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Follicular helper T cells (TFH cells) and follicular regulatory T cells (TFR cells) regulate the quantity and quality of humoral immunity. Although both cell types express the costimulatory receptor ICOS and require the transcription factor Bcl-6 for their differentiation, the ICOS-dependent pathways that coordinate their responses are not well understood. Here we report that activation of ICOS in CD4+ T cells promoted interaction of the p85α regulatory subunit of the signaling kinase PI(3)K and intracellular osteopontin (OPN-i), followed by translocation of OPN-i to the nucleus, its interaction with Bcl-6 and protection of Bcl-6 from ubiquitin-dependent proteasome degradation. Post-translational protection of Bcl-6 by OPN-i was essential for sustained responses of TFH cells and TFR cells and regulation of the germinal center B cell response to antigen. Thus, the p85α-OPN-i axis represents a molecular bridge that couples activation of ICOS to Bcl-6-dependent functional differentiation of TFH cells and TFR cells; this suggests new therapeutic avenues to manipulate the responses of these cells.

Lipotoxicity plays a vital role in development and progression of type 2 diabetes. Prolonged elevation of free fatty acids especially the palmitate leads to pancreatic [beta]-cell dysfunction and apoptosis. Curcumin (diferuloylmethane), a polyphenol from the curry spice turmeric, is considered to be a broadly cytoprotective agent. The present study was designed to determine the protective effect of curcumin on palmitate-induced apoptosis in [beta]-cells and investigate underlying mechanisms. Our results showed that curcumin improved cell viability and enhanced glucose-induced insulin secretory function in MIN6 pancreatic [beta]-cells. Palmitate incubation evoked chromatin condensation, DNA nick end labeling and activation of caspase-3 and -9. Curcumin treatment inhibited palmitate-induced apoptosis, relieved mitochondrial depolarization and up-regulated Bcl-2/Bax ratio. Palmitate induced the generation of reactive oxygen species and inhibited activities of antioxidant enzymes, which could be neutralized by curcumin treatment. Moreover, curcumin could promote rapid phosphorylation of Akt and nuclear exclusion of FoxO1 in MIN6 cells under lipotoxic condition. Phosphatidylinositol 3-kinase and Akt specific inhibitors abolished the anti-lipotoxic effect of curcumin and stimulated FoxO1 nuclear translocation. These findings suggested that curcumin protected MIN6 pancreatic [beta]-Cells against apoptosis through activation of Akt, inhibition of nuclear translocation of FoxO1 and mitochondrial survival pathway.

Formation of multi-component signaling complex necrosomes is essential for tumor necrosis factor α (TNF)-induced programmed necrosis (also called necroptosis). However, the mechanisms of necroptosis are still largely unknown. We isolated a TNF-resistant L929 mutant cell line generated by retrovirus insertion and identified that disruption of the guanine nucleotide-binding protein γ 10 (Gγ10) gene is responsible for this phenotype. We further show that Gγ10 is involved in TNF-induced necroptosis and G[beta]2 is the partner of Gγ10. Src is the downstream effector of G[beta]2γ10 in TNF-induced necroptosis because TNF-induced Src activation was impaired upon Gγ10 knockdown. Gγ10 does not affect TNF-induced activation of NF-κB and MAPKs and the formation of necrosomes, but is required for trafficking of necrosomes to their potential functioning site, an unidentified subcellular organelle that can be fractionated into heterotypic membrane fractions. The TNF-induced G[beta]γ-Src signaling pathway is independent of RIP1/RIP3 kinase activity and necrosome formation, but is required for the necrosome to function.

SignificanceAside from its undisputed role in the import of newly synthesized proteins into the endoplasmic reticulum (ER), the Sec61 translocon was proposed to ensure the reverse transport of misfolded proteins to the cytosol. Based on this model, Sec61 was also proposed to be the channel exporting internalized antigens from endosomes to the cytosol, for degradation and cross-presentation. Establishing Sec61’s contribution to these connected trafficking pathways has nevertheless proven difficult, due to a technical incapacity to blunt its activity acutely. Here, we took advantage of a recently identified Sec61 blocker to determine whether or not Sec61 can mediate retrograde protein transport. Both ER-to-cytosol and endosome-to-cytosol protein export were intact in mycolactone-treated cells, which argues against Sec61 operating as a retrotranslocon. Although antigen cross-presentation in dendritic cells (DCs) is critical to the initiation of most cytotoxic immune responses, the intracellular mechanisms and traffic pathways involved are still unclear. One of the most critical steps in this process, the export of internalized antigen to the cytosol, has been suggested to be mediated by Sec61. Sec61 is the channel that translocates signal peptide-bearing nascent polypeptides into the endoplasmic reticulum (ER), and it was also proposed to mediate protein retrotranslocation during ER-associated degradation (a process called ERAD). Here, we used a newly identified Sec61 blocker, mycolactone, to analyze Sec61’s contribution to antigen cross-presentation, ERAD, and transport of internalized antigens into the cytosol. As shown previously in other cell types, mycolactone prevented protein import into the ER of DCs. Mycolactone-mediated Sec61 blockade also potently suppressed both antigen cross-presentation and direct presentation of synthetic peptides to CD8+ T cells. In contrast, it did not affect protein export from the ER lumen or from endosomes into the cytosol, suggesting that the inhibition of cross-presentation was not related to either of these trafficking pathways. Proteomic profiling of mycolactone-exposed DCs showed that expression of mediators of antigen presentation, including MHC class I and β2 microglobulin, were highly susceptible to mycolactone treatment, indicating that Sec61 blockade affects antigen cross-presentation indirectly. Together, our data suggest that the defective translocation and subsequent degradation of Sec61 substrates is the cause of altered antigen cross-presentation in Sec61-blocked DCs.

Integrin [beta]4 and its Y-1494 phosphorylation play an important role in cell signaling. We found a small molecule, ethyl1-(3-(4-chlorophenoxy)-2-hydroxypropyl)-3-(4-chlorophenyl)-1H-pyrazole-5-carboxylate (ECPC), that could elevate the levels of KIT ligand (KITLG), interleukin 8 (IL-8), prostaglandin-endoperoxide synthase 2 (PTGS2) and activating transcription factor 3 (ATF3) and promote apoptosis in vascular endothelial cells (VECs) through integrin [beta]4. We investigated the underlying mechanism of integrin [beta]4 participating in this process. ECPC treatment increased the phosphorylation of Y-1494 in the integrin [beta]4 cytoplasmic domain via a well-known receptor tyrosine kinase, fibroblast growth factor receptor 1 (FGFR1), and integrin [beta]4 translocated from the cytoplasm to nucleus. With suppression of Y-1494 phosphorylation by FGF-2 or siRNA of FGFR1, ECPC failed to promote integrin [beta]4 nuclear translocation and could not increase the expression of KITLG, IL-8, PTGS2 or ATF3. Y-1494 phosphorylation and nuclear translocation of integrin [beta]4 may be important during ECPC-induced apoptosis in VECs.[PUBLICATION ABSTRACT]

Mitochondrial dysfunction contributing to the pathogenesis of glaucomatous neurodegeneration has stimulated considerable interest recently. In this study, we explored the role of peroxisome proliferator activated receptor-γ co-activator 1[alpha] (PGC-1[alpha]) in resveratrol-triggered mitochondrial biogenesis for preventing apoptosis in a retinal ganglion cell line RGC-5. Our results showed that serum deprivation induced cell apoptosis in a time-dependent manner. Applying resveratrol maintained the normal mitochondrial membrane potential, decreased the levels of both total and cleaved caspase-3, and inhibited the release of cytochrome c, which subsequently enhanced cell survival. Moreover, resveratrol stimulated mitochondrial biogenesis by increasing the absolute quantity of mitochondria as well as their DNA copies. Treatment with resveratrol promoted the protein expression of SIRT1, but not PGC-1[alpha]; instead, resveratrol facilitated PGC-1[alpha] translocation from the cytoplasm to the nucleus and up-regulated NRF1 and TFAM, which were blocked by nicotinamide. Collectively, we demonstrate that the SIRT1-dependent PGC-1[alpha] subcellular translocation following resveratrol application potentially attenuates serum deprivation-elicited RGC-5 cell death, thereby raising the possibility of mitigating glaucomatous retinopathy by enhancement of mitochondrial biogenesis.[PUBLICATION ABSTRACT]

In bacteria, translocation of most soluble secreted proteins (and outer membrane proteins in Gram-negative bacteria) across the cytoplasmic membrane by the Sec machinery is mediated by the essential ATPase SecA. At its core, this machinery consists of SecA and the integral membrane proteins SecYEG, which form a protein conducting channel in the membrane. Proteins are recognised by the Sec machinery by virtue of an internally encoded targeting signal, which usually takes the form of an N-terminal signal sequence. In addition, substrate proteins must be maintained in an unfolded conformation in the cytoplasm, prior to translocation, in order to be competent for translocation through SecYEG. Recognition of substrate proteins occurs via SecA-either through direct recognition by SecA or through secondary recognition by a molecular chaperone that delivers proteins to SecA. Substrate proteins are then screened for the presence of a functional signal sequence by SecYEG. Proteins with functional signal sequences are translocated across the membrane in an ATP-dependent fashion. The current research investigating each of these steps is reviewed here.

The Sec61 complex forms a protein-conducting channel in the endoplasmic reticulum membrane that is required for secretion of soluble proteins and production of many membrane proteins. Several natural and synthetic small molecules specifically inhibit Sec61, generating cellular effects that are useful for therapeutic purposes, but their inhibitory mechanisms remain unclear. Here we present near-atomic-resolution structures of human Sec61 inhibited by a comprehensive panel of structurally distinct small molecules—cotransin, decatransin, apratoxin, ipomoeassin, mycolactone, cyclotriazadisulfonamide and eeyarestatin. All inhibitors bind to a common lipid-exposed pocket formed by the partially open lateral gate and plug domain of Sec61. Mutations conferring resistance to the inhibitors are clustered at this binding pocket. The structures indicate that Sec61 inhibitors stabilize the plug domain in a closed state, thereby preventing the protein-translocation pore from opening. Our study provides the atomic details of Sec61–inhibitor interactions and the structural framework for further pharmacological studies and drug design. Itskanov and Wang et al. determined high-resolution structures of the human Sec61 channel inhibited by several structurally distinct small molecules and revealed the common inhibitor-binding site in Sec61 and molecular interactions in atomic detail.