-
PloS One 2024SlyD is a widely-occurring prokaryotic FKBP-family prolyl isomerase with an additional chaperone domain. Often, such as in Escherichia coli, a third domain is found at...
SlyD is a widely-occurring prokaryotic FKBP-family prolyl isomerase with an additional chaperone domain. Often, such as in Escherichia coli, a third domain is found at its C-terminus that binds nickel and provides it for nickel-enzyme biogenesis. SlyD has been found to bind signal peptides of proteins that are translocated by the Tat pathway, a system for the transport of folded proteins across membranes. Using peptide arrays to analyze these signal peptide interactions, we found that SlyD interacted only with positively charged peptides, with a preference for arginines over lysines, and large hydrophobic residues enhanced binding. Especially a twin-arginine motif was recognized, a pair of highly conserved arginines adjacent to a stretch of hydrophobic residues. Using isothermal titration calorimetry (ITC) with purified SlyD and a signal peptide-containing model Tat substrate, we could show that the wild type twin-arginine signal peptide was bound with higher affinity than an RR>KK mutated variant, confirming that positive charges are recognized by SlyD, with a preference of arginines over lysines. The specific role of negative charges of the chaperone domain surface and of hydrophobic residues in the chaperone active site was further analyzed by ITC of mutated SlyD variants. Our data show that the supposed key hydrophobic residues of the active site are indeed crucial for binding, and that binding is influenced by negative charges on the chaperone domain. Recognition of positive charges is likely achieved by a large negatively charged surface region of the chaperone domain, which is highly conserved although individual positions are variable.
Topics: Escherichia coli Proteins; Peptidylprolyl Isomerase; Escherichia coli; Protein Binding; Molecular Chaperones; Protein Sorting Signals; Hydrophobic and Hydrophilic Interactions; Calorimetry; Arginine; Amino Acid Sequence
PubMed: 38917203
DOI: 10.1371/journal.pone.0305823 -
Microbiology Spectrum Jun 2024Protein acetylation and deacetylation are key epigenetic modifications that regulate the initiation and development of several diseases. In the context of infection with...
UNLABELLED
Protein acetylation and deacetylation are key epigenetic modifications that regulate the initiation and development of several diseases. In the context of infection with (), these processes are essential for host-pathogen interactions and immune responses. However, the specific effects of acetylation and deacetylation on cellular functions during infection are not fully understood. This study employed Tandem Mass Tag (TMT) labeling for quantitative proteomic profiling to examine the acetylproteome (acetylome) profiles of noninfected and -infected macrophages. We identified 715 acetylated peptides from 1,072 proteins and quantified 544 lysine acetylation sites (Kac) in 402 proteins in noninfected and -infected macrophages. Our research revealed a link between acetylation events and metabolic changes during infection. Notably, the deacetylation of heat shock protein 60 (HSP60), a key chaperone protein, was significantly associated with this process. Specifically, the deacetylation of HSP60 at K96 by sirtuin3 (SIRT3) enhances macrophage apoptosis, leading to the elimination of intracellular . These findings underscore the pivotal role of the SIRT3-HSP60 axis in the host immune response to . This study offers a new perspective on host protein acetylation and suggests that targeting host-directed therapies could be a promising approach for tuberculosis immunotherapy.
IMPORTANCE
Protein acetylation is crucial for the onset, development, and outcome of tuberculosis (TB). Our study comprehensively investigated the dynamics of lysine acetylation during infection, shedding light on the intricate host-pathogen interactions that underlie the pathogenesis of tuberculosis. Using an advanced quantitative lysine proteomics approach, different profiles of acetylation sites and proteins in macrophages infected with were identified. Functional enrichment and protein-protein network analyses revealed significant associations between acetylated proteins and key cellular pathways, highlighting their critical role in the host response to infection. Furthermore, the deacetylation of HSP60 and its influence on macrophage-mediated clearance of underscore the functional significance of acetylation in tuberculosis pathogenesis. In conclusion, this study provides valuable insights into the regulatory mechanisms governing host immune responses to infection and offers promising avenues for developing novel therapeutic interventions against TB.
PubMed: 38916288
DOI: 10.1128/spectrum.00749-24 -
[Analysis of enzyme activity and substrate specificity of dolichyl-phosphate β-glucosyltransferase].Sheng Wu Gong Cheng Xue Bao = Chinese... Jun 2024Protein folding and quality control processes primarily occur in the endoplasmic reticulum (ER). ER-resident molecular chaperones play a crucial role in guiding nascent...
Protein folding and quality control processes primarily occur in the endoplasmic reticulum (ER). ER-resident molecular chaperones play a crucial role in guiding nascent polypeptides towards their correct tertiary structures. Some of these chaperones specifically recognize glucosylated -glycan moieties on peptide. It is of great significance to study the -glycan biosynthetic pathway and glycoprotein quality control system by analyzing the sugar donor of ER luminal glucosyltransferases, known as dolichol phosphate glucose (Dol-P-Glc), or its analogues . In this study, we investigated a range of dolichol analogues to synthesize lipid phosphate glucose, which served as substrates for dolichyl-phosphate β-glucosyltransferase E (Alg5E) derived from . The results demonstrated that the recombinant Alg5E, expressed in , exhibited strong catalytic activity and the ability to recognize lipid phosphate glucose with varying chain lengths. Interestingly, the enzyme's catalytic reaction was found to be faster with longer carbon chains in the substrate. Additionally, Alg5E showed a preference for branched chain methyl groups in the lipid structure. Furthermore, our study confirmed the importance of divalent metal ions in the binding of the crucial DXD motif, which is essential for the enzyme's catalytic function. These findings lay the groundwork for future research on glucosyltransferases Alg6, Alg8, and Alg10 in the synthesis pathway of dolichol-linked oligosaccharide (DLO).
Topics: Glucosyltransferases; Substrate Specificity; Escherichia coli; Trichomonas vaginalis; Recombinant Proteins; Dolichol Phosphates; Endoplasmic Reticulum
PubMed: 38914494
DOI: 10.13345/j.cjb.230737 -
Journal of Applied Biomedicine Jun 2024Resveratrol (RSV) is a polyphenol antioxidant that has been shown to have neuroprotective effects. We sought molecular mechanisms that emphasize the anti-inflammatory...
Resveratrol (RSV) is a polyphenol antioxidant that has been shown to have neuroprotective effects. We sought molecular mechanisms that emphasize the anti-inflammatory activity of RSV in traumatic brain injury (TBI) in mice associated with endoplasmic reticulum stress (ERS). After establishing three experimental groups (sham, TBI, and TBI+RSV), we explored the results of RSV after TBI on ERS and caspase-12 apoptotic pathways. The expression levels of C/EBP homologous protein (CHOP), glucose regulated protein 78kD (GRP78), caspase-3, and caspase-12 in cortical brain tissues were assessed by western blotting. The qPCR analysis was also performed on mRNA expression of tumor necrosis factor (TNF)-α and interleukin (IL)-1β in cortical brain tissue. In addition, the expression of GRP78 in microglia (ionized calcium binding adaptor molecule 1; Iba-1) and neurons (neuronal nuclei; NeuN) was identified by immunofluorescence staining. The neurological function of mice was assessed by modified neurological severity scores (mNSS). After drug treatment, the expression of CHOP, GRP78, caspase-3 and caspase-12 decreased, and qPCR results showed that TNF-α and IL-1β were down-regulated. Immunofluorescence staining showed down-regulation of Iba-1+/GRP78+ and NeuN+/GRP78+ cells after RSV treatment. The mNSS analysis confirmed improvement after RSV treatment. RSV improved apoptosis by downregulating the ERS signaling pathway and improved neurological prognosis in mice with TBI.
Topics: Animals; Brain Injuries, Traumatic; Resveratrol; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Mice; Male; Apoptosis; Prognosis; Neuroprotective Agents; Neurons; Interleukin-1beta; Caspase 12; Heat-Shock Proteins; Tumor Necrosis Factor-alpha; Mice, Inbred C57BL; Cell Death; Microglia; Transcription Factor CHOP
PubMed: 38912865
DOI: 10.32725/jab.2024.008 -
JCI Insight Jun 2024Spermatogenesis requires precise posttranslational control in the endoplasmic reticulum (ER), but the mechanism remains largely unknown. The protein disulfide isomerase...
Spermatogenesis requires precise posttranslational control in the endoplasmic reticulum (ER), but the mechanism remains largely unknown. The protein disulfide isomerase (PDI) family is a group of thiol oxidoreductases responsible for catalyzing the disulfide bond formation of nascent proteins. In this study, we generated 14 strains of KO mice lacking the PDI family enzymes and found that only PDI deficiency caused spermatogenesis defects. Both inducible whole-body PDI-KO (UBC-Cre/Pdifl/fl) mice and premeiotic PDI-KO (Stra8-Cre/Pdifl/fl) mice experienced a significant decrease in germ cells, testicular atrophy, oligospermia, and complete male infertility. Stra8-Cre/Pdifl/fl spermatocytes had significantly upregulated ER stress-related proteins (GRP78 and XBP1) and apoptosis-related proteins (Cleaved caspase-3 and BAX), together with cell apoptosis. PDI deletion led to delayed DNA double-strand break repair and improper crossover at the pachytene spermatocytes. Quantitative mass spectrometry indicated that PDI deficiency downregulated vital proteins in spermatogenesis such as HSPA4L, SHCBP1L, and DDX4, consistent with the proteins' physical association with PDI in normal testes tissue. Furthermore, PDI served as a thiol oxidase for disulfide bond formation of SHCBP1L. Thus, PDI plays an essential role in protein quality control for spermatogenesis in mice.
Topics: Animals; Male; Spermatogenesis; Protein Disulfide-Isomerases; Mice; Mice, Knockout; Testis; Endoplasmic Reticulum Chaperone BiP; Infertility, Male; Apoptosis; Spermatocytes; Endoplasmic Reticulum Stress; Oligospermia
PubMed: 38912589
DOI: 10.1172/jci.insight.177743 -
Journal of Cancer Research and Clinical... Jun 2024Glioblastoma (GBM) is a high-grade and heterogeneous subtype of glioma that presents a substantial challenge to human health, characterized by a poor prognosis and low...
BACKGROUND
Glioblastoma (GBM) is a high-grade and heterogeneous subtype of glioma that presents a substantial challenge to human health, characterized by a poor prognosis and low survival rates. Despite its known involvement in regulating leukemia and melanoma, the function and mechanism of DNAJC1 in GBM remain poorly understood.
METHODS
Utilizing data from the TCGA, CGGA, and GEO databases, we investigated the expression pattern of DNAJC1 and its correlation with clinical characteristics in GBM specimens. Loss-of-function experiments were conducted to explore the impact of DNAJC1 on GBM cell lines, with co-culture experiments assessing macrophage infiltration and functional marker expression.
RESULTS
Our analysis demonstrated frequent overexpression of DNAJC1 in GBM, significantly associated with various clinical characteristics including WHO grade, IDH status, chromosome 1p/19q codeletion, and histological type. Moreover, Kaplan‒Meier and ROC analyses revealed DNAJC1 as a negative prognostic predictor and a promising diagnostic biomarker for GBM patients. Functional studies indicated that silencing DNAJC1 impeded cell proliferation and migration, induced cell cycle arrest, and enhanced apoptosis. Mechanistically, DNAJC1 was implicated in stimulating extracellular matrix reorganization, triggering the epithelial-mesenchymal transition (EMT) process, and initiating immunosuppressive macrophage infiltration.
CONCLUSIONS
Our findings underscore the pivotal role of DNAJC1 in GBM pathogenesis, suggesting its potential as a diagnostic and therapeutic target for this challenging disease.
Topics: Humans; Glioblastoma; Brain Neoplasms; Macrophages; Disease Progression; Extracellular Matrix; Prognosis; HSP40 Heat-Shock Proteins; Cell Line, Tumor; Animals; Male; Female; Mice; Biomarkers, Tumor; Cell Proliferation; Epithelial-Mesenchymal Transition; Cell Movement; Gene Expression Regulation, Neoplastic; Apoptosis; Middle Aged
PubMed: 38909166
DOI: 10.1007/s00432-024-05823-1 -
Cell Stress & Chaperones Jun 2024Anaplasma phagocytophilum is an intracellular tick-transmitted bacterial pathogen which infects neutrophils in mammals and causes granulocytic anaplasmosis. In this...
Anaplasma phagocytophilum is an intracellular tick-transmitted bacterial pathogen which infects neutrophils in mammals and causes granulocytic anaplasmosis. In this study, we investigated the molecular chaperones ClpB and DnaK from A. phagocytophilum. In E. coli, ClpB cooperates with DnaK and its co-chaperones DnaJ and GrpE in ATP-dependent reactivation of aggregated proteins. Since ClpB is not produced in metazoans, it is a promising target for developing antimicrobial therapies, which generates interest in studies on that chaperone's role in pathogenic bacteria. We found that ClpB and DnaK are transcriptionally upregulated in A. phagocytophilum 3-5 days after infection of human HL-60 and tick ISE6 cells, which suggests an essential role of the chaperones in supporting the pathogen's intracellular life cycle. Multiple sequence alignments show that A. phagocytophilum ClpB and DnaK contain all structural domains that were identified in their previously studied orthologs from other bacteria. Both A. phagocytophilum ClpB and DnaK display ATPase activity, which is consistent with their participation in the ATP-dependent protein disaggregation system. However, despite a significant sequence similarity between the chaperones from A. phagocytophilum and those from E. coli, the former were not as effective as their E. coli orthologs during reactivation of aggregated proteins in vitro and in supporting survival of E. coli cells under heat stress. We conclude that the A. phagocytophilum chaperones might have evolved with distinct biochemical properties to maintain the integrity of pathogenic proteins under unique stress conditions of an intracellular environment of host cells.
PubMed: 38908470
DOI: 10.1016/j.cstres.2024.06.003 -
Molecular Cell Jun 2024Protein folding is assisted by molecular chaperones that bind nascent polypeptides during mRNA translation. Several structurally distinct classes of chaperones promote...
Protein folding is assisted by molecular chaperones that bind nascent polypeptides during mRNA translation. Several structurally distinct classes of chaperones promote de novo folding, suggesting that their activities are coordinated at the ribosome. We used biochemical reconstitution and structural proteomics to explore the molecular basis for cotranslational chaperone action in bacteria. We found that chaperone binding is disfavored close to the ribosome, allowing folding to precede chaperone recruitment. Trigger factor recognizes compact folding intermediates that expose an extensive unfolded surface, and dictates DnaJ access to nascent chains. DnaJ uses a large surface to bind structurally diverse intermediates and recruits DnaK to sequence-diverse solvent-accessible sites. Neither Trigger factor, DnaJ, nor DnaK destabilize cotranslational folding intermediates. Instead, the chaperones collaborate to protect incipient structure in the nascent polypeptide well beyond the ribosome exit tunnel. Our findings show how the chaperone network selects and modulates cotranslational folding intermediates.
PubMed: 38908370
DOI: 10.1016/j.molcel.2024.06.002 -
Communications Biology Jun 2024Pod is an important organ for seed production in soybean. Pod size varies among soybean cultivars, but the mechanism is largely unknown. Here we reveal one of the...
Pod is an important organ for seed production in soybean. Pod size varies among soybean cultivars, but the mechanism is largely unknown. Here we reveal one of the factors for pod size regulation. We investigate pod size differences between two cultivars. The longer pod of 'Tachinagaha' is due to more cell number than in the short pod of 'Iyodaizu'. POD SIZE OF SOYBEAN 8 (GmPSS8), a member of the heat shock protein 70 (HSP70) family, is identified as a candidate gene for determining pod length in a major QTL for pod length. Expression of GmPSS8 in pods is higher in 'Tachinagaha' than 'Iyodaizu' and is highest in early pod development. The difference in expression is the result of an in/del polymorphism which includes an enhancer motif. Treatment with an HSP70 inhibitor reduces pod length and cell number in the pod. Additionally, shorter pods in Arabidopsis hsp70-1/-4 double mutant are rescued by overexpression of GmPSS8. Our results identify GmPSS8 as a target gene for pod length, which regulates cell number during early pod development through regulation of transcription in soybean. Our findings provide the mechanisms of pod development and suggest possible strategies enhancing yield potential in soybean.
Topics: Glycine max; HSP70 Heat-Shock Proteins; Cell Proliferation; Gene Expression Regulation, Plant; Plant Proteins; Quantitative Trait Loci; Seeds; Arabidopsis
PubMed: 38906939
DOI: 10.1038/s42003-024-06443-8 -
Life Science Alliance Sep 2024H3.1 histone is predominantly synthesized and enters the nucleus during the G1/S phase of the cell cycle, as a new component of duplicating nucleosomes. Here, we found...
H3.1 histone is predominantly synthesized and enters the nucleus during the G1/S phase of the cell cycle, as a new component of duplicating nucleosomes. Here, we found that p53 is necessary to secure the normal behavior and modification of H3.1 in the nucleus during the G1/S phase, in which p53 increases C-terminal domain nuclear envelope phosphatase 1 (CTDNEP1) levels and decreases enhancer of zeste homolog 2 (EZH2) levels in the H3.1 interactome. In the absence of p53, H3.1 molecules tended to be tethered at or near the nuclear envelope (NE), where they were predominantly trimethylated at lysine 27 (H3K27me3) by EZH2, without forming nucleosomes. This accumulation was likely caused by the high affinity of H3.1 toward phosphatidic acid (PA). p53 reduced nuclear PA levels by increasing levels of CTDNEP1, which activates lipin to convert PA into diacylglycerol. We moreover found that the cytosolic H3 chaperone HSC70 attenuates the H3.1-PA interaction, and our molecular imaging analyses suggested that H3.1 may be anchored around the NE after their nuclear entry. Our results expand our knowledge of p53 function in regulation of the nuclear behavior of H3.1 during the G1/S phase, in which p53 may primarily target nuclear PA and EZH2.
Topics: Histones; Tumor Suppressor Protein p53; Cell Nucleus; Humans; Enhancer of Zeste Homolog 2 Protein; G1 Phase; S Phase; Nuclear Envelope; Methylation; Animals; Nucleosomes
PubMed: 38906678
DOI: 10.26508/lsa.202402835