Canonical and unconventional purposes and mechanisms

Selective autophagic degradation of cellular components underlies many of the important physiological and pathological implications that autophagy has for mammalian cells. Cytoplasmic vesicles, just like other intracellular items, can be subjected to conventional autophagic events where double-membrane autophagosomes specifically isolate and deliver them for lysosomal destruction. However, intracellular membranes appear to constitute common platforms for unconventional versions of the autophagic pathway, a notion that has become apparent during the past few years. For instance, in many cases of autophagy directed against bacterial phagosomes, subversion of the process results in multimembrane vacuoles that promote bacterial replication instead of the usual degradative outcome. In a different atypical modality, single-membrane vesicles can be labeled with LC3 to direct their contents for lysosomal degradation. In fact, single-membrane compartments of various kinds often provide an assembly site for the autophagic machinery to perform unanticipated nondegradative activities that range from localized secretion of lysosomal contents to melanosome function. Interestingly, many of these unconventional processes seem to be initiated through engagement of relevant nodes of the autophagic signaling network that, once activated, promote LC3 decoration of the targeted membrane, and some cases of inducer/receptor proteins that specifically engage those important signaling hubs have recently been described. Here we review the available examples of all autophagic variants involving membranous compartments, with a main focus on the more recently discovered unconventional phenomena where the usual degradation purpose of autophagy or its canonical mechanistic features are not completely conserved.Funding was provided by grants from the Ministerio de Ciencia e Innovación of the Spanish Government (Refs. SAF2008-00350 and SAF2011-23714), the Fundación Solórzano, the Junta de Castilla y León local government (Consejería de Educación, Ref. CSI001A10-2, and Consejería de Sanidad) and the Consejo Superior de Investigaciones Científicas (CSIC; Ref. 200720I026). Additional funding comes from the FEDER program of the European Union. EB is a graduate student funded by a predoctoral fellowship from the FPU program (Ministerio de Educación, MEC, Spanish Government).Peer Reviewe

TMEM59 defines a novel ATG16L1-binding motif that promotes local activation of LC3

Selective autophagy underlies many of the important physiological roles that autophagy plays in multicellular organisms, but the mechanisms involved in cargo selection are poorly understood. Here we describe a molecular mechanism that can target conventional endosomes for autophagic degradation. We show that the human transmembrane protein TMEM59 contains a minimal 19-amino-acid peptide in its intracellular domain that promotes LC3 labelling and lysosomal targeting of its own endosomal compartment. Interestingly, this peptide defines a novel protein motif that mediates interaction with the WD-repeat domain of ATG16L1, thus providing a mechanistic basis for the activity. The motif is represented with the same ATG16L1-binding ability in other molecules, suggesting a more general relevance. We propose that this motif may play an important role in targeting specific membranous compartments for autophagic degradation, and therefore it may facilitate the search for adaptor proteins that promote selective autophagy by engaging ATG16L1. Endogenous TMEM59 interacts with ATG16L1 and mediates autophagy in response to Staphylococcus aureus infection.This work was funded by grants from the Ministerio de Ciencia e Innovación of the Spanish Government (Refs SAF2008‐00350 and SAF2011‐23714), Fundación Solórzano, Junta de Castilla y León (Consejería de Educación, Ref. CSI001A10‐2, and Consejería de Sanidad) and Consejo Superior de Investigaciones Científicas (CSIC; Ref. 200720I026). Additional funding comes from the FEDER programme of the European Union. EB is a graduate student funded by a predoctoral fellowship from the FPU programme (Ministerio de Educación, MEC, Spanish Government). ML is funded by JAE‐Doc and Juan de la Cierva postdoctoral contracts (MEC and Social European Fund of the European Union, 2007–2013). AF is funded by a long‐term EMBO postdoctoral fellowship and a Juan de la Cierva contract. KP is a graduate student funded by an FPI fellowship (MEC).Peer Reviewe

Biochemical analyses reveal amino acid residues critical for cell cycle-dependent phosphorylation of human Cdc14A phosphatase by cyclin-dependent kinase 1

Cdc14 enzymes compose a family of highly conserved phosphatases that are present in a wide range of organisms, including yeast and humans, and that preferentially reverse the phosphorylation of Cyclin-Dependent Kinase (Cdk) substrates. The budding yeast Cdc14 orthologue has essential functions in the control of late mitosis and cytokinesis. In mammals, however, the two Cdc14 homologues, Cdc14A and Cdc14B, do not play a prominent role in controlling late mitotic events, suggesting that some Cdc14 functions are not conserved across species. Moreover, in yeast, Cdc14 is regulated by changes in its subcellular location and by phosphorylation events. In contrast, little is known about the regulation of human Cdc14 phosphatases. Here, we have studied how the human Cdc14A orthologue is regulated during the cell cycle. We found that Cdc14A is phosphorylated on Ser411, Ser453 and Ser549 by Cdk1 early in mitosis and becomes dephosphorylated during late mitotic stages. Interestingly, in vivo and in vitro experiments revealed that, unlike in yeast, Cdk1-mediated phosphorylation of human Cdc14A did not control its catalytic activity but likely modulated its interaction with other proteins in early mitosis. These findings point to differences in Cdk1-mediated mechanisms of regulation between human and yeast Cdc14 orthologues.We thank S. Andrés for technical assistance and other members of laboratory for helpful discussions. We are grateful to Dr. I. García-Higuera and S. Moreno (IBFG, Salamanca) for Cdh1 plasmids and anti-Plk1 antibodies, Dr. J. Dong (Univ. Nebraska Medical Center, Nebraska) for the KIBRA plasmids, Dr. C. Guerrero (IBMCC, Salamanca) for the anti-PP2A antibody and Dr. Jallepalli (Memorial Sloan Kettering Cancer Center, New York) for the RPE Cdc14A − / − cells. We are grateful to the proteomics facility of Centro de Investigación del Cáncer, Salamanca, Spain, where the proteomic analysis was performed, Grant PRB2 (IPT13/0001 - ISCIII-SGEFI / FEDER). This work was funded by grants from the Spanish Ministry of Economy, Industry and Competitiveness (MINECO; BFU2015-69709-P and SAF2015-69920-R). S.O. was supported by a FPU fellowship from the Spanish Ministry of Education and P.A. was supported by a JAE-Predoctoral fellowship from the Spanish National research Council (CSIC).S

Physical and functional interaction between A20 and ATG16L1-WD40 domain in the control of intestinal homeostasis

Prevention of inflammatory bowel disease (IBD) relies on tight control of inflammatory, cell death and autophagic mechanisms, but how these pathways are integrated at the molecular level is still unclear. Here we show that the anti-inflammatory protein A20 and the critical autophagic mediator Atg16l1 physically interact and synergize to regulate the stability of the intestinal epithelial barrier. A proteomic screen using the WD40 domain of ATG16L1 (WDD) identified A20 as a WDD-interacting protein. Loss of A20 and Atg16l1 in mouse intestinal epithelium induces spontaneous IBD-like pathology, as characterized by severe inflammation and increased intestinal epithelial cell death in both small and large intestine. Mechanistically, absence of A20 promotes Atg16l1 accumulation, while elimination of Atg16l1 or expression of WDD-deficient Atg16l1 stabilizes A20. Collectively our data show that A20 and Atg16l1 cooperatively control intestinal homeostasis by acting at the intersection of inflammatory, autophagy and cell death pathways

Bcl-XL specifically activates Bak to induce swelling and restructuring of the endoplasmic reticulum

Bcl-2 family members Bak and Bax constitute a mitochondrial gateway for multiple death pathways. Both proteins are also present in the endoplasmic reticulum where they control apoptosis through the regulation of calcium levels. We show here that reticular Bak has the additional capacity of modulating the structure of this organelle. Coexpression of Bak and Bcl-XL provokes extensive swelling and vacuolization of reticular cisternae. A Bak version lacking the BH3 domain suffices to induce this phenotype, and reticular targeting of this mutant retains the activity. Expression of upstream BH3-only activators in similar conditions recapitulates ER swelling and vacuolization if ryanodine receptor calcium channel activity is inhibited. Experiments with Bak and Bax-deficient mouse embryonic fibroblasts show that endogenous Bak mediates the effect, whereas Bax is mainly irrelevant. These results reveal a previously unidentified role of Bak in regulating reticular conformation. Because this activity is absent in Bax, it constitutes one of the first examples of functional divergence between the two multidomain homologues. © The Rockefeller University Press.This work was funded by grants SAF2002-00193 and GEN2003-20239-C06-05 from the Ministerio de Ciencia y Tecnología (Spanish Government). M. Klee is the recipient of a predoctoral fellowship from the Formación de Profesorado Universitario program of the Spanish Ministry of Education. F.X. Pimentel-Muiños is an investigator affiliated with the University of Salamanca and funded by the Ramón y Cajal program of the Spanish Government.Peer Reviewe

Nuevas funciones génicas en apoptosis y autofagia identificadas mediante genómica funcional

Resumen del trabajo presentado al XXXXVIII Congreso de la Sociedad Española de Bioquímica y Biología Molecular (SEBBM), celebrado en Valencia del 7 al 10 de septiembre de 2015.Previamente en el laboratorio hemos diseñado un sistema de alto rendimiento para identificar funcionalmente moléculas capaces de inducir diferentes modalidades de muerte celular al ser sobreexpresadas. Este abordaje se basa en el uso de sistemas robóticos que facilitan el manejo sistemático de genotecas de cDNA clonadas en vectores de expresión, y en la transfección de los clones en células susceptibles y en combinación con GFP. Tras el escrutinio de 135.000 clones de una genoteca humana, hemos identificado un total de 90 elementos genéticos cuya expresión induce muerte celular. De forma interesante, aunque la mayoría son capaces de provocar muerte celular apoptótica, otros inducen formas de muerte celular morfológicamente atípica, diferentes de la apoptosis. Dentro de los clones apoptóticos hemos identificado el transportador mitocondrial de fosfato. Un trabajo adicional indica que esta molécula forma parte del poro de transición de permeabilidad que media la liberación de citocromo c y la activación de las caspasas en algunos paradigmas de apoptosis. Por otro lado, dentro de los clones atípicos hemos descubierto una nueva proteína transmembrana denominada TMEM59. Estudios adicionales indican que esta molécula induce un fenómeno atípico de autofagia celular implicado en defensa contra agentes infecciosos, y no parece tener un papel fisiológico en muerte celular. Por tanto, a través de un sistema no sesgado de identificación funcional hemos podido adscribir funciones celulares nuevas, bien a genes conocidos que previamente no se habían implicado en una determinada función (transportador de fosfato), o a genes nuevos cuya función fisiológica se desconocía por completo (TMEM59), demostrando el potencial de este tipo de aproximaciones para anotar funcionalmente el genoma.Peer reviewe

Impaired function of the WD40 domain of ATG16L1 caused by the T300A Crohn’s disease risk polymorphism

Resumen del póster presentado al XXXIX Congreso de la Sociedad Española de Bioquímica y Biología Molecular, celebrado en Salamanca del 5 al 8 de septiembre de 2016.A coding allele of human ATG16L1 (rs2241880; T300A) increases the risk of Crohn’s disease, but the underlying molecular defects introduced by this mutation are not fully understood. We show that T300A alters the ability of the C-terminal WD40-repeat domain of ATG16L1 to interact with an amino acid motif that recognizes this region. This alteration impairs the unconventional autophagic activity of TMEM59, a transmembrane protein that contains the WD40 domain-binding motif, and disrupts its normal intracellular trafficking and ability to engage ATG16L1 in response to bacterial infection. Notably, these defects are independent of ATG16L1 T300A caspase cleavage. In addition, TMEM59-induced autophagy is blunted in cells expressing the fragments generated by caspase 3 cleavage of the risk allele, whereas canonical autophagy remains unaffected. These results suggest that the T300A polymorphism alters the function of motif-containing molecules that engage ATG16L1 through the WD40 domain, either by influencing this interaction under non-stressful conditions or by inhibiting their downstream autophagic signalling after caspase-mediated cleavage.Peer reviewe