Rab7b modulates autophagic flux by interacting with Atg4B

Autophagy (macroautophagy) is a highly conserved eukaryotic degradation pathway in which cytosolic components and organelles are sequestered by specialized autophagic membranes and degraded through the lysosomal system. The autophagic pathway maintains basal cellular homeostasis and helps cells adapt during stress; thus, defects in autophagy can cause detrimental effects. It is therefore crucial that autophagy is properly regulated. In this study, we show that the cysteine protease Atg4B, a key enzyme in autophagy that cleaves LC3, is an interactor of the small GTPase Rab7b. Indeed, Atg4B interacts and co‐localizes with Rab7b on vesicles. Depletion of Rab7b increases autophagic flux as indicated by the increased size of autophagic structures as well as the magnitude of macroautophagic sequestration and degradation. Importantly, we demonstrate that Rab7b regulates LC3 processing by modulating Atg4B activity. Taken together, our findings reveal Rab7b as a novel negative regulator of autophagy through its interaction with Atg4B

Analysis of ATG4C function in vivo

Despite the great advances in macroautophagy/autophagy research in the last years, the in vivo role of the different members of the four mammalian orthologs of yeast Atg4 protease (ATG4A-D) remain unclear. To gain further insights into the functional relevance of Atg4 orthologs, we have generated mutant mice deficient in Atg4c. These mice are viable and fertile, and do not display any obvious abnormalities, indicating that they are able to develop the autophagic response required during the early neonatal period. However, they show tissue-specific autophagy alterations, including reduced autophagic flux in diaphragm and show decreased breathing and locomotor activity after fasting. In addition, atg4c-/- mice show reduced number of circulating T and B lymphocytes, which is associated with accumulation of apoptotic cells in the spleen and an increased susceptibility to develop chemically-induced fibrosarcomas. Moreover, through the analysis of cells and mice simultaneously deficient for ATG4C and ATG4D proteases we also reveal a role for ATG4C in mATG8 proteins delipidation.This work was supported by grants from Ministerio Ciencia eInnovación (Spain) (PID2021-127534OB-I00), the South-Eastern 1315 Norway Regional Health Authority (2021088 to N.E.) and Instituto de Salud Carlos III (RTICC Spain). Jesús Prieto-Lloret is funded by Programa Estrategico IBGM, Escalera de Excelencia, ref. CCVC8485, Consejería de Educación, Junta de Castilla y León (Spain). Funding for open Access Charge: Roche Farma”, as the aricle will be published via Open access and the OA costs will be funded by Roche Farma.Peer reviewe

Chloroquine inhibits autophagic flux by decreasing autophagosome-lysosome fusion

Macroautophagy/autophagy is a conserved transport pathway where targeted structures are sequestered by phagophores, which mature into autophagosomes, and then delivered into lysosomes for degradation. Autophagy is involved in the pathophysiology of numerous diseases and its modulation is beneficial for the outcome of numerous specific diseases. Several lysosomal inhibitors such as bafilomycin A(1) (BafA(1)), protease inhibitors and chloroquine (CQ), have been used interchangeably to block autophagy in in vitro experiments assuming that they all primarily block lysosomal degradation. Among them, only CQ and its derivate hydroxychloroquine (HCQ) are FDA-approved drugs and are thus currently the principal compounds used in clinical trials aimed to treat tumors through autophagy inhibition. However, the precise mechanism of how CQ blocks autophagy remains to be firmly demonstrated. In this study, we focus on how CQ inhibits autophagy and directly compare its effects to those of BafA(1). We show that CQ mainly inhibits autophagy by impairing autophagosome fusion with lysosomes rather than by affecting the acidity and/or degradative activity of this organelle. Furthermore, CQ induces an autophagy-independent severe disorganization of the Golgi and endo-lysosomal systems, which might contribute to the fusion impairment. Strikingly, HCQ-treated mice also show a Golgi disorganization in kidney and intestinal tissues. Altogether, our data reveal that CQ and HCQ are not bona fide surrogates for other types of late stage lysosomal inhibitors for in vivo experiments. Moreover, the multiple cellular alterations caused by CQ and HCQ call for caution when interpreting results obtained by blocking autophagy with this drug

Assessing autophagy in archived tissue or how to capture autophagic flux from a tissue snapshot

Autophagy is a highly conserved degradation mechanism that is essential for maintaining cellular homeostasis. In human disease, autophagy pathways are frequently deregulated and there is immense interest in targeting autophagy for therapeutic approaches. Accordingly, there is a need to determine autophagic activity in human tissues, an endeavor that is hampered by the fact that autophagy is characterized by the flux of substrates whereas histology informs only about amounts and localization of substrates and regulators at a single timepoint. Despite this challenging task, considerable progress in establishing markers of autophagy has been made in recent years. The importance of establishing clear-cut autophagy markers that can be used for tissue analysis cannot be underestimated. In this review, we attempt to summarize known techniques to quantify autophagy in human tissue and their drawbacks. Furthermore, we provide some recommendations that should be taken into consideration to improve the reliability and the interpretation of autophagy biomarkers in human tissue samples.This work was supported by grants from the Bernese Cancer League, “Stiftung für klinisch-experimentelle Tumorforschung”, and the Werner and Hedy Berger-Janser Foundation for Cancer Research (to M.H.); by Institute of Health Carlos III (ISCIII) and FEDER funds from the EU (PI14/01085 and PI17/00093) and supported by Miguel Servet contract by ISCIII and FSE funds (CPII16/00023) (to M.M.); from the Spanish Ministry of Science, Innovation and Universities (RTI2018-096748-B-100 to N.A.); from the University Professor Training Fellowship, Ministry of Science, Innovation and University, Government of Spain (FPU17/00026) (to P.C.O); from the ISCIII (PI16/00090 and PI19/01266) and the Andalusian Government (Consejería de Igualdad, Salud y Políticas Sociales, PI-0198-2016) for their financial support, and from the Biomedical Research Network Center for Liver and Digestive Diseases (CIBERehd) founded by the ISCIII and co-financed by European Development Regional Fund (EDRF) “A way to achieve Europe” for their financial support (to J.M.), from Breakthrough Cancer Research, Ireland funding (to S.L.M); from the PI18/00442 grant integrated into the State Plan for R & D + I2013-2016 and funded by the ISCIII and the ERDF, a way to make Europe (to G.V.); from the Luxembourg National Research Fund (C18/BM/12670304/COMBATIC to B.J.); from the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, by the European Regional Development Fund (FEDER), through the Competitiveness Factors Operational Programme (COMPETE) (NORTE-01-0145-FEDER-000013) and from the projects POCI-01-0145-FEDER-028159 and POCI-01-0145-FEDER-030782 by FEDER, through the COMPETE (to P.L.); from National funds, through the Foundation for Science and Technology (FCT) (to P.L.); from ARRS—the Slovenian research agency, programme P1-0140: Proteolysis and its regulation (led by B. Turk) (to E.Ž.); from the Swiss Cancer Research (KFS-3360-02-2014) (to A.P, and M.P.T.) (KFS-3409-02-2014), and the Swiss National Science Foundation (31003A_173219) (to M.P.T.)

A Novel Role of Listeria monocytogenes Membrane Vesicles in Inhibition of Autophagy and Cell Death

Bacterial membrane vesicle (MV) production has been mainly studied in Gram-negative species. In this study, we show that Listeria monocytogenes, a Gram-positive pathogen that causes the food-borne illness listeriosis, produces MVs both in vitro and in vivo. We found that a major virulence factor, the pore-forming hemolysin listeriolysin O (LLO), is tightly associated with the MVs, where it resides in an oxidized, inactive state. Previous studies have shown that LLO may induce cell death and autophagy. To monitor possible effects of LLO and MVs on autophagy, we performed assays for LC3 lipidation and LDH sequestration as well as analysis by confocal microscopy of HEK293 cells expressing GFP-LC3. The results revealed that MVs alone did not affect autophagy whereas they effectively abrogated autophagy induced by pure LLO or by another pore-forming toxin from Vibrio cholerae, VCC. Moreover, Listeria monocytogenes MVs significantly decreased Torin1-stimulated macroautophagy. In addition, MVs protected against necrosis of HEK293 cells caused by the lytic action of LLO. We explored the mechanisms of LLO-induced autophagy and cell death and demonstrated that the protective effect of MVs involves an inhibition of LLO-induced pore formation resulting in inhibition of autophagy and the lytic action on eukaryotic cells. Further, we determined that these MVs help bacteria to survive inside eukaryotic cells (mouse embryonic fibroblasts). Taken together, these findings suggest that intracellular release of MVs from L. monocytogenes may represent a bacterial strategy to survive inside host cells, by its control of LLO activity and by avoidance of destruction from the autophagy system during infection

The Lactate Dehydrogenase Sequestration Assay - A Simple and Reliable Method to Determine Bulk Autophagic Sequestration Activity in Mammalian Cells

Bulk autophagy is characterized by the sequestration of large portions of cytoplasm into double/multi-membrane structures termed autophagosomes. Here a simple protocol to monitor this process is described. Moreover, typical results and experimental validation of the method under autophagy-inducing conditions in various types of cultured mammalian cells are provided. During bulk autophagy, autophagosomes sequester cytosol, and thereby also soluble cytosolic proteins, alongside other autophagic cargo. LDH is a stable and highly abundant, soluble cytosolic enzyme that is non-selectively sequestered into autophagosomes. The amount of LDH sequestration therefore reflects the amount of bulk autophagic sequestration. To efficiently and accurately determine LDH sequestration in cells, we employ an electrodisruption-based fractionation protocol that effectively separates sedimentable from cytosolic LDH, followed by measurement of enzymatic activity in sedimentable fractions versus whole-cell samples. Autophagic sequestration is determined by subtracting the proportion of sedimentable LDH in untreated cells from that in treated cells. The advantage of the LDH sequestration assay is that it gives a quantitative measure of the autophagic sequestration of endogenous cargo, as opposed to other methods that either involve ectopic expression of sequestration probes or semi-quantitative protease protection analyses of autophagy markers or receptors