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

Implication of Bcl-2 family members in apoptosis signalling pathways triggered by growth factor deprivation

Apoptosis or programmed cell death is a conserved cellular suicide program that selectively eliminates excess or potentially dangerous cells. To elucidate the signalling pathways implicated in the regulation of apoptosis and proliferation, cytokine withdrawal was used as a physiological stimulus to trigger programmed cell death. By means of representational difference analysis, the short isoform of the integral membrane protein (ITM2Bs) was identified in the present study as a novel regulator of cell death that was upregulated upon IL-2 deprivation. ITM2Bs was shown to belong to the BH3-only members of the Bcl-2 family and promoted apoptosis by antagonizing the pro-survival function of Bcl-2 protein. Biochemical and confocal studies demonstrated that association of ITM2Bs to mitochondria correlated with loss of mitochondrial membrane potential, release of cytochrome c into the cytosol as well as activation of up- and downstream caspases suggesting signalling via mi! tochondrial-mediated pathways. Functional interactions between proteins play a pivotal role in signal transduction. In this work, a novel function has been uncovered for the lymphoid-specific transcription factor Aiolos that was found to be associated with the pro-survival Bcl-xL protein in IL-4-deprived, but not IL-4-stimulated T-lymphocytes. The phosphorylation status appeared to be important since dephosphorylation of Aiolos and Bcl-xL increased their binding affinity following IL-4 withdrawal. These data propose a model, in which the anti-apoptotic effects of Bcl-xL are blocked through its sequestration by unphosphorylated Aiolos. Moreover, the anti-apoptotic proteins Bcl-xL and Bcl-w were identified as targeting subunits of the serine/threonine phosphatase PP1alpha in IL-4-stimulated T-lymphocytes as well as in freshly isolated thymocytes, replacing Bcl-2 as regulatory subunit of PP1alpha in IL-2-stimulated cells. Using co-immunoprecipitation assays, Bcl-xL or Bcl-w were shown to interact with PP1alpha and Bad forming two types of trimolecular complexes, Bcl-xL/PP1alpha/Bad and Bcl-w/PP1alpha/Bad. Depletion of Bcl-xL and Bcl-w resulted in loss of the interaction of PP1alpha and its substrate Bad, thus preventing dephosphorylation of Bad and subsequently programmed cell death. In addition to phosphorylation, the function of the pro-apoptotic Bad is also regulated by subcellular localization. It was demonstrated that a major fraction of Bad was localized in rafts upon IL-4 stimulation. This pool of Bad was dephosphorylated, but it was unable to exert its cell death-inducing function due to its sequestration in these plasma membrane microdomains. Confocal microscopy studies provided evidence that IL-4 deprivation triggered disorganization of lipids rafts, resulting in the segregation of dephosphorylated Bad from the plasma membrane and subsequent translocation to mitochondria correlating with T-lymphocyte death

Implication of Bcl-2 family members in apoptosis signalling pathways triggered by growth factor deprivation

Apoptosis or programmed cell death is a conserved cellular suicide program that selectively eliminates excess or potentially dangerous cells. To elucidate the signalling pathways implicated in the regulation of apoptosis and proliferation, cytokine withdrawal was used as a physiological stimulus to trigger programmed cell death. By means of representational difference analysis, the short isoform of the integral membrane protein (ITM2Bs) was identified in the present study as a novel regulator of cell death that was upregulated upon IL-2 deprivation. ITM2Bs was shown to belong to the BH3-only members of the Bcl-2 family and promoted apoptosis by antagonizing the pro-survival function of Bcl-2 protein. Biochemical and confocal studies demonstrated that association of ITM2Bs to mitochondria correlated with loss of mitochondrial membrane potential, release of cytochrome c into the cytosol as well as activation of up- and downstream caspases suggesting signalling via mi! tochondrial-mediated pathways. Functional interactions between proteins play a pivotal role in signal transduction. In this work, a novel function has been uncovered for the lymphoid-specific transcription factor Aiolos that was found to be associated with the pro-survival Bcl-xL protein in IL-4-deprived, but not IL-4-stimulated T-lymphocytes. The phosphorylation status appeared to be important since dephosphorylation of Aiolos and Bcl-xL increased their binding affinity following IL-4 withdrawal. These data propose a model, in which the anti-apoptotic effects of Bcl-xL are blocked through its sequestration by unphosphorylated Aiolos. Moreover, the anti-apoptotic proteins Bcl-xL and Bcl-w were identified as targeting subunits of the serine/threonine phosphatase PP1alpha in IL-4-stimulated T-lymphocytes as well as in freshly isolated thymocytes, replacing Bcl-2 as regulatory subunit of PP1alpha in IL-2-stimulated cells. Using co-immunoprecipitation assays, Bcl-xL or Bcl-w were shown to interact with PP1alpha and Bad forming two types of trimolecular complexes, Bcl-xL/PP1alpha/Bad and Bcl-w/PP1alpha/Bad. Depletion of Bcl-xL and Bcl-w resulted in loss of the interaction of PP1alpha and its substrate Bad, thus preventing dephosphorylation of Bad and subsequently programmed cell death. In addition to phosphorylation, the function of the pro-apoptotic Bad is also regulated by subcellular localization. It was demonstrated that a major fraction of Bad was localized in rafts upon IL-4 stimulation. This pool of Bad was dephosphorylated, but it was unable to exert its cell death-inducing function due to its sequestration in these plasma membrane microdomains. Confocal microscopy studies provided evidence that IL-4 deprivation triggered disorganization of lipids rafts, resulting in the segregation of dephosphorylated Bad from the plasma membrane and subsequent translocation to mitochondria correlating with T-lymphocyte death

Methods of screening apoptosis modulating compounds, compounds identified by said methods and use of said compounds as therapeutic agents

Filing Date: 2002-03-07.The invention relates to the modulation of apoptosis in mammalian cells. More particularly, the invention provides methods for identifying novel pro-apoptotic or anti-apoptotic cellular polypeptides, methods of screening compounds which modulate apoptosis, and method of detecting early events of the apoptotic process

Generation of Mouse and Human Induced Pluripotent Stem Cells (iPSC) from Primary Somatic Cells

Cellular reprogramming consists of the conversion of differentiated cells into pluripotent cells; the so-called induced Pluripotent Stem Cells. iPSC are amenable to in vitro manipulation and, in theory, direct production of any differentiated cell type. Furthermore, iPSC can be obtained from sick individuals and subsequently used for disease modeling, drug discovery and regenerative treatments. iPSC production was first achieved by transducing, with the use of retroviral vectors, four specific transcription factors: Oct4, Klf4, Sox2 and c-Myc (OKSM), into primary cells in culture Takahashi and Yamanaka, (Cell 126(4):663-676, 2006). Many alternative protocols have since been proposed: repeated transfections of expression plasmids containing the four pluripotency-associated genes Okita et al. (Science 322(5903):949-953, 2008), lentiviral delivery of the four factors Sommer et al. (Stem Cells 27(3):543-549, 2009), Sendai virus delivery Fusaki et al. (Proceedings of the Japan Academy. Series B, Physical and Biological Sciences 85(8):348-362, 2009), removal of the reprogramming vectors by 'piggyBac' transposition Woltjen et al. (Nature 458(7239):766-770, 2009); Kaji et al. (Nature 458(7239):771-775, 2009), Cre-recombinase excisable viruses Soldner et al. (Cell 136(5):964-977, 2009), episomal vectors Yu et al. (Science 324(5928):797-801, 2009), cell-penetrating reprogramming proteins Zhou et al. (Stem Cells 4(5):381-384, 2009), mammalian artificial chromosomes Hiratsuka et al. (PLoS One 6(10):e25961, 2011) synthetically modified mRNAs Warren et al. (Scientific Reports 2:657, 2012), miRNA Anokye-Danso et al. (Cell Stem Cell 8(4):376-388, 2009); however, although some of these methods are commercially available, in general they still need to attain the reproducibility and reprogramming efficiency required for routine applications Mochiduki and Okita (Biotechnol Journal 7(6):789-797, 2012). Herein we explain, in four detailed protocols, the isolation of mouse and human somatic cells and their reprogramming into iPSC. All-encompassing instructions, not previously published in a single document, are provided for mouse and human iPSC colony isolation and derivation. Although mouse and human iPSC share similarities in the cellular reprogramming process and culture, both cell types need to be handled differently. © 2012 Springer Science+Business Media New YorkThis work was supported by the MICINN-JDC and MICINN PLE2009-0091 and IPT-2011-1402-900000 grantsPeer reviewe

Double and inducible suicide gene construct and its use in gene therapy

The present invention provides a double and inducible suicide gene construct that combines the cell death-inducing capacity of the two suicide systems HSV-TK/GCV and iCaspase9/AP20187 in a single polycistronic cassette. The invention also relates to an expression vector comprising said construct, a cell comprising said construct or said vector, pharmaceutical compositions comprising these products and their applications as a medicament for the genetic safety for inducing the specific and directed cell death of specific cell populations that are causing adverse effects in the interior of an organism. The present invention is applicable, for example, in cancer treatments or in transplant situations where the transplanted cells are giving rise to immune rejection or to the formation of tumors. [ES]Peer reviewedConsejo Superior de Investigaciones Científicas (CSIC)A1 Solicitud de patente con informe sobre el estado de la técnic

Generation of a human iPSC line (IMEDEAi008-A) derived from natural homozygous CCR5-Δ32 PBMCs enriched in the pro-erythroblast population

A 32 base pair deletion in the C-C chemokine receptor type gene (CCR5-Δ32), the main Human Immunodeficiency Virus (HIV) co-receptor results in a non-functional protein. Individuals homozygous for the CCR5-Δ32 mutation are resistant to HIV infection. Here we report the generation, from pro-erythroblast enriched Peripheral Blood Mononuclear Cells (PBMCs) from a naturally occurring CCR5-Δ32/Δ32 individual, of the fully characterized iPSC line IMEDEAi008-A. The new line has normal karyotype, carry the Δ32 mutation in homozygosity, is free of plasmid integrations, express high levels of pluripotency markers and can differentiate into all three germ layers

Generation of one iPSC line (IMEDEAi007-A) by Sendai Virus transduction of PBMCs from a Psoriasis donor

Psoriasis is a chronic inflammatory skin disease that speeds up the life cycle of skin cells, forming scales and red patches that are itchy and sometimes painful. It is a complex disease of autoimmune origin and genetic predisposition with more than 10 different loci associated. Here we described the production of an iPSC line generated by Sendai Virus (Klf4, Oct3/4, Sox2 and c-Myc) reprogramming of Peripheral Blood Mononuclear Cells (PBMCs) from a Psoriasis patient. The iPSC line generated has normal 46XY karyotype, is free of SeV genome and transgenes insertions, express high levels of pluripotency markers and can differentiate into all three germ layers.Funding was provided by the Spanish Ministry for Science and Innovation (RTC-2016-5324-1). JMMF was a postdoctoral Berrikertu fellow from the Basque government. AF was a recipient of Juan de la Cierva (JCI-2006-2675) and Torres Quevedo (PTQ-16-08496) postdoctoral fellowships from the Spanish Ministry for Science and Innovation

A high-throughput screening for mammalian cell death effectors identifies the mitochondrial phosphate carrier as a regulator of cytochrome c release

Functional annotation of complex genomes requires the development of novel experimental platforms with increased capacity. Here, we describe a high-throughput system designed to identify cDNAs whose overexpression induces morphologically distinct cell death modalities. The methodology incorporates two robotized steps, and relies on coexpression of library clones with GFP to reveal the morphological features presented by the dying cells. By using this system we screened 135 000 cDNA clones and obtained 90 independent molecules. Interestingly, three death categories were identified, namely; apoptotic, vacuolated and autophagic. Among the pro-apoptotic clones, we found four members of the mitochondrial carrier family: the phosphate and adenine nucleotide (type 3) transporters, and the mitochondrial carrier homologs (MTCHs) 1 and 2. Expression of these molecules induced cytochrome c release and caspase-9-dependent death. One of them, the phosphate carrier, was able to interact with members of the permeability transition pore complex ANT1 and VDAC1, and its binding to ANT1 was stabilized in the presence of apoptotic activators. Depletion of this carrier by siRNA delayed cytochrome c mobilization and apoptosis. These results attribute a previously undescribed apoptotic function to the phosphate carrier and, more generally, suggest that a common property of various mitochondrial transporters was exploited during evolution to regulate apoptosis. © 2008 Nature Publishing Group All rights reserved.This work was funded by Grants SAF2002-00193, SAF2005- 01208 and GEN2003-20239-C06-05 from the Spanish MEC. Additional funding came from the FEDER program of the European Union. SA, MK and JF hold predoctoral fellowships from the Junta de Castilla y León (SA)or the FPU program of the Spanish Government (MK and JF). AF holds a long-term EMBO postdoctoral fellowship. FXP is an investigator affiliated to the University of Salamanca and funded by the Ramón y Cajal program (Spanish Government).Peer Reviewe