Mecanismos autofágicos no convencionales implicados en la regulación del tráfico endocítico y la señalización celular

[ES] La autofagia es una ruta de degradación celular que actúa como un mecanismo de control de calidad del citoplasma, eliminando agregados proteicos, orgánulos dañados y microorganismos invasores. Aunque la degradación aleatoria de citoplasma es una respuesta común a estrés nutricional, en los últimos años se han descrito modalidades selectivas de autofagia, responsables de la eliminación específica de componentes potencialmente dañinos. Consecuentemente, alteraciones en esta ruta están implicadas en la patogénesis de distintos desórdenes. Recientemente se ha descrito que el incremento de la susceptibilidad a enfermedad de Crohn está asociado, entre otros factores, al polimorfismo A300 en el efector autofágico ATG16L1. La molécula ATG16L1 no sólo es un efector esencial de la ruta autofágica, sino que también se encuentra implicada en funciones autofágicas no convencionales, esto es, no ligadas a esta vía degradativa. Entre las funciones no convencionales de ATG16L1 destacan el control inflamatorio, el tráfico de vesículas secretoras en determinados tipos celulares o la eliminación de microorganismos invasores. Resultados previos del laboratorio han demostrado que ATG16L1 interacciona con la proteína transmembrana TMEM59, desencadenando un fenómeno autofágico atípiconcuyo estudio ha contribuido a dilucidar algunas de las funciones no convencionales en las que se encuentra implicado ATG16L1. En el caso de TMEM59, la activación de ATG16L1 conduce al marcaje con LC3 de endosomas de membrana sencilla en los que se localiza la propia molécula, siendo dirigidos más eficazmente al compartimento lisosómico. TMEM59 interacciona con el dominio WDD de ATG16L1, a través de un motivo aminoacídico que está presente en diversidad de moléculas de distinta entidad biológica. Esta interacción se altera en presencia del alelo de riesgo A300, ralentizándose el tráfico intracelular de TMEM59 y reduciéndose la respuesta xenofágica frente a Staphylococcus aureus. Entre las moléculas que interaccionan con ATG16L1 destaca, asimismo, A20/TNFAIP3, que modula la respuesta antiinflamatoria regulando la vía NF- κB. La interacción entre ATG16L1 y A20 había sido descrita previamente en el laboratorio. Adicionalmente, ensayos preliminares de microarrays peptídicos habían contribuido a identificar un elevado número de proteínas transmembrana que presentan diferentes versiones del motivo de unión al dominio WDD de ATG16L1 en sus regiones intracelulares. Entre el conjunto de moléculas identificado, destacan una colección de receptores de citoquinas pro y antiinflamatorias. Los ensayos moleculares y funcionales efectuados en esta tesis doctoral han permitido evaluar el impacto que el dominio WDD de ATG16L1 ejerce sobre las actividades biológicas de las moléculas más relevantes constituyentes del denominado “interactoma” de ATG16L1

The T300A Crohn’s disease risk polymorphism impairs function of the WD40 domain of ATG16L1

Article number: 11821 (2016)[EN]A coding polymorphism of human ATG16L1 (rs2241880; T300A) increases the risk of Crohn’s disease and it has been shown to enhance susceptibility of ATG16L1 to caspase cleavage. Here we show that T300A also alters the ability of the C-terminal WD40-repeat domain of ATG16L1 to interact with an amino acid motif that recognizes this region. Such 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 its ability to engage ATG16L1 in response to bacterial infection. TMEM59-induced autophagy is blunted in cells expressing the fragments generated by caspase processing of the ATG16L1-T300A 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

Implementación de flipped learning y evaluación entre iguales en la enseñanza de la genética clínica a través de las redes sociales

Memoria ID2022-109. Ayudas de la Universidad de Salamanca para la innovación docente, curso 2022-2023

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

Integración de las redes sociales Twitter e Instagram en la plataforma Studium para la enseñanza de la genética

Memoria ID-101. Ayudas de la Universidad de Salamanca para la innovación docente, curso 2021-2022

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

Unconventional autophagy mediated by the WD40 domain of ATG16L1 is derailed by the T300A Crohn disease risk polymorphism

A coding polymorphism of the critical autophagic effector ATG16L1 (T300A) increases the risk of Crohn disease, but how this mutation influences the function of ATG16L1 has remained unclear. In a recent report, we showed that the A300 allele alters the ability of the C-terminal WD40 domain of ATG16L1 to interact with proteins containing a specific amino acid motif able to recognize this region. This defect impairs the capacity of the motif-containing transmembrane molecule TMEM59 to induce the unconventional autophagic labeling of the same single-membrane vesicles where this protein is located. Such alteration derails the intracellular trafficking of TMEM59 and the xenophagic response against bacterial infection. In contrast, canonical autophagy remains unaffected in the presence of ATG16L1. These data argue that the T300A polymorphism impairs the unconventional autophagic activities carried out by the WD40 domain, a region of ATG16L1 whose function has remained poorly understood.Funding was obtained from the Ministerio de Ciencia e Innovacion (Ref. SAF2011-23714) and the Ministerio de Economia y Competitividad (Ref. SAF2014-53320-R) of the Spanish Government, the Broad Medical Research Program (IBD-0369), the Junta de Castilla y Leon local government (Department of Education (CSI001A10-2, FIC016U14) and Department of Health (SAN11-FXP)) and the Fundacion Solorzano (FS/1-2009 and FS/18-2014). Additional funding was received from the FEDER program of the European Union.Peer Reviewe

ATG16L1 WD40 domain-dependent IL10R (interleukin 10 receptor) signaling is insensitive to the T300A Crohn disease risk polymorphism

A coding allele of ATG16L1 that increases the risk of Crohn disease (T300A; rs2241880) impairs the interaction between the C-terminal WD40 domain (WDD) and proteins containing a WDD-binding motif, thus specifically inhibiting the unconventional autophagic activities of ATG16L1. In a recent publication we described a novel atypical role of ATG16L1 in the regulation of IL10R (interleukin 10 receptor) trafficking and signaling, an activity that involves direct interaction between the WDD and a target motif present in IL10RB (interleukin 10 receptor subunit beta). Here we show that, unexpectedly, neither the ability of ATG16L1 to interact with IL10RB nor its role in supporting IL10 signaling are altered by the T300A mutation. These results indicate that the ATG16L1T300A allele selectively impairs the interaction between the WDD and a subset of WDD-binding motif versions, suggesting that only a fraction of the unconventional activities mediated by ATG16L1 are required to prevent Crohn disease.This work was supported by the Ministerio de Economía, Industria y Competitividad, Gobierno de España [SAF2017-88390-R, PID2020-114699RB-100]; Consejería de Educación, Junta de Castilla y León [SA042P17] and [CLC-2017-01]; Crohn’s and Colitis Foundation of America [IBD-0369]; Fundación Solórzano (USAL) [FS/18-2014]

Unconventional WD40 domain-dependent role of ATG16L1 in the regulation of IL10R (interleukin 10 receptor) endocytosis, trafficking and signaling

ATG16L1 is a critical mediator of macroautophagy/autophagy required for LC3 lipidation and autophagosome formation. However, ATG16L1 has a C-terminal domain including 7 WD40-type repetitions (WD40 domain, WDD) that is unnecessary for the conventional autophagic pathway. Instead, this domain mediates unconventional activities where LC3 is lipidated in atypical subcellular localizations unrelated to canonical double-membrane autophagosomes. The WDD provides a docking surface for molecules including a specific amino acid motif, thus engaging the LC3 lipidation capabilities of ATG16L1 in single-membrane structures. The physiological implications of such atypical activities are poorly characterized. In a recent report we described the improvement of the WDD-binding motif and the identification of transmembrane molecules that harbor this element in their intracellular region. One of them, IL10RB (interleukin 10 receptor subunit beta), binds the WDD after IL10 activation to facilitate endocytosis, early trafficking and signaling of IL10-IL10R complexes without influencing their degradation rate. These results reveal a novel unconventional role of ATG16L1 in cytokine signaling that does not entail a degradative purpose, thus contributing to catalog the physiological roles played by unconventional activities of the autophagic machinery.This work was funded by grants from the Ministerio de Ciencia e Innovación of the Spanish Government (Refs. SAF2014-53320-R and SAF2017-88390-R), the Junta de Castilla y León local government (Ref. SA042P17), the Broad Medical Research Program (Crohn’s and Colitis Foundation of America, CCFA; Ref. IBD-0369) and the Fundación Solórzano (Ref. FS/18-2014). The Centro de Investigación del Cáncer is supported by the Programa de Apoyo a Planes Estratégicos de Investigación de Estructuras de Investigación de Excelencia cofunded by the Castilla y León autonomous government and the European Regional Development Fund (CLC–2017–01). Additional funding comes from the Fondo Europeo de Desarrollo Regional (FEDER) program of the European Union. R.V. and A.F. are the recipients of predoctoral contracts from the Junta de Castilla y León and the University of Salamanca, respectively. I.S.G. was supported by predoctoral fellowships from the Fundación Moraza and the Estrategia Regional de Investigación e Innovación (Junta de Castilla y León and FEDER; Ref. CLC-2017-01). E.T. holds a predoctoral contract from the Formación de Doctores program of the Ministerio de Ciencia, Innovación y Universidades (Spanish Government). Rachid Taouil Hammouti acknowledges a predoctoral grant from the Government of Andorra (Ref. ATC0XX - AND-2019/2020)