PRKCI negatively regulates autophagy via PIK3CA/AKT-MTOR signaling.

The atypical protein kinase C isoform PRKC iota (PRKCI) plays a key role in cell proliferation, differentiation, and carcinogenesis, and it has been shown to be a human oncogene. Here, we show that PRKCI overexpression in U2OS cells impaired functional autophagy in normal or cell stress conditions, as characterized by decreased levels of light chain 3B-II protein (LC3B-II) and weakened degradation of endogenous and exogenous autophagic substrates. Conversely, PRKCI knockdown by small interference RNA resulted in opposite effects. Additionally, we identified two novel PRKCI mutants, PRKCI(L485M) and PRKCI(P560R), which induced autophagy and exhibited dominant negative effects. Further studies indicated that PRKCI knockdown-mediated autophagy was associated with the inactivation of phosphatidylinositol 3-kinase alpha/AKT-mammalian target of rapamycin (PIK3CA/AKT-MTOR) signaling. These data underscore the importance of PRKCI in the regulation of autophagy. Moreover, the finding may be useful in treating PRKCI-overexpressing carcinomas that are characterized by increased levels of autophagy.

Transmembrane protein 106A is silenced by promoter region hypermethylation and suppresses gastric cancer growth by inducing apoptosis.

Inactivation of tumour suppressor genes by promoter methylation plays an important role in the initiation and progression of gastric cancer (GC). Transmembrane 106A gene (TMEM106A) encodes a novel protein of previously unknown function. This study analysed the biological functions, epigenetic changes and the clinical significance of TMEM106A in GC. Data from experiments indicate that TMEM106A is a type II membrane protein, which is localized to mitochondria and the plasma membrane. TMEM106A was down-regulated or silenced by promoter region hypermethylation in GC cell lines, but expressed in normal gastric tissues. Overexpression of TMEM106A suppressed cell growth and induced apoptosis in GC cell lines, and retarded the growth of xenografts in nude mice. These effects were associated with the activation of caspase-2, caspase-9, and caspase-3, cleavage of BID and inactivation of poly (ADP-ribose) polymerase (PARP). In primary GC samples, loss or reduction of TMEM106A expression was associated with promoter region hypermethylation. TMEM106A was methylated in 88.6% (93/105) of primary GC and 18.1% (2/11) in cancer adjacent normal tissue samples. Further analysis suggested that TMEM106A methylation in primary GCs was significantly correlated with smoking and tumour metastasis. In conclusion, TMEM106A is frequently methylated in human GC. The expression of TMEM106A is regulated by promoter hypermethylation. TMEM106A is a novel functional tumour suppressor in gastric carcinogenesis.

PDCD5 negatively regulates autoimmunity by upregulating FOXP3(+) regulatory T cells and suppressing Th17 and Th1 responses.

Maintenance of FOXP3 protein expression is crucial for differentiation and maturation of regulatory T (Treg) cells, which play important roles in immune homeostasis and immune tolerance. We demonstrate here that PDCD5 interacts with FOXP3, increases acetylation of FOXP3 in synergy with Tip60 and enhances the repressive function of FOXP3. In PDCD5 transgenic (PDCD5tg) mice, overexpression of PDCD5 enhanced the level of FOXP3 protein and percentage of CD4(+)CD25(+)FOXP3(+) cells. Naïve CD4(+) T cells from PDCD5tg mice were more sensitive to TGF-ß-induced Treg polarization and expansion. These induced Tregs retained normal suppressive function in vitro. Severity of experimentally-induced autoimmune encephalomyelitis (EAE) in PDCD5tg mice was significantly reduced relative to that of wild-type mice. The beneficial effect of PDCD5 likely resulted from increases of Treg cell frequency, accompanied by a reduction of the predominant pathogenic Th17/Th1 response. Activation-induced cell death enhanced by PDCD5 was also linked to this process. This is the first report revealing that PDCD5 activity in T cells suppresses autoimmunity by modulating Tregs. This study suggests that PDCD5 serves as a guardian of immunological functions and that the PDCD5-FOXP3-Treg axis may be a therapeutic target for autoimmunity.

TMEM189 negatively regulates the stability of ULK1 protein and cell autophagy.

ULK1 is crucial for initiating autophagosome formation and its activity is tightly regulated by post-translational modifications and protein-protein interactions. In the present study, we demonstrate that TMEM189 (Transmembrane protein 189), also known as plasmanylethanolamine desaturase 1 (PEDS1), negatively regulates the proteostasis of ULK1 and autophagy activity. In TMEM189-overexpressed cells, the formation of autophagesome is impaired, while TMEM189 knockdown increases cell autophagy. Further investigation reveals that TMEM189 interacts with and increases the instability of ULK1, as well as decreases its kinase activities. The TMEM189 N-terminal domain is required for the interaction with ULK1. Additionally, TMEM189 overexpression can disrupt the interaction between ULK1 and TRAF6, profoundly impairs K63-linked polyubiquitination of ULK1 and self-association, leading to the decrease of ULK1 stability. Moreover, in vitro and in vivo experiments suggest that TMEM189 deficiency results in the inhibition of tumorigenicity of gastric cancer. Our findings provide a new insight into the molecular regulation of autophagy and laboratory evidence for investigating the physiological and pathological roles of TMEM189.

COVID-19: the epidemiology and treatment.

After initially emerging in late 2019, coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spread rapidly to cause a global pandemic. SARS-CoV-2 is a betacoronavirus that is closely related to severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome coronavirus, all of which can cause severe lung injury, respiratory distress and cytokine storm. While mortality rates associated with SARS-CoV-2 are lower than those associated with severe acute respiratory syndrome coronavirus or Middle East respiratory syndrome coronavirus, it is more contagious and spreads more rapidly than these other viruses. This article summarises the epidemiology and potential options for treating COVID-19 to give a foundation for future studies of the diagnosis, treatment and prevention of this deadly disease.

Deletion of Pdcd5 in mice led to the deficiency of placenta development and embryonic lethality.

Programmed cell death 5 (PDCD5) is an apoptosis promoter molecule that displays multiple biological activities. However, the function of PDCD5 in vivo has not yet been investigated. Here, we generated a Pdcd5 knockout mouse model to study the physiological role of PDCD5 in vivo. Knockout of the Pdcd5 gene resulted in embryonic lethality at mid-gestation. Histopathological analysis revealed dysplasia in both the LZs and JZs in Pdcd5-/- placentas with defects in spongiotrophoblasts and trophoblast giant cells. Furthermore, Pdcd5-/- embryos had impaired transplacental passage capacity. We also found that Pdcd5-/- embryos exhibited cardiac abnormalities and defective liver development. The growth defect is linked to impaired placental development and may be caused by insufficient oxygen and nutrient transfer across the placenta. These findings were verified in vitro in Pdcd5 knockout mouse embryonic fibroblasts, which showed increased apoptosis and G0/G1 phase cell cycle arrest. Pdcd5 knockout decreased the Vegf and hepatocyte growth factor (Hgf) levels, downregulated the downstream Pik3ca-Akt-Mtor signal pathway and decreased cell survival. Collectively, our studies demonstrated that Pdcd5 knockout in mouse embryos results in placental defects and embryonic lethality.

MARCH2 regulates autophagy by promoting CFTR ubiquitination and degradation and PIK3CA-AKT-MTOR signaling.

MARCH2 (membrane-associated RING-CH protein 2), an E3 ubiquitin ligase, is mainly associated with the vesicle trafficking. In the present study, for the first time, we demonstrated that MARCH2 negatively regulates autophagy. Our data indicated that overexpression of MARCH2 impaired autophagy, as evidenced by attenuated levels of LC3B-II and impaired degradation of endogenous and exogenous autophagic substrates. By contrast, loss of MARCH2 expression had the opposite effects. In vivo experiments demonstrate that MARCH2 knockout mediated autophagy results in an inhibition of tumorigenicity. Further investigation revealed that the induction of autophagy by MARCH2 deficiency was mediated through the PIK3CA-AKT-MTOR signaling pathway. Additionally, we found that MARCH2 interacts with CFTR (cystic fibrosis transmembrane conductance regulator), promotes the ubiquitination and degradation of CFTR, and inhibits CFTR-mediated autophagy in tumor cells. The functional PDZ domain of MARCH2 is required for the association with CFTR. Thus, our study identified a novel negative regulator of autophagy and suggested that the physical and functional connection between the MARCH2 and CFTR in different conditions will be elucidated in the further experiments.

EVA1A/TMEM166 Regulates Embryonic Neurogenesis by Autophagy.

Self-renewal and differentiation of neural stem cells is essential for embryonic neurogenesis, which is associated with cell autophagy. However, the mechanism by which autophagy regulates neurogenesis remains undefined. Here, we show that Eva1a/Tmem166, an autophagy-related gene, regulates neural stem cell self-renewal and differentiation. Eva1a depletion impaired the generation of newborn neurons, both in vivo and in vitro. Conversely, overexpression of EVA1A enhanced newborn neuron generation and maturation. Moreover, Eva1a depletion activated the PIK3CA-AKT axis, leading to the activation of the mammalian target of rapamycin and the subsequent inhibition of autophagy. Furthermore, addition of methylpyruvate to the culture during neural stem cell differentiation rescued the defective embryonic neurogenesis induced by Eva1a depletion, suggesting that energy availability is a significant factor in embryonic neurogenesis. Collectively, these data demonstrated that EVA1A regulates embryonic neurogenesis by modulating autophagy. Our results have potential implications for understanding the pathogenesis of neurodevelopmental disorders caused by autophagy dysregulation.

TMEM166/EVA1A interacts with ATG16L1 and induces autophagosome formation and cell death.

The formation of the autophagosome is controlled by an orderly action of ATG proteins. However, how these proteins are recruited to autophagic membranes remain poorly clarified. In this study, we have provided a line of evidence confirming that EVA1A (eva-1 homolog A)/TMEM166 (transmembrane protein 166) is associated with autophagosomal membrane development. This notion is based on dotted EVA1A structures that colocalize with ZFYVE1, ATG9, LC3B, ATG16L1, ATG5, STX17, RAB7 and LAMP1, which represent different stages of the autophagic process. It is required for autophagosome formation as this phenotype was significantly decreased in EVA1A-silenced cells and Eva1a KO MEFs. EVA1A-induced autophagy is independent of the BECN1-PIK3C3 (phosphatidylinositol 3-kinase, catalytic subunit type 3) complex but requires ATG7 activity and the ATG12-ATG5/ATG16L1 complex. Here, we present a molecular mechanism by which EVA1A interacts with the WD repeats of ATG16L1 through its C-terminal and promotes ATG12-ATG5/ATG16L1 complex recruitment to the autophagic membrane and enhances the formation of the autophagosome. We also found that both autophagic and apoptotic mechanisms contributed to EVA1A-induced cell death while inhibition of autophagy and apoptosis attenuated EVA1A-induced cell death. Overall, these findings provide a comprehensive view to our understanding of the pathways involved in the role of EVA1A in autophagy and programmed cell death.

Anti-inflammatory effects of recombinant human PDCD5 (rhPDCD5) in a rat collagen-induced model of arthritis.

Programmed cell death 5 (PDCD5) was first identified as a gene upregulated in cells undergoing apoptosis. We recently demonstrated the inhibitory effect of PDCD5 on experimentally induced autoimmune encephalomyelitis. In this study, we investigated the anti-inflammatory effects of recombinant human PDCD5 (rhPDCD5) in a rat collagen-induced arthritis (CIA) model. We find that vaccination of collagen II (CII) induced CIA rats with rhPDCD5 significantly delayed the occurrence and reduced the severity of CIA rats. rhPDCD5 also restored the loss of Foxp3(+) regulatory T (Treg) cells and decreased the population of Th1 and Th17 in CIA rats. Simultaneously, rhPDCD5 treatment suppressed the production of pro-inflammatory cytokines (interleukin (IL)-6, IL-17A, tumor necrosis factor-α (TNF-α), and interferon gamma (IFN-γ)) and increased the secretion of anti-inflammatory cytokines (transforming growth factor beta 1 (TGF-ß1) and IL-10) in CIA rats. In addition, rhPDCD5 inhibited the ability of CII to induce proliferation of splenocytes and lymph node cells (LNCs) and promoted the CII-activated CD4(+) cell apoptosis. These results of rhPDCD5-treated CIA rats were similar with those of recombinant human TNF-α receptor IgG Fc (rhTNFR:Fc). Thus, to our knowledge, we provide the first evidence that rhPDCD5 may be an efficient approach to diminishing exacerbated immune responses in CIA, indicating its therapeutic potential in the treatment of rheumatoid arthritis and other autoimmune diseases.

PHF23 (plant homeodomain finger protein 23) negatively regulates cell autophagy by promoting ubiquitination and degradation of E3 ligase LRSAM1.

Autophagy is a multistep process that involves the degradation and digestion of intracellular components by the lysosome. It has been proved that many core autophagy-related molecules participate in this event. However, new component proteins that regulate autophagy are still being discovered. At present, we report PHF23 (PHD finger protein 23) with a PHD-like zinc finger domain that can negatively regulate autophagy. Data from experiments indicated that the overexpression of PHF23 impaired autophagy, as characterized by decreased levels of LC3B-II and weakened degradation of endogenous and exogenous autophagic substrates. Conversely, knockdown of PHF23 resulted in opposite effects. Molecular mechanism studies suggested that PHF23 interacts with LRSAM1, which is an E3 ligase key for ubiquitin-dependent autophagy against invading bacteria. PHF23 promotes the ubiquitination and proteasome degradation of LRSAM1. We also show that the PHD finger of PHF23 is a functional domain needed for the interaction with LRSAM1. Altogether, our results indicate that PHF23 is a negative regulator associated in autophagy via the LRSAM1 signaling pathway. The physical and functional connection between the PHF23 and LRSAM1 needs further investigation.

A novel ER-localized transmembrane protein, EMC6, interacts with RAB5A and regulates cell autophagy.

Autophagy is mediated by a unique organelle, the autophagosome, which encloses a portion of the cytoplasm for delivery to the lysosome. Phosphatidylinositol 3-phosphate (PtdIns3P) produced by the class III phosphatidylinositol 3-kinase (PtdIns3K) complex is essential for canonical autophagosome formation. RAB5A, a small GTPase localized to early endosomes, has been shown to associate with the class III PtdIns3K complex, regulate its activity and promote autophagosome formation. However, little is known about how endosome-localized RAB5A functions with the class III PtdIns3K complex. Here we identified a novel endoplasmic reticulum (ER)-localized transmembrane protein, ER membrane protein complex subunit 6 (EMC6), which interacted with both RAB5A and BECN1/Beclin 1 and colocalized with the omegasome marker ZFYVE1/DFCP1. It was shown to regulate autophagosome formation, and its deficiency caused the accumulation of autophagosomal precursor structures and impaired autophagy. Our study showed for the first time that EMC6 is a novel regulator involved in autophagy.

Transmembrane protein 208: a novel ER-localized protein that regulates autophagy and ER stress.

Autophagy and endoplasmic reticulum (ER) stress are both tightly regulated cellular processes that play central roles in various physiological and pathological conditions. Recent reports have indicated that ER stress is a potent inducer of autophagy. However, little is known about the underlying molecular link between the two processes. Here we report a novel human protein, transmembrane protein 208 (TMEM208) that can regulate both autophagy and ER stress. When overexpressed, TMEM208 impaired autophagy as characterized by the decrease of the accumulation of LC3-II, decreased degradation of autophagic substrates, and reduced expression of critical effectors and vital molecules of the ER stress and autophagy processes. In contrast, knockdown of the TMEM208 gene promoted autophagy, as demonstrated by the increase of LC3-II, increased degradation of autophagic substrates, and enhanced expression levels for genes key in the ER stress and autophagic processes. Taken together, our results reveal that this novel ER-located protein regulates both ER stress and autophagy, and represents a possible link between the two different cellular processes.