PINK1 regulated mitophagy is evident in skeletal muscles

PINK1, mutated in familial forms of Parkinson’s disease, initiates mitophagy following mitochondrial depolarization. However, it is difficult to monitor this pathway physiologically in mice as loss of PINK1 does not alter basal mitophagy levels in most tissues. To further characterize this pathway in vivo, we used mito-QC mice in which loss of PINK1 was combined with the mitochondrial-associated POLGD257A mutation. We focused on skeletal muscle as gene expression data indicates that this tissue has the highest PINK1 levels. We found that loss of PINK1 in oxidative hindlimb muscle significantly reduced mitophagy. Of interest, the presence of the POLGD257A mutation, while having a minor effect in most tissues, restored levels of muscle mitophagy caused by the loss of PINK1. Although our observations highlight that multiple mitophagy pathways operate within a single tissue, we identify skeletal muscle as a tissue of choice for the study of PINK1-dependant mitophagy under basal conditions

Greining a hlutverki ATG7 ísóforma

ATG7, an essential protein for cellular homeostasis, plays a pivotal role in regulating autophagy. The autophagy process ensures the degradation and recycling of cellular components for proper cell function. ATG7 facilitates the lipidation of ATG8 proteins, a vital step in autophagosome formation. Notably, ATG7 is expressed as two mRNA splice variants, referred to as ATG7(1) and ATG7(2). The corresponding ATG7(2) protein lacks an essential domain rendering it incapable of carrying out the canonical autophagy-related functions. While ATG7(1) is well-known for its involvement in autophagy, the role, and regulatory mechanisms of ATG7(2) remain unknown. Studies have established a connection between ATG7 and tumorigenesis. Notably, previous research by the team revealed an association of an ATG7 variant (D522E) with increased risk of hepatic cancer. ATG7 is also connected to glucose and lipid metabolism, exerting influence over metabolic pathways. However, significant knowledge gaps persist, particularly regarding the non-autophagic functions of ATG7. We started by investigating how the D522 residue in ATG7 impacts the risk of liver cancer development using protein interactome analysis of mutant variants. We observed interactions between the ATG7-D522 variants, and proteins involved in cytoskeletal organization. Then, we discovered that D522 is located in a conserved vertebrate specific region (VSR) of ATG7. Subsequently, we created cell models with ΔVSR mutants to study their biological functions and found that the VSR is required for the lipidating activity and potentially affects cytoskeletal organization. However, as the most substantial differences were observed within ATG7(2), our research efforts were directed towards characterizing this specific isoform. We found that ATG7(2) expression correlated with malignancy, being more prevalent in primary tumors and influencing survival in cancer patients. Comparing protein interactions of the two isoforms, we found that ATG7(1) primarily interacts with autophagy-related proteins, while ATG7(2) interacts with mitochondrial membrane proteins and crucial glycolytic enzymes. Functional analysis revealed that ATG7(2) exerts a repressive effect on glycolysis and mitochondrial activities. Our research sheds light on the non-canonical ATG7(2) isoform, revealing its associations with malignancy, and distinct protein interactions, ultimately impacting glycolysis and mitochondrial activities. Unraveling the complexities of ATG7(2) will enhance our understanding of the diverse roles of ATG7 in cellular processes beyond autophagy.ATG7 próteinið gegnir lykilhlutverki í sjálfsáti í frumum. Sjálfsát er niðurbrotsferli sem tryggir að ekki safnist upp skemmd frumulíffæri og stórsameindir innan frumu, og er þannig mikilvægt fyrir heilbrigða frumustarfsemi. Ferlið er einnig mikilvægt viðbragð frumu til þess að komast af við erfiðar umhverfisaðstæður eins og súrefnis- eða næringarskort. ATG7 hvatar nauðsynlegt skref í myndun sjálfsátsbóla, með því að hvata lípíðbindingu annars sjálfsátspróteins, ATG8. Tvö ATG7 mRNA eru hæst tjáð í vefjum mannslíkamans, ATG7(1) og ATG7(2). Í samanburði við ATG7(1), vantar svæði í ATG7(2) próteinið, sem er nauðsynlegt fyrir sjálfsátsvirkni þess. Hlutverk ATG7(1) í sjálfsáti er vel skilgreint, en lítið er vitað um hlutverk ATG7(2). Áhrif ATG7 í krabbameinum hafa einkum verið rannsökuð í músum, en fyrri niðurstöður rannsóknarhópsins, höfðu sýnt að erfðabreytileiki í ATG7 (D522E) hefur fylgni við auknar líkur á lifrar- og gallvegakrabbameini. Komið hefur í ljós að ATG7 hefur margvísleg áhrif á glúkósa og lípíð efnaskipti bæði í æxlum og heilbrigðum vef. Við byrjuðum á að rannsaka hvaða hlutverki D522 amínósýran í ATG7 gegnir í krabbameinum. Við gerðum massagreiningu á prótein bindifélögum ATG7 villigerðar og stökkbreytts próteins. Í ljós kom að ATG7 með stökkbreytingu í D522 binst frymisgrindarpróteinum. Þróunarfræðigreining leiddi í ljós að D522 er staðsett í hluta próteinsins sem eingöngu er til staðar í hryggdýrum (VSR svæði). Við könnuðum áhrif þessa svæðis með því að tjá prótein sem vantar þetta svæði (∆VSR) og í ljós kom að svæðið er nauðsynlegt fyrir sjálfsátsvirkni ATG7 og hefur áhrif á frymisgrindina. Áhrifin voru þó mest þegar svæðið vantar í ATG7(2). Við ákváðum því að leggja áherslu á að skilgreina hlutverk ATG7(2). Greining á tjáningu á ATG7 í heilbrigðum vef og æxlum leiddi í ljós aukningu á ATG7(2) í æxlum og áhrif tjáningar þess á lifun. Þegar bornir voru saman prótein bindifélagar ísóformanna kom í ljós að ATG7(1) binst sjálfsátspróteinum eins og skilgreint hefur verið áður. Hins vegar binst ATG7(2) lykilpróteinum í glýkólýsu og himnupróteinum hvatbera. Frekari rannsóknir sýndu að ATG7(2) hindrar glýkólýsu og hvatberavirkni. Rannsóknir okkar hafa leitt í ljós áður óskilgreinda virkni ATG7. ATG7(2) hefur áhrif á lifun í krabbameinum, binst efnaskiptapróteinum í stað sjálfsátspróteina og hefur áhrif á glýkólýsu og hvatberavirkni. Mikilvægt verður að skilgreina þetta hlutverk nánar og varpa þannig ljósi á hlutverk ATG7 og sjálfsáts í krabbameinum

EB1 Restricts Breast Cancer Cell Invadopodia Formation and Matrix Proteolysis via FAK

International audienceRegulation of microtubule dynamics by plus-end tracking proteins (+TIPs) plays an essential role in cancer cell migration. However, the role of +TIPs in cancer cell invasion has been poorly addressed. Invadopodia, actin-rich protrusions specialized in extracellular matrix degradation, are essential for cancer cell invasion and metastasis, the leading cause of death in breast cancer. We, therefore, investigated the role of the End Binding protein, EB1, a major hub of the +TIP network, in invadopodia functions. EB1 silencing increased matrix degradation by breast cancer cells. This was recapitulated by depletion of two additional +TIPs and EB1 partners, APC and ACF7, but not by the knockdown of other +TIPs, such as CLASP1/2 or CLIP170. The knockdown of Focal Adhesion Kinase (FAK) was previously proposed to similarly promote invadopodia formation as a consequence of a switch of the Src kinase from focal adhesions to invadopodia. Interestingly, EB1-, APC-, or ACF7-depleted cells had decreased expression/activation of FAK. Remarkably, overexpression of wild type FAK, but not of FAK mutated to prevent Src recruitment, prevented the increased degradative activity induced by EB1 depletion. Overall, we propose that EB1 restricts invadopodia formation through the control of FAK and, consequently, the spatial regulation of Src activity

A novel region within a conserved domain in ATG7 emerged in vertebrates

The E1-like enzyme ATG7 belongs to a group of ATG proteins that mediate the autophagy process. Autophagy is a highly conserved degradation pathway important for maintaining homeostasis in eukaryotic cells. Here, we study the evolution of E1 enzymes and specifically describe a region of ATG7 that emerged early in vertebrates. This vertebrate-specific region (VSR) is situated within the adenylation domain of the protein, which is the most conserved domain of E1 enzymes and is of prokaryotic origin. A comparative analysis revealed that ATG7 is unique in this respect, as in other E1 enzyme family members this domain is highly conserved from yeast to humans and has not experienced insertions of extra amino acids. The function of the VSR domain is unknown, but two residues within the region, D522 and S531 have been linked with cancer in humans. Analysis of natural selection indicates positive selection on S531 only on the mammalian clade. Notably, this was the only residue in ATG7 showing this signal. Interestingly, structural analysis of ATG7 predicted that the VSR may be intrinsically disordered and could harbor a macro-molecular binding site. Analysis of cells expressing ATG7 lacking the VSR indicated that these cells are unable to facilitate the lipidation of LC3, suggesting an important role of this region in autophagy. Abbreviations: aBSREL - an adaptive branch-site random effects likelihood; AD - adenylation domain; ATGs - autophagy-related genes; Baf-A1 - Bafilomycin-A1; EV - empty-vector; CTD - C-terminal domain; ECTD - extreme C-terminal domain; EMT - epithelial-mesenchymal transition; FEL - fixed effects likelihood; GABARAP - gamma-aminobutyric acid receptor-associated protein; LC3 - microtubule-associated protein 1A/1B-light chain 3; MEFs - mouse embryonic fibroblasts; MOCS3 - molybdenum cofactor synthesis 3; NTD - N-terminal domain; UBL ubiquitin like protein; VSR - vertebrate specific regio

PINK1 regulated mitophagy is evident in skeletal muscles

ABSTRACTPINK1, mutated in familial forms of Parkinson’s disease, initiates mitophagy following mitochondrial depolarization. However, it is difficult to monitor this pathway physiologically in mice as loss of PINK1 does not alter basal mitophagy levels in most tissues. To further characterize this pathway in vivo, we used mito-QC mice in which loss of PINK1 was combined with the mitochondrial-associated POLGD257A mutation. We focused on skeletal muscle as gene expression data indicates that this tissue has the highest PINK1 levels. We found that loss of PINK1 in oxidative hindlimb muscle significantly reduced mitophagy. Of interest, the presence of the POLGD257A mutation, while having a minor effect in most tissues, restored levels of muscle mitophagy caused by the loss of PINK1. Although our observations highlight that multiple mitophagy pathways operate within a single tissue, we identify skeletal muscle as a tissue of choice for the study of PINK1-dependant mitophagy under basal conditions

miR-9 Does Not Regulate Lamin A Expression in Metastatic Cells from Lung Adenocarcinoma

International audienceIn lung adenocarcinoma, low lamin A expression in pleural metastatic cells has been proposed as a pejorative factor. miR-9 physiologically inhibits the expression of lamin A in neural cells and seems to be a central actor in the carcinogenesis and the metastatic process in lung cancer. Thus, it could be a good candidate to explain the reduction of lamin A expression in lung adenocarcinoma cells. miR-9 expression was analyzed in 16 pleural effusions containing metastatic cells from lung adenocarcinoma and was significantly reduced in patients from the ‘Low lamin A expression’ group compared to patients from the ‘High lamin A expression’ group. Then, carcinoma cells selection by fluorescence-activated cell sorting (FACS) was performed according to epithelial membrane antigen (EMA) expression, reflecting lamin A expression. miR-9 was underexpressed in lamin A− carcinoma cells compared to lamin A+ carcinoma cells in patients from the ‘Low lamin A expression’ group, whereas there was no difference of miR-9 expression between lamin A+ and lamin A− carcinoma cells in patients from the ‘High lamin A expression’ group. These results suggest that miR-9 does not regulate lamin A expression in metastatic cells from lung adenocarcinoma. On the contrary, miR-9 expression was shown to be reduced in lamin A-negative carcinoma cells

A proximity-labeling proteomic approach to investigate invadopodia molecular landscape in breast cancer cells

International audienceMetastatic progression is the leading cause of mortality in breast cancer. Invasive tumor cells develop invadopodia to travel through basement membranes and the interstitial matrix. Substantial efforts have been made to characterize invadopodia molecular composition. However, their full molecular identity is still missing due to the difficulty in isolating them. To fill this gap, we developed a non-hypothesis driven proteomic approach based on the BioID proximity biotinylation technology, using the invadopodia-specific protein Tks5α fused to the promiscuous biotin ligase BirA* as bait. In invasive breast cancer cells, Tks5α fusion concentrated to invadopodia and selectively biotinylated invadopodia components, in contrast to a fusion which lacked the membrane-targeting PX domain (Tks5β). Biotinylated proteins were isolated by affinity capture and identified by mass spectrometry. We identified known invadopodia components, revealing the pertinence of our strategy. Furthermore, we observed that Tks5 newly identified close neighbors belonged to a biologically relevant network centered on actin cytoskeleton organization. Analysis of Tks5β interactome demonstrated that some partners bound Tks5 before its recruitment to invadopodia. Thus, the present strategy allowed us to identify novel Tks5 partners that were not identified by traditional approaches and could help get a more comprehensive picture of invadopodia molecular landscape