Graphical Abstract Highlights d Islet C3 expression is upregulated in human T2D and rodent models of diabetes d C3 is present within the cytosol and binds autophagy-related protein 16-1 (ATG16L1) d b cells lacking C3 have impaired autophagy d Intracellular C3 protects b cells from palmitic acid/IAPPmediated apoptosis In Brief King et al. show that the main complement protein, C3, is expressed intracellularly in pancreatic b cells. C3 binds ATG16L1, thus regulating autophagy and protecting b cells from death from various insults. These findings highlight a novel intracellular protective role of this major immunological protein. SUMMARYWe show here that human pancreatic islets highly express C3, which is both secreted and present in the cytosol. Within isolated human islets, C3 expression correlates with type 2 diabetes (T2D) donor status, HbA1c, and inflammation. Islet C3 expression is also upregulated in several rodent diabetes models. C3 interacts with ATG16L1, which is essential for autophagy. Autophagy relieves cellular stresses faced by b cells during T2D and maintains cellular homeostasis. C3 knockout in clonal b cells impaired autophagy and led to increased apoptosis after exposure of cells to palmitic acid and IAPP. In the absence of C3, autophagosomes do not undergo fusion with lysosomes. Thus, C3 may be upregulated in islets during T2D as a cytoprotective factor against b cell dysfunction caused by impaired autophagy. Therefore, we revealed a previously undescribed intracellular function for C3, connecting the complement system directly to autophagy, with a broad potential importance in other diseases and cell types.
CD46 is a cell surface complement inhibitor widely expressed in human tissues, in contrast to mice, where expression is limited to the testes. In humans, it has been identified as an important T cell costimulatory receptor, and patients deficient in CD46 or its endogenous ligands are unable to mount effective Th1 T cell responses. Stimulation of human CD4+ T cells with CD3 and CD46 also leads to the differentiation of a ‘switched’ Th1 population, which shuts down IFN-γ secretion and upregulates IL-10, and is believed to be important for negative feedback regulation of the Th1 response. We show here that CD46 costimulation leads to amplified microRNA expression changes in human CD4+ T cells, with associated increases in activation more potent than that mediated by the ‘classic’ costimulator CD28. Blockade of cell-surface CD46 inhibited CD28-mediated costimulation, identifying autocrine CD46 signaling as downstream of CD28. We also identify a downregulation of microRNA-150 in CD46-costimulated T cells, and identify the glucose transporter-1 (GLUT1) encoding transcript SLC2A1 as a target of microRNA-150 regulation, connecting microRNA-150 with modulation of glucose uptake. We also investigated microRNA expression profiles of CD46-induced ‘switched’ IL-10-secreting Th1 T cells and found increased expression of microRNA-150, compared to IFN-γ-secreting Th1 cells. Knockdown of microRNA-150 led to a reduction in IL-10 but not IFN-γ. CD46 therefore controls both Th1 activation and regulation via a miRNA-150-dependent mechanism.
CD59 is a glycosylphosphatidylinositol (GPI)‐anchored cell surface inhibitor of the complement membrane attack complex (MAC). We showed previously that CD59 is highly expressed in pancreatic islets but is down‐regulated in rodent models of diabetes. CD59 knockdown but not enzymatic removal of cell surface CD59 led to a loss of glucose‐stimulated insulin secretion (GSIS), suggesting that an intracellular pool of CD59 is required. In this current paper, we now report that non‐GPI‐anchored CD59 is present in the cytoplasm, colocalizes with exocytotic protein vesicle‐associated membrane protein 2, and completely rescues GSIS in cells lacking endogenous CD59 expression. The involvement of cytosolic non–GPI‐anchored CD59 in GSIS is supported in phosphatidylinositol glycan class A knockout GPI anchor–deficient β‐cells, in which GSIS is still CD59 dependent. Furthermore, site‐directed mutagenesis demonstrated different structural requirements of CD59 for its 2 functions, MAC inhibition and GSIS. Our results suggest that CD59 is retrotranslocated from the endoplasmic reticulum to the cytosol, a process mediated by recognition of trimmed N‐linked oligosaccharides, supported by the partial glycosylation of non‐GPI‐anchored cytosolic CD59 as well as the failure of N‐linked glycosylation site mutant CD59 to reach the cytosol or rescue GSIS. This study thus proposes the previously undescribed existence of non–GPI‐anchored cytosolic CD59, which is required for insulin secretion.—Golec, E., Rosberg, R., Zhang, E., Renström, E., Blom, A. M., King, B. C. A cryptic non–GPI‐anchored cytosolic isoform of CD59 controls insulin exocytosis in pancreatic β‐cells by interaction with SNARE proteins. FASEB J. 33, 12425–12434 (2019). http://www.fasebj.org
Protein kinase RNA-activated (PKR) is a cytoplasmic receptor for dsRNA, and as such is involved in detection of viral infection. On binding dsRNA, PKR dimerizes, autophosphorylates, and then phosphorylates its substrate, eukaryotic translation initiation factor 2 subunit α (eIF2α), causing inhibition of mRNA translation and shutdown of viral protein production. However, active PKR has also been found to be involved in the NF-κB signaling pathway by inducing phosphorylation of IκBα. PKR is regulated by the noncoding RNA nc886, which has altered expression in cancer. We have found that expression of nc886 is highly upregulated during activation of human CD4+ T cells. As has been described in other cell types, nc886 bound to PKR in human T cell lysates, preventing PKR phosphorylation by polyinosinic:polycytidylic acid or HIV trans-activation response element RNA in lysates of T cell lines or primary human CD4+ T cells. Using clonal human T cell lines, we found that nc886 expression was strictly required for IFN-γ and IL-2 expression and secretion after T cell activation but did not affect proliferation or activation-induced cell death. In stimulated human PBMCs, nc886 expression strongly correlated with IFN-γ expression. Although nc886 inhibited PKR activation by dsRNA, it was required for PKR phosphorylation during T cell stimulation, with subsequent NF-κB signaling and CREB phosphorylation. nc886 also regulated PKR phosphorylation during human monocyte-derived macrophage activation. We have therefore identified nc886 as a noncoding RNA marker of T cell activation and regulator of PKR-dependent signaling.
Yeast cells originating from one single colony are considered genotypically and phenotypically identical. However, taking into account the cellular heterogeneity, it seems also important to monitor cell-to-cell variations within a clone population. In the present study, a comprehensive yeast karyotype screening was conducted using single chromosome comet assay. Chromosome-dependent and mutation-dependent changes in DNA (DNA with breaks or with abnormal replication intermediates) were studied using both single-gene deletion haploid mutants (bub1, bub2, mad1, tel1, rad1 and tor1) and diploid cells lacking one active gene of interest, namely BUB1/bub1, BUB2/bub2, MAD1/mad1, TEL1/tel1, RAD1/rad1 and TOR1/tor1 involved in the control of cell cycle progression, DNA repair and the regulation of longevity. Increased chromosome fragility and replication stress-mediated chromosome abnormalities were correlated with elevated incidence of genomic instability, namely aneuploid events—disomies, monosomies and to a lesser extent trisomies as judged by in situ comparative genomic hybridization (CGH). The tor1 longevity mutant with relatively balanced chromosome homeostasis was found the most genomically stable among analyzed mutants. During clonal yeast culture, spontaneously formed abnormal chromosome structures may stimulate changes in the ploidy state and, in turn, promote genomic heterogeneity. These alterations may be more accented in selected mutated genetic backgrounds, namely in yeast cells deficient in proper cell cycle regulation and DNA repair.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.