TRIM27 is an autophagy substrate facilitating mitochondria clustering and mitophagy via phosphorylated TBK1

Tripartite motif-containing protein 27 (TRIM27/also called RFP) is a multifunctional ubiquitin E3 ligase involved in numerous cellular functions, such as proliferation, apoptosis, regulation of the NF-kB pathway, endosomal recycling and the innate immune response. TRIM27 interacts directly with TANK-binding kinase 1 (TBK1) and regulates its stability. TBK1 in complex with autophagy receptors is recruited to ubiquitin chains assembled on the mitochondrial outer membrane promoting mitophagy. Here, we identify TRIM27 as an autophagy substrate, depending on ATG7, ATG9 and autophagy receptors for its lysosomal degradation. We show that TRIM27 forms ubiquitylated cytoplasmic bodies that co-localize with autophagy receptors. Surprisingly, we observed that induced expression of EGFP-TRIM27 in HEK293 FlpIn TRIM27 knockout cells mediates mitochondrial clustering. TRIM27 interacts with autophagy receptor SQSTM1/p62, and the TRIM27-mediated mitochondrial clustering is facilitated by SQSTM/p62. We show that phosphorylated TBK1 is recruited to the clustered mitochondria. Moreover, induced mitophagy activity is reduced in HEK293 FlpIn TRIM27 knockout cells, while re-introduction of EGFP-TRIM27 completely restores the mitophagy activity. Inhibition of TBK1 reduces mitophagy in HEK293 FlpIn cells and in the reconstituted EGFP-TRIM27-expressing cells, but not in HEK293 FlpIn TRIM27 knockout cells. Altogether, these data reveal novel roles for TRIM27 in mitophagy, facilitating mitochondrial clustering via SQSTM1/p62 and mitophagy via stabilization of phosphorylated TBK1 on mitochondria

High-resolution visualization and assessment of basal and OXPHOS-induced mitophagy in H9c2 cardiomyoblasts

Mitochondria are susceptible to damage resulting from their activity as energy providers. Damaged mitochondria can cause harm to the cell and thus mitochondria are subjected to elaborate quality-control mechanisms including elimination via lysosomal degradation in a process termed mitophagy. Basal mitophagy is a house-keeping mechanism fine-tuning the number of mitochondria according to the metabolic state of the cell. However, the molecular mechanisms underlying basal mitophagy remain largely elusive. In this study, we visualized and assessed the level of mitophagy in H9c2 cardiomyoblasts at basal conditions and after OXPHOS induction by galactose adaptation. We used cells with a stable expression of a pH-sensitive fluorescent mitochondrial reporter and applied state-of-the-art imaging techniques and image analysis. Our data showed a significant increase in acidic mitochondria after galactose adaptation. Using a machine-learning approach we also demonstrated increased mitochondrial fragmentation by OXPHOS induction. Furthermore, super-resolution microscopy of live cells enabled capturing of mitochondrial fragments within lysosomes as well as dynamic transfer of mitochondrial contents to lysosomes. Applying correlative light and electron microscopy we revealed the ultrastructure of the acidic mitochondria confirming their proximity to the mitochondrial network, ER and lysosomes. Finally, exploiting siRNA knockdown strategy combined with flux perturbation with lysosomal inhibitors, we demonstrated the importance of both canonical as well as non-canonical autophagy mediators in lysosomal degradation of mitochondria after OXPHOS induction. Taken together, our high-resolution imaging approaches applied on H9c2 cells provide novel insights on mitophagy during physiologically relevant conditions. The implication of redundant underlying mechanisms highlights the fundamental importance of mitophagy

Regulation of expression of autophagy genes by Atg8a-interacting partners Sequoia, YL-1 and Sir2 in Drosophila

Autophagy is the degradation of cytoplasmic material through the lysosomal pathway. One of the most studied autophagy-related proteins is LC3. Despite growing evidence that LC3 is enriched in the nucleus, its nuclear role is poorly understood. Here, we show that Drosophila Atg8a protein, homologous to mammalian LC3, interacts with the transcription factor Sequoia in a LIR motif-dependent manner. We show that Sequoia depletion induces autophagy in nutrient-rich conditions through the enhanced expression of autophagy genes. We show that Atg8a interacts with YL-1, a component of a nuclear acetyltransferase complex, and that it is acetylated in nutrient-rich conditions. We also show that Atg8a interacts with the deacetylase Sir2, which deacetylates Atg8a during starvation to activate autophagy. Our results suggest a mechanism of regulation of the expression of autophagy genes by Atg8a, which is linked to its acetylation status and its interaction with Sequoia, YL-1, and Sir2

FKBP8 and the autophagy-inducing Class-III PI3K Complex Roles of LIR dependent interactions

Autophagy (Greek meaning “self eating”) is a renovation process in cells. Dysfunctional autophagy plays a major role in human diseases including cancer, inflammatory and neurodegenerative diseases. Misfolded or aggregated proteins, damaged organelles, such as mitochondria, endoplasmic reticulum and peroxisomes, as well as intracellular pathogens are degraded by autophagy. During the process, the components that are going to be degraded are encapsulated by a double membrane structure called the autophagosome, which delivers its contents to the lysosome for degradation. Mitochondria is an important cellular organelle, providing energy to the cell by generating the energy molecule adenosine triphosphate through oxidative phosphorylation. This process generates reactive oxygen species that may lead to DNA damage and mutations, as byproducts. Therefore, a healthy population of mitochondria is critical for the well-being of cells. Mitophagy is the selective degradation of mitochondria by autophagy, mediated by specific proteins sitting on the mitochondria outer surface. These proteins cooperate with autophagosomal membrane proteins to deliver the damaged or surplus mitochondria to the lysosome. The PhD thesis work of Zambarlal Bhujabal carried out in the Molecular Cancer Research Group, under guidance of Professor Terje Johansen, focuses on the process of mitophagy and the formation of autophagosomes. This work identifies the outer mitochondrial protein FKBP8 as a mitophagy-mediating protein. FKBP8 was found to cooperate with the autophagosomal membrane protein to clear damaged mitochondria. Interestingly, FKBP8 was further identified to stimulate autophagy in general, probably mediated by interactions with specific proteins at different steps of the autophagy process. In addition, some of the important proteins involved in the formation the autophagosome forming a Phosphatidylinositol kinase complex required for generation of autophagosomes were studied. It was found that ATG14, Beclin-1 and VPS34 bound to the autophagosomal membrane protein GABARAP via short sequence motifs. Notably, during this work, FKBP8 was also found to interact with the Phosphatidylinositol kinase complex member, Beclin-1

The immunophilin Zonda controls regulated exocytosis in endocrine and exocrine tissues

Exocytosis is a fundamental process in physiology, that ensures communication between cells, organs and even organisms. Hormones, neuropeptides and antibodies, among other cargoes are packed in exocytic vesicles that need to reach and fuse with the plasma membrane to release their content to the extracellular milieu. Hundreds of proteins participate in this process and several others in its regulation. We report here a novel component of the exocytic machinery, the Drosophila transmembrane immunophilin Zonda (Zda), previously found to participate in autophagy. Zda is highly expressed in secretory tissues, and regulates exocytosis in at least three of them: the ring gland, insulin-producing cells and the salivary gland. Using the salivary gland as a model system, we found that Zda is required at final steps of the exocytic process for fusion of secretory granules to the plasma membrane. In a genetic screen we identified the small GTPase RalA as a crucial regulator of secretory granule exocytosis that is required, similarly to Zda, for fusion between the secretory granule and the plasma membrane.Fil: de la Riva Carrasco, Rocío Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Perez Pandolfo, Sebastián. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Suarez, Sofia Belen. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Romero, Nuria Magdalena. Centre National de la Recherche Scientifique; Francia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Bhujabal, Zambarlal. University of Tromso; NoruegaFil: Johansen, Terje. University of Tromso; NoruegaFil: Wappner, Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Melani, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentin

CALCOCO1 acts with VAMP ‐associated proteins to mediate ER ‐phagy

The endoplasmic reticulum (ER) plays important roles in protein synthesis and folding, and calcium storage. The volume of the ER and expression of its resident proteins are increased in response to nutrient stress. ER‐phagy, a selective form of autophagy, is involved in the degradation of the excess components of the ER to restore homeostasis. Six ER‐resident proteins have been identified as ER‐phagy receptors so far. In this study, we have identified CALCOCO1 as a novel ER‐phagy receptor for the degradation of the tubular ER in response to proteotoxic and nutrient stress. CALCOCO1 is a homomeric protein that binds directly to ATG8 proteins via LIR‐ and UDS‐interacting region (UIR) motifs acting co‐dependently. CALCOCO1‐mediated ER‐phagy requires interaction with VAMP‐associated proteins VAPA and VAPB on the ER membranes via a conserved FFAT‐like motif. Depletion of CALCOCO1 causes expansion of the ER and inefficient basal autophagy flux. Unlike the other ER‐phagy receptors, CALCOCO1 is peripherally associated with the ER. Therefore, we define CALCOCO1 as a soluble ER‐phagy receptor

TRIM32, but not its muscular dystrophy-associated mutant, positively regulates and is targeted to autophagic degradation by p62/SQSTM1

The tripartite motif (TRIM) proteins constitute a family of ubiquitin E3 ligases involved in a multitude of cellular processes, including protein homeostasis and autophagy. TRIM32 is characterized by six protein–protein interaction domains termed NHL, various point mutations in which are associated with limb-girdle-muscular dystrophy 2H (LGMD2H). Here, we show that TRIM32 is an autophagy substrate. Lysosomal degradation of TRIM32 was dependent on ATG7 and blocked by knockout of the five autophagy receptors p62 (also known as SQSTM1), NBR1, NDP52 (also known as CALCOCO2), TAX1BP1 and OPTN, pointing towards degradation by selective autophagy. p62 directed TRIM32 to lysosomal degradation, while TRIM32 mono-ubiquitylated p62 on lysine residues involved in regulation of p62 activity. Loss of TRIM32 impaired p62 sequestration, while reintroduction of TRIM32 facilitated p62 dot formation and its autophagic degradation. A TRIM32LGMD2H disease mutant was unable to undergo autophagic degradation and to mono-ubiquitylate p62, and its reintroduction into the TRIM32-knockout cells did not affect p62 dot formation. In light of the important roles of autophagy and p62 in muscle cell proteostasis, our results point towards impaired TRIM32-mediated regulation of p62 activity as a pathological mechanisms in LGMD2H

CALCOCO

The endoplasmic reticulum (ER) plays important roles in protein synthesis and folding, and calcium storage. The volume of the ER and expression of its resident proteins are increased in response to nutrient stress. ER‐phagy, a selective form of autophagy, is involved in the degradation of the excess components of the ER to restore homeostasis. Six ER‐resident proteins have been identified as ER‐phagy receptors so far. In this study, we have identified CALCOCO1 as a novel ER‐phagy receptor for the degradation of the tubular ER in response to proteotoxic and nutrient stress. CALCOCO1 is a homomeric protein that binds directly to ATG8 proteins via LIR‐ and UDS‐interacting region (UIR) motifs acting co‐dependently. CALCOCO1‐mediated ER‐phagy requires interaction with VAMP‐associated proteins VAPA and VAPB on the ER membranes via a conserved FFAT‐like motif. Depletion of CALCOCO1 causes expansion of the ER and inefficient basal autophagy flux. Unlike the other ER‐phagy receptors, CALCOCO1 is peripherally associated with the ER. Therefore, we define CALCOCO1 as a soluble ER‐phagy receptor

The immunophilin Zonda controls regulated exocytosis in endocrine and exocrine tissues

Exocytosis is a fundamental process in physiology, that ensures communication between cells, organs and even organisms. Hormones, neuropeptides and antibodies, among other cargoes are packed in exocytic vesicles that need to reach and fuse with the plasma membrane to release their content to the extracellular milieu. Hundreds of proteins participate in this process and several others in its regulation. We report here a novel component of the exocytic machinery, the Drosophila transmembrane immunophilin Zonda (Zda), previously found to participate in autophagy. Zda is highly expressed in secretory tissues, and regulates exocytosis in at least three of them: the ring gland, insulin-producing cells and the salivary gland. Using the salivary gland as a model system, we found that Zda is required at final steps of the exocytic process for fusion of secretory granules to the plasma membrane. In a genetic screen we identified the small GTPase RalA as a crucial regulator of secretory granule exocytosis that is required, similarly to Zda, for fusion between the secretory granule and the plasma membrane

Membrane Atg8ylation, stress granule formation, and MTOR regulation during lysosomal damage

The functions of mammalian Atg8 proteins (mATG8s) expand beyond canonical autophagy and include processes collectively referred to as Atg8ylation. Global modulation of protein synthesis under stress conditions is governed by MTOR and liquid-liquid phase separated condensates containing ribonucleoprotein particles known as stress granules (SGs). We report that lysosomal damage induces SGs acting as a hitherto unappreciated inhibitor of protein translation via EIF2A/eIF2α phosphorylation while favoring an ATF4-dependent integrated stress response. SGs are induced by lysosome-damaging agents, SARS-CoV-2 open reading frame 3a protein (ORF3a) expression, Mycobacterium tuberculosis infection, and exposure to proteopathic MAPT/tau. Proteomic studies revealed recruitment to damaged lysosomes of the core SG proteins NUFIP2 and G3BP1 along with the GABARAPs of the mATG8 family. The recruitment of these proteins is independent of SG condensates or canonical autophagy. GABARAPs interact directly with NUFIP2 and G3BP1 whereas Atg8ylation is needed for their recruitment to damaged lysosomes. At the lysosome, NUFIP2 contributes to MTOR inactivation together with LGALS8 (galectin 8) via the Ragulator-RRAGA-RRAGB complex. The separable functions of NUFIP2 and G3BP1 in SG formation vis-a-vis their role in MTOR inactivation are governed by GABARAP and Atg8ylation. Thus, cells employ membrane Atg8ylation to control and coordinate SG and MTOR responses to lysosomal damage