Endoplasmic reticulum stress inhibits cell cycle progression via induction of p27 in melanoma cells

The accumulation of unfolded proteins in the endoplasmic reticulum (ER) triggers the unfolded protein response (UPR), a stress signaling pathway. The UPR coordinates the induction of ER chaperones with decreased protein synthesis and growth arrest in G1 phase of the cell cycle. However, the molecular mechanism underlying UPR-induced G1 cell cycle arrest remains largely unknown. Here we report that activation of the UPR response by tunicamycin (TM), an ER stress inducer, leads to accumulation of p27 and G1 cell cycle arrest in melanoma cells. This accumulation of p27 is due to the inhibition on its polyubiquitination and subsequent degradation upon TM treatment. Correlated with p27 stabilization, the levels of Skp2, an E3 ligase for p27, are decreased in response to TM treatment. More importantly, knockdown of p27 greatly reduces TM-induced G1 cell cycle arrest. Taken together, these data implicate p27 as a critical mediator of ER stress-induced growth arrest.     

PERK-dependent compartmentalization of ERAD and unfolded protein response machineries during ER stress

Accumulation of misfolded proteins in the endoplasmic reticulum (ER) activates the ER membrane kinases PERK and IRE1 leading to the unfolded protein response (UPR). We show here that UPR activation triggers PERK and IRE1 segregation from BiP and their sorting with misfolded proteins to the ER-derived quality control compartment (ERQC), a pericentriolar compartment that we had identified previously. PERK phosphorylates translation factor eIF2alpha, which then accumulates on the cytosolic side of the ERQC. Dominant negative PERK or eIF2alpha(S51A) mutants prevent the compartmentalization, whereas eIF2alpha(S51D) mutant, which mimics constitutive phosphorylation, promotes it. This suggests a feedback loop where eIF2alpha phosphorylation causes pericentriolar concentration at the ERQC, which in turn amplifies the UPR. ER-associated degradation (ERAD) is an UPR-dependent process; we also find that ERAD components (Sec61beta, HRD1, p97/VCP, ubiquitin) are recruited to the ERQC, making it a likely site for retrotranslocation. In addition, we show that autophagy, suggested to play a role in elimination of aggregated proteins, is unrelated to protein accumulation in the ERQC.     

未折叠蛋白响应的激活机制

未折叠蛋白在内质网（endoplasmic reticulum,ER）腔中累积造成ER应激，此时细胞启动未折叠蛋白响应（unfolded protein response,UPR）以恢复蛋白质稳态。目前已知有三种UPR感受器，即IRE1、PERK和ATF6，它们均为ER跨膜蛋白，在ER应激时被激活并启动下游UPR信号通路。虽然UPR感受器最早是在研究细胞如何应对ER应激时发现的，但它们如何感知ER应激至今未得到完满的回答。随着研究的深入，人们发现UPR的功能不仅限于维持蛋白质稳态，而UPR感受器也不是只对未折叠蛋白累积作出响应。本文对UPR的发现及其经典通路作一介绍，着重阐述目前已知的UPR感受器的激活机制，并就UPR和ER应激关系以及该领域存在的问题进行讨论。     

细胞内质网应激与糖脂代谢紊乱的机制关联

在真核细胞中,内质网是蛋白质合成、折叠、加工及其质量监控的重要场所。当内质网难以承担蛋白折叠的高负荷时则引发内质网应激(ER stress),激活细胞的未折叠蛋白响应(unfoldedprotein response,UPR)。细胞通过内质网跨膜蛋白ATF6、PERK和IRE1介导的三条极为关键的UPR信号通路,调控下游相关基因的表达,以增强内质网对蛋白折叠的处理能力。因此,UPR通路在细胞的稳态平衡中具有举足轻重的作用,而这一动态过程的调控对于维持机体的正常生理功能至关重要。近来大量研究表明,在哺乳动物中内质网应激与机体的营养感应和糖脂代谢的调控过程密切相关。在肝脏、脂肪、胰岛以及下丘脑等不同的组织器官中,内质网应激均影响代谢通路的调节机制,因此在糖脂代谢紊乱的发生发展中扮演重要的角色。综上所述,进一步深入了解内质网应激引发代谢异常的生理学机制,可以为肥胖、脂肪肝及2型糖尿病等相关代谢性疾病的防治提供新的潜在药物靶点和重要的理论线索。     

Intrinsic capacities of molecular sensors of the unfolded protein response to sense alternate forms of endoplasmic reticulum stress

The unfolded protein response (UPR) regulates the protein-folding capacity of the endoplasmic reticulum (ER) according to cellular demand. In mammalian cells, three ER transmembrane components, IRE1, PERK, and ATF6, initiate distinct UPR signaling branches. We show that these UPR components display distinct sensitivities toward different forms of ER stress. ER stress induced by ER Ca2+ release in particular revealed fundamental differences in the properties of UPR signaling branches. Compared with the rapid response of both IRE1 and PERK to ER stress induced by thapsigargin, an ER Ca2+ ATPase inhibitor, the response of ATF6 was markedly delayed. These studies are the first side-by-side comparisons of UPR signaling branch activation and reveal intrinsic features of UPR stress sensor activation in response to alternate forms of ER stress. As such, they provide initial groundwork toward understanding how ER stress sensors can confer different responses and how optimal UPR responses are achieved in physiological settings.     

FAD-linked presenilin-1 mutants impede translation regulation under ER stress

FAD mutations in presenilin-1 (PS1) cause attenuation of the induction of the endoplasmic reticulum (ER)-resident chaperone GRP78/BiP under ER stress, due to disturbed function of IRE1, the sensor for accumulation of unfolded protein in the ER lumen. PERK, an ER-resident transmembrane protein kinase, is also a sensor for the unfolded protein response (UPR), causing phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha) to inhibit translation initiation. Here, we report that the FAD mutant PS1 disturbs the UPR by attenuating both the activation of PERK and the phosphorylation of eIF2alpha. Consistent with the results of a disturbed UPR, inhibition of protein synthesis under ER stress was impaired in cells expressing PS1 mutants. These results suggest that mutant PS1 impedes general translational attenuation regulated by PERK and eIF2alpha, resulting in an increased load of newly synthesized proteins into the ER and subsequently increasing vulnerability to ER stress.     

Phenylbutyric acid induces the cellular senescence through an Akt/p21(WAF1) signaling pathway

It has been well known that three sentinel proteins - PERK, ATF6 and IRE1 - initiate the unfolded protein response (UPR) in the presence of misfolded or unfolded proteins in the ER. Recent studies have demonstrated that upregulation of UPR in cancer cells is required to survive and proliferate. Here, we showed that long exposure to 4-phenylbutyric acid (PBA), a chemical chaperone that can reduce retention of unfolded and misfolded proteins in ER, induced cellular senescence in cancer cells such as MCF7 and HT1080. In addition, we found that treatment with PBA activates Akt, which results in p21(WAF1) induction. Interestingly, the depletion of PERK but not ATF6 and IRE1 also induces cellular senescence, which was rescued by additional depletion of Akt. This suggests that Akt pathway is downstream of PERK in PBA induced cellular senescence. Taken together, these results show that PBA induces cellular senescence via activation of the Akt/p21(WAF1) pathway by PERK inhibition.     

Disruption of the ribosomal P complex leads to stress-induced autophagy

The human ribosomal P complex, which consists of the acidic ribosomal P proteins RPLP0, RPLP1, and RPLP2 (RPLP proteins), recruits translational factors, facilitating protein synthesis. Recently, we showed that overexpression of RPLP1 immortalizes primary cells and contributes to transformation. Moreover, RPLP proteins are overexpressed in human cancer, with the highest incidence in breast carcinomas. It is thought that disruption of the P complex would directly affect protein synthesis, causing cell growth arrest and eventually apoptosis. Here, we report a distinct mechanism by which cancer cells undergo cell cycle arrest and induced autophagy when RPLP proteins are downregulated. We found that absence of RPLP0, RPLP1, or RPLP2 resulted in reactive oxygen species (ROS) accumulation and MAPK1/ERK2 signaling pathway activation. Moreover, ROS generation led to endoplasmic reticulum (ER) stress that involved the EIF2AK3/PERK-EIF2S1/eIF2α-EIF2S2-EIF2S3-ATF4/ATF-4- and ATF6/ATF-6-dependent arms of the unfolded protein response (UPR). RPLP protein-deficient cells treated with autophagy inhibitors experienced apoptotic cell death as an alternative to autophagy. Strikingly, antioxidant treatment prevented UPR activation and autophagy while restoring the proliferative capacity of these cells. Our results indicate that ROS are a critical signal generated by disruption of the P complex that causes a cellular response that follows a sequential order: first ROS, then ER stress/UPR activation, and finally autophagy. Importantly, inhibition of the first step alone is able to restore the proliferative capacity of the cells, preventing UPR activation and autophagy. Overall, our results support a role for autophagy as a survival mechanism in response to stress due to RPLP protein deficiency.     

Endoplasmic reticulum stress causes insulin resistance by inhibiting delivery of newly synthesized insulin receptors to the cell surface

Accumulation of unfolded proteins in the endoplasmic reticulum (ER) causes ER stress and activates a signaling network known as the unfolded protein response (UPR). Here we characterize how ER stress and the UPR inhibit insulin signaling. We find that ER stress inhibits insulin signaling by depleting the cell surface population of the insulin receptor. ER stress inhibits proteolytic maturation of insulin proreceptors by interfering with transport of newly synthesized insulin proreceptors from the ER to the plasma membrane. Activation of AKT, a major target of the insulin signaling pathway, by a cytosolic, membrane-bound chimera between the AP20187-inducible F_V2E dimerization domain and the cytosolic protein tyrosine kinase domain of the insulin receptor was not affected by ER stress. Hence, signaling events in the UPR, such as activation of the JNK mitogen-activated protein (MAP) kinases or the pseudokinase TRB3 by the ER stress sensors IRE1α and PERK, do not contribute to inhibition of signal transduction in the insulin signaling pathway. Indeed, pharmacologic inhibition and genetic ablation of JNKs, as well as silencing of expression of TRB3, did not restore insulin sensitivity or rescue processing of newly synthesized insulin receptors in ER-stressed cells.     

The cargo receptor ERGIC-53 is a target of the unfolded protein response

The accumulation of unfolded proteins in the ER triggers a signaling response known as unfolded protein response (UPR). In yeast the UPR affects several hundred genes that encode ER chaperones and proteins operating at later stages of secretion. In mammalian cells the UPR appears to be more limited to chaperones of the ER and genes assumed to be important after cell recovery from ER stress that are not important for secretion. Here, we report that the mRNA of lectin ERGIC-53, a cargo receptor for the transport of glycoproteins from ER to ERGIC, and of its related protein VIP36 is induced by the known inducers of ER stress, tunicamycin and thapsigargin. In parallel, the rate of synthesis of the ERGIC-53 protein was induced by these agents. The response was due to the UPR since it was also triggered by castanospermine, a specific inducer of UPR, and inhibited by genistein. Thapsigargin-induced upregulation of ERGIC-53 could be fully accounted for by the ATF6 pathway of UPR. The results suggest that in mammalian cells the UPR also affects traffic from and beyond the ER.