Beyond autophagy: The role of UVRAG in membrane trafficking

Autophagy is a lysosome-directed membrane trafficking event for the degradation of cytoplasmic components, including organelles. The past few years have seen a great advance in our understanding of the cellular machinery of autophagosome biogenesis, the hallmark of autophagy. However, our global understanding of autophagosome maturity remains relatively poor and fragmented. The topological similarity of autophagosome and endosome delivery to lysosomes suggests that autophagic and endosomal maturation may have evolved to share associated machinery to promote the lysosomal delivery of their cargoes. We have recently discovered that UVRAG, originally identified as a Beclin 1-binding autophagy protein, appears to be an important factor in autophagic and endosomal trafficking through its interaction with the class C Vps tethering complex. Given the ability of UVRAG to bind Beclin 1 and the class C Vps complex in a genetically and functionally separable manner, it may serve as an important regulator for the spatial and/or temporal control of diverse cellular trafficking events. As more non-autophagic functions of UVRAG are unveiled, our understanding of seemingly different cellular processes may move a step further.

Addendum to: Liang C, Lee JS, Inn KS, Gack MU, Li Q, Roberts EA, Vergne I, Deretic V, Feng P, Akazawa C, Jung JU. Beclin1-binding UVRAG targets the class C Vps complex to coordinate autophagosome maturation and endocytic trafficking. Nat Cell Biol 2008; 10:776–87.     

Beyond autophagy: the role of UVRAG in membrane trafficking

Autophagy is a lysosome-directed membrane trafficking event for the degradation of cytoplasmic components, including organelles. The past few years have seen a great advance in our understanding of the cellular machinery of autophagosome biogenesis, the hallmark of autophagy. However, our global understanding of autophagosome maturity remains relatively poor and fragmented. The topological similarity of autophagosome and endosome delivery to lysosomes suggests that autophagic and endosomal maturation may have evolved to share associated machinery to promote the lysosomal delivery of their cargoes. We have recently discovered that UVRAG, originally identified as a Beclin 1-binding autophagy protein, appears to be an important factor in autophagic and endosomal trafficking through its interaction with the class C Vps tethering complex. Given the ability of UVRAG to bind Beclin 1 and the class C Vps complex in a genetically and functionally separable manner, it may serve as an important regulator for the spatial and/or temporal control of diverse cellular trafficking events. As more non-autophagic functions of UVRAG are unveiled, our understanding of seemingly different cellular processes may move a step further.     

UVRAG: At the crossroad of autophagy and genomic stability

UVRAG is a promoter of the autophagy pathway, and its deficiency may fuel the development of cancers. Intriguingly, our recent study has demonstrated that this protein also mediates the repair of damaged DNA and patrols centrosome stability, mechanisms that commonly prevent cancer progression, in a manner independent of its role in autophagy signaling. Given the central role of UVRAG in genomic stability and autophagic cleaning, it is speculated that UVRAG is a bona fide genome protector and that the decrease in UVRAG seen in some cancers may render these cells vulnerable to chromosomal damage, making UVRAG an appealing target for cancer therapy.     

A critical role for UVRAG in apoptosis

Autophagy and apoptosis are tightly regulated biological processes that are crucial for cell growth, development and tissue homeostasis. UVRAG (UV radiation resistance-associated gene), a mammalian homolog of yeast Vps38, activates the Beclin 1/PtdIns3KC3 (class III phosphatidylinositol-3-kinase) complex, which promotes autophagosome formation. Moreover, UVRAG promotes autophagosome maturation by recruiting class C Vps complexes (HOPS complexes) and Rab7 of the late endosome. We found that UVRAG has anti-apoptotic activity during tumor therapy through interactions with Bax. UVRAG inhibits Bax translocation from the cytosol to mitochondria during chemotherapy- or UV irradiation-induced apoptosis of human tumor cells. Moreover, deletion of the UVRAG C2 domain abolishes Bax binding and anti-apoptotic activity. These results suggest that, in addition to its previously recognized pro-autophagy activity in response to starvation, UVRAG has cytoprotective functions in the cytosol that control the localization of Bax in tumor cells exposed to apoptotic stimuli.     

A critical role for UVRAG in apoptosis

Autophagy and apoptosis are tightly regulated biological processes that are crucial for cell growth, development and tissue homeostasis. UVRAG (UV radiation resistance-associated gene), a mammalian homolog of yeast Vps38, activates the Beclin 1/PtdIns3KC3 (class III phosphatidylinositol-3-kinase) complex, which promotes autophagosome formation. Moreover, UVRAG promotes autophagosome maturation by recruiting class C Vps complexes (HOPS complexes) and Rab7 of the late endosome. We found that UVRAG has anti-apoptotic activity during tumor therapy through interactions with Bax. UVRAG inhibits Bax translocation from the cytosol to mitochondria during chemotherapy- or UV irradiation-induced apoptosis of human tumor cells. Moreover, deletion of the UVRAG C2 domain abolishes Bax binding and anti-apoptotic activity. These results suggest that, in addition to its previously recognized pro-autophagy activity in response to starvation, UVRAG has cytoprotective functions in the cytosol that control the localization of Bax in tumor cells exposed to apoptotic stimuli.     

Essential role for nuclear PTEN in maintaining chromosomal integrity

A broad spectrum of mutations in PTEN, encoding a lipid phosphatase that inactivates the P13-K/AKT pathway, is found associated with primary tumors. Some of these mutations occur outside the phosphatase domain, suggesting that additional activities of PTEN function in tumor suppression. We report a nuclear function for PTEN in controlling chromosomal integrity. Disruption of Pten leads to extensive centromere breakage and chromosomal translocations. PTEN was found localized at centromeres and physically associated with CENP-C, an integral component of the kinetochore. C-terminal PTEN mutants disrupt the association of PTEN with centromeres and cause centromeric instability. Furthermore, Pten null cells exhibit spontaneous DNA double-strand breaks (DSBs). We show that PTEN acts on chromatin and regulates expression of Rad51, which reduces the incidence of spontaneous DSBs. Our results demonstrate that PTEN plays a fundamental role in the maintenance of chromosomal stability through the physical interaction with centromeres and control of DNA repair. We propose that PTEN acts as a guardian of genome integrity.     

A dual role for Ca(2+) in autophagy regulation

Autophagy is a cellular process responsible for delivery of proteins or organelles to lysosomes. It participates not only in maintaining cellular homeostasis, but also in promoting survival during cellular stress situations. It is now well established that intracellular Ca²⁺ is one of the regulators of autophagy. However, this control of autophagy by intracellular Ca²⁺ signaling is the subject of two opposite views. On the one hand, the available evidence indicates that intracellular Ca²⁺ signals, and mainly inositol 1,4,5-trisphosphate receptors (IP₃Rs), suppress autophagy. On the other hand, elevated cytosolic Ca²⁺ concentrations ([Ca²⁺]_cyt) were also shown to promote the autophagic process. Here, we will provide a critical overview of the literature and discuss both hypotheses. Moreover, we will suggest a model explaining how changes in intracellular Ca²⁺ signaling can lead to opposite outcomes, depending on the cellular state.     

UVRAG in autophagy,inflammation, and cancer

ABSTRACT

Macroautophagy/autophagy deregulation has been observed in perpetuated inflammation and the proliferation of tumor cells. However, the mechanisms underlying these changes have yet to be well-identified. UVRAG is one of the key players of autophagy, but its role in vivo remained puzzling. Our recent study utilized a mouse model with inducible expression of a cancer-derived frameshift (FS) mutation in UVRAG that dominant-negatively inhibits wild-type UVRAG, resulting in impaired stimulus-induced autophagy. The systemically compromised autophagy, particularly mitophagy, notably increases inflammation and associated pathologies. Furthermore, our discovery indicates that time-dependent autophagy suppression and ensuing CTNNB1/β-catenin activation may serve as one tumor-promoting mechanism underpinning age-related cancer susceptibility.     

The role of autophagy in maintaining intestinal mucosal barrier

The intestinal mucosal barrier is the first line to defense against luminal content penetration and performs numerous biological functions. The intestinal epithelium contains a huge surface that is lined by a monolayer of intestinal epithelial cells (IECs). IECs are dominant mediators in maintaining intestinal homeostasis that drive diverse functions including nutrient absorption, physical segregation, secretion of antibacterial peptides, and modulation of immune responses. Autophagy is a cellular self-protection mechanism in response to various stresses, and accumulating studies have revealed its importance in participating physiological processes of IECs. The regulatory effects of autophagy depend on the specific IEC types. This review aims to elucidate the myriad roles of autophagy in regulating the functions of different IECs (stem cells, enterocytes, goblet cells, and Paneth cells), and present the progress of autophagy-targeting therapy in intestinal diseases. Understanding the involved mechanisms can provide new preventive and therapeutic strategies for gastrointestinal dysfunction and diseases.     

Antioxidant role of autophagy in maintaining the integrity of glomerular capillaries

Autophagy is a lysosomal degradation system by which cytosolic materials and damaged organelles are broken down into basic components. To explore the physiological role of autophagy in glomerular endothelial cells (GEnCs), we compared the autophagic flux among cells in the kidney under starvation. Inhibition of autophagy by chloroquine administration significantly increased the number of autophagosomes or autolysosomes in GEnCs and proximal tubular cells, but not in podocytes, suggesting that the GEnCs exhibit substantial autophagic activity. Next, we analyzed endothelial and hematopoietic cell-specific atg5-deficient mice (atg5-conditional KO [cKO] mice). Glomeruli of 4-wk-old atg5-cKO mice exhibited slightly distended capillary loops accompanied by an accumulation of reactive oxygen species (ROS). Glomeruli of 8-wk-old atg5-cKO mice showed a lobular pattern with thickening of the capillary loops and mesangial matrix expansion; however, the vasculature of other organs was preserved. The atg5-cKO mice died by 12 wk of age, presumably due to pancytopenia resulting from the defect in their hematopoietic lineages. Therefore, we subjected 4-wk atg5-cKO mice to irradiation followed by bone marrow transplantation from normal littermates. Transplanted mice recapitulated the glomerular phenotypes of the atg5-cKO mice with no obvious histological changes in other organs. Twelve-mo-old transplanted mice developed mesangiolysis and glomerulosclerosis with significant deterioration of kidney function. Administration of N-acetyl-l-cysteine, a ROS scavenger, to atg5-cKO mice rescued the glomerular phenotypes. These data suggest that endothelial autophagy protects glomeruli from oxidative stress and maintains the integrity of glomerular capillaries. Enhancing endothelial autophagy may provide a novel therapeutic approach to minimizing glomerular diseases.     

UVRAG: a new player in autophagy and tumor cell growth

Autophagy has a well-documented role in the maintenance of homeostasis and the response to stressful environments and it is often deregulated in various human diseases including cancer. The regulation of the Beclin 1-PI3KC3 complex lipid kinase activity is a critical element in the autophagy signaling pathway. Previous studies(1) have demonstrated that Beclin 1-PI3KC3-mediated autophagy is negatively regulated by a proto-oncogene Bcl-2. We have recently identified a novel coiled-coil UVRAG tumor suppressor candidate, which positively engages in Beclin 1-dependent autophagy. UVRAG interacts with Beclin 1, leading to activation of autophagy and thereof inhibition of tumorigenesis. This finding adds a new player to the emerging picture of the autophagy network, under-scoring the importance of the coordinated activity between Bcl-2 and UVRAG in the regulation of Beclin 1-PI3KC3-mediated autophagy and tumor cell control.     

Essential role for autophagy protein VMP1 in maintaining neuronal homeostasis and preventing axonal degeneration

Vacuole membrane protein 1 (VMP1), the endoplasmic reticulum (ER)-localized autophagy protein, plays a key role during the autophagy process in mammalian cells. To study the impact of VMP1-deficiency on midbrain dopaminergic (mDAergic) neurons, we selectively deleted VMP1 in the mDAergic neurons of VMP1^fl/fl/DAT^CreERT2 bigenic mice using a tamoxifen-inducible CreERT2/loxp gene targeting system. The VMP1^fl/fl/DAT^CreERT2 mice developed progressive motor deficits, concomitant with a profound loss of mDAergic neurons in the substantia nigra pars compacta (SNc) and a high presynaptic accumulation of α-synuclein (α-syn) in the enlarged terminals. Mechanistic studies showed that VMP1 deficiency in the mDAergic neurons led to the increased number of microtubule-associated protein 1 light chain 3-labeled (LC3) puncta and the accumulation of sequestosome 1/p62 aggregates in the SNc neurons, suggesting the impairment of autophagic flux in these neurons. Furthermore, VMP1 deficiency resulted in multiple cellular abnormalities, including large vacuolar-like structures (LVSs), damaged mitochondria, swollen ER, and the accumulation of ubiquitin⁺ aggregates. Together, our studies reveal a previously unknown role of VMP1 in modulating neuronal survival and maintaining axonal homeostasis, which suggests that VMP1 deficiency might contribute to mDAergic neurodegeneration via the autophagy pathway.Subject terms: Neuroscience, Pathogenesis     

A protective role for autophagy in vitiligo

A growing number of studies supports the existence of a dynamic interplay between energetic metabolism and autophagy, whose induction represents an adaptive response against several stress conditions. Autophagy is an evolutionarily conserved and a highly orchestrated catabolic recycling process that guarantees cellular homeostasis. To date, the exact role of autophagy in vitiligo pathogenesis is still not clear. Here, we provide the first evidence that autophagy occurs in melanocytes and fibroblasts from non-lesional skin of vitiligo patients, as a result of metabolic surveillance response. More precisely, this study is the first to reveal that induction of autophagy exerts a protective role against the intrinsic metabolic stress and attempts to antagonize degenerative processes in normal appearing vitiligo skin, where melanocytes and fibroblasts are already prone to premature senescence.Subject terms: Macroautophagy, Translational research     

A role for mitochondria in autophagy regulation

Understanding the functional relationship between mitochondria and autophagy is critical for understanding the molecular mechanisms underlying aging and neurodegeneration. Autophagy functions in both cellular homeostasis and in quality control in the selective removal of dysfunctional mitochondria. A current working model in the field is that impaired autophagy results in a cell-damaging accumulation of dysfunctional mitochondria over time. We described our findings that respiratory-deficient mitochondria can inhibit general (macro) autophagy in Saccharomyces cerevisiae by conserved signaling pathways during amino acid starvation. These data point to an interdependence of mitochondrial function and autophagy and raise the possibility that negative regulation of autophagy by dysfunctional mitochondria is a critical contributing factor in many diseases.     

A role for diacylglycerol in antibacterial autophagy

Antibacterial autophagy is understood to be a key cellular immune response to invading microbes. However, the mechanism(s) by which bacteria are selected as targets of autophagy remain unclear. We recently identified diacylglycerol as a novel signaling molecule that targets bacteria to the autophagy pathway, and show that it acts via protein kinase C activation. We also found that Pkc1 is required for autophagy in yeast, indicating that this kinase plays a conserved role in autophagy regulation.Key words: bacteria, Salmonella, innate immunity, adaptor, lipid second messenger, diacylglycerol, ubiquitin, NDP52, p62, SQSTM1The mechanism by which bacteria and other subcellular targets are identified and degraded by the autophagy pathway is an area of intense research. Ubiquitin has been recently found to act as an essential signal required for the autophagy of bacteria and proteins. We have previously observed ubiquitin on autophagy-targeted Salmonella enterica serovar Typhimurium (S. Typhimurium) but were surprised to see that only 50% of these bacteria were positive for ubiquitin. This indicated the possibility that an alternate signal was required for efficient autophagic targeting of the nonubiquitinated population of these bacteria.We initially performed a screen quantifying the colocalization of different lipid second messengers (diacylglycerol (DAG), PtdIns(3)P, PtdIns(4,5)P₂, PtdIns(3,4) P₂, and PtdIns(3,4,5)P₃) with autophagytargeted (i.e., LC3⁺) S. Typhimurium. We observed that DAG preferentially localizes with LC3⁺ bacteria. A kinetic analysis revealed that maximal DAG colocalization with bacteria (45 min post-infection) precedes maximal autophagy of the bacteria (60 min post-infection). Using pharmacological agents, siRNA and dominant negative constructs we were able to determine that DAG localization to the bacteria requires the action of phospholipase D (PLD; phosphatidylcholine to phosphatidic acid conversion) and phosphatidic acid phosphatase (PAP; phosphatidic acid to DAG conversion). We observed that inhibition of these pathways significantly reduces DAG localization to bacteria as well as concomitant autophagy of the bacteria, indicating a role for this lipid second messenger in the regulation of this process.Having determined that DAG is necessary for autophagy of bacteria we subsequently wanted to identify the effector through which it was signaling. Conventional and novel isoforms of the protein kinase C (PKC) family contain DAG-binding C1 domains. Accordingly, we targeted PKC isoforms using pharmacological agents, siRNA and knockout cell lines and were able to determine that DAG is signaling through the δ isoform of PKC. Inhibition of this serine/threonine kinase results in significant inhibition of antibacterial autophagy. Furthermore, bacterial replication in PKCδ knockout mouse embryonic fibroblasts is significantly higher compared to control fibroblasts, consistent with previous observations demonstrating that autophagy impairs intracellular replication of S. Typhimurium (Birmingham et al. 2006).We addressed the possibility that DAG and ubiquitin are functioning in a cooperative manner to target Salmonella for degradation by autophagy. We simultaneously inhibited both pathways using siRNA or pharmacological agents and observed additive inhibitory effects on autophagy of the bacteria. While this is indicative of two independent pathways, we cannot discount the possibility that there is still cooperation between the two pathways, especially as we did observe a small population of bacteria that were positive for both DAG and ubiquitin (Fig. 1). There are also a number of technical limitations in the methods we used, such as detection levels of the probes and antibodies that warrant caution in concluding that the two pathways are completely independent. Nonetheless, our studies clearly demonstrate a role for both DAG (Shahnazari et al. 2010) and ubiquitin (Zheng et al. 2009) in autophagy of S. Typhimurium. Future studies are required to further examine how these signals contribute to regulation of antibacterial autophagy.Open in a separate windowFigure 1Autophagic targeting of Salmonella Typhimurium. Invading S. Typhimurium can be targeted to the autophagy pathway by two independent signaling mechanisms. The first requires ubiquitin and the autophagy adaptors p62 and NDP52. The second requires DAG generation and PKCδ function. DAG generation on the SCV may occur through interaction of the SCV with DAG-positive endocytic vesicles (pathway 1) or through direct DAG production on the SCV (pathway 2). SCV, Salmonella-containing vacuole; PA, phosphatidic acid; DAG, diacylglycerol; PAP, phosphatidic acid phosphatase; PKCδ, protein kinase C delta; Ub, ubiquitin.Having characterized this pathway in antibacterial autophagy we were interested in determining whether these components were required for general autophagy. We therefore tested whether DAG localizes with rapamycin-induced autophagosomes. We observed DAG on these compartments and also found a requirement for PAP and PKCδ in this process. Other PKC isoforms are involved in alternate types of autophagy including ER stress-induced autophagy (Sakaki et al. 2008) as well as hypoxia-induced autophagy (Chen et al. 2009). As a result, we were interested in determining whether PKC function in autophagy was evolutionarily conserved. We therefore tested a role for the yeast ortholog, Pkc1, in this process and observed that it is required for starvation-induced autophagy in Saccharomyces cerevisiae.Having identified and characterized a novel signal and effector for antibacterial autophagy, further work still remains to be done in order to obtain a complete picture of this process. This includes additional study of the mechanism by which DAG is generated and the subcellular localization of PLD and PAP during this process. It is possible that DAG⁺ endocytic vesicles fuse with the Salmonella-containing vacuole (SCV) coating this compartment with DAG (pathway 1, see Fig. 1). It is also possible that both PLD and PAP function directly on the SCV, converting phosphatidylcholine to DAG via the phosphatidic acid intermediate (pathway 2, Fig. 1).More work also needs to be done to dissect DAG and ubiquitin signaling contributions to this pathway. Questions to be answered include the identification of the ubiquitinated protein(s) on the SCV, which may be host or bacterial proteins. Additionally, while we know that DAG is present on the SCV we do not yet know the signal that induces its generation. One intriguing possibility is that DAG generation occurs in response to bacterial-induced damage to the SCV during invasion. To date, PKC has been implicated in at least three different types of autophagy, and the possibility exists that other PKC isoforms (DAG responsive or not) are also involved in this process.     

Base excision repair and its role in maintaining genome stability

For all living organisms, genome stability is important, but is also under constant threat because various environmental and endogenous damaging agents can modify the structural properties of DNA bases. As a defense, organisms have developed different DNA repair pathways. Base excision repair (BER) is the predominant pathway for coping with a broad range of small lesions resulting from oxidation, alkylation, and deamination, which modify individual bases without large effect on the double helix structure. As, in mammalian cells, this damage is estimated to account daily for 10(4) events per cell, the need for BER pathways is unquestionable. The damage-specific removal is carried out by a considerable group of enzymes, designated as DNA glycosylases. Each DNA glycosylase has its unique specificity and many of them are ubiquitous in microorganisms, mammals, and plants. Here, we review the importance of the BER pathway and we focus on the different roles of DNA glycosylases in various organisms.     

A role for mitochondria in autophagy regulation

Understanding the functional relationship between mitochondria and autophagy is critical for understanding the molecular mechanisms underlying aging and neurodegeneration. Autophagy functions in both cellular homeostasis and in quality control in the selective removal of dysfunctional mitochondria. A current working model in the field is that impaired autophagy results in a cell-damaging accumulation of dysfunctional mitochondria over time. We described our findings that respiratory-deficient mitochondria can inhibit general (macro) autophagy in Saccharomyces cerevisiae by conserved signaling pathways during amino acid starvation. These data point to an interdependence of mitochondrial function and autophagy and raise the possibility that negative regulation of autophagy by dysfunctional mitochondria is a critical contributing factor in many diseases.     

A novel role for autophagy in neurodevelopment

We recently showed that Ambra 1, a WD40-containing approximately 130 KDa protein, is a novel activating molecule in Beclin 1-regulated autophagy and plays a role in the development of the nervous system. Ambra 1 binds to Beclin 1 and favors Beclin 1/Vps34 interaction. At variance with these factors, Ambra 1 is highly conserved among vertebrates only, and its expression is mostly confined to the neuroepithelium during early neurogenesis. Ambra 1 functional inactivation in mouse led to lethality in utero (starting from embryonic day 14.5), characterized by severe neural tube defects associated with autophagy impairment, unbalanced cell proliferation, accumulation of ubiquitinated proteins, and excessive apoptosis. We also demonstrated that hyperproliferation was the earliest detectable abnormality in the developing neuroepithelium, followed by a wave of caspase-dependent cell death. These findings provided in vivo evidence supporting the existence of a complex interplay between autophagy, cell proliferation and cell death during neural development in mammals. In this article, we review our findings in the contexts of autophagy and neurodevelopment and consider some of the issues raised.