment as described in the case of calpain 2. However, EPRS, LeuRS, ArgRS, and p43 failed to be co-immunoprecipitated with FLAG-calpain 1, and only GlnRS and LysRS could be weakly co-immunoprecipitated with calpain 1 D. This r2+ and completely inhibited by EDTA , calpains could hydrolyze free ArgRS contaminated in the MSC preparation obtained by immunoprecipitation of the overexpressed ArgRS and generate the short form of ArgRS (A and B). However, when p43 was used as the bait for MSC purification (B), ArgRS was in the complex and was resistant to proteolysis by calpains (C and D). The results indicated that in the MSC ArgRS was resistant to calpain cleavage as was p18. From a recent structural analysis of the ArgRS-GlnRS-p43 complex, this resistance might reflect some steric hindrance of calpain attack that might originate from the N terminus of p43 or the catalytic domain of GlnRS that is in the vicinity of the N terminus of ArgRS in the MSC  and overexpressed it in 293T cells independently. Human MSCs were purified by co-immunoprecipitation with anti-FLAG M2 affinity gel. After SDS-PAGE, we observed that the isolated MSCs contained all components as shown by Coomassie Blue staining . We then by EDTA  or calpe by EDTA . Notably the MSC A, calpaiof ArgRS , A and Bfication B, ArgRS calpains , C and D the MSC .6 tag in 293T cells and co-purified AspRS, MetRS, and EPRS with anti-FLAG affinity gel and subjected them to calpain digestion. After digestion by calpains, MetRS produced a large fragment with a deleted peptide of about 20 kDa, probably its N-terminal GST domain (A and B); AspRS was truncated in its N-terminal helix (A and B); and EPRS produced two major fragments from the N terminus of ∼100 and ∼120 kDa, indicating that the cleavage sites are between the WHEP domains , matching perfectly with the sequence of p43 from residues 107 to 118. From the full-length p43 with a molecular mass of 40 kDa, the result showed that calpains cleaved p43 between Ser106 and Ser107 to form the C-terminal fragment, precisely the same as the previously discovered p43-derived apoptosis-released factor in U937 cells (199TDRRTAKDVVEN210 (A), and the cleavage site was between Glu198 and Thr199. The truncated GlnRS, named GlnRSΔN198, had completely lost its eukaryote-specific N-terminal extension, leaving the intact catalytic core corresponding to bacterial GlnRS  . These results confirmed that the first 63 residues in the N terminus of ArgRS were critical for its incorporation into the MSC. With p43 and GlnRS, we performed a more comprehensive analysis. We found that p43-N106 (residues 1–106) could interact with the other tested MSC components, including EPRS, LeuRS, ArgRS, LysRS, and p38 (C), in agreement with a previous study (D).To explore the effect of calpain cleavage on assembly of the MSC rgRSΔN63 B. These  and p38 C, in agrus study ; howeverus study D.A). Under the same conditions, the aminoacylation activity of full-length GlnRS and GlnRSΔN198 was assayed. The rate of glutaminylation of tRNAGln catalyzed by GlnRSΔN198 was obviously decreased (B). Their kinetic parameters were also measured  of GlnRSΔN198 was caused by an elevation of the mK value.GlnRS was stable after an unusually large deletion by calpain cleavage, and this prompted us to assay the catalytic efficiency of the truncated GlnRS. By alignment of full-length eukaryotic GlnRS and bacterial GlnRS, human GlnRSΔN198 generated here is comparable in size to the bacterial GlnRS A. Under ecreased B. Their measured . CompareC). The full-length GlnRS was mainly located in the cytoplasm with a small amount in the nucleus; however, both GlnRS-N198 and GlnRSΔN198 were accumulated in the nucleus (D). The result suggested that the hydrolysis of GlnRS by calpains had a great effect on its cellular distribution, and these generated fragments might be involved in some nuclear processes, such as signaling or transcription, as observed for TyrRS . To validate that the p43ΔN106 came from calpain cleavage, the gene encoding calpain 1, calpain 2, or regulatory subunit CAPNS1 was overexpressed in 293T cells. P43ΔN106 could be detected when calpain 1, but not calpain 2, was overexpressed probably because the Ca2+ concentration in 293T could not reach the mm concentration for activation of calpain 2 (B). In addition, partial autolysis in the N terminus of calpain 1 was observed (B), which might indicate its activation as described previously  and then secreted into the culture medium . Under serum starvation conditions, we observed that the production (cell lysate) and secretion (culture medium) of p43ΔN106 were completely inhibited by calpeptin. p43 was also reported previously to be secreted with some cytokine function . Forskolin, an activator of calpain proteins, acting through cAMP-Ca2+-calpain cycle signaling . Moreover, calpain 1 or 2 could not be detected in the culture medium, indicating that the cleavage of p43 did not occur after secretion into the starvation medium but occurred earlier in the cytosol . Previous reports have shown that serum starvation could increase cytosolic Ca2+ levels , which consequently induced a decrease in p43ΔN106 formation in U937 cells and total loss of the secretion into the culture medium (F). Unambiguously, this result showed that the p43ΔN106 fragment, which is exactly the same as p43-derived apoptosis-released factor, was formed by calpain cleavage in vivo and indicated that calpains could remodel the MSC and release their fragments with new regulatory properties under specific conditions.To further confirm the t CAPNS1 . Knockdot CAPNS1 E, which e medium F. Unambiin vivo  were slightly higher  and ArgRSΔN72, could play similar roles in the cell despite their different derivation processes (Among the fragments generated by calpains, p43ΔN106 could bind tRNA with higher affinity than native p43 . Mirande2+-calpain-Gsα cycle (–Starvation could stimulate calpain hydrolytic activity, which is necessary for starvation-induced autophagy , 41. Thesα cycle . Considesα cycle –45, the Four major intracellular proteolytic systems exist in organisms, including the ubiquitin-proteasome system, the autophagy-lysosome system, the caspase system, and the calpain family . ActivatH.-Y. L. and E.-D. W. designed the research. H.-Y. L. performed the research. H.-Y. L., X.-L. Z., Z.-R. R., W.-C. S., G. E., and E.-D. W. contributed to data analysis and proper discussion. H.-Y. L. and E.-D. W. wrote the paper.


Escherichia coli produces the iron-chelating compound enterobactin to enable growth under iron-limiting conditions. After biosynthesis, enterobactin is released from the cell. However, the enterobactin export system is not fully understood. Previous studies have suggested that the outer membrane channel TolC is involved in enterobactin export. There are several multidrug efflux transporters belonging to resistance-nodulation-cell division (RND) family that require interaction with TolC to function. Therefore, several RND transporters may be responsible for enterobactin export. In this study, we investigated whether RND transporters are involved in enterobactin export using deletion mutants of multidrug transporters in E. coli. Single deletions of acrB, acrD, mdtABC, acrEF, or mdtEF did not affect the ability of E. coli to excrete enterobactin, whereas deletion of tolC did affect enterobactin export. We found that multiple deletion of acrB, acrD, and mdtABC resulted in a significant decrease in enterobactin export and that plasmids carrying the acrAB, acrD, or mdtABC genes restored the decrease in enterobactin export exhibited by the ΔacrB acrD mdtABC mutant. These results indicate that AcrB, AcrD, and MdtABC are required for the secretion of enterobactin. Multidrug efflux transporters cause serious problems in cancer chemotherapy and in the treatment of bacterial infections. In gram-negative bacteria, transporters belonging to the resistance-nodulation-cell division (RND) family are particularly effective in generating resistance because they form a tripartite complex with periplasmic proteins and an outer membrane protein channel. The RND transporters have wide substrate specificity E. coli, including antibiotics E. coli to expel structurally diverse molecules. In E. coli, AcrB, AcrD, AcrEF, MdtABC, and MdtEF belong to the RND transporters and require TolC to function TolC plays an important role in the excretion of a wide range of molecules in E. coli can produce the catecholate siderophore enterobactin , which is a cyclic triester of 2, 3-dihydroxybenzoylserine (DHBS) Iron is an essential element for many biological processes, such as amino acid and nucleotide synthesis, electron transport, and peroxide reduction ybdA gene product) Salmonella enterica serovar Typhimurium et al. showed that the outer membrane channel TolC is involved in enterobactin export from the periplasm to the culture medium The systems responsible for enterobactin synthesis and uptake are well characterized, as presented above. In contrast, the enterobactin export system is not fully understood. Previously, enterobactin export across the cytoplasmic membrane was shown to be dependent on the major-facilitator transporter EntS  glucose, 0.005% (wt/vol) nicotinic acid, 1% (wt/vol) thiamine before inoculation, and 150 µM 2,2′-dipyridyl. Deferrated casamino acids were prepared using the following methods. Twenty-five ml of 10% (wt/vol) casamino acids were mixed with 0.75 g hydroxyquinoline and 25 ml chloroform. Thereafter, the supernatant was again mixed with 25 ml chloroform and its supernatant was collected as deferrated casamino acids. Aliquots of iron-restricted T medium were inoculated with 0.1% (v/v) of overnight cultures of strains grown in Luria-Bertani (LB) broth  Bacterial strains and plasmids used in this study are listed in tolC, ΔacrB, ΔacrD, ΔmdtABC, ΔmdtEF, ΔacrB acrD, ΔacrB mdtABC, and ΔacrB acrD mdtABC were constructed as previously described acrE-P1 (TTGGGTAAATAACGCGCTTTTGGTTTTTTGAGGAATAGTAGTGTAGGCTGGAGCTGCTTC) and acrF-P2 (AAATAATAAAGGCACCCGAAAGCGCCTTTATGTTTCTGATCATATGAATATCCTCCTTAG) were used to construct the ΔacrEF mutant. The kanamycin resistance gene aph, flanked by Flp recognition sites, was amplified by polymerase chain reaction (PCR) using the primers listed above. The resulting PCR products were used to transform the recipient MG1655 strain harboring the plasmid pKD46, which expresses lambda Red recombinase. The chromosomal structures of the mutated loci were verified by PCR using the primers acrE-F (GTTAAATAAATAATATATATTATTTACCTA) and acrF-R (CGTGAGCACAGCCCGCCAGCAATGCGGTGA), and K-1 (CAGTCATAGCCGAATAGCCT) and K-2 (CGGTGCCCTGAATGAACTGC). To construct ΔacrD mdtABC, ΔacrB acrD mdtABC mdtEF, and ΔacrB acrD mdtABC mdtEF acrEF mutants, the individual deletions were transferred to a fresh isolate of MG1655 by P1 transduction. The cat or aph genes were eliminated using plasmid pCP20, as previously described entS mutant, precise in-frame deletions were generated using crossover PCR. The oligonucleotide primers entS-No (CGCGGATCCAAAGGCAACAATCAATGAGGC) and entS-Ni (CACGCAATAACCTTCACACTCCAAATTTATAACCATTACAATGCCTTGCCATC), plus entS-Ci (GTTATAAATTTGGAGTGTGAAGGTTATTGCGTGTAATGCTTAAAACAGCGCCTTAAGCC), and entS-Co (CGCGTCGACGACGACAAAACGTGGCAGTC) were used. Then the fragment containing the deletion was cloned into the BamHI site of vector pKO3, after which the deletion was introduced into the chromosome by gene replacement, as previously described To construct the drug efflux mutants, gene disruption was performed as described by Datsenko and Wanner acrAB and acrD genes were subcloned from pHSGacrAB and pHSGacrD into vector pTrc99A using BamHI and HindIII, and HindIII and SacI, respectively mdtABC-EcoRI (CGCGAATTCAAGAAATCCTTTCTTAGAGAA) and mdtABC-KpnI (CGCGGTACCTTACTCGGTTACCGTTTGTTT) was used to amplify mdtABC from the chromosomal DNA of E. coli MG1655. This process introduced the restriction enzyme recognition site present in the multicloning region of the pTrc99A vector. The DNA fragments were digested with restriction enzymes and then ligated into the multicloning region of pTrc99A.The 9 cells of each strain. Supernatants of 13 h cultures were acidified with 50 µl 12 N HCl per 10 ml and extracted twice with 5 ml ethyl acetate. Thereafter, the ethyl acetate phase was concentrated by evaporation . Dried residues were resuspended in 500 µl methanol and analyzed by reverse-phase (RP) high-performance liquid chromatography (HPLC).Enterobactin was prepared from supernatants separated from approximately 1018, 4.6×250 mm, 5 µm: Waters Corp, Milford, MA, USA) using a LaChrom Elite instrument  containing a D-2000 interface, a L-2130 pump, and a L-2400 UV detector. The mobile phase consisted of 0.075% (vol/vol) trifluoroacetic acid in H2O (pH 2) and acetonitrile. The flow rate was adjusted to 1 ml min−1, and 20 µl of each supernatant extraction was injected and separated as described by the manufacture of the standard, with monitoring at 250 nm. Peaks were identified using HPLC-grade enterobactin standards . The amount of enterobactin exported was calculated from peak areas and normalized to that of the wild-type using HITACHI Model D-2000 Elite HPLC System Manager software.RP HPLC analysis was performed on a Symmetry C18 Column . A recent study by Ruiz and Levy also showed that inactivation of the enterobactin biosynthetic genes affects the expression level of acrABThe AcrAB–TolC system is constitutively expressed, but the expression levels of


HLA-A*24:06156Trp and HLA-A*24:13156Leu showed high levels of cell surface expression while HLA-A*24:02156Gln was expressed at low levels in tapasin deficient cells. Peptides presented by these allelic variants showed distinct differences in features and repertoire. Immunoprecipitation experiments demonstrated all the HLA-A*24/156 variants to associate at similar levels with tapasin when present. Structurally, HLA-A*24:02 contains the residue triad Met97/His114/Gln156 and a Trp156 or Leu156 polymorphism provides tapasin independence by stabilizing these triad residues, thus generating an energetically stable and a more peptide receptive environment. Micropolymorphisms at position 156 can influence the generic peptide loading pathway for HLA-A*24 by altering their tapasin dependence for peptide selection. The trade-off for this tapasin independence could be the presentation of unusual ligands by these alleles, imposing significant risk following hematopoietic stem cell transplantation (HSCT).While many HLA class I molecules interact directly with the peptide loading complex (PLC) for conventional loading of peptides certain class I molecules are able to present peptides in a way that circumvents the PLC components. We investigated micropolymorphisms at position 156 of HLA-A Allogeneic hematopoietic stem cell transplantation (HSCT) is an established treatment of various haematological malignancies and certain congenital metabolic disorders. The chance of finding a human leucocyte antigen- (HLA-) identical donor-recipient pair amongst siblings is only one in four. Therefore, stem cells from unrelated donors are most frequently used for transplantation . However+ T-cells [HLA class I molecules play an important role in binding and presentation of high affinity peptides to the cell surface for its subsequent recognition by CD8 T-cells . For its T-cells . Calnexi T-cells . The tra T-cells . The tra T-cells . TPN bri T-cells , thereby T-cells  and help*44:02 [*15:10 [*27:05 [*44:05 [*15:01, and HLA-B*15:18 [α2 helix within the HLA class I hc to be an important determinant for its association with TPN. The great impact of position 156 on HLA function is reflected by its large HLA class I polymorphism and its influence in peptide binding. By structural analysis residue 156 could be described to contact position 3 of a bound peptide in almost all structures (>70%) and position 4, 5, 6, or 7 in 5–70% of peptide-HLA structures available [*44 allotypes [*44:02156Asp and B*44:03156Leu complexed with the same, natural high affinity ligand [*44 allotypes for optimal peptide loading and cell surface expression. Our results illustrated that TPN independent peptide loading resulted in the presentation of exclusive set of peptides with distinct features and lengths [HLA class I polymorphisms primarily concentrated in the peptide binding region (PBR) not only influence the features and repertoire of peptides presented to the cell surface but also affect their requirement on the PLC components for antigen processing and presentation –19. Most*44:02 , 20, and [*15:10  are high [*15:10 , 21. Oth [*27:05 , HLA-B*4 [*44:05 , HLA-B*1-B*15:18 , 17, 22.vailable  and acutllotypes . Structuy ligand . Recentl*24:02 ; within the HLA-A*24 group are 3 naturally occurring variants that differ exclusively at residue 156: HLA-A*24:02156Gln, A*24:06156Trp, and A*24:13156Leu. Since HLA-A*24:02 was described to process and present the immediate early-1 antigen (IE-1) derived peptides of HCMV [*24:02 is able to load peptides independently of the PLC; however the presentation of viral peptides seems to be orchestrated by the interaction of HLA and HCMV immune evasions [*24/156 allotypes are independent of TPN for cell surface expression, they might have very less ER retention time and might facilitate the export of HLA-A*24 molecules loaded with IE-1 peptides to the cell surface during HCMV infection.One of the most common occurring HLA class I variants worldwide is HLA-A of HCMV , it becoevasions . Peptideevasions . However* group, the aim of the present study was to investigate the mode of peptide recruitment and interaction of HLA-A*24 variants and the PLC with regard to micropolymorphism at position 156 in the HLA heavy chain.To understand how residue 156 impacts on the function within this common HLA-AHuman B-LCL 721.220 is lacking functional TPN and both HLA-A and HLA-B genes  while 72*24:02 positive donor (exon 1 through exon 7) was amplified by PCR and products were cloned using TOPO-TA-Cloning Kit . HLA-A*24:06 and HLA-A*24:13 variants were generated by mutating Glu at residue 156 to Trp or Leu, respectively, by SDM . HLA-A*24 inserts were subsequently cloned into pRRL.PPT.SFFV.mcs.pre vector.cDNA from a HLA-A*24/156 variants were generated from pRRL.PPT.SFFV.mcs.pre/A*24 vectors by SDM to introduce a stop codon after exon 4.Soluble HLA-ATo construct shRNA-expressing lentiviral vectors for silencing of TPN, stable short hairpin RNA (shRNA) expression cassettes were cloned into the pLVTHm/si vector containing eGFP as reporter .Lentiviral particles were produced and B-LCLs transduction was performed as described earlier.*24 in transduced cells or TPN in TPN-silenced cells were measured by real time PCR using StepOnePlus Real-Time PCR system (Applied Biosystems).mRNA levels of HLA-A*24/156 variants was assessed by flow cytometry using anti-HLA-A9-FITC (OneLambda) or w6/32-PE (Biolegend)abs. Cells were analyzed using FACS Canto A (BD Biosciences) and FLOWJO software.Surface expression of HLA-A CELLine  and protein levels were determined by sandwich ELISA using w6/32  [ HiTrap columns coupled to mAb w6/32.sHLA producing clones were grown in bioreactorsGermany) , 32 mono*24/156 complexes  were differentiated into low and high binding peptides. The trimeric elution fractions were filtered through a 10 kDa MWCO membrane  and peptides detected in the flow-through were considered to be low binding. The retentate containing sHLA-A*24 trimeric complexes was treated with 0.1% trifluoroacetic acid (TFA) to elute high binding peptides. Fractions containing low or high affinity peptides were subjected to an Eksigent Nano-LC Ultra 2D HPLC coupled to an Orbitrap ion trap . Database queries were carried out using Mascot software [Peptides from sHLA-Asoftware .*24/156 expressing cells were immunoprecipitated with protein A-sepharose beads  covalently coupled to rabbit polyclonal TAP1 (Enzo Life Sciences) or PaSta1  abs. Where TAP1 was used for immunoprecipitation, mouse mAb to ERp57 and CRT and rabbit polyclonal TPN conjugated to HRP (Enzo Life Sciences) were used as primary abs. For immunoprecipitations with PaSta1, rabbit polyclonal to ERp57 and TAP1 and CRT (Enzo Life Sciences) were used as primary abs. Mouse anti-HLA-A mAb (Santa Cruz) was used to detect the heavy chain. Blots were incubated with respective HRP conjugated secondary abs.Lysates of HLA-A*24:02 structure (PDB 3I6L) [http://mordred.bioc.cam.ac.uk/sdm/sdm.php) [http://www.pymol.org/) was used to generate all structure figures.We modeled the AA exchanges at 156 on the HLA-ADB 3I6L)  using thsdm.php)  and the sdm.php) . The gra*24/156 variants demonstrated all the allelic variants to be expressed at high levels on the surface of HLA-A*24 transduced 721.221 cells  or 721.221 (HLA-/TPN+) cells and their derived peptides were categorized as low or high binding peptides  (*24:02156Gln in the presence of TPN (721.221 cells) demonstrated the majority of peptides being of low binding (~82%). In sHLA-A*24/156 expressing 721.221 cells, we obtained 113, 215, and 165 low binding peptides from sHLA-A*24:02156Gln, sHLA-A*24:06156Trp, or sHLA-A*24:13156Leu, respectively. Upon TFA treatment, we were able to acquire 25 high binding peptides derived from sHLA-A*24:02156Gln, 148 from sHLA-A*24:06156Trp, and 64 from sHLA-A*24:13156Leu. We found that sHLA-A*24:06156Trp in the absence of TPN (721.220 cells) presented a significantly higher percentage of high binding peptides compared to low binding ones. From sHLA-A*24/220 cells, we were able to recover 42 low binding and 2 high binding peptides unique to sHLA-A*24:02156Gln, 90 low binding and 136 high binding peptides from sHLA-A*24:06156Trp, and 100 low binding and 21 high binding peptides from sHLA-A*24:13156Leu.Mass spectrometric analysis used in this study had a high mass accuracy in the range of 10 ppm. Soluble HLA- (sHLA-) AMethods) . Analysi*24/156 variants in the presence of TPN were found to be of canonical length (8–10 AA) (*24:02156Gln (77%) and HLA-A*24:06156Trp (79%). We also identified peptides of extraordinary lengths (≥15 AA) from all the 3 subtypes (The majority of peptides presented by sHLA-A8–10 AA) . Absencesubtypes .*24/156 variants  or E (8%) at p2. Absence of TPN resulted in change in the binding preference for HLA-A*24:02 at p2 from Y (12.8%) to S (15.3%). In addition, they also preferred to bind K (10.2%), L (10.2%), F, I, and V (7.6%) at p2. Among low binding peptides eluted from sHLA-A*24:06, L was found to occur at higher frequency in A*24:06 (9.4%) compared to A*24:02 (2.7%) and A*24:13 (2.9%). Low binding peptides eluted from sHLA-A*24:06/220 demonstrated significantly higher frequency of L (16%) and A (8%) occurring at p2 compared to sHLA-A*24:02  and sHLA-A*24:13 . We also found W (5%) at p2 unique to peptides derived from sHLA-A*24:06 in the absence of TPN. Among the high affinity peptides presented in the absence of TPN, Y (47%) and F  occurred frequently at p2 in addition to L (9.8% for A*24:06 and 5% for A*24:13) and A (6.8% for A*24:06 and 11.7% for A*24:13).Analysis of peptides presented in both the presence and the absence of TPN demonstrated Y to occur most frequently at p2 in all the HLA-Avariants . In addi*24/156 variants. Low binding peptides derived from sHLA-A*24:02 in the absence of TPN showed preference for D (30.7%) and A (10.2%) in addition to L (20.5%) and F (10.2%). Among high binding peptides eluted from sHLA-A*24:02, D (12%) and I (8%) were also found to occur more frequently. K (11.2%) at pΩ occurred more frequently among low binding peptides derived from sHLA-A*24:06 in both the presence and the absence of TPN.Comparison of AA frequencies at pΩ demonstrated F or L to occur most frequently among peptides derived from sHLA-A*24/156 variants both in the presence and in the absence of TPN. Among the peptides eluted from sHLA-A*24/221 cells, ~3.5% of low binding and 1.9% of high binding peptides were shared between all the 3 variants. In the absence of TPN, only 0.8% of low binding peptides and 0.2% of high binding peptides were shared between them. Also, the shared peptide percentage of HLA-A*24:02156Gln in the presence and absence of TPN  was found to be lower than the peptide repertoire shared by HLA-A*24:06156Trp  and HLA-A*24:13156Leu .A small percentage of overall peptide repertoire was found to be shared among all the three HLA-A*24:02 contains the residue triad Met97/His114/Gln156 that appears to influence the stability of the PBR and dictate PLC independent peptide loading. The measure of surface expression is low for HLA-A*24:02156Gln but high for HLA-A*24:06156Trp and HLA-A*24:13156Leu allotypes in the absence of TPN (721.220 cells). To investigate the structural basis of this TPN dependency in HLA-A*24 alleles we generated molecular models using the structure of HLA-A*24:02156Gln as a template and mutating residue 156 to HLA-A*24:06156Trp or HLA-A*24:13156Leu (*24:06156Trp or HLA-A*24:13156Leu polymorphisms on the protein structure we used the Site Directed Mutator (SDM) server (http://mordred.bioc.cam.ac.uk/sdm/sdm.php) [G) is negative when a mutation is destabilizing while a positive score indicates that a particular mutation is stabilizing thereby allowing us to predict the effect that a particular mutation will have on the stability of proteins. The HLA-A*24:06156Trp or HLA-A*24:13156Leu polymorphisms both illustrated high stability score values of 2.37 and 3.11, respectively, suggesting that they have increased stability. To confirm the predicted increase in stability we also calculated the predicted difference in free energies between wild type and mutant proteins  using FoldX [*24:06156Trp or HLA-A*24:13156Leu polymorphisms both illustrated ΔΔG values of −0.18 and −1.44, respectively, consistent with increased stability and suggesting that these mutations are likely to provide enhanced TPN independence through generating an energetically stable and a more peptide receptive environment.HLA-A13156Leu . To predsdm.php) . The SDMng FoldX . Again tClinical outcome of unrelated bone marrow transplantation over the last years demonstrated the value of knowledge about the impact of distinct HLA class I mismatches. The feasibility of using mismatched transplants is appreciated by the extensive datasets that have been collected over the years, including clinical data, high resolution sequencing, peptide sequencing, and T-cell studies.Given the fact that every HLA allele has an individual peptide repertoire, it becomes obvious how many single peptide-HLA molecules are available for an antigenic T-cell. For that reason it has been proposed that whether GvHD and GvL can be related to the recipient specific peptide repertoire and its set of shared peptides by the donor HLA molecule should be assessed . While tThe conventional peptide selection and presentation is known to be driven by several proteins including TAP and TPN which import and edit peptides for being loaded and presented by the individual HLA class I molecule. A key component of the PLC is the protein TPN that orchestrates peptide selection by facilitating the stabilization of TAP and promoting the binding and translocation of peptides by TAP. The absence of TPN abrogates the binding of class I molecules to TAP and results in unstable pHLA complexes with short half-life times making it one of the main targets for viruses to interfere with the presentation of viral peptides to CTLs. Despite its dedicated function certain HLA alleles are able to circumvent the PLC and can therefore present an alternate repertoire of peptides due to their PLC independence. These TPN independent alleles are therefore advantageous in viral infections such as hepatitis C (HCV)  or HIV .*44 variants to influence PLC association and therefore peptide repertoire and selection [* group, we carefully choose A*24, an allelic group that is relatively common in the Caucasian population (A*24:02 ~20%). Since TPN independence might permit distinct alleles to present poorly tolerated self-peptides to the self-immune sytem, a probability of autoimmune reactions should be considered. In the view of this context, it becomes interesting that HLA-A*24:02 is found to be a predisposing factor for type 1 diabetes [*24:02 [*24:02 with autoimmune disorders and its ability to present viral peptides, we wanted to investigate if polymorphisms occurring at position 156 influence PLC dependence, structure and property of the PBR, and subsequently peptide features and repertoire presented by these allotypes.We recently described AA position 156 in the heavy chain of Bdiabetes  and proldiabetes . TPN-ind [*24:02 . Given t*24:02156Gln, HLA-A*24:06156Trp, and HLA-A*24:13156Leu. Interestingly, a difference in function for A*24/156 variants' PLC association and peptide selection could be observed. The peptide binding motifs at p2 and pΩ for A*24/156 derived peptides show in the absence of TPN high promiscuity, particularly the low binding peptides  in the absence of TPN, while A*24:02 exhibited partial independence. However no difference in association with TPN or TAP for all the allelic variants could be detected, although their peptides clearly showed different patterns and features in presence or absence of TPN. This observation demonstrates that also TPN independent alleles preferentially select and present peptides via the classical PLC pathway.We systematically investigated HLA-A Figures . Remarka Figures , suggest*24:06 and A*24:13 more independent of TPN than A*24:02. To understand this, the three molecules were analyzed based on their structural features. Structurally, HLA-A*24:02 contains the residue triad Met97/His114/Gln156 .The further reduction of HSCT associated risks relies on our understanding of how successful clinical outcomes can be achieved despite patient-donor allelic mismatches. The clinical importance of single mismatches at specific residues like position 156 is well known and is associated with acute Graft versus Host Disease in the Bic group  and stro in A*02 –43, B*35*35 [*44  subtypes*24:02, A*24:06 or A*24:13 restricted self-peptides of low or high affinity acquired in the presence (source LCL 721.221 cells) or absence (source LCL 721.220 cells) of Tapasin.Supplementary Tables 1, 2, and 3 present HLA-A


Objective: This study aimed to detect the association between polymorphisms and haplotype in the ATP-binding cassette transporter A1 (ABCA1) gene and overweight/obese Uyghur patients in China. Methods: A total of 259 overweight/obese patients and 276 normal weight subjects, which were randomly selected from among 3049 adult Uyghurs, were matched for age. We genotyped ABCA1 single nucleotide polymorphisms of rs2515602, rs3890182, rs2275542, rs2230806, rs1800976, and rs4149313. Results: (1) The genotypic and allelic frequencies of rs2515602 and rs4149313 differed between the control group and case group. The genotypic frequency of rs2275542 also differed between the control group and case group (p < 0.05); (2) rs2515602, rs2230806, and rs4149313 polymorphisms were significantly related to risk of overweight/obese; (3) a significant linkage disequilibrium (LD) was observed between the ABCA1 gene rs2275542 with rs3890182 and rs2515602 with rs4149313. (4) the C-C-C-A-G-G, T-C-G-A-G-G, and T-T-G-G-G-A haplotypes were significant in normal weight and overweight/obese subjects (p < 0.05); (5) the levels of HDL-C  in normal weight subjects were different among the genotypes (p < 0.05); the levels of TC, LDL-C and TG (rs1800976) in overweight/obese subjects were different among the genotypes (p < 0.05). Conclusions: The rs2515602, rs4149313, and rs2275542 polymorphisms were associated with overweight/obese conditions among Uyghurs. Strong LD was noted between rs2275542 with rs3890182 and rs2515602 with rs4149313. The C-C-C-A-G-G and T-C-G-A-G-G haplotypes may serve as risk factors of overweight/obesity among Uyghurs. The T-T-G-G-G-A haplotype may serve as a protective factor of overweight/obesity among Uyghurs. Rs2515602, rs2275542, rs4149313, and rs1800976 polymorphisms in the ABCA1 gene may influence lipid profiles. ABCA1 get al.’s  studies et al.’s . Whetheret al.’s ,11. A caThe protocol was approved by the Institutional Ethics Review Board (IERB) of the First Affiliated Hospital of Shihezi University School of Medicine (IERB No. SHZ2010LL01). Written informed consent was obtained from each participant. Standard university hospital guidelines, including informed consent, confidentiality, voluntary participation, and anonymity were followed. All participants gave written informed consent before the study began.The total subjects in this study consisted of 535 unrelated adults who reside in Jiashi County, Xinjiang Uyghur Autonomous Region, People’s Republic of China. They were randomly selected from our previous stratified randomized cluster samples . A total2). After overnight fasting, venous blood samples (5 mL) were drawn from the forearm vein of all participants. A part of the blood (3 mL) was collected into glass tubes and used to determine the serum lipid levels. Another part of the blood (2 mL) was transferred into tubes and used to extract DNA. The concentrations of triglycerides (TGs), total cholesterol (TC), HDL-C, and low -density lipoprotein-cholesterol (LDL-C) in serum were measured with a DXC-800 automatic biochemical analyzer  at the Clinical Science Experiment Center of the First Affiliated Hospital of Shihezi University School of Medicine.Information on demographic and personal lifestyles was collected with a self-developed questionnaire during face-to-face interviews. Blood pressure, height, weight, waist circumference, and hip circumference were measured according to standardized methods . Body ma2; respectively [The diagnostic criteria of overweight, obesity and normal weight were defined as a BMI 24–28, >28, and <24 kg/mectively . Low HDLectively . Hyperte260/OD280) of 1.7–2.0 were considered acceptable. Samples that met these criteria were diluted to 10–30 ng/µL with double-distilled water and then stored at −80 °C.Fasting venous blood (200 µL) was taken from each study subject, and a blood genomic DNA isolation kit  was used to extract the whole blood genomic DNA. The extracted DNA was verified by gel electrophoresis (0.7% agarose). A NanoDrop spectrophotometer  was used for quantitative determination of DNA concentration and purity: concentration ≥30 ng/µL and purity levels  was used to remove excess dNTPs from samples after PCR. This step served to ensure the accuracy of single-base extension. The final SAP reaction volumes were 5.0 mL, which included 0.5 µL 10 × SAP buffer, 2 uL of PCR product, 2 µL of double-distilled water, and 0.5 µL of SAP enzyme. The reactions were conducted by incubation at 37 °C for 40 min, followed by incubation at 85 °C for 5 min. The reaction products were stored at 4 °C.For single-base extension reactions, the final reaction volumes were 6.0 µL, which included 0.5 μL of SNaPshot reagent, 2.5 µL of water, 1 µL of primer mix, and 2 uL of purified PCR products. The reaction conditions were as follows: denaturation at 94 °C for 30 s, followed by 40 cycles of 94 °C for 5 s, 52 °C for 5 s, and finally 52 °C for 5 s. The reaction products were stored at 4 °C.Genotyping analysis was as follows: about 1 µL of reaction product and 9 µL HIDI were obtained. Denaturation was conducted at 95 °C for 3 min. Samples were then immediately chilled in an ice-water bath. All representative SNP genotyping experiments were performed via TaqMan technology on an ABI3730XL system . GeneMapper was used to complete the classification and output the results.t-Test. Single factor logistic regression analysis was used to assess risk factors of overweight/obesity ), independent variable included sex , age , hypertension , high TC , high TG , high LDL-C , smoker , drinker , rs2515602 , rs3890182 , rs2275542 , rs2230806 , rs1800976 , rs4149313 . Frequency table and statistical analysis were used with SPSS 17.0  statistical package. SHEsis software was used to analysis haplotype and LD [p value of 0.05 was used to define the level of significance.Epidata 3.02 software was used to establish a database, and the double entry method was used for data input and logic error detection. Non-normally distributed continuous variables such as TG, TC and LDL-C are shown as median and interquartile range , while HDL-C concentrations, age, height, weight, BMI, waist circumference, hip circumference, waist-to-hip ratio, systolic blood pressure, diastolic blood pressure and Pulse pressure are presented as mean ± standard deviation. The frequency of the ABCA1 alleles was determined by gene counting. Chi-square tests were used to compare the differences in percentages and to assess Hardy-Weinberg expectations. The Kruskal-Wallis H statistic or One Way ANOVA were used to compare continuous variables among the three genotype groups, while the Kolmogorov-Smirnov Z test or the Student’s p < 0.05). The levels of serum HDL-C were higher in overweight/obese subjects than those in normal weight subjects (p < 0.001). No significant differences were found in the values of average age, smokers, and drinkers between overweight/obese and normal weight subjects (p > 0.05).p > 0.05). The genotypic and allelic frequencies of rs3890182, rs2230806 and rs1800976 between the control group and case group showed no significant differences (p > 0.05). For rs2515602 polymorphism, we observed a lower frequency of the T allele  in overweight/obese patients compared with normal weight subjects. Rs2275542 genotypes were significance differences between normal weight and overweight/obese subjects (p = 0.033). Moreover, the frequency of rs4149313 G allele in case group was higher than in control group .The genotype and allele frequencies of the SNPs between normal weight and overweight/obese subjects are presented in The relationship of the six SNPs in ABCA1 and overweight/obese are summarized in 2 ranging from 0.000 to 0.889. We can found that a strong pairwise LD was present in the ABCA1 for the following SNPs pairs: rs2275542 with rs3890182  and rs2515602 with rs4149313 .Pairwise LD performed for all six SNPs identified in normal weight and overweight/obese individuals is shown in p < 0.001). C-C-C-A-G-G and T-C-G-A-G-G haplotypes were significantly more frequent in the case group than in the control group, whereas the T-T-G-G-G-A haplotypes was less frequent in the overweight/obese group than in the normal weight group (p < 0.05).The results of haplotype analysis of the six SNPs are shown in p < 0.05); serum HDL-C levels was higher in control according to rs2275542 with CC/CT genotype than in TT genotype (p < 0.05); in cases, the levels of TG, TC, and LDL-C were higher according to rs1800976 with CG genotype than in GG/CC genotype (p < 0.05); serum HDL-C level was higher in control group according to rs4149313 with GG genotype than in AA genotype (p < 0.05).The results of the present study showed that the levels of TC, TG, and LDL-C were lower in normal weight than those in overweight/obese subjects; whereas the level of HDL-C was higher. These findings were line with those of previous studies ,17,18. EOur study showed no significant difference between case group and the control group in age, and the genotypic frequency was consistent with Hardy-Weinberg Equilibrium, with group representative. The frequency distribution of the six SNPs also varied in different research groups. Our research showed that rs2515602 C allele frequency (42.1%) was higher than European 26.3%), but was lower than that in Han Chinese (73.7%) .3%, but . We founSerum lipid levels are affected by exogenous and endogenous factors, while increasing rates of overweight and obesity may partly be attributed to dyslipidemia . The ABCInformation about the association of ABCA1 gene haplotype and overweight/obesity is limited. In this study, we observed that C-C-C-A-G-G and T-C-G-A-G-G haplotypes were significantly more frequent in the case group than in the control group, whereas the T-T-G-G-G-A haplotypes was less frequent in the overweight/obese group than in the normal weight group. These findings suggested that the C-C-C-A-G-G and T-C-G-A-G-G haplotypes may serve as risk factors of overweight/obesity more than the C-T-G-G-G-A haplotype among Uyghurs, whereas the T-T-G-G-G-A haplotype may serve as a protective factor of overweight/obesity more than the C-T-G-G-G-A haplotype among Uyghurs.rs2230806 and rs4149313 are located in the exon region, whereas rs2275542 and 3890182 are located in the intron region and rs1800976 is located in promoter . These SOur study had several potential limitations. First, the sample size in our study was small. No individuals with the rs3890182 AA genotype were detected in our case group, and the number of subjects with rs3890182 AA genotype in control group was also small. Second, although we have discussed the relationship between ABCA1 six SNPs polymorphisms and overweight/obese, the mechanism of ABCA1 gene polymorphism and overweight/obesity remains unclear. Thus, further studies are required to understand the role of ABCA1 gene polymorphisms in developing overweight/obese. Finally, overweight/obese is affected by multiple environmental and genetic factors and their interactions. Many environmental and genetic factors and their interactions remain unclear and undetected.The present study shows that there is no significant difference in the genotypic and allelic frequencies of ABCA1 rs2230806, rs3890182 and rs1800976 polymorphisms between a normal weight group and an overweight/obese group. We also found that rs2515602, rs2230806 and rs4149313 in ABCA1 gene variants were significantly related to risk of overweight/obesity. Finally, we determined that C-C-C-A-G-G and T-C-G-A-G-G haplotypes may serve as risk factors of overweight/obesity among Uyghurs, whereas the T-T-G-G-G-A haplotype may serve as a protective factor of overweight/obesity among Uyghurs. The differences in serum lipid levels between normal weight and overweight/obese subjects might partly result from ABCA1 gene variant.


B, an alternative transcription factor, controls the response of the cell to a variety of environmental stresses in Bacillus cereus. Previously, we reported that RsbM negatively regulates σB through the methylation of RsbK, a hybrid sensor kinase, on a signaling helix (S-helix). However, RsbK comprises a C-terminal receiver (REC) domain whose function remains unclear. In this study, deletion of the C-terminal REC domain of RsbK resulted in high constitutive σB expression independent of environmental stimuli. Thus, the REC domain may serve as an inhibitory element. Mutagenic substitution was employed to modify the putative phospho-acceptor residue D827 in the REC domain of RsbK. The expression of RsbKD827N and RsbKD827E exhibited high constitutive σB, indicating that D827, if phosphorylatable, possibly participates in σB regulation. Bacterial two-hybrid analyses demonstrated that RsbK forms a homodimer and the REC domain interacts mainly with the histidine kinase (HK) domain and partly with the S-helix. In particular, co-expression of RsbM strengthens the interaction between the REC domain and the S-helix. Consistently, our structural model predicts a significant interaction between the HK and REC domains of the RsbK intradimer. Here, we demonstrated that coordinated the methylatable S-helix and the REC domain of RsbK is functionally required to modulate σB-mediated stress response in B. cereus and maybe ubiquitous in microorganisms encoded RsbK-type sensor kinases.σ B found in low-GC Gram-positive bacteria such as Bacillus subtilis, Staphylococcus aureus, and Listeria monocytogenes controls σB-dependent regulon expression mediated by signaling cascades to cope with a variety of environmental stresses, including changes in temperature, pH, ethanol levels and osmolarity . The cells were disrupted with glass beads  in a Fast Prep 120 homogenizer (Savant), and the cell extract was obtained after centrifugation. Next, 0.7 ml of Z buffer and 200 μl of 2 mg ml−1 2-nitrophenyl-β-D-galactoside (Sigma) for the β-galactosidase assay were added to 100 μl of cell extract. The mixture was incubated at 37°C, and the reaction was stopped by the addition of 0.2 ml of 2 mM Na2CO3. Subsequently, the optical density of the reaction mixture was measured at 405 nm. The protein content was measured using the Bio-Rad protein assay with bovine serum albumin as the standard. Specific activities are expressed in the units of β-galactosidase per milligram of protein (Miller units) using the following formula: [O.D.405 nm x 378]/[time (min) x volume of cell extract (ml) x protein concentration (mg/ml)].To measure β-galactosidase activity in a bacterial assay, exponentially growing cells (2 ml) were harvested and resuspended in 0.5 ml of Z buffer  and sonicated. Soluble proteins were separated from the cell debris by centrifugation at 15000 x g for 30 min at 4°C, and the supernatant was passed through a 0.22-μm filter (Millipore) and loaded onto a nickel column . Bound proteins were eluted according to the manufacturer’s instruction.For the purification of RsbM, E. coli BL21 (λDE3) cells were transformed with pET21b-rsbK. As previously described, the cells were harvested by centrifugation at 6000 x g for 10 min at 4°C after IPTG induction, washed with cold PBS and then re-suspended in methylation buffer . The collected cells were disrupted by sonication to prepare whole cell lysates, and 100 μg of each cell lysate was incubated with purified recombinant RsbM protein in 45 μl methylation buffer supplemented with 500 nCi of S-adenosyl-L-(methyl-14C) methionine  (radioactive methylation) for 1 h at room temperature. The reactions were stopped by adding 3X 10% SDS-PAGE sample buffer  SDS, 10% glycerol, 0.03% (w/v) bromophenol blue, 1.25 M DTT, pH 6.8) followed by heating at 95°C for 10 min. The samples were analyzed by 12% SDS-PAGE and stained with Coomassie blue. The gel was treated with Amplify Fluorographic Reagent  for 30 min, according to the manufacturer’s instruction, and dried by a vacuum. The radioactivity was visualized by exposing the gel to an X-ray film at -80°C for one week.For the overproduction of RsbK, Amino acid sequences of the RsbK homologs were extracted from NCBI GenBank using PSI-Blast . These sThermotoga maritima class I HK853 and its cognate response regulator, RR468 (PDB ID: 3DGE) [Structures of the RsbK cytoplasmic domains HK and REC were created using MODELLER 9v12 . The cryD: 3DGE) , was useD: 3DGE) . To sampD: 3DGE)  and the D: 3DGE)  in the SD: 3DGE) . The strD: 3DGE) . The prosigB cluster in B. cereus  on the S-helix is required to recruit the REC domain and thereby repress σB before environmental stress.In addition to the inhibitory function of the REC domain, methylation of the S-helix of RsbK by RsbM has been shown essential to repress σB . It needn mutant . The forlag-rsbM , and the high σB , indicatE. coli growth of blue colonies on M63 selection medium and quantification of β-galactosidase activity. Bacterial growth were comparable to measurements of β-galactosidase activity showing that the REC domain could interact with full-length RsbK (E. coli BHT101 cells (data not shown).To test the above hypothesized mechanism, BACTH was employed to analyze whether the REC domain interacted with other RsbK subdomains, particularly the S-helix. The pKT25 plasmid containing the REC domain was constructed, and full-length RsbK or RsbK sequences with successive subdomain truncations such as REC-truncated RsbK, REC-CA-truncated RsbK, REC-CA-HK-truncated RsbK, REC-CA-HK-S-truncated RsbK and RsbK with only HK or S-helix deletion were separately constructed in pKT18 plasmids . The phygth RsbK -#1, REC-gth RsbK -#2, REC-gth RsbK -#3, REC-gth RsbK -#4 and Rgth RsbK -#8. Howegth RsbK -#9. Moregth RsbK -#5. Thesgth RsbK -#4, #6. gth RsbK -#5, #7. B in response to environmental stress. Two complementary plasmids, pHT304-rsbKD827N-rsbM and pHT304-rsbKD827E-rsbM, carrying RsbK mutants with site-directed mutagenesis at D827 were generated  in the HK domain and an aspartate residue (D827) predicted as a putative phospho-acceptor in the C-terminal REC domain. We examined whether D827 regulates σenerated . Residueectively , 44. Thetivation . Our resD827N or RsbKD827E constitutively activates σB  [To study the function of the REC domain of RsbK from the structural point of view, we took advantage of the resolved crystal structure of the complex formed by the D: 3DGE) . The strD: 3DGE)  showed 1D: 3DGE) . The quaD: 3DGE) , and theD: 3DGE) . PROCHECT. maritima HK853 . Therefore, we present the model structure in the trans binding mode, in which the HK domain of one monomer interacts with the REC domain from the other monomer.We built an RsbK HK dimer based on the homodimeric structure of ma HK853 . In the 2. At the interface, there are 13 ± 4 hydrogen bonds and 4 ± 2 salt bridges; the calculated solvation free energy gain upon formation of the interface is approximately -12 ± 2 kcal/mol. PISA calculates a complexation significance score (CSS), which indicates the probability that an interface might represent a 'real' interface on a scale from 0 to 1. The CSS for the HK-REC interface of RsbK is 1.0, suggesting that the interaction between the HK and REC domains may be significant.We used the PISA server  to assesB. In fact, truncation of the REC domain of RsbK led to constitutive high σB activation  at the Lβα1 loop and the oxygen in the backbone of the conserved Met residue (Met55 in RR468 and Met829 in RsbK) at the Lβα3 loop may coordinate the Mg2+ ion during the reaction . Rather, the expression of D827 mutants caused constitutive high σB under unstressed conditions (B regulation. Because the net charge of D827N (0) is less than that of D827 (-1), our result indicated that loss of the negative charge at D827 appears to increase σB expression . Both RsbKD827E and RsbKD827N resulted in constitutive high σB inconsistent with the expectation that either D827 variant may activate σB. Actually, different substitutions at a critical residue causing the same biological effect were sometimes observed. For example, reversal frequency was induced by RomR phosphorylation, whereas both RomRD53E and RomRD53N led to lower reversal frequency in Myxococcus xanthus [The S-helix in sensor kinases, as an adaptation subdomain, has been reported to function as a switch to prevent the constitutive activation of downstream signaling domains . Moreovenditions , suggestpression . In fact xanthus .trans binding mode  Construction of the complementary plasmid pHT304-rsbK-rsbM and its derivatives. The DNA fragment, including the partial bc1009 and rsbK-rsbM sequences, was inserted into the vector pHT304 to construct the complementary plasmid pHT304-rsbK-rsbM. The other complementary plasmids were constructed by inserting DNA segments lacking either the REC domain or rsbM. As described in the experimental procedures, the pHT304-rsbKΔrec-recflag and pHT304-rsbKΔrec-recflag-rsbM plasmids were designed to express the Flag-tagged REC domain in trans. The restriction sites are indicated.(A) Deletion of the (TIF)Click here for additional data file.S2 FigT. maritima HK853. (B) CA domain of RsbK compared with that in T. maritima HK853. (C) REC domain of RsbK compared with that in T. maritima RR468. The sequences of ten RsbK homologs from different species were also included in the alignments to demonstrate the amino acid conservation of each residue along the RsbK sequence. Information about these RsbK homologs is summarized in 2+-binding Asn625 is marked by a cyan triangle, and residues involved in ATP binding are marked by orange triangles. In alignment C, the phosphoacceptor Asp827 is marked by a magenta triangle, and residues inside the active site are marked by green triangles. Throughout the three alignments, residues participating in van der Waals interactions are marked by orange circles. The residues forming hydrogen bonds and salt bridges are marked by cyan and magenta, respectively.(A) The HK domain of RsbK compared with that in (TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


In summary, this study demonstrates that advanced age, atherosclerosis and type 2 diabetes mellitus reduce the functional potency of MSCs. Optimizing the criteria for the selection of MSC donors could enhance the results of cell-based therapies.To this end human MSCs were isolated from adipose tissue and the MSC:CD4The online version of this article (doi:10.1186/s13287-015-0127-9) contains supplementary material, which is available to authorized users. Age and age- associated conditions (type 2 diabetes and athrosclerosis) decrease the immunomodulatory capacity of MSCs.Optimal donor selection and proper immunological characterization of MSCs are critical for the successful therapeutic use of MSCs.+ T cells seem to be critical to accelerate atherogenesis and to promote plaque instability [+ T cells and the extent of ATH in carotid and coronary arteries, and by the larger number of these cells in unstable plaques compared to those from patients with stable coronary artery disease [Atherosclerotic ischemic heart disease is the leading cause of death in developed countries. The prevalence, incidence, and severity of atherosclerosis (ATH) markedly increase with chronological age and in the context of age-associated chronic inflammatory conditions such as type 2 diabetes mellitus (T2DM) . Chronic disease , 4. In v disease . The fun disease , 7). Imm disease . A criti disease , 10. The+ T-cell suppression was assessed in co-cultures [+ T cells were plotted and the suppressive effect of MSCs on T cells was determined by comparing maximal proliferation  versus proliferation in co-cultures (MSCs + T cells) . All hypotheses tests were two-sided and performed at a significance level of 0.05.The McGill University Health Center Ethics Review Board approved the study and participants provided written informed consent. Subcutaneous adipose tissue was obtained from patients undergoing programmed cardiovascular surgery. Table cultures . Prolife+ T cells. The suppressive effect of A-MSCs and E-MSCs on CD4+ T-cell proliferation was dose-dependent. At a MSC:T cell ratio of 1:8  (p < 0.003). Similar results were obtained at MSC:T cell ratios of 1:14 and 1:20. A-MSCs at 1:14 ratio  had similar inhibitory capacity as E-MSCs at 1:8 ratio, highlighting the magnitude of the E-MSC defect , with increasing mean CD4+ T-cell proliferation of 0.5 %  0.1–1.0) for any 1-year increase in age.The immunomodulatory capacity of adult MSCs  and elderly MSCs  was examined by analyzing their ability to inhibit the proliferation of anti-CD3/CD28-activated CD41:8 Fig. , A-MSCs ect Fig. . MSC donect Fig. , indicat+ T cells compared to MSCs from ATH age-matched controls   than those without ATH . Similarly, subjects with T2DM had a higher mean percentage of proliferating CD4+ T-cells . There was no statistically significant interaction either between ATH and T2DM or between each of them and age. This suggests that the effect of ATH is independent of T2DM and that these effects are also independent of age.The effect of biological aging on human MSC-mediated T-cell suppression was tested by evaluating MSCs from donors with ATH and T2DM, diseases associated with chronic inflammation and premature aging. MSCs from patients undergoing valve replacement surgery who had a normal pre-surgical coronary angiography  had a higher ability to suppress activated CD418) Fig. . Moreove18) Fig. . Unadjus18) Fig. . In the The PRECISE trial demonstrated the feasibility, safety and potential therapeutic benefit of the transendocardial administration of autologous adipose-derived MSCs in no-option patients with ischemic cardiomyopathy . This stRecent evidence confirmed that early-passage  and freshly harvested MSCs have better in vitro T-cell suppression capacity and are associated with more clinical benefits than late-passage and freeze/thawed MSCs . Our finin vivo ischemic and inflammatory environment. However, this system predicts the MSC immunomodulatory potency, which is the most relevant mechanism for the therapeutic effect of MSCs [The overall efficacy of stem cell transplantation relies on the activity of donor cells and tissue environment. Our co-culture model is limited to MSCs and PBMCs and does not fully simulate all the components of the  of MSCs . The fin of MSCs . A reduc of MSCs , an incr of MSCs  and highIn summary, our results indicate that age and age-associated conditions (T2DM and ATH) decrease the immunomodulatory capacity of MSCs, highlighting the relevance of donor selection and the need for proper immunological characterization of MSCs. Understanding the interplay between aging, MSC function and their clinical implications remains the only rational path to the successful therapeutic use of MSCs.Additional file 1:Supplementary materials and methods. (DOCX 46 kb)Additional file 2: Figure S1.+ T cells was assessed in a 4-day allogeneic co-culture system. (A) PBMCs expanded for 4 days were used as controls . (B) MSCs from different donors were co-cultured for 4 days with primary monocyte-depleted PBMCs obtained from a single unrelated donor. At day 4, PBMCs were stained with 7-aminoactinomycin D (7-AAD), and CD4-APC and flow cytometry was performed. The expansion index of 7AAD−CD4+ T cells was calculated with FlowJo. The percentage of CD4+ T-cell proliferation was calculated according to the following formula: % of Proliferation = (X – Control) /  × 100, where X = Expansion index of MSC-CD4+ T cell co-culture for each sample, Control = Expansion index of non-stimulated CD4+ T cells, and Maximal Proliferation = Expansion index of CD4+ T cells stimulated with anti-CD3/CD28 beads in the absence of MSCs. (PPT 538 kb)Gating strategy for the MSC:T cell suppression assay. The capacity of MSCs to suppress proliferative responses on activated CD4Additional file 3: Figure S2.+ T-cell suppression. (A) The capacity of MSCs from non-elderly adult donors  to suppress CD8+ T cells is higher than that of elderly donors  . (B) The E-MSC:CD8+ T-cell suppressive potency at 1:8 ratio is similar to that of A-MSCs at 1:14 ratio. (C) Age-dependent decline in MSC:CD8+ T-cell suppression in ATH patients . (D) Correlation of the suppressive effect of MSCs on CD4+ and CD8+ T cells . (PPT 142 kb)Age-associated reduction in MSC-mediated CD8