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1.
Cell ; 141(4): 564-6, 2010 May 14.
Artículo en Inglés | MEDLINE | ID: mdl-20478247

RESUMEN

During starvation, the cell recycles cytoplasmic material by sequestering it in double-membrane organelles, called autophagosomes, which eventually fuse with lysosomes. In this issue, Hailey et al. (2010) identify the outer membrane of mitochondria as a new source of autophagosomal membranes during starvation.

2.
Proc Natl Acad Sci U S A ; 119(28): e2113465119, 2022 07 12.
Artículo en Inglés | MEDLINE | ID: mdl-35867735

RESUMEN

The role of autophagy in cancer is complex. Both tumor-promoting and tumor-suppressive effects are reported, with tumor type, stage and specific genetic lesions dictating the role. This calls for analysis in models that best recapitulate each tumor type, from initiation to metastatic disease, to specifically understand the contribution of autophagy in each context. Here, we report the effects of deleting the essential autophagy gene Atg7 in a model of pancreatic ductal adenocarcinoma (PDAC), in which mutant KrasG12D and mutant Trp53172H are induced in adult tissue leading to metastatic PDAC. This revealed that Atg7 loss in the presence of KrasG12D/+ and Trp53172H/+ was tumor promoting, similar to previous observations in tumors driven by embryonic KrasG12D/+ and deletion of Trp53. However, Atg7 hemizygosity also enhanced tumor initiation and progression, even though this did not ablate autophagy. Moreover, despite this enhanced progression, fewer Atg7 hemizygous mice had metastases compared with animals wild type for this allele, indicating that ATG7 is a promoter of metastasis. We show, in addition, that Atg7+/- tumors have comparatively lower levels of succinate, and that cells derived from Atg7+/- tumors are also less invasive than those from Atg7+/+ tumors. This effect on invasion can be rescued by ectopic expression of Atg7 in Atg7+/- cells, without affecting the autophagic capacity of the cells, or by treatment with a cell-permeable analog of succinate. These findings therefore show that ATG7 has roles in invasion and metastasis that are not related to the role of the protein in the regulation of autophagy.


Asunto(s)
Proteína 7 Relacionada con la Autofagia , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Animales , Proteína 7 Relacionada con la Autofagia/genética , Proteína 7 Relacionada con la Autofagia/metabolismo , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/secundario , Línea Celular Tumoral , Ratones , Mutación , Invasividad Neoplásica , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Proteínas Proto-Oncogénicas p21(ras)/genética , Succinatos/metabolismo , Succinatos/farmacología
3.
Mol Cell ; 57(1): 39-54, 2015 Jan 08.
Artículo en Inglés | MEDLINE | ID: mdl-25498145

RESUMEN

The lysosome is the final destination for degradation of endocytic cargo, plasma membrane constituents, and intracellular components sequestered by macroautophagy. Fusion of endosomes and autophagosomes with the lysosome depends on the GTPase Rab7 and the homotypic fusion and protein sorting (HOPS) complex, but adaptor proteins that link endocytic and autophagy pathways with lysosomes are poorly characterized. Herein, we show that Pleckstrin homology domain containing protein family member 1 (PLEKHM1) directly interacts with HOPS complex and contains a LC3-interacting region (LIR) that mediates its binding to autophagosomal membranes. Depletion of PLEKHM1 blocks lysosomal degradation of endocytic (EGFR) cargo and enhances presentation of MHC class I molecules. Moreover, genetic loss of PLEKHM1 impedes autophagy flux upon mTOR inhibition and PLEKHM1 regulates clearance of protein aggregates in an autophagy- and LIR-dependent manner. PLEKHM1 is thus a multivalent endocytic adaptor involved in the lysosome fusion events controlling selective and nonselective autophagy pathways.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Lisosomas/metabolismo , Fusión de Membrana/genética , Glicoproteínas de Membrana/genética , Proteínas Asociadas a Microtúbulos/genética , Fagosomas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Reguladoras de la Apoptosis , Autofagia , Proteínas Relacionadas con la Autofagia , Endosomas/metabolismo , Regulación de la Expresión Génica , Células HeLa , Humanos , Glicoproteínas de Membrana/antagonistas & inhibidores , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Transgénicos , Proteínas Asociadas a Microtúbulos/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Transporte de Proteínas , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Alineación de Secuencia , Transducción de Señal , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión a GTP rab7
4.
EMBO Rep ; 21(3): e48412, 2020 03 04.
Artículo en Inglés | MEDLINE | ID: mdl-32009292

RESUMEN

The intracellular trafficking pathway, macroautophagy, is a recycling and disposal service that can be upregulated during periods of stress to maintain cellular homeostasis. An essential phase is the elongation and closure of the phagophore to seal and isolate unwanted cargo prior to lysosomal degradation. Human ATG2A and ATG2B proteins, through their interaction with WIPI proteins, are thought to be key players during phagophore elongation and closure, but little mechanistic detail is known about their function. We have identified a highly conserved motif driving the interaction between human ATG2 and GABARAP proteins that is in close proximity to the ATG2-WIPI4 interaction site. We show that the ATG2A-GABARAP interaction mutants are unable to form and close phagophores resulting in blocked autophagy, similar to ATG2A/ATG2B double-knockout cells. In contrast, the ATG2A-WIPI4 interaction mutant fully restored phagophore formation and autophagy flux, similar to wild-type ATG2A. Taken together, we provide new mechanistic insights into the requirements for ATG2 function at the phagophore and suggest that an ATG2-GABARAP/GABARAP-L1 interaction is essential for phagophore formation, whereas ATG2-WIPI4 interaction is dispensable.


Asunto(s)
Autofagosomas , Proteínas de la Membrana , Proteínas Reguladoras de la Apoptosis/metabolismo , Autofagosomas/metabolismo , Autofagia , Proteínas Relacionadas con la Autofagia/genética , Proteínas Relacionadas con la Autofagia/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Transporte de Proteínas , Proteínas de Transporte Vesicular/metabolismo
5.
EMBO Rep ; 18(8): 1382-1396, 2017 08.
Artículo en Inglés | MEDLINE | ID: mdl-28655748

RESUMEN

Through the canonical LC3 interaction motif (LIR), [W/F/Y]-X1-X2-[I/L/V], protein complexes are recruited to autophagosomes to perform their functions as either autophagy adaptors or receptors. How these adaptors/receptors selectively interact with either LC3 or GABARAP families remains unclear. Herein, we determine the range of selectivity of 30 known core LIR motifs towards individual LC3s and GABARAPs. From these, we define a G ABARAP I nteraction M otif (GIM) sequence ([W/F]-[V/I]-X2-V) that the adaptor protein PLEKHM1 tightly conforms to. Using biophysical and structural approaches, we show that the PLEKHM1-LIR is indeed 11-fold more specific for GABARAP than LC3B. Selective mutation of the X1 and X2 positions either completely abolished the interaction with all LC3 and GABARAPs or increased PLEKHM1-GIM selectivity 20-fold towards LC3B. Finally, we show that conversion of p62/SQSTM1, FUNDC1 and FIP200 LIRs into our newly defined GIM, by introducing two valine residues, enhances their interaction with endogenous GABARAP over LC3B. The identification of a GABARAP-specific interaction motif will aid the identification and characterization of the expanding array of autophagy receptor and adaptor proteins and their in vivo functions.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Secuencias de Aminoácidos , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Reguladoras de la Apoptosis , Autofagia , Proteínas Relacionadas con la Autofagia , Células HEK293 , Células HeLa , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Unión Proteica , Dominios y Motivos de Interacción de Proteínas
6.
J Biol Chem ; 291(17): 9025-41, 2016 Apr 22.
Artículo en Inglés | MEDLINE | ID: mdl-26929408

RESUMEN

The covalent conjugation of ubiquitin-fold modifier 1 (UFM1) to proteins generates a signal that regulates transcription, response to cell stress, and differentiation. Ufmylation is initiated by ubiquitin-like modifier activating enzyme 5 (UBA5), which activates and transfers UFM1 to ubiquitin-fold modifier-conjugating enzyme 1 (UFC1). The details of the interaction between UFM1 and UBA5 required for UFM1 activation and its downstream transfer are however unclear. In this study, we described and characterized a combined linear LC3-interacting region/UFM1-interacting motif (LIR/UFIM) within the C terminus of UBA5. This single motif ensures that UBA5 binds both UFM1 and light chain 3/γ-aminobutyric acid receptor-associated proteins (LC3/GABARAP), two ubiquitin (Ub)-like proteins. We demonstrated that LIR/UFIM is required for the full biological activity of UBA5 and for the effective transfer of UFM1 onto UFC1 and a downstream protein substrate both in vitro and in cells. Taken together, our study provides important structural and functional insights into the interaction between UBA5 and Ub-like modifiers, improving the understanding of the biology of the ufmylation pathway.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Procesamiento Proteico-Postraduccional/fisiología , Proteínas/metabolismo , Enzimas Activadoras de Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Secuencias de Aminoácidos , Proteínas Reguladoras de la Apoptosis , Células HEK293 , Humanos , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/genética , Estructura Secundaria de Proteína , Proteínas/química , Proteínas/genética , Relación Estructura-Actividad , Enzimas Activadoras de Ubiquitina/química , Enzimas Activadoras de Ubiquitina/genética , Enzimas Ubiquitina-Conjugadoras/química , Enzimas Ubiquitina-Conjugadoras/genética
7.
Mol Cell ; 34(3): 259-69, 2009 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-19450525

RESUMEN

Ubiquitination is the hallmark of protein degradation by the 26S proteasome. However, the proteasome is limited in its capacity to degrade oligomeric and aggregated proteins. Removal of harmful protein aggregates is mediated by autophagy, a mechanism by which the cell sequesters cytosolic cargo and delivers it for degradation by the lysosome. Identification of autophagy receptors, such as p62/SQSTM1 and NBR1, which simultaneously bind both ubiquitin and autophagy-specific ubiquitin-like modifiers, LC3/GABARAP, has provided a molecular link between ubiquitination and autophagy. This review explores the hypothesis that ubiquitin represents a selective degradation signal suitable for targeting various types of cargo, ranging from protein aggregates to membrane-bound organelles and microbes.


Asunto(s)
Autofagia/fisiología , Ubiquitina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Histona Desacetilasa 6 , Histona Desacetilasas/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Mitocondrias/metabolismo , Peroxisomas/metabolismo , Fagosomas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Transporte de Proteínas , Proteínas/metabolismo , Ribosomas/metabolismo , Ubiquitinación
8.
Mol Cell ; 33(4): 505-16, 2009 Feb 27.
Artículo en Inglés | MEDLINE | ID: mdl-19250911

RESUMEN

Autophagy is a catabolic process where cytosolic cellular components are delivered to the lysosome for degradation. Recent studies have indicated the existence of specific receptors, such as p62, which link ubiquitinated targets to autophagosomal degradation pathways. Here we show that NBR1 (neighbor of BRCA1 gene 1) is an autophagy receptor containing LC3- and ubiquitin (Ub)-binding domains. NBR1 is recruited to Ub-positive protein aggregates and degraded by autophagy depending on an LC3-interacting region (LIR) and LC3 family modifiers. Although NBR1 and p62 interact and form oligomers, they can function independently, as shown by autophagosomal clearance of NBR1 in p62-deficient cells. NBR1 was localized to Ub-positive inclusions in patients with liver dysfunction, and depletion of NBR1 abolished the formation of Ub-positive p62 bodies upon puromycin treatment of cells. We propose that NBR1 and p62 act as receptors for selective autophagosomal degradation of ubiquitinated targets.


Asunto(s)
Autofagia , Proteínas/metabolismo , Ubiquitina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Sitios de Unión , Células Cultivadas , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular , Ratones , Microscopía Confocal , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas/análisis , Proteína Sequestosoma-1 , Especificidad por Sustrato
9.
J Cell Sci ; 127(Pt 1): 3-9, 2014 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-24345374

RESUMEN

Continuous synthesis of all cellular components requires their constant turnover in order for a cell to achieve homeostasis. To this end, eukaryotic cells are endowed with two degradation pathways - the ubiquitin-proteasome system and the lysosomal pathway. The latter pathway is partly fed by autophagy, which targets intracellular material in distinct vesicles, termed autophagosomes, to the lysosome. Central to this pathway is a set of key autophagy proteins, including the ubiquitin-like modifier Atg8, that orchestrate autophagosome initiation and biogenesis. In higher eukaryotes, the Atg8 family comprises six members known as the light chain 3 (LC3) or γ-aminobutyric acid (GABA)-receptor-associated protein (GABARAP) proteins. Considerable effort during the last 15 years to decipher the molecular mechanisms that govern autophagy has significantly advanced our understanding of the functioning of this protein family. In this Cell Science at a Glance article and the accompanying poster, we present the current LC3 protein interaction network, which has been and continues to be vital for gaining insight into the regulation of autophagy.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Células Eucariotas/metabolismo , Homeostasis/genética , Proteínas de Microfilamentos/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Autofagia/genética , Familia de las Proteínas 8 Relacionadas con la Autofagia , Células Eucariotas/citología , Regulación de la Expresión Génica , Humanos , Lisosomas/metabolismo , Proteínas de Microfilamentos/genética , Proteínas Asociadas a Microtúbulos/genética , Fagosomas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Mapeo de Interacción de Proteínas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transducción de Señal , Ubiquitinación
11.
Biochem J ; 454(3): 459-66, 2013 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-23805866

RESUMEN

Selective autophagy is mediated by the interaction of autophagy modifiers and autophagy receptors that also bind to ubiquitinated cargo. Optineurin is an autophagy receptor that plays a role in the clearance of cytosolic Salmonella. The interaction between receptors and modifiers is often relatively weak, with typical values for the dissociation constant in the low micromolar range. The interaction of optineurin with autophagy modifiers is even weaker, but can be significantly enhanced through phosphorylation by the TBK1 {TANK [TRAF (tumour-necrosis-factor-receptor-associated factor)-associated nuclear factor κB activator]-binding kinase 1}. In the present study we describe the NMR and crystal structures of the autophagy modifier LC3B (microtubule-associated protein light chain 3 beta) in complex with the LC3 interaction region of optineurin either phosphorylated or bearing phospho-mimicking mutations. The structures show that the negative charge induced by phosphorylation is recognized by the side chains of Arg¹¹ and Lys5¹ in LC3B. Further mutational analysis suggests that the replacement of the canonical tryptophan residue side chain of autophagy receptors with the smaller phenylalanine side chain in optineurin significantly weakens its interaction with the autophagy modifier LC3B. Through phosphorylation of serine residues directly N-terminally located to the phenylalanine residue, the affinity is increased to the level normally seen for receptor-modifier interactions. Phosphorylation, therefore, acts as a switch for optineurin-based selective autophagy.


Asunto(s)
Autofagia , Proteínas Asociadas a Microtúbulos/química , Salmonella/fisiología , Factor de Transcripción TFIIIA/química , Secuencias de Aminoácidos , Sustitución de Aminoácidos , Proteínas de Ciclo Celular , Cristalografía por Rayos X , Interacciones Huésped-Patógeno , Humanos , Enlace de Hidrógeno , Proteínas de Transporte de Membrana , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Resonancia Magnética Nuclear Biomolecular , Fosforilación , Unión Proteica , Procesamiento Proteico-Postraduccional , Estructura Secundaria de Proteína , Termodinámica , Factor de Transcripción TFIIIA/genética
12.
J Cell Biol ; 222(12)2023 12 04.
Artículo en Inglés | MEDLINE | ID: mdl-37796195

RESUMEN

Cells harness multiple pathways to maintain lysosome integrity, a central homeostatic process. Damaged lysosomes can be repaired or targeted for degradation by lysophagy, a selective autophagy process involving ATG8/LC3. Here, we describe a parallel ATG8/LC3 response to lysosome damage, mechanistically distinct from lysophagy. Using a comprehensive series of biochemical, pharmacological, and genetic approaches, we show that lysosome damage induces non-canonical autophagy and Conjugation of ATG8s to Single Membranes (CASM). Following damage, ATG8s are rapidly and directly conjugated onto lysosome membranes, independently of ATG13/WIPI2, lipidating to PS (and PE), a molecular hallmark of CASM. Lysosome damage drives V-ATPase V0-V1 association, direct recruitment of ATG16L1 via its WD40-domain/K490A, and is sensitive to Salmonella SopF. Lysosome damage-induced CASM is associated with formation of dynamic, LC3A-positive tubules, and promotes robust LC3A engagement with ATG2, a lipid transfer protein central to lysosome repair. Together, our data identify direct ATG8 conjugation as a rapid response to lysosome damage, with important links to lipid transfer and dynamics.


Asunto(s)
Familia de las Proteínas 8 Relacionadas con la Autofagia , Autofagia , Lisosomas , Autofagia/genética , Lisosomas/genética , Lisosomas/metabolismo , Macroautofagia/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Salmonella , Familia de las Proteínas 8 Relacionadas con la Autofagia/genética , Familia de las Proteínas 8 Relacionadas con la Autofagia/metabolismo
13.
Cell Death Dis ; 14(7): 436, 2023 07 15.
Artículo en Inglés | MEDLINE | ID: mdl-37454104

RESUMEN

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common genetic cause of Parkinson's disease (PD), with growing importance also for Crohn's disease and cancer. LRRK2 is a large and complex protein possessing both GTPase and kinase activity. Moreover, LRRK2 activity and function can be influenced by its phosphorylation status. In this regard, many LRRK2 PD-associated mutants display decreased phosphorylation of the constitutive phosphorylation cluster S910/S935/S955/S973, but the role of these changes in phosphorylation status with respect to LRRK2 physiological functions remains unknown. Here, we propose that the S910/S935/S955/S973 phosphorylation sites act as key regulators of LRRK2-mediated autophagy under both basal and starvation conditions. We show that quadruple LRRK2 phosphomutant cells (4xSA; S910A/S935A/S955A/S973A) have impaired lysosomal functionality and fail to induce and proceed with autophagy during starvation. In contrast, treatment with the specific LRRK2 kinase inhibitors MLi-2 (100 nM) or PF-06447475 (150 nM), which also led to decreased LRRK2 phosphorylation of S910/S935/S955/S973, did not affect autophagy. In explanation, we demonstrate that the autophagy impairment due to the 4xSA LRRK2 phospho-dead mutant is driven by its enhanced LRRK2 kinase activity. We show mechanistically that this involves increased phosphorylation of LRRK2 downstream targets Rab8a and Rab10, as the autophagy impairment in 4xSA LRRK2 cells is counteracted by expression of phosphorylation-deficient mutants T72A Rab8a and T73A Rab10. Similarly, reduced autophagy and decreased LRRK2 phosphorylation at the constitutive sites were observed in cells expressing the pathological R1441C LRRK2 PD mutant, which also displays increased kinase activity. These data underscore the relation between LRRK2 phosphorylation at its constitutive sites and the importance of increased LRRK2 kinase activity in autophagy regulation and PD pathology.


Asunto(s)
Autofagia , Proteínas de Unión al GTP rab , Fosforilación/fisiología , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismo , Mutación , Autofagia/genética , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismo
14.
Blood Adv ; 7(7): 1190-1203, 2023 04 11.
Artículo en Inglés | MEDLINE | ID: mdl-36044386

RESUMEN

Leukemia cells reciprocally interact with their surrounding bone marrow microenvironment (BMM), rendering it hospitable to leukemia cell survival, for instance through the release of small extracellular vesicles (sEVs). In contrast, we show here that BMM deficiency of pleckstrin homology domain family M member 1 (PLEKHM1), which serves as a hub between fusion and secretion of intracellular vesicles and is important for vesicular secretion in osteoclasts, accelerates murine BCR-ABL1+ B-cell acute lymphoblastic leukemia (B-ALL) via regulation of the cargo of sEVs released by BMM-derived mesenchymal stromal cells (MSCs). PLEKHM1-deficient MSCs and their sEVs carry increased amounts of syntenin and syndecan-1, resulting in a more immature B-cell phenotype and an increased number/function of leukemia-initiating cells (LICs) via focal adhesion kinase and AKT signaling in B-ALL cells. Ex vivo pretreatment of LICs with sEVs derived from PLEKHM1-deficient MSCs led to a strong trend toward acceleration of murine and human BCR-ABL1+ B-ALL. In turn, inflammatory mediators such as recombinant or B-ALL cell-derived tumor necrosis factor α or interleukin-1ß condition murine and human MSCs in vitro, decreasing PLEKHM1, while increasing syntenin and syndecan-1 in MSCs, thereby perpetuating the sEV-associated circuit. Consistently, human trephine biopsies of patients with B-ALL showed a reduced percentage of PLEKHM1+ MSCs. In summary, our data reveal an important role of BMM-derived sEVs for driving specifically BCR-ABL1+ B-ALL, possibly contributing to its worse prognosis compared with BCR-ABL1- B-ALL, and suggest that secretion of inflammatory cytokines by cancer cells in general may similarly modulate the tumor microenvironment.


Asunto(s)
Linfoma de Burkitt , Células Madre Mesenquimatosas , Leucemia-Linfoma Linfoblástico de Células Precursoras B , Humanos , Animales , Ratones , Sindecano-1/metabolismo , Sinteninas/metabolismo , Comunicación Celular , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Linfoma de Burkitt/patología , Células Madre Mesenquimatosas/metabolismo , Microambiente Tumoral
15.
Autophagy Rep ; 2(1)2023 Dec 31.
Artículo en Inglés | MEDLINE | ID: mdl-38214012

RESUMEN

The Atg8 family of ubiquitin-like proteins play pivotal roles in autophagy and other processes involving vesicle fusion and transport where the lysosome/vacuole is the end station. Nuclear roles of Atg8 proteins are also emerging. Here, we review the structural and functional features of Atg8 family proteins and their protein-protein interaction modes in model organisms such as yeast, Arabidopsis, C. elegans and Drosophila to humans. Although varying in number of homologs, from one in yeast to seven in humans, and more than ten in some plants, there is a strong evolutionary conservation of structural features and interaction modes. The most prominent interaction mode is between the LC3 interacting region (LIR), also called Atg8 interacting motif (AIM), binding to the LIR docking site (LDS) in Atg8 homologs. There are variants of these motifs like "half-LIRs" and helical LIRs. We discuss details of the binding modes and how selectivity is achieved as well as the role of multivalent LIR-LDS interactions in selective autophagy. A number of LIR-LDS interactions are known to be regulated by phosphorylation. New methods to predict LIR motifs in proteins have emerged that will aid in discovery and analyses. There are also other interaction surfaces than the LDS becoming known where we presently lack detailed structural information, like the N-terminal arm region and the UIM-docking site (UDS). More interaction modes are likely to be discovered in future studies.

16.
EMBO Rep ; 11(1): 45-51, 2010 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-20010802

RESUMEN

Autophagy is the cellular homeostatic pathway that delivers large cytosolic materials for degradation in the lysosome. Recent evidence indicates that autophagy mediates selective removal of protein aggregates, organelles and microbes in cells. Yet, the specificity in targeting a particular substrate to the autophagy pathway remains poorly understood. Here, we show that the mitochondrial protein Nix is a selective autophagy receptor by binding to LC3/GABARAP proteins, ubiquitin-like modifiers that are required for the growth of autophagosomal membranes. In cultured cells, Nix recruits GABARAP-L1 to damaged mitochondria through its amino-terminal LC3-interacting region. Furthermore, ablation of the Nix:LC3/GABARAP interaction retards mitochondrial clearance in maturing murine reticulocytes. Thus, Nix functions as an autophagy receptor, which mediates mitochondrial clearance after mitochondrial damage and during erythrocyte differentiation.


Asunto(s)
Autofagia/fisiología , Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Familia de las Proteínas 8 Relacionadas con la Autofagia , Sitios de Unión , Western Blotting , Células COS , Células Cultivadas , Chlorocebus aethiops , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Ratones , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Mitocondriales/química , Proteínas Mitocondriales/genética , Datos de Secuencia Molecular , Unión Proteica , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/genética , Receptores de GABA-A/metabolismo , Reticulocitos/citología , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidad por Sustrato , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/genética , Ubiquitina-Proteína Ligasas/metabolismo
17.
FEBS J ; 289(22): 7113-7127, 2022 11.
Artículo en Inglés | MEDLINE | ID: mdl-34783437

RESUMEN

Communication between organelles is an essential process that helps maintain cellular homeostasis and organelle contact sites have recently emerged as crucial mediators of this communication. The emergence of a class of molecular bridges that span the inter-organelle gaps has now been shown to direct the flow of lipid traffic from one lipid bilayer to another. One of the key components of these molecular bridges is the presence of an N-terminal Chorein/VPS13 domain. This is an evolutionarily conserved domain present in multiple proteins within the endocytic and autophagy trafficking pathways. Herein, we discuss the current state-of-the-art of this class of proteins, focusing on the role of these lipid transporters in the autophagy and endocytic pathways. We discuss the recent biochemical and structural advances that have highlighted the essential role Chorein-N domain containing ATG2 proteins play in driving the formation of the autophagosome and how lipids are transported from the endoplasmic reticulum to the growing phagophore. We also consider the VPS13 proteins, their role in organelle contacts and the endocytic pathway and highlight how disease-causing mutations disrupt these contact sites. Finally, we open the door to discuss other Chorein_N domain containing proteins, for instance, UHRF1BP1/1L, their role in disease and look towards prokaryote examples of Chorein_N-like domains. Taken together, recent advances have highlighted an exciting opportunity to delve deeper into inter-organelle communication and understand how lipids are transported between membrane bilayers and how this process is disrupted in multiple diseases.


Asunto(s)
Autofagosomas , Retículo Endoplásmico , Autofagosomas/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas/metabolismo , Transporte Biológico , Lípidos
18.
FEBS J ; 289(13): 3752-3769, 2022 07.
Artículo en Inglés | MEDLINE | ID: mdl-35060334

RESUMEN

Macroautophagy is a membrane-trafficking process that delivers cytoplasmic material to lysosomes for degradation. The process preserves cellular integrity by removing damaged cellular constituents and can promote cell survival by providing substrates for energy production during hiatuses of nutrient availability. The process is also highly responsive to other forms of cellular stress. For example, DNA damage can induce autophagy and this involves up-regulation of the Damage-Regulated Autophagy Modulator-1 (DRAM-1) by the tumor suppressor p53. DRAM-1 belongs to an evolutionarily conserved protein family, which has five members in humans and we describe here the initial characterization of two members of this family, which we term DRAM-4 and DRAM-5 for DRAM-Related/Associated Member 4/5. We show that the genes encoding these proteins are not regulated by p53, but instead are induced by nutrient deprivation. Similar to other DRAM family proteins, however, DRAM-4 principally localizes to endosomes and DRAM-5 to the plasma membrane and both modulate autophagy flux when over-expressed. Deletion of DRAM-4 using CRISPR/Cas-9 also increased autophagy flux, but we found that DRAM-4 and DRAM-5 undergo compensatory regulation, such that deletion of DRAM-4 does not affect autophagy flux in the absence of DRAM-5. Similarly, deletion of DRAM-4 also promotes cell survival following growth of cells in the absence of amino acids, serum, or glucose, but this effect is also impacted by the absence of DRAM-5. In summary, DRAM-4 and DRAM-5 are nutrient-responsive members of the DRAM family that exhibit interconnected roles in the regulation of autophagy and cell survival under nutrient-deprived conditions.


Asunto(s)
Proteínas de la Membrana , Proteína p53 Supresora de Tumor , Apoptosis/fisiología , Autofagia/fisiología , Supervivencia Celular/genética , Humanos , Proteínas de la Membrana/metabolismo , Nutrientes , Proteína p53 Supresora de Tumor/genética
19.
Biol Open ; 10(12)2021 12 15.
Artículo en Inglés | MEDLINE | ID: mdl-34779483

RESUMEN

The small GTPase ARF family member ARL15 gene locus is associated in population studies with increased risk of type 2 diabetes, lower adiponectin and higher fasting insulin levels. Previously, loss of ARL15 was shown to reduce insulin secretion in a human ß-cell line and loss-of-function mutations are found in some lipodystrophy patients. We set out to understand the role of ARL15 in adipogenesis and showed that endogenous ARL15 palmitoylated and localised in the Golgi of mouse liver. Adipocyte overexpression of palmitoylation-deficient ARL15 resulted in redistribution to the cytoplasm and a mild reduction in expression of some adipogenesis-related genes. Further investigation of the localisation of ARL15 during differentiation of a human white adipocyte cell line showed that ARL15 was predominantly co-localised with a marker of the cis face of Golgi at the preadipocyte stage and then translocated to other Golgi compartments after differentiation was induced. Finally, co-immunoprecipitation and mass spectrometry identified potential interacting partners of ARL15, including the ER-localised protein ARL6IP5. Together, these results suggest a palmitoylation dependent trafficking-related role of ARL15 as a regulator of adipocyte differentiation via ARL6IP5 interaction. This article has an associated First Person interview with the first author of the paper.


Asunto(s)
Diabetes Mellitus Tipo 2 , Proteínas de Unión al GTP Monoméricas , Factores de Ribosilacion-ADP/genética , Factores de Ribosilacion-ADP/metabolismo , Adipocitos/metabolismo , Adipogénesis , Animales , Diabetes Mellitus Tipo 2/metabolismo , Aparato de Golgi/metabolismo , Humanos , Ratones , Proteínas de Unión al GTP Monoméricas/metabolismo
20.
R Soc Open Sci ; 8(7): 202333, 2021 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-34295519

RESUMEN

We report here that RUFY4, a newly characterized member of the 'RUN and FYVE domain-containing' family of proteins previously associated with autophagy enhancement, is highly expressed in alveolar macrophages (AM). We show that RUFY4 interacts with mitochondria upon stimulation by microbial-associated molecular patterns of AM and dendritic cells. RUFY4 interaction with mitochondria and other organelles is dependent on a previously uncharacterized OmpH domain located immediately upstream of its C-terminal FYVE domain. Further, we demonstrate that rufy4 messenger RNA can be translated from an alternative translation initiation codon, giving rise to a N-terminally truncated form of the molecule lacking most of its RUN domain and with enhanced potential for its interaction with mitochondria. Our observations point towards a role of RUFY4 in selective mitochondria clearance in activated phagocytes.

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