Endoplasmic reticulum stress and fungal pathogenesis

The gateway to the secretory pathway is the endoplasmic reticulum (ER), an organelle that is responsible for the accurate folding, post-translational modification and final assembly of up to a third of the cellular proteome. When secretion levels are high, errors in protein biogenesis can lead to the accumulation of abnormally folded proteins, which threaten ER homeostasis. The unfolded protein response (UPR) is an adaptive signaling pathway that counters a buildup in misfolded and unfolded proteins by increasing the expression of genes that support ER protein folding capacity. Fungi, like other eukaryotic cells that are specialized for secretion, rely upon the UPR to buffer ER stress caused by fluctuations in secretory demand. However, emerging evidence is also implicating the UPR as a central regulator of fungal pathogenesis. In this review, we discuss how diverse fungal pathogens have adapted ER stress response pathways to support the expression of virulence-related traits that are necessary in the host environment.     

内质网应激偶联炎症反应与慢性病发病机制

内质网是合成细胞内分泌蛋白和膜蛋白并进行蛋白折叠的主要细胞器。新近研究证明,当内质网蛋白质合成与折叠的负担增加、非折叠或错误折叠蛋白质堆积,可激活内质网的几组特定信号转导通路,将这些应激信号传递到细胞浆和细胞核,引起未/错误折叠蛋白反应。这对维持细胞动态平衡和生物体的发育具有重要意义。更为重要的是,未/错误折叠蛋白反应能够与细胞内炎症反应信号转导通路偶联,是非感染性致病原引发炎症反应的主要原因。因此,内质网应激-未/错误折叠蛋白反应-炎症反应在特定的细胞发生偶联是许多炎症疾病的发病机制。本文综述该领域的研究进展,并介绍了内质网应激信号和炎症反应偶联参与一些慢性病发病的分子细胞机制。这些研究不仅加深人们对这些慢性病发病机制的了解,也有助于对调节内质网应激-炎症反应的药物的研发。     

Unfolded protein response

In eukaryotic cells, the endoplasmic reticulum (ER) is a membrane-enclosed interconnected organelle responsible for the synthesis, folding, modification, and quality control of numerous secretory and membrane proteins. The processes of protein folding and maturation are highly assisted and scrutinized but are also sensitive to changes in ER homeostasis, such as Ca(2+) depletion, oxidative stress, hypoxia, energy deprivation, metabolic stimulation, altered glycosylation, activation of inflammation, as well as increases in protein synthesis or the expression of misfolded proteins or unassembled protein subunits. Only properly folded proteins can traffic to the Golgi apparatus, whereas those that misfold are directed to ER-associated degradation (ERAD) or to autophagy. The accumulation of unfolded/misfolded proteins in the ER activates signaling events to orchestrate adaptive cellular responses. This unfolded protein response (UPR) increases the ER protein-folding capacity, reduces global protein synthesis, and enhances ERAD of misfolded proteins.     

The endoplasmic reticulum and unfolded protein response in the control of mammalian recombinant protein production

The endoplasmic reticulum (ER) of eukaryotic cells is involved in the synthesis and processing of proteins and lipids in the secretory pathway. These processing events that proteins undergo in the ER may present major limiting steps for recombinant protein production. Increased protein synthesis, accumulation of improperly processed or mis-folded protein can induce ER stress. To cope with ER stress, the ER has quality control mechanisms, such as the unfolded protein response (UPR) and ER-associated degradation to restore homeostasis. ER stress and UPR activation trigger multiple physiological cellular changes. Here we review cellular mechanisms that cope with ER stress and illustrate how this knowledge can be applied to increase the efficiency of recombinant protein expression.     

Redox signaling loops in the unfolded protein response

The endoplasmic reticulum (ER) is the first compartment of secretory pathway. It plays a major role in ER chaperone-assisted folding and quality control, including post-translational modification such as disulfide bond formation of newly synthesized secretory proteins. Protein folding and assembly takes place in the ER, where redox conditions are distinctively different from the other organelles and are favorable for disulfide formation. These reactions generate the production of reactive oxygen species (ROS) as a byproduct of thiol/disulfide exchange reaction among ER oxidoreductin 1 (Ero1), protein disulfide isomerase (PDI) and ER client proteins, during the formation of disulfide bonds in nascent or incorrectly folded proteins. When uncontrolled, this phenomenon perturbs ER homeostasis, thus aggravating the accumulation of improperly folded or unfolded proteins in this compartment (ER stress). This results in the activation of an adaptive mechanism named the unfolded protein response (UPR). In mammalian cells, the UPR is mediated by three ER-resident membrane proteins (PERK, IRE1 and ATF6) and regulates the expression of the UPR target genes, which themselves encode ER chaperones, folding enzymes, pro-apoptotic proteins and antioxidants, with the objective of restoring ER homeostatic balance. In this review, we will describe redox dependent activation (ER) and amplification (cytosol) loops that control the UPR and the consequences these regulatory loops have on cell fate and physiology.     

The impact of the unfolded protein response on human disease

A central function of the endoplasmic reticulum (ER) is to coordinate protein biosynthetic and secretory activities in the cell. Alterations in ER homeostasis cause accumulation of misfolded/unfolded proteins in the ER. To maintain ER homeostasis, eukaryotic cells have evolved the unfolded protein response (UPR), an essential adaptive intracellular signaling pathway that responds to metabolic, oxidative stress, and inflammatory response pathways. The UPR has been implicated in a variety of diseases including metabolic disease, neurodegenerative disease, inflammatory disease, and cancer. Signaling components of the UPR are emerging as potential targets for intervention and treatment of human disease.     

Activation of mammalian unfolded protein response is compatible with the quality control system operating in the endoplasmic reticulum

Newly synthesized secretory and transmembrane proteins are folded and assembled in the endoplasmic reticulum (ER) where an efficient quality control system operates so that only correctly folded molecules are allowed to move along the secretory pathway. The productive folding process in the ER has been thought to be supported by the unfolded protein response (UPR), which is activated by the accumulation of unfolded proteins in the ER. However, a dilemma has emerged; activation of ATF6, a key regulator of mammalian UPR, requires intracellular transport from the ER to the Golgi apparatus. This suggests that unfolded proteins might be leaked from the ER together with ATF6 in response to ER stress, exhibiting proteotoxicity in the secretory pathway. We show here that ATF6 and correctly folded proteins are transported to the Golgi apparatus via the same route and by the same mechanism under conditions of ER stress, whereas unfolded proteins are retained in the ER. Thus, activation of the UPR is compatible with the quality control in the ER and the ER possesses a remarkable ability to select proteins to be transported in mammalian cells in marked contrast to yeast cells, which actively utilize intracellular traffic to deal with unfolded proteins accumulated in the ER.     

Cotranslocational degradation protects the stressed endoplasmic reticulum from protein overload

The ER's capacity to process proteins is limited, and stress caused by accumulation of unfolded and misfolded proteins (ER stress) contributes to human disease. ER stress elicits the unfolded protein response (UPR), whose components attenuate protein synthesis, increase folding capacity, and enhance misfolded protein degradation. Here, we report that P58(IPK)/DNAJC3, a UPR-responsive gene previously implicated in translational control, encodes a cytosolic cochaperone that associates with the ER protein translocation channel Sec61. P58(IPK) recruits HSP70 chaperones to the cytosolic face of Sec61 and can be crosslinked to proteins entering the ER that are delayed at the translocon. Proteasome-mediated cytosolic degradation of translocating proteins delayed at Sec61 is cochaperone dependent. In P58(IPK-/-) mice, cells with a high secretory burden are markedly compromised in their ability to cope with ER stress. Thus, P58(IPK) is a key mediator of cotranslocational ER protein degradation, and this process likely contributes to ER homeostasis in stressed cells.     

Kar2p availability defines distinct forms of endoplasmic reticulum stress in living cells

Accumulation of misfolded secretory proteins in the endoplasmic reticulum (ER) activates the unfolded protein response (UPR) stress pathway. To enhance secretory protein folding and promote adaptation to stress, the UPR upregulates ER chaperone levels, including BiP. Here we describe chromosomal tagging of KAR2, the yeast homologue of BiP, with superfolder green fluorescent protein (sfGFP) to create a multifunctional endogenous reporter of the ER folding environment. Changes in Kar2p-sfGFP fluorescence levels directly correlate with UPR activity and represent a robust reporter for high-throughput analysis. A novel second feature of this reporter is that photobleaching microscopy (fluorescence recovery after photobleaching) of Kar2p-sfGFP mobility reports on the levels of unfolded secretory proteins in individual cells, independent of UPR status. Kar2p-sfGFP mobility decreases upon treatment with tunicamycin or dithiothreitol, consistent with increased levels of unfolded proteins and the incorporation of Kar2p-sfGFP into slower-diffusing complexes. During adaptation, we observe a significant lag between down-regulation of the UPR and resolution of the unfolded protein burden. Finally, we find that Kar2p-sfGFP mobility significantly increases upon inositol withdrawal, which also activates the UPR, apparently independent of unfolded protein levels. Thus Kar2p mobility represents a powerful new tool capable of distinguishing between the different mechanisms leading to UPR activation in living cells.     

The endoplasmic reticulum and the unfolded protein response

The endoplasmic reticulum (ER) is the site where proteins enter the secretory pathway. Proteins are translocated into the ER lumen in an unfolded state and require protein chaperones and catalysts of protein folding to attain their final appropriate conformation. A sensitive surveillance mechanism exists to prevent misfolded proteins from transiting the secretory pathway and ensures that persistently misfolded proteins are directed towards a degradative pathway. In addition, those processes that prevent accumulation of unfolded proteins in the ER lumen are highly regulated by an intracellular signaling pathway known as the unfolded protein response (UPR). The UPR provides a mechanism by which cells can rapidly adapt to alterations in client protein-folding load in the ER lumen by expanding the capacity for protein folding. In addition, a variety of insults that disrupt protein folding in the ER lumen also activate the UPR. These include changes in intralumenal calcium, altered glycosylation, nutrient deprivation, pathogen infection, expression of folding-defective proteins, and changes in redox status. Persistent protein misfolding initiates apoptotic cascades that are now known to play fundamental roles in the pathogenesis of multiple human diseases including diabetes, atherosclerosis and neurodegenerative diseases.     

ER stress and its functional link to mitochondria: role in cell survival and death

The endoplasmic reticulum (ER) is the primary site for synthesis and folding of secreted and membrane-bound proteins. Proteins are translocated into ER lumen in an unfolded state and require protein chaperones and catalysts of protein folding to assist in proper folding. Properly folded proteins traffic from the ER to the Golgi apparatus; misfolded proteins are targeted to degradation. Unfolded protein response (UPR) is a highly regulated intracellular signaling pathway that prevents accumulation of misfolded proteins in the ER lumen. UPR provides an adaptive mechanism by which cells can augment protein folding and processing capacities of the ER. If protein misfolding is not resolved, the UPR triggers apoptotic cascades. Although the molecular mechanisms underlying ER stress-induced apoptosis are not completely understood, increasing evidence suggests that ER and mitochondria cooperate to signal cell death. Mitochondria and ER form structural and functional networks (mitochondria-associated ER membranes [MAMs]) essential to maintain cellular homeostasis and determine cell fate under various pathophysiological conditions. Regulated Ca(2+) transfer from the ER to the mitochondria is important in maintaining control of prosurvival/prodeath pathways. We discuss the signaling/communication between the ER and mitochondria and focus on the role of the mitochondrial permeability transition pore in these complex processes.     

未折叠蛋白反应的信号转导

在内质网中,分泌性蛋白、跨膜蛋白和内质网驻留蛋白折叠成天然构象,经过修饰后,形成有活性的功能性蛋白质。如果蛋白质在内质网内的折叠受到抑制,造成未折叠蛋白聚集,将引起内质网应激。激活未折叠蛋白反应（unfolded protein response,UPR）,使蛋白质的生物合成减少,内质网的降解功能增强,从而降低内质网负担,维持细胞内的稳态。如果内质网应激持续存在,则可能诱发细胞凋亡。研究表明,未折叠蛋白反应能在多种肿瘤细胞中发生,并能促进肿瘤细胞的生长。本文对未折叠蛋白反应与肿瘤研究的最新进展进行综述。