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The Journal of Biological Chemistry Sep 2002Tunicamycin is a reversible inhibitor of polyprenol-phosphate: N-acetylhexosamine-1-phosphate translocases and is produced by several Streptomyces species. We have...
Tunicamycin is a reversible inhibitor of polyprenol-phosphate: N-acetylhexosamine-1-phosphate translocases and is produced by several Streptomyces species. We have examined tunicamycin biosynthesis, an important but poorly characterized biosynthetic pathway. Biosynthetic precursors have been identified by incorporating radioactive and stable isotopes, and by determining the labeling pattern using electrospray ionization-collision induced dissociation-mass spectrometry (ESI-CID-MS), and proton, deuterium, and C-13 nuclear magnetic resonance (NMR) spectroscopy. Preparation and analysis of [uracil-5-(2)H]-labeled tunicamycin established the complete ESI-CID-MS fragmentation pathway for the major components of the tunicamycin complex. Competitive metabolic experiments indicate that 7 deuteriums incorporate into tunicamycin from [6,6'-(2)H,(2)H]-labeled D-glucose, 6 of which arise from D-GlcNAc and 1 from uridine and/or D-ribose. Inverse correlation NMR experiments (heteronuclear single-quantum coherence (HSQC)) of (13)C-labeled tunicamycin enriched from D-[1-(13)C]glucose suggest that the unique tunicamine 11-carbon dialdose sugar backbone arises from a 5-carbon furanose precursor derived from uridine and a 6-carbon N-acetylamino-pyranose precursor derived from UDP-D-N-acetylglucosamine. The equivalent incorporation of (13)C into both the alpha-1" and beta-11' anomeric carbons of tunicamycin supports a direct biosynthesis via 6-carbon metabolism. It also indicates that the tunicamine motif and the alpha-1"-linked GlcNAc residue are both derived from the same metabolic pool of UDP-GlcNAc, without significant differential metabolic processing. A biosynthetic pathway is therefore proposed for tunicamycin for the first time: an initial formation of the 11-carbon tunicamine sugar motif from uridine and UDP-GlcNAc via uridine-5'-aldehyde and UDP-4-keto-6-ene-N-acetylhexosamine, respectively, and subsequent formation of the anomeric-to-anomeric alpha, beta-1",11'-glycosidic bond.
Topics: Carbohydrate Sequence; Carbon; Chromatography, High Pressure Liquid; Disaccharides; Galactosamine; Nuclear Magnetic Resonance, Biomolecular; Spectrometry, Mass, Electrospray Ionization; Tunicamycin
PubMed: 12093793
DOI: 10.1074/jbc.M201345200 -
Biochemistry Feb 2019C1q/TNF-related protein 12 (CTRP12) is a secreted regulator of glucose and lipid metabolism. It circulates in plasma as a full-length protein or as a cleaved isoform...
C1q/TNF-related protein 12 (CTRP12) is a secreted regulator of glucose and lipid metabolism. It circulates in plasma as a full-length protein or as a cleaved isoform generated by furin/PCSK3 cleavage. These isoforms preferentially activate different signaling pathways, and their ratio in plasma is altered in obesity and diabetes. Here, we show that three conserved asparagine residues (Asn-39, Asn-287, and Asn-297) play important roles in modulating CTRP12 cleavage, secretion, and stability. Mass spectrometry analysis provided direct evidence of Asn-39 glycosylation. When N-linked glycosylation was inhibited by tunicamycin or abolished by the N39Q, N39A, or T41A mutation, CTRP12 cleavage was enhanced. Complex-type N-glycans on CTRP12 blocked cleavage by the Golgi-localized furin. In N-acetylglucosaminyltransferase I (GnTI)-deficient cells that could not form hybrid and complex-type N-glycans in the Golgi, CTRP12 cleavage was enhanced, and re-expressing GnTI reduced cleavage. Replacing the nonglycosylated Asn-297 with glutamine or alanine also increased CTRP12 cleavage. Both Asn-39 and Asn-297 contributed independently to CTRP12 cleavage: maximum cleavage was observed in the double mutant. In addition, CTRP12 cleavage was abolished in furin-deficient cells and restored by furin re-expression. Replacing the nonglycosylated Asn-287 with glutamine or alanine resulted in protein misfolding and aggregation, leading to retention in the endoplasmic reticulum. Cycloheximide chase analyses indicated reduced protein stability for N39Q, T41A, and N297Q mutants. Lastly, we show that increasing the flux through the hexosamine biosynthesis pathway by exogenous glucosamine, known to disrupt protein glycosylation, also promoted CTRP12 cleavage. Combined, these data highlight glycosylation-dependent and -independent mechanisms regulating CTRP12 cleavage, secretion, and protein stability.
Topics: Adipokines; Animals; Asparagine; Cell Line, Tumor; Furin; Glucosamine; Glycosylation; HEK293 Cells; Humans; Mice; Protein Stability; Proteolysis; Tunicamycin
PubMed: 30566828
DOI: 10.1021/acs.biochem.8b00528 -
Analytical Chemistry Sep 2022Protein structures are decisive for their activities and interactions with other molecules. Global analysis of protein structures and conformational changes cannot be...
Protein structures are decisive for their activities and interactions with other molecules. Global analysis of protein structures and conformational changes cannot be achieved by commonly used abundance-based proteomics. Here, we integrated cysteine covalent labeling, selective enrichment, and quantitative proteomics to study protein structures and structural changes on a large scale. This method was applied to globally investigate protein structures in HEK293T cells and protein structural changes in the cells with the tunicamycin (Tm)-induced endoplasmic reticulum (ER) stress. We quantified several thousand cysteine residues, which contain unprecedented and valuable information of protein structures. Combining this method with pulsed stable isotope labeling by amino acids in cell culture, we further analyzed the folding state differences between pre-existing and newly synthesized proteins in cells under the Tm treatment. Besides newly synthesized proteins, unexpectedly, many pre-existing proteins were found to become unfolded upon ER stress, especially those related to gene transcription and protein translation. Furthermore, the current results reveal that N-glycosylation plays a more important role in the folding process of the tertiary and quaternary structures than the secondary structures for newly synthesized proteins. Considering the importance of cysteine in protein structures, this method can be extensively applied in the biological and biomedical research fields.
Topics: Cysteine; Endoplasmic Reticulum Stress; HEK293 Cells; Humans; Proteins; Proteomics; Tunicamycin
PubMed: 36108266
DOI: 10.1021/acs.analchem.2c03076 -
International Journal of Molecular... Feb 2023Female infertility is caused by premature ovarian failure (POF), which is triggered by the endoplasmic reticulum (ER) stress-mediated apoptosis of granulosa cells. The...
Female infertility is caused by premature ovarian failure (POF), which is triggered by the endoplasmic reticulum (ER) stress-mediated apoptosis of granulosa cells. The ER unfolded protein response (UPR) is initiated to promote cell survival by alleviating excessive ER stress, but cellular apoptosis is induced by persistent or strong ER stress. Recent studies have reported that reticulophagy is initiated by ER stress. Whether reticulophagy is activated in the ER stress-mediated apoptosis of granulosa cells and which pathway is initiated to activate reticulophagy during the apoptosis of granulosa cells are unknown. Therefore, the role of reticulophagy in granulosa cell death and the relationship between ER stress and reticulophagy were investigated in this work. Our results suggest that the ER stress inducer tunicamycin causes POF in mice, which is attributed to the apoptosis of granulosa cells and is accompanied by the activation of UPR and reticulophagy. Furthermore, granulosa cells were treated with tunicamycin, and granulosa cell apoptosis was triggered and increased the expression of UPR and reticulophagy molecules. The expression of ATF4 was then downregulated by RNAi, which decreased the levels of autophagy and the reticulophagy receptor CCGP1. Furthermore, ATF4 targets MAP1LC3A, as revealed by the ChIP sequencing results, and co-IP results demonstrated that MAP1LC3A interacts with CCPG1. Therefore, reticulophagy was activated by ER stress through the ATF4-MAP1LC3A-CCPG1 pathway to mitigate ER stress. Additionally, the role of reticulophagy in granulosa cells was investigated by the knockdown of CCPG1 with RNAi. Interestingly, only a small number of granulosa cells died by apoptosis, whereas the death of most granulosa cells occurred by necroptosis triggered by STAT1 and STAT3 to impair ER proteostasis and the ER protein quality control system UPR. Taken together, the results indicate that the necroptosis of granulosa cells is triggered by up- and downregulating the reticulophagy receptor CCPG1 through STAT1/STAT3-(p)RIPK1-(p)RIPK3-(p)MLKL and that reticulophagy is activated by ER stress through the ATF4-MAP1LC3A-CCPG1 pathway.
Topics: Female; Mice; Animals; Tunicamycin; Necroptosis; Endoplasmic Reticulum Stress; Unfolded Protein Response; Autophagy; Apoptosis; Granulosa Cells
PubMed: 36769070
DOI: 10.3390/ijms24032749 -
Cell Sep 2006Glycans are central to many biological processes, but efforts to define their functions at the molecular level have been frustrated by a lack of suitable technologies.... (Review)
Review
Glycans are central to many biological processes, but efforts to define their functions at the molecular level have been frustrated by a lack of suitable technologies. Here we highlight chemical tools that are beginning to address this need.
Topics: Carbohydrate Conformation; Carbohydrate Sequence; Enzyme Inhibitors; Glycoproteins; Glycosylation; Glycosyltransferases; Green Fluorescent Proteins; Molecular Sequence Data; Photoaffinity Labels; Protein Array Analysis; Protein Conformation; Protein Processing, Post-Translational; Proteomics; Tunicamycin
PubMed: 16959565
DOI: 10.1016/j.cell.2006.08.017 -
Biochemical and Biophysical Research... Nov 2022Increased endoplasmic reticulum (ER) stress is strongly associated with the phenotypic switching of vascular smooth muscle cells (VSMCs) in atherosclerosis. Depletion of...
BACKGROUND AND AIMS
Increased endoplasmic reticulum (ER) stress is strongly associated with the phenotypic switching of vascular smooth muscle cells (VSMCs) in atherosclerosis. Depletion of the ER Ca content is one of the leading causes of increased ER stress in VSMCs. The ryanodine receptor (RyR) is a major Ca release channel in the sarcoplasmic reticulum membrane. Calmodulin (CaM), which binds to RyR (CaM-RyR), stabilizes the closed state of RyR in the resting state in normal cells. Defective CaM-RyR interactions can cause abnormal Ca leakage through RyR, resulting in decreased Ca content, indicating that defective CaM-RyR interactions may be a cause of increased ER stress. Herein, we used a mouse VSMCs to assess whether CaM-RyR plays a pivotal role in VSMCs phenotypic switching, which is caused by ER stress, and whether dantrolene, which enhances the binding affinity of CaM to RyR, affects VSMCs phenotypic switching.
METHODS AND RESULTS
Tunicamycin was used to mimic ER stress in vitro. Tunicamycin-induced ER stress caused CaM to dissociate from the RyR and translocate to the nucleus, which stimulated phenotypic switching through the activation of MEF2 and KLF5. Dantrolene suppressed tunicamycin-induced apoptosis, ER stress (restoring ER Ca content), and phenotypic switching of VSMCs. Suramin, which directly unbinds CaM from RyR, promoted nuclear CaM accumulation with parallel VSMCs phenotypic switching, and dantrolene prevented these effects.
CONCLUSIONS
We observed that ER stress causes CaM translocation to the nucleus and drives the phenotypic switching of VSMCs. Thus, restoration of the binding affinity of CaM to RyR may be a therapeutic target for atherosclerosis.
Topics: Animals; Atherosclerosis; Calmodulin; Dantrolene; Endoplasmic Reticulum Stress; Mice; Muscle, Smooth, Vascular; Ryanodine Receptor Calcium Release Channel; Suramin; Tunicamycin
PubMed: 36099691
DOI: 10.1016/j.bbrc.2022.08.069 -
Journal of Healthcare Engineering 2021Triple negative breast cancer (TNBC) has significantly threatened human health. Many aspects of TNBC are closely related to Wnt/-catenin pathway, and cell apoptosis...
Triple negative breast cancer (TNBC) has significantly threatened human health. Many aspects of TNBC are closely related to Wnt/-catenin pathway, and cell apoptosis induced by endoplasmic reticulum stress (ER stress) in TNBC may act as a potential target of non-chemotherapy treatment. However, how ER stress interacts with this pathway in TNBC has not yet been understood. Here, the tunicamycin and LiCl have been applied to MDA-MB-231. The related proteins' expression was measured by western blotting. Moreover, acridine orange/ethidium bromide (AO/EB) staining was applied to test the apoptosis degree of the cells, and cell viability was tested by MTT experiment. Then, we found the ER stress and apoptosis degree of MDA-MB-231 were induced after treatment with tunicamycin. Besides, tunicamycin dose dependently inhibited both Wnt/-catenin pathway and cells viability. Licl, an activator of Wnt/-catenin signaling pathway, could significantly inhibit cell apoptosis. In conclusion, our study found that the activation of ER stress could promote the MDA-MB-231 apoptosis by repressing Wnt/-catenin pathway, which provides some promising prospects and basic mechanism to the further research.
Topics: Apoptosis; Cell Line, Tumor; Cell Proliferation; Endoplasmic Reticulum Stress; Humans; Triple Negative Breast Neoplasms; Tunicamycin; Wnt Signaling Pathway
PubMed: 34336161
DOI: 10.1155/2021/6394514 -
PloS One 2021Endoplasmic reticulum (ER) stress is associated with acute kidney injury (AKI) caused by various mechanisms, including antibiotics, non-steroidal anti-inflammatory...
Endoplasmic reticulum (ER) stress is associated with acute kidney injury (AKI) caused by various mechanisms, including antibiotics, non-steroidal anti-inflammatory drugs, cisplatin, and radiocontrast. Tunicamycin (TM) is a nucleoside antibiotic that induces ER stress and is a commonly used model of AKI. 4-phenylbutyrate (4-PBA) is a chemical chaperone and histone deacetylase (HDAC) inhibitor and has been shown to protect the kidney from ER stress, apoptosis, and structural damage in a tunicamycin model of AKI. The renal protection provided by 4-PBA is attributed to its ability to prevent misfolded protein aggregation and inhibit ER stress; however, the HDAC inhibitor effects of 4-PBA have not been examined in the TM-induced model of AKI. As such, the main objective of this study was to determine if histone hyperacetylation provides any protective effects against TM-mediated AKI. The FDA-approved HDAC inhibitor vorinostat was used, as it has no ER stress inhibitory effects and therefore the histone hyperacetylation properties alone could be investigated. In vitro work demonstrated that vorinostat inhibited histone deacetylation in cultured proximal tubular cells but did not prevent ER stress or protein aggregation induced by TM. Vorinostat induced a significant increase in cell death, and exacerbated TM-mediated total cell death and apoptotic cell death. Wild type male mice were treated with TM (0.5 mg/kg, intraperitoneal injection), with or without vorinostat (50 mg/kg/day) or 4-PBA (1 g/kg/day). Mice treated with 4-PBA or vorinostat exhibited similar levels of histone hyperacetylation. Expression of the pro-apoptotic protein CHOP was induced with TM, and not inhibited by vorinostat. Further, vorinostat did not prevent any renal damage or decline in renal function caused by tunicamycin. These data suggest that the protective mechanisms found by 4-PBA are primarily due to its molecular chaperone properties, and the HDAC inhibitors used did not provide any protection against renal injury caused by ER stress.
Topics: Acute Kidney Injury; Animals; Cell Line; Disease Models, Animal; Endoplasmic Reticulum Stress; Histone Deacetylase Inhibitors; Male; Mice; Protein Aggregation, Pathological; Tunicamycin; Vorinostat
PubMed: 34847196
DOI: 10.1371/journal.pone.0260519 -
Genome Research Dec 2016Gene-by-environment (GxE) interactions determine common disease risk factors and biomedically relevant complex traits. However, quantifying how the environment modulates...
Gene-by-environment (GxE) interactions determine common disease risk factors and biomedically relevant complex traits. However, quantifying how the environment modulates genetic effects on human quantitative phenotypes presents unique challenges. Environmental covariates are complex and difficult to measure and control at the organismal level, as found in GWAS and epidemiological studies. An alternative approach focuses on the cellular environment using in vitro treatments as a proxy for the organismal environment. These cellular environments simplify the organism-level environmental exposures to provide a tractable influence on subcellular phenotypes, such as gene expression. Expression quantitative trait loci (eQTL) mapping studies identified GxE interactions in response to drug treatment and pathogen exposure. However, eQTL mapping approaches are infeasible for large-scale analysis of multiple cellular environments. Recently, allele-specific expression (ASE) analysis emerged as a powerful tool to identify GxE interactions in gene expression patterns by exploiting naturally occurring environmental exposures. Here we characterized genetic effects on the transcriptional response to 50 treatments in five cell types. We discovered 1455 genes with ASE (FDR < 10%) and 215 genes with GxE interactions. We demonstrated a major role for GxE interactions in complex traits. Genes with a transcriptional response to environmental perturbations showed sevenfold higher odds of being found in GWAS. Additionally, 105 genes that indicated GxE interactions (49%) were identified by GWAS as associated with complex traits. Examples include GIPR-caffeine interaction and obesity and include LAMP3-selenium interaction and Parkinson disease. Our results demonstrate that comprehensive catalogs of GxE interactions are indispensable to thoroughly annotate genes and bridge epidemiological and genome-wide association studies.
Topics: Alleles; Caffeine; Cell Line; Gene Expression Profiling; Gene Expression Regulation; Gene-Environment Interaction; Genome-Wide Association Study; Human Umbilical Vein Endothelial Cells; Humans; Melanocytes; Quantitative Trait Loci; Selenium; Tunicamycin
PubMed: 27934696
DOI: 10.1101/gr.209759.116 -
Journal of Cellular and Molecular... Jan 2015The endoplasmic reticulum (ER) is an organelle important for protein synthesis and folding, lipid synthesis and Ca(2+) homoeostasis. Consequently, ER stress or...
The endoplasmic reticulum (ER) is an organelle important for protein synthesis and folding, lipid synthesis and Ca(2+) homoeostasis. Consequently, ER stress or dysfunction affects numerous cellular processes and has been implicated as a contributing factor in several pathophysiological conditions. Tunicamycin induces ER stress in various cell types in vitro as well as in vivo. In mice, a hallmark of tunicamycin administration is the development of fatty livers within 24-48 hrs accompanied by hepatic ER stress. We hypothesized that tunicamycin would induce ER stress in adipose tissue that would lead to increased lipolysis and subsequently to fatty infiltration of the liver and hepatomegaly. Our results show that intraperitoneal administration of tunicamycin rapidly induced an ER stress response in adipose tissue that correlated with increased circulating free fatty acids (FFAs) and glycerol along with decreased adipose tissue mass and lipid droplet size. Furthermore, we found that in addition to fatty infiltration of the liver as well as hepatomegaly, lipid accumulation was also present in the heart, skeletal muscle and kidney. To corroborate our findings to a clinical setting, we examined adipose tissue from burned patients where increases in lipolysis and the development of fatty livers have been well documented. We found that burned patients displayed significant ER stress within adipose tissue and that ER stress augments lipolysis in cultured human adipocytes. Our results indicate a possible role for ER stress induced lipolysis in adipose tissue as an underlying mechanism contributing to increases in circulating FFAs and fatty infiltration into other organs.
Topics: Adipose Tissue; Animals; Burns; Cell Separation; Cells, Cultured; Endoplasmic Reticulum Stress; Fatty Acids; Hepatocytes; Humans; Lipolysis; Male; Mice, Inbred BALB C; Organ Specificity; Tunicamycin
PubMed: 25381905
DOI: 10.1111/jcmm.12384