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Redox Biology Aug 2022Blood-testis barrier (BTB) damage promotes spermatogenesis dysfunction, which is a critical cause of male infertility. Dyslipidemia has been correlated with male...
Blood-testis barrier (BTB) damage promotes spermatogenesis dysfunction, which is a critical cause of male infertility. Dyslipidemia has been correlated with male infertility, but the major hazardous lipid and the underlying mechanism remains unclear. In this study, we firstly discovered an elevation of palmitic acid (PA) and a decrease of inhibin B in patients with severe dyszoospermia, which leaded us to explore the effects of PA on Sertoli cells. We observed a damage of BTB by PA. PA penetration to endoplasmic reticulum (ER) and its damage to ER structures were exhibited by microimaging and dynamic observation, and consequent ER stress was proved to mediate PA-induced Sertoli cell barrier disruption. Remarkably, we demonstrated a critical role of aberrant protein palmitoylation in PA-induced Sertoli cell barrier dysfunction. An ER protein, Calnexin, was screened out and was demonstrated to participate in this process, and suppression of its palmitoylation showed an ameliorating effect. We also found that ω-3 poly-unsaturated fatty acids down-regulated Calnexin palmitoylation, and alleviated BTB dysfunction. Our results indicate that dysregulated palmitoylation induced by PA plays a pivotal role in BTB disruption and subsequent spermatogenesis dysfunction, suggesting that protein palmitoylation might be therapeutically targetable in male infertility.
Topics: Blood-Testis Barrier; Calnexin; Humans; Infertility, Male; Lipoylation; Male; Palmitic Acid; Spermatogenesis
PubMed: 35803125
DOI: 10.1016/j.redox.2022.102380 -
Autophagy Oct 2022The endolysosomal system not only is an integral part of the cellular catabolic machinery that processes and recycles nutrients for synthesis of biomaterials, but also...
The endolysosomal system not only is an integral part of the cellular catabolic machinery that processes and recycles nutrients for synthesis of biomaterials, but also acts as signaling hub to sense and coordinate the energy state of cells with growth and differentiation. Lysosomal dysfunction adversely influences vesicular transport-dependent macromolecular degradation and thus causes serious problems for human health. In mammalian cells, loss of the lysosome associated membrane proteins LAMP1 and LAMP2 strongly affects autophagy and cholesterol trafficking. Here we show that the previously uncharacterized Lamp1 is a ortholog of vertebrate LAMP1 and LAMP2. Surprisingly and in contrast to double-mutant mice, Lamp1 is not required for viability or autophagy, suggesting that fly and vertebrate LAMP proteins acquired distinct functions, or that autophagy defects in mutants may have indirect causes. However, Lamp1 deficiency results in an increase in the number of acidic organelles in flies. Furthermore, we find that mutant larvae have defects in lipid metabolism as they show elevated levels of sterols and diacylglycerols (DAGs). Because DAGs are the main lipid species used for transport through the hemolymph (blood) in insects, our results indicate broader functions of Lamp1 in lipid transport. Our findings make an ideal model to study the role of LAMP proteins in lipid assimilation without the confounding effects of their storage and without interfering with autophagic processes.: aa: amino acid; AL: autolysosome; AP: autophagosome; APGL: autophagolysosome; AV: autophagic vacuole (i.e. AP and APGL/AL); AVi: early/initial autophagic vacuoles; AVd: late/degradative autophagic vacuoles; : autophagy-related; CMA: chaperone-mediated autophagy; Cnx99A: Calnexin 99A; DAG: diacylglycerol; eMI: endosomal microautophagy; ESCRT: endosomal sorting complexes required for transport; FB: fat body; HDL: high-density lipoprotein; Hrs: Hepatocyte growth factor regulated tyrosine kinase substrate; LAMP: lysosomal associated membrane protein; LD: lipid droplet; LDL: low-density lipoprotein; Lpp: lipophorin; LTP: Lipid transfer particle; LTR: LysoTracker Red; MA: macroautophagy; MCC: Manders colocalization coefficient; MEF: mouse embryonic fibroblast MTORC: mechanistic target of rapamycin kinase complex; PV: parasitophorous vacuole; SNARE: soluble N-ethylmaleimide sensitive factor attachment protein receptor; Snap: Synaptosomal-associated protein; st: starved; TAG: triacylglycerol; TEM: transmission electron microscopy; TFEB/Mitf: transcription factor EB; TM: transmembrane domain; tub: tubulin; UTR: untranslated region.
Topics: Amino Acids; Animals; Autophagy; Biocompatible Materials; Calnexin; Diglycerides; Drosophila; Drosophila Proteins; Endosomal Sorting Complexes Required for Transport; Ethylmaleimide; Fibroblasts; Hepatocyte Growth Factor; Humans; Lipoproteins, HDL; Lipoproteins, LDL; Lysosomal Membrane Proteins; Lysosomes; Mammals; Mice; Protein-Tyrosine Kinases; SNARE Proteins; Sirolimus; Sterols; Triglycerides; Tubulin; Untranslated Regions
PubMed: 35266854
DOI: 10.1080/15548627.2022.2038999 -
Autophagy Dec 2022Amino acids play crucial roles in the MTOR (mechanistic target of rapamycin kinase) complex 1 (MTORC1) pathway. However, the underlying mechanisms are not fully...
Amino acids play crucial roles in the MTOR (mechanistic target of rapamycin kinase) complex 1 (MTORC1) pathway. However, the underlying mechanisms are not fully understood. Here, we establish a cell-free system to mimic the activation of MTORC1, by which we identify CANX (calnexin) as an essential regulator for leucine-stimulated MTORC1 pathway. CANX translocates to lysosomes after leucine deprivation, and its loss of function renders either the MTORC1 activity or the lysosomal translocation of MTOR insensitive to leucine deprivation. We further find that CANX binds to LAMP2 (lysosomal associated membrane protein 2), and LAMP2 is required for leucine deprivation-induced CANX interaction with the Ragulator to inhibit Ragulator activity toward RRAG GTPases. Moreover, leucine deprivation promotes the lysine (K) 525 crotonylation of CANX, which is another essential condition for the lysosomal translocation of CANX. Finally, we find that KAT7 (lysine acetyltransferase 7) mediates the K525 crotonylation of CANX. Loss of KAT7 renders the MTORC1 insensitivity to leucine deprivation. Our findings provide new insights for the regulatory mechanism of the leucine-stimulated MTORC1 pathway. CALR: calreticulin; CANX: calnexin; CLF: crude lysosome fraction; EIF4EBP1: eukaryotic translation initiation factor 4E binding protein 1; ER: endoplasmic reticulum; GST: glutathione S-transferase; HA: hemagglutinin; HEK293T: human embryonic kidney-293T; KAT7: lysine acetyltransferase 7; Kcr; lysine crotonylation; KO: knockout; LAMP2: lysosomal associated membrane protein 2; LAMTOR/Ragulator: late endosomal/lysosomal adaptor: MAPK and MTOR activator; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; PDI: protein disulfide isomerase; PTM: post-translational modification; RPS6KB1/p70S6 kinase 1: ribosomal protein S6 kinase B1; RPTOR: regulatory associated protein of MTOR complex 1; SESN2: sestrin 2; TMEM192: transmembrane protein 192; ULK1: unc-51 like autophagy activating kinase 1.
Topics: Humans; Autophagy; Calnexin; HEK293 Cells; Leucine; Lysine; Lysine Acetyltransferases; Lysosomal-Associated Membrane Protein 2; Lysosomes; Mechanistic Target of Rapamycin Complex 1; Signal Transduction
PubMed: 35266843
DOI: 10.1080/15548627.2022.2047481 -
Nature Medicine Dec 2014Proper function of the endoplasmic reticulum (ER) and mitochondria is crucial for cellular homeostasis, and dysfunction at either site has been linked to...
Proper function of the endoplasmic reticulum (ER) and mitochondria is crucial for cellular homeostasis, and dysfunction at either site has been linked to pathophysiological states, including metabolic diseases. Although the ER and mitochondria play distinct cellular roles, these organelles also form physical interactions with each other at sites defined as mitochondria-associated ER membranes (MAMs), which are essential for calcium, lipid and metabolite exchange. Here we show that in the liver, obesity leads to a marked reorganization of MAMs resulting in mitochondrial calcium overload, compromised mitochondrial oxidative capacity and augmented oxidative stress. Experimental induction of ER-mitochondria interactions results in oxidative stress and impaired metabolic homeostasis, whereas downregulation of PACS-2 or IP3R1, proteins important for ER-mitochondria tethering or calcium transport, respectively, improves mitochondrial oxidative capacity and glucose metabolism in obese animals. These findings establish excessive ER-mitochondrial coupling as an essential component of organelle dysfunction in obesity that may contribute to the development of metabolic pathologies such as insulin resistance and diabetes.
Topics: Animals; Calcium; Calnexin; Disease Models, Animal; Down-Regulation; Endoplasmic Reticulum; Endoplasmic Reticulum Stress; GTP Phosphohydrolases; Glucose; Hepatocytes; Inositol 1,4,5-Trisphosphate Receptors; Lipid Metabolism; Liver; Mice; Microscopy, Electron, Transmission; Mitochondria; Obesity; Oxidative Stress; Vesicular Transport Proteins
PubMed: 25419710
DOI: 10.1038/nm.3735 -
Cells Jan 2023Calnexin is a type I integral endoplasmic reticulum (ER) membrane protein with an N-terminal domain that resides in the lumen of the ER and a C-terminal domain that... (Review)
Review
Calnexin is a type I integral endoplasmic reticulum (ER) membrane protein with an N-terminal domain that resides in the lumen of the ER and a C-terminal domain that extends into the cytosol. Calnexin is commonly referred to as a molecular chaperone involved in the folding and quality control of membrane-associated and secreted proteins, a function that is attributed to its ER- localized domain with a structure that bears a strong resemblance to another luminal ER chaperone and Ca-binding protein known as calreticulin. Studies have discovered that the cytosolic C-terminal domain of calnexin undergoes distinct post-translational modifications and interacts with a variety of proteins. Here, we discuss recent findings and hypothesize that the post-translational modifications of the calnexin C-terminal domain and its interaction with specific cytosolic proteins play a role in coordinating ER functions with events taking place in the cytosol and other cellular compartments.
Topics: Calnexin; Molecular Chaperones; Endoplasmic Reticulum; Membrane Proteins; Cytosol
PubMed: 36766745
DOI: 10.3390/cells12030403 -
The Journal of Cell Biology Oct 2023In mammalian cells, misfolded glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are cleared out of the ER to the Golgi via a constitutive and a...
In mammalian cells, misfolded glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are cleared out of the ER to the Golgi via a constitutive and a stress-inducible pathway called RESET. From the Golgi, misfolded GPI-APs transiently access the cell surface prior to rapid internalization for lysosomal degradation. What regulates the release of misfolded GPI-APs for RESET during steady-state conditions and how this release is accelerated during ER stress is unknown. Using mutants of prion protein or CD59 as model misfolded GPI-APs, we demonstrate that inducing calnexin degradation or upregulating calnexin-binding glycoprotein expression triggers the release of misfolded GPI-APs for RESET. Conversely, blocking protein synthesis dramatically inhibits the dissociation of misfolded GPI-APs from calnexin and subsequent turnover. We demonstrate an inverse correlation between newly synthesized calnexin substrates and RESET substrates that coimmunoprecipitate with calnexin. These findings implicate competition by newly synthesized substrates for association with calnexin as a key factor in regulating the release of misfolded GPI-APs from calnexin for turnover via the RESET pathway.
Topics: Animals; Calnexin; Cell Membrane; Glycosylphosphatidylinositols; Mammals; Molecular Chaperones; Prions; Endoplasmic Reticulum; Golgi Apparatus; Protein Folding; GPI-Linked Proteins
PubMed: 37702712
DOI: 10.1083/jcb.202108160 -
The FEBS Journal Oct 2020The endoplasmic reticulum (ER) is the major folding compartment for secreted and membrane proteins and is the site of a specific chaperone system, the calnexin cycle,... (Review)
Review
The endoplasmic reticulum (ER) is the major folding compartment for secreted and membrane proteins and is the site of a specific chaperone system, the calnexin cycle, for folding N-glycosylated proteins. Recent structures of components of the calnexin cycle have deepened our understanding of quality control mechanisms and protein folding pathways in the ER. In the calnexin cycle, proteins carrying monoglucosylated glycans bind to the lectin chaperones calnexin and calreticulin, which recruit a variety of function-specific chaperones to mediate protein disulfide formation, proline isomerization, and general protein folding. Upon trimming by glucosidase II, the glycan without an inner glucose residue is no longer able to bind to the lectin chaperones. For proteins that have not yet folded properly, the enzyme UDP-glucose:glycoprotein glucosyltransferase (UGGT) acts as a checkpoint by adding a glucose back to the N-glycan. This allows the misfolded proteins to re-associate with calnexin and calreticulin for additional rounds of chaperone-mediated refolding and prevents them from exiting the ERs. Here, we review progress in structural studies of the calnexin cycle, which reveal common features of how lectin chaperones recruit function-specific chaperones and how UGGT recognizes misfolded proteins.
Topics: Animals; Calnexin; Endoplasmic Reticulum; Humans; Molecular Chaperones
PubMed: 32285592
DOI: 10.1111/febs.15330 -
Nature Communications Oct 2022Virus infection affects cellular proteostasis and provides an opportunity to study this cellular process under perturbation. The proteostasis network in the endoplasmic...
Virus infection affects cellular proteostasis and provides an opportunity to study this cellular process under perturbation. The proteostasis network in the endoplasmic reticulum (ER) is composed of the calnexin cycle, and the two protein degradation pathways ER-associated protein degradation (ERAD) and ER-to-lysosome-associated degradation (ERLAD/ER-phagy/reticulophagy). Here we show that calnexin and calreticulin trigger Zaire Ebolavirus (EBOV) glycoprotein GP misfolding. Misfolded EBOV-GP is targeted by ERAD machinery, but this results in lysosomal instead of proteasomal degradation. Moreover, the ER Ub ligase RNF185, usually associated with ERAD, polyubiquitinates EBOV-GP on lysine 673 via ubiquitin K27-linkage. Polyubiquinated GP is subsequently recruited into autophagosomes by the soluble autophagy receptor sequestosome 1 (SQSTM1/p62), in an ATG3- and ATG5-dependent manner. We conclude that EBOV hijacks all three proteostasis mechanisms in the ER to downregulate GP via polyubiquitination and show that this increases viral fitness. This study identifies linkages among proteostasis network components previously thought to function independently.
Topics: Autophagy; Calnexin; Calreticulin; Endoplasmic Reticulum-Associated Degradation; Hemorrhagic Fever, Ebola; Humans; Ligases; Lysine; Mitochondrial Proteins; Molecular Chaperones; Proteostasis; Sequestosome-1 Protein; Ubiquitin; Ubiquitin-Protein Ligases
PubMed: 36224200
DOI: 10.1038/s41467-022-33805-9 -
The EMBO Journal Aug 2021Efficient degradation of by-products of protein biogenesis maintains cellular fitness. Strikingly, the major biosynthetic compartment in eukaryotic cells, the...
Efficient degradation of by-products of protein biogenesis maintains cellular fitness. Strikingly, the major biosynthetic compartment in eukaryotic cells, the endoplasmic reticulum (ER), lacks degradative machineries. Misfolded proteins in the ER are translocated to the cytosol for proteasomal degradation via ER-associated degradation (ERAD). Alternatively, they are segregated in ER subdomains that are shed from the biosynthetic compartment and are delivered to endolysosomes under control of ER-phagy receptors for ER-to-lysosome-associated degradation (ERLAD). Demannosylation of N-linked oligosaccharides targets terminally misfolded proteins for ERAD. How misfolded proteins are eventually marked for ERLAD is not known. Here, we show for ATZ and mutant Pro-collagen that cycles of de-/re-glucosylation of selected N-glycans and persistent association with Calnexin (CNX) are required and sufficient to mark ERAD-resistant misfolded proteins for FAM134B-driven lysosomal delivery. In summary, we show that mannose and glucose processing of N-glycans are triggering events that target misfolded proteins in the ER to proteasomal (ERAD) and lysosomal (ERLAD) clearance, respectively, regulating protein quality control in eukaryotic cells.
Topics: Animals; Calnexin; Endoplasmic Reticulum-Associated Degradation; Fibroblasts; Glucosyltransferases; Humans; Lysosomal-Associated Membrane Protein 1; Lysosomes; Membrane Proteins; Membrane Transport Proteins; Mice; Oligosaccharides; Polysaccharides; Procollagen; Protein Folding; alpha 1-Antitrypsin
PubMed: 34152647
DOI: 10.15252/embj.2020107240 -
Molecules (Basel, Switzerland) May 2021ERp57, a member of the protein disulfide isomerase family, is a ubiquitous disulfide catalyst that functions in the oxidative folding of various clients in the mammalian...
ERp57, a member of the protein disulfide isomerase family, is a ubiquitous disulfide catalyst that functions in the oxidative folding of various clients in the mammalian endoplasmic reticulum (ER). In concert with ER lectin-like chaperones calnexin and calreticulin (CNX/CRT), ERp57 functions in virtually all folding stages from co-translation to post-translation, and thus plays a critical role in maintaining protein homeostasis, with direct implication for pathology. Here, we present mechanisms by which Ca regulates the formation of the ERp57-calnexin complex. Biochemical and isothermal titration calorimetry analyses revealed that ERp57 strongly interacts with CNX via a non-covalent bond in the absence of Ca. The ERp57-CNX complex not only promoted the oxidative folding of human leukocyte antigen heavy chains, but also inhibited client aggregation. These results suggest that this complex performs both enzymatic and chaperoning functions under abnormal physiological conditions, such as Ca depletion, to effectively guide proper oxidative protein folding. The findings shed light on the molecular mechanisms underpinning crosstalk between the chaperone network and Ca.
Topics: Calcium; Calnexin; Disulfides; Humans; Models, Biological; Oxidation-Reduction; Protein Aggregates; Protein Binding; Protein Disulfide-Isomerases; Protein Folding; Thermodynamics
PubMed: 34064874
DOI: 10.3390/molecules26102853