Stress and the nonsense-mediated RNA decay pathway

Cells respond to internal and external cellular stressors by activating stress-response pathways that re-establish homeostasis. If homeostasis is not achieved in a timely manner, stress pathways trigger programmed cell death (apoptosis) to preserve organism integrity. A highly conserved stress pathway is the unfolded protein response (UPR), which senses excessive amounts of unfolded proteins in the ER. While a physiologically beneficial pathway, the UPR requires tight regulation to provide a beneficial outcome and avoid deleterious consequences. Recent work has demonstrated that a conserved and highly selective RNA degradation pathway—nonsense-mediated RNA decay (NMD)—serves as a major regulator of the UPR pathway. NMD degrades mRNAs encoding UPR components to prevent UPR activation in response to innocuous ER stress. In response to strong ER stress, NMD is inhibited by the UPR to allow for a full-magnitude UPR response. Recent studies have indicated that NMD also has other stress-related functions, including promoting the timely termination of the UPR to avoid apoptosis; NMD also regulates responses to non-ER stressors, including hypoxia, amino-acid deprivation, and pathogen infection. NMD regulates stress responses in species across the phylogenetic scale, suggesting that it has conserved roles in shaping stress responses. Stress pathways are frequently constitutively activated or dysregulated in human disease, raising the possibility that “NMD therapy” may provide clinical benefit by downmodulating stress responses.     

Endoplasmic reticulum stress responses

In homeostasis, cellular processes are in a dynamic equilibrium. Perturbation of homeostasis causes stress. In this review I summarize how perturbation of three major functions of the endoplasmic reticulum (ER) in eukaryotic cells–protein folding, lipid and sterol biosynthesis, and storing intracellular Ca²⁺ – causes ER stress and activates signaling pathways collectively termed the unfolded protein response (UPR). I discuss how the UPR reestablishes homeostasis, and summarize our current understanding of how the transition from protective to apoptotic UPR signaling is controlled, and how the UPR induces inflammatory signaling. Received 21 August 2007; received after revision 26 October 2007; accepted 29 October 2007     

Genetically determined epithelial dysfunction and its consequences for microflora–host interactions

The intestinal epithelium forms a highly active functional interface between the relatively sterile internal body surfaces and the enormously complex and diverse microbiota that are contained within the lumen. Genetic models that allow for manipulation of genes specifically in the intestinal epithelium have provided an avenue to understand the diverse set of pathways whereby intestinal epithelial cells (IECs) direct the immune state of the mucosa associated with homeostasis versus either productive or non-productive inflammation as occurs during enteropathogen invasion or inflammatory bowel disease (IBD), respectively. These pathways include the unfolded protein response (UPR) induced by stress in the endoplasmic reticulum (ER), autophagy, a self-cannibalistic pathway important for intracellular bacterial killing and proper Paneth cell function as well as the interrelated functions of NOD2/NF-κB signaling which also regulate autophagy induction. Multiple genes controlling these IEC pathways have been shown to be genetic risk factors for human IBD. This highlights the importance of these pathways not only for proper IEC function but also suggesting that IECs may be one of the cellular originators of organ-specific and systemic inflammation as in IBD.     

Plant ribosome-inactivating proteins type II induce the unfolded protein response in human cancer cells

Cytotoxic ribosome-inactivating proteins (RIPs) of type II such as ricin were investigated as anti-cancer agents, but also pose a threat as biological weapons. The molecular mechanism leading to their toxic effects is, however, not yet clear. The current paradigm, which states that the irreversible depurination of 28S rRNA results in a general translational arrest eventually leading to cell death, has been questioned. Using micro-array, qRT-PCR and Western blot, we identified the unfolded protein response (UPR), a cellular mechanism activated in response to endoplasmic reticulum stress, that is induced in HCT116 and MDA-MB-231 cells exposed to the plant type II RIPs ricin, riproximin and volkensin. Apoptosis was induced by concentrations at which translation of UPR-related genes still occurred, despite concomitant ribosomal depurination. We conclude that UPR induction represents a model that better describes the cellular effects of RIP exposure at concentrations at which selected proteins are translated despite ribosomal depurination.     

Autophagy: molecular mechanisms,physiological functions and relevance in human pathology

Autophagy is a degradative mechanism mainly involved in the recycling and turnover of cytoplasmic constituents from eukaryotic cells. Over the last years, yeast genetic screens have considerably increased our knowledge about the molecular mechanisms of autophagy, and a number of genes involved in fundamental steps of the autophagic pathway have been identified. Most of these autophagy genes are present in higher eukaryotes indicating that this process has been evolutionarily conserved. In yeast, autophagy is mainly involved in adaptation to starvation, but in multicellular organisms this route has emerged as a multifunctional pathway involved in a variety of additional processes such as programmed cell death, removal of damaged organelles and development of different tissue-specific functions. Furthermore, autophagy is associated with a growing number of pathological conditions, including cancer, myopathies and neurodegenerative disorders. The physiological and pathological roles of autophagy, as well as the molecular mechanisms underlying this multifunctional pathway, are discussed in this review.Received 12 January 2004; received after revision 29 January 2004; accepted 4 February 2004     

A small GTPase, human Rab32, is required for the formation of autophagic vacuoles under basal conditions

Here we show that a small GTPase, Rab32, is a novel protein required for the formation of autophagic vacuoles. We found that the wild-type or GTP-bound form of human Rab32 expressed in HeLa and COS cells is predominantly localized to the endoplasmic reticulum (ER), and overexpression induces the formation of autophagic vacuoles containing an autophagosome marker protein LC3, the ER-resident protein calnexin and endosomal/lysosomal membrane protein LAMP-2, even under nutrient-rich conditions. The recruitment of Rab32 to the ER membrane was necessary for autophagic vacuole formation, suggesting involvement of the ER as a source of autophagosome membranes. In contrast, the expression of the inactive form of, or siRNA-specific for, Rab32 caused the formation of p62/SQSTM1 and ubiquitinated protein-accumulating aggresome-like structures and significantly prevented constitutive autophagy. We postulate that Rab32 facilitates the formation of autophagic vacuoles whose membranes are derived from the ER and regulates the clearance of aggregated proteins by autophagy. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Mechanisms of autophagy and relevant small-molecule compounds for targeted cancer therapy

Autophagy is an evolutionarily conserved, multi-step lysosomal degradation process for the clearance of damaged or superfluous proteins and organelles. Accumulating studies have recently revealed that autophagy is closely related to a variety of types of cancer; however, elucidation of its Janus role of either tumor-suppressive or tumor-promoting still remains to be discovered. In this review, we focus on summarizing the context-dependent role of autophagy and its complicated molecular mechanisms in different types of cancer. Moreover, we discuss a series of small-molecule compounds targeting autophagy-related proteins or the autophagic process for potential cancer therapy. Taken together, these findings would shed new light on exploiting the intricate mechanisms of autophagy and relevant small-molecule compounds as potential anti-cancer drugs to improve targeted cancer therapy.     

Anatomy of autophagy: from the beginning to the end

Autophagy is a highly regulated process in eukaryotes to maintain homeostasis and manage stress responses. Understanding the regulatory mechanisms and key players involved in autophagy will provide critical insights into disease-related pathogenesis and potential clinical treatments. In this review, we describe the hallmark events involved in autophagy, from its initiation, to the final destruction of engulfed targets. Furthermore, based on structural and biochemical data, we evaluate the roles of key players in these processes and provide rationale as to how they control autophagic events in a highly ordered manner.     

Pathogenic mutation in the ALS/FTD gene, <Emphasis Type="Italic">CCNF,</Emphasis> causes elevated Lys48-linked ubiquitylation and defective autophagy

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal neurodegenerative disorders that have common molecular and pathogenic characteristics, such as aberrant accumulation and ubiquitylation of TDP-43; however, the mechanisms that drive this process remain poorly understood. We have recently identified CCNF mutations in familial and sporadic ALS and FTD patients. CCNF encodes cyclin F, a component of an E3 ubiquitin–protein ligase (SCF^{cyclin F}) complex that is responsible for ubiquitylating proteins for degradation by the ubiquitin–proteasome system. In this study, we examined the ALS/FTD-causing p.Ser621Gly (p.S621G) mutation in cyclin F and its effect upon downstream Lys48-specific ubiquitylation in transfected Neuro-2A and SH-SY5Y cells. Expression of mutant cyclin F^S621G caused increased Lys48-specific ubiquitylation of proteins in neuronal cells compared to cyclin F^WT. Proteomic analysis of immunoprecipitated Lys48-ubiquitylated proteins from mutant cyclin F^S621G-expressing cells identified proteins that clustered within the autophagy pathway, including sequestosome-1 (p62/SQSTM1), heat shock proteins, and chaperonin complex components. Examination of autophagy markers p62, LC3, and lysosome-associated membrane protein 2 (Lamp2) in cells expressing mutant cyclin F^S621G revealed defects in the autophagy pathway specifically resulting in impairment in autophagosomal–lysosome fusion. This finding highlights a potential mechanism by which cyclin F interacts with p62, the receptor responsible for transporting ubiquitylated substrates for autophagic degradation. These findings demonstrate that ALS/FTD-causing mutant cyclin F^S621G disrupts Lys48-specific ubiquitylation, leading to accumulation of substrates and defects in the autophagic machinery. This study also demonstrates that a single missense mutation in cyclin F causes hyper-ubiquitylation of proteins that can indirectly impair the autophagy degradation pathway, which is implicated in ALS pathogenesis.     

Autophagy and other vacuolar protein degradation mechanisms.

Autophagic degradation of cytoplasm (including protein, RNA etc.) is a non-selective bulk process, as indicated by ultrastructural evidence and by the similarity in autophagic sequestration rates of various cytosolic enzymes with different half-lives. The initial autophagic sequestration step, performed by a poorly-characterized organelle called a phagophore, is subject to feedback inhibition by purines and amino acids, the effect of the latter being potentiated by insulin and antagonized by glucagon. Epinephrine and other adrenergic agonists inhibit autophagic sequestration through a prazosin-sensitive alpha 1-adrenergic mechanism. The sequestration is also inhibited by cAMP and by protein phosphorylation as indicated by the effects of cyclic nucleotide analogues, phosphodiesterase inhibitors and okadaic acid. Asparagine specifically inhibits autophagic-lysosomal fusion without having any significant effects on autophagic sequestration, on intralysosomal degradation or on the endocytic pathway. Autophaged material that accumulates in prelysosomal vacuoles in the presence of asparagine is accessible to endocytosed enzymes, revealing the existence of an amphifunctional organelle, the amphisome. Evidence from several cell types suggests that endocytosis may be coupled to autophagy to a variable extent, and that the amphisome may play a central role as a collecting station for material destined for lysosomal degradation. Protein degradation can also take place in a 'salvage compartment' closely associated with the endoplasmic reticulum (ER). In this compartment unassembled protein chains are degraded by uncharacterized proteinases, while resident proteins return to the ER and assembled secretory and membrane proteins proceed through the Golgi apparatus. In the trans-Golgi network some proteins are proteolytically processed by Ca(2+)-dependent proteinases; furthermore, this compartment sorts proteins to lysosomes, various membrane domains, endosomes or secretory vesicles/granules. Processing of both endogenous and exogenous proteins can occur in endosomes, which may play a particularly important role in antigen processing and presentation. Proteins in endosomes or secretory compartments can either be exocytosed, or channeled to lysosomes for degradation. The switch mechanisms which decide between these options are subject to bioregulation by external agents (hormones and growth factors), and may play an important role in the control of protein uptake and secretion.     

Chaperone-mediated autophagy: machinery,regulation and biological consequences

Degradation of dysfunctional intracellular components in the lysosome system can occur through three different pathways, i.e., macroautophagy, microautophagy and chaperone-mediated autophagy (CMA). In this review, we focus on CMA, a type of autophagy distinct from the other two autophagic pathways owing to its selectivity, saturability and competitivity by which a subset of long-lived cytosolic soluble proteins are directly delivered into the lysosomal lumen via specific receptors. CMA participates in quality control to maintain normal cell functions by clearing “old” proteins and provides energy to cells under nutritional stress. Deregulation of CMA has recently been shown to underlie some diseases, especially neurodegenerative disorders for which the decline with age in the activity of CMA may become a major aggravating factor. Therefore, targeting aberrant alteration in CMA under pathological conditions could serve as a potential therapeutic strategy for treating related diseases.     

Many mechanisms, one entrance: membrane protein translocation into the nucleus

The inner nuclear membrane harbors a unique set of membrane proteins, many of which interact with nuclear intermediate filaments and chromatin components and thus play an important role in nuclear organization and gene expression regulation. These membrane proteins have to be constantly transported into the nucleus from their sites of synthesis in the ER to match the growth of the nuclear membrane during interphase. Many mechanisms have evolved to enable translocation of these proteins to the nucleus. The full range of mechanisms goes from rare autophagy events to regulated translocation using the nuclear pore complexes. Though mechanisms involving nuclear pores are predominant, within this group an enormous mechanistic range is observed from free diffusion through the peripheral channels to many distinct mechanisms involving different nucleoporins and other components of the soluble protein transport machinery in the central channels. This review aims to provide a comprehensive insight into this mechanistic diversity.     

Autophagy in diabetic kidney disease: regulation,pathological role and therapeutic potential

Diabetic kidney disease, a leading cause of end-stage renal disease, has become a serious public health problem worldwide and lacks effective therapies. Autophagy is a highly conserved lysosomal degradation pathway that removes protein aggregates and damaged organelles to maintain cellular homeostasis. As important stress-responsive machinery, autophagy is involved in the pathogenesis of various diseases. Emerging evidence has suggested that dysregulated autophagy may contribute to both glomerular and tubulointerstitial pathologies in kidneys under diabetic conditions. This review summarizes the recent findings regarding the role of autophagy in the pathogenesis of diabetic kidney disease and highlights the regulation of autophagy by the nutrient-sensing pathways and intracellular stress signaling in this disease. The advances in our understanding of autophagy in diabetic kidney disease will facilitate the discovery of a new therapeutic target for the prevention and treatment of this life-threatening diabetes complication.     

Basal autophagy is involved in the degradation of the ERAD component EDEM1

Little is known about the fate of machinery proteins of the protein quality control and endoplasmic reticulum(ER)-associated degradation (ERAD). We investigated the degradation of the ERAD component EDEM1, which directs overexpressed misfolded glycoproteins to degradation. Endogenous EDEM1 was studied since EDEM1 overexpression not only resulted in inappropriate occurrence throughout the ER but also caused cytotoxic effects. Proteasome inhibitors had no effect on the clearance of endogenous EDEM1 in non-starved cells. However, EDEM1 could be detected by immunocytochemistry in autophagosomes and biochemically in LC3 immuno-purified autophagosomes. Furthermore, influencing the lysosome-autophagy pathway by vinblastine or pepstatin A/E64d and inhibiting autophagosome formation by 3-methyladenine or ATGs short interfering RNA knockdown stabilized EDEM1. Autophagic degradation involved removal of cytosolic Triton X-100-insoluble deglycosylated EDEM1, but not of EDEM1-containing ER cisternae. Our studies demonstrate that endogenous EDEM1 in cells not stressed by the expression of a transgenic misfolded protein reaches the cytosol and is degraded by basal autophagy. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Received 15 January 2009; received after revision 16 February 2009; accepted 17 February 2009 V. Le Fourn, K. Gaplovska-Kysela: These authors equally contributed to this work.