Tension-induced autophagy

Impairment of autophagy in patients and animal models severely affects mechanically strained tissues such as skeletal muscle, heart, lung and kidney, leading for example to muscle dystrophy, cardiomyopathy and renal injury. However, the reason for this high reliance on autophagy remained largely elusive. Recent work in our lab now provides a possible explanation. We identified chaperone-assisted selective autophagy (CASA) as a tension-induced autophagy pathway essential for mechanotransduction in mammalian cells.     

Bacterial autophagy

Autophagy is a vital process through which cellular material and dysfunctional organelles are degraded and recycled, and it is inhibited by the metabolic checkpoint kinase MTOR. Autophagy also targets intracellular bacteria (a process termed xenophagy) for lysosomal degradation, thereby playing a key role in innate immunity. In the past few years, the identification of molecules, such as CALCOCO2/NDP52, SQSTM1/p62 and ubiquitin, implicated in the specific targeting of intracellular bacteria, received considerable attention. However, it remains unclear how xenophagy is initiated, since this process commonly occurs in metabolically replete cells. In a recent study, we demonstrated that infection with Shigella and Salmonella triggered an early state of intracellular amino acid (AA) starvation causing MTOR dissociation from endomembranes, downregulation of MTOR activity and activation of the EIF2AK4/GCN2-EIF2S1/eIF2α/ATF3 signaling axis. We also observed that AA starvation was caused by host membrane damage, which appeared to be transient in the case of Salmonella and sustained in Shigella-infected cells, thus highlighting the existence of key timing disparities in xenophagy triggering, depending on the bacterial pathogen. Together, our findings demonstrate that xenophagy is only one arm of a more general metabolic switch geared toward AA starvation in bacteria-infected cells.     

Hypoxia-induced autophagy

A major challenge in formulating an effective immunotherapy is to overcome the mechanisms of tumor escape from immunosurveillance. We showed that hypoxia-induced autophagy impairs cytotoxic T-lymphocyte (CTL)-mediated tumor cell lysis by regulating phospho-STAT3 in target cells. Autophagy inhibition in hypoxic cells decreases phospho-STAT3 and restores CTL-mediated tumor cell killing by a mechanism involving the ubiquitin proteasome system and SQSTM1/p62. Simultaneously boosting the CTL-response, using a TRP-peptide vaccination strategy, and targeting autophagy in hypoxic tumors, improves the efficacy of cancer vaccines and promotes tumor regression in vivo. Overall, in addition to its immunosuppressive effect, the hypoxic microenvironment also contributes to immunoresistance and can be detrimental to antitumor effector cell functions.     

Non-selective autophagy

Autophagy is the major process by which cells degrade their own cytoplasm. Autophagy begins with the sequestration of a portion of the cytoplasm by a membraneous organelle called a phagophore. The resulting vacuole (autophagosome) can fuse with an endocytic vacuole to form am amphisome, which subsequently fuses with a lysosome to have its mixed autophagic/endocytic content degraded by lysosomal enzymes. Autophagy is a non-selective bulk process as indicated by the fact that hepatocytic cytosol enzymes with widely different half-lives are sequestered at the same rate. Regulation of autophagy is exerted at the sequestration step by amino acids, purines, ATP-depleting metabolites, cyclic nucleotides, phosphorylation, and hormones like insulin, glucagon and alpha-adrenergic agonists.     

Finding autophagy

To tell the truth, I find it difficult to work when flying, or even when sitting in an airport for an extended period of time. So, typically I take along a book to read. And when I truly cannot concentrate, for example when a flight is considerably delayed, I have even been known to resort to word puzzles. Depending on the type, they do not require much attention (that is, you can pick up right where you left off after you glance at the flight status screen for the twentieth or so time, even though you know nothing has changed), or effort (although you need to use a pen or pencil, not a keyboard), but nonetheless they can keep your mind somewhat occupied. I even rationalize doing them based on the assumption that they are sharpening my observational/pattern-finding skills. One type of word puzzle that is particularly mindless, but for that very reason I still enjoy in the above circumstances, is a word search; you are given a grid with letters and/or numbers, and a list of “hidden” terms, and you circle them within the grid, crossing them off the list as you go along. I do admit that the categories of terms used in the typical word searches can become rather mundane (breeds of dog, types of food, words that are followed by “stone,” words associated with a famous movie star, words from a particular television show, etc.). Therefore, on one of my last seminar trips I decided to generate my own word search, using the category of autophagy.     

Cardiovascular autophagy

Despite recent scientific and technological advances, cardiovascular disease remains the leading cause of morbidity and mortality worldwide. Autophagy, an evolutionarily ancient response to cellular stress, has been implicated in the pathogenesis of a wide range of heart pathologies. However, the precise role of autophagy in these contexts remains obscure owing to its multifarious actions. Here, we review recently derived insights regarding the role of autophagy in multiple manifestations of cardiac plasticity and disease.     

Inducing autophagy

Autophagy is a lysosomal-mediated catabolic process, which through degradation of different cytoplasmic components aids in maintaining cellular homeostasis and survival during exposure to extra- or intracellular stresses. Ammonia is a potential toxic and stress-inducing byproduct of glutamine catabolism, which has recently been found to induce autophagy in an MTOR independent way and support cancer cell survival. In this study, quantitative phosphoproteomics was applied to investigate the initial signaling events linking ammonia to the induction of autophagy. The MTOR inhibitor rapamycin was used as a reference treatment to emphasize the differences between an MTOR-dependent and -independent autophagy-induction. By this means 5901 phosphosites were identified of which 626 were treatment-specific regulated and 175 were coregulated. Investigation of the ammonia-specific regulated sites supported that MTOR activity was not affected, but indicated increased MAPK3 activity, regulation of proteins involved in Rho signal transduction, and a novel phosphorylation motif, serine-proline-threonine (SPT), which could be linked to cytoskeleton-associated proteins. MAPK3 could not be identified as the primary driver of ammonia-induced autophagy but instead the data suggested an upregulation of AMPK and the unfolded protein response (UPR), which might link ammonia to autophagy induction. Support of UPR induction was further obtained from the finding of increased protein levels of the ER stress markers DDIT3/CHOP and HSPA5 during ammonia treatment. The large-scale data set presented here comprises extensive high-quality quantitative information on phosphoprotein regulation in response to 2 very different autophagy inducers and should therefore be considered a general resource for the community.     

Endosome-mediated autophagy

Activation of TLR signaling has been shown to induce autophagy in antigen-presenting cells (APCs). Using high-resolution microscopy approaches, we show that in LPS-stimulated dendritic cells (DCs), autophagosomes emerge from MHC class II compartments (MIICs) and harbor both the molecular machinery for antigen processing and the autophagosome markers LC3 and ATG16L1. This ENdosome-Mediated Autophagy (ENMA) appears to be the major type of autophagy in DCs, as similar structures were observed upon established autophagy-inducing conditions (nutrient deprivation, rapamycin) and under basal conditions in the presence of bafilomycin A1. Autophagosome formation was not significantly affected in DCs expressing ATG4B^C74A mutant and atg4b^−/− bone marrow DCs, but the degradation of the autophagy substrate SQSTM1/p62 was largely impaired. Furthermore, we demonstrate that the previously described DC aggresome-like LPS-induced structures (DALIS) contain vesicular membranes, and in addition to SQSTM1 and ubiquitin, they are positive for LC3. LC3 localization on DALIS is independent of its lipidation. MIIC-driven autophagosomes preferentially engulf the LPS-induced SQSTM1-positive DALIS, which become later degraded in autolysosomes. DALIS-associated membranes also contain ATG16L1, ATG9 and the Q-SNARE VTI1B, suggesting that they may represent (at least in part) a membrane reservoir for autophagosome expansion. We propose that ENMA constitutes an unconventional, APC-specific type of autophagy, which mediates the processing and presentation of cytosolic antigens by MHC class II machinery, and/or the selective clearance of toxic by-products of elevated ROS/RNS production in activated DCs, thereby promoting their survival.     

Targeting autophagy

In recent years, tremendous progress has been made toward unveiling the mechanism of autophagy and its exploitation by many different cancer types. This year the American Association for the Advancement of Science held a one day Symposium on Autophagy: An Emerging Therapeutic Target in Human Disease in Vancouver, British Columbia and brought together experts in cell biology, drug discovery, and clinical translation to share their research findings and prospects. Currently, autophagy is being investigated on several fronts, from modulation of gene expression to in vivo studies, and more recently clinical trials in cancer. Key topics of discussion were determining which stage of autophagy would be the ideal target for inhibition to produce the highest impact, and which cancers or cancer subtypes would be the most sensitive to autophagy inhibitors; the answers to these questions may be a turning point in cancer therapy research.     

Mechanisms of chaperone-mediated autophagy

Chaperone-mediated autophagy is one of several lysosomal pathways of proteolysis. This pathway is activated by physiological stresses such as prolonged starvation. Cytosolic proteins with particular peptide sequence motifs are recognized by a complex of molecular chaperones and delivered to lysosomes. No vesicular traffic is required for this protein degradation pathway, so it differs from microautophagy and macroautophagy. Protein substrates bind to a receptor in the lysosomal membrane, the lysosome-associated membrane protein (lamp) type 2a. Levels of lamp2a in the lysosomal membrane are controlled by alterations in the lamp2a half-life as well as by the dynamic distribution of the protein between the lysosomal membrane and the lumen. Substrate proteins are unfolded before transport into the lysosome lumen, and the transport of substrate proteins requires a molecular chaperone within the lysosomal lumen. The exact roles of this lysosomal chaperone remain to be defined. The mechanisms of chaperone-mediated autophagy are similar to mechanisms of protein import into mitochondria, chloroplasts, and the endoplasmic reticulum.     

Viral evasion of autophagy

Restriction of both bacterial and viral pathogen growth by autophagy has been documented in vitro. In this issue of Cell Host & Microbe, Orvedahl et al. demonstrate for the first time that inhibition of autophagy by a viral gene product is essential for neurovirulence of herpesviruses.     

Pathophysiology of chaperone-mediated autophagy

In contrast to the classically described “in bulk” lysosomal degradation, the first evidence for selective degradation of cytosolic proteins in lysosomes was presented more than 20 years ago. Throughout this time, we have gained a better understanding about this process, now known as chaperone-mediated autophagy (CMA). The identification of new substrates for CMA and novel components, in both the cytosol and the lysosomes, along with better insights on how CMA is regulated, have all helped to shape the possible physiological roles of CMA. We review here different intracellular functions of CMA that arise from its unique characteristics when compared to other forms of autophagy. In view of these functions, we discuss the relevance of the changes in CMA activity in aging and in different pathological conditions.     

Cardioprotection through autophagy

Interventions that reduce infarct size in animal models have largely failed to improve outcome in patients suffering acute myocardial infarction (MI), or ‘heart attack’. Our group recently reported a reduction of infarct size by chloramphenicol treatment in a porcine in vivo model of acute MI, through a mechanism involving the induction of autophagy. Since 2005 several studies have implicated autophagy as a target for cardioprotection.     

The autophagy community

There are various definitions of community. A definition that I found in one of my dictionaries is the following: “A social, religious, occupational, or other group sharing common characteristics or interests and perceived or perceiving itself as distinct in some respect from the larger society within which it exists.” Thus, I think it is fair to say that there is a worldwide autophagy community. That is, there is a group of researchers (our occupation), whose members share an interest in autophagy (our common characteristic), and that group is distinct from the larger society (I do not want to begin describing the many ways this applies). But do we feel like a community, and do we need a community? I suggest that a community is indeed beneficial, and I propose one mechanism for enhancing the development of the autophagy community.