El papel del colesterol en la funcionalidad de los lipid rafts y su repercusión en la adipogénesis

El colesterol es un componente esencial de las membranas de mamíferos. En este sentido, es esencial para mantener las propiedades de los microdominios de membrana denominados ?lipid rafts? o balsas lipídicas. Son zonas con alto grado de orden que se describen como plataformas de señalización que albergan multitud de proteínas implicadas en este proceso. Son especialmente abundantes en adipocitos; durante la diferenciación a estas células, existe un tráfico del colesterol de los ?lipid rafts? a la gota lipídica. Esto sugiere una relación entre el colesterol de membrana y la fisiología del adipocito. Así, nos planteamos el estudio de su papel en el proceso de la adipogénesis mediante el análisis del efecto producido por distintos inhibidores de la biosíntesis de colesterol sobre la capacidad de las células 3T3-L1 (preadipocitos de ratón) de diferenciarse a adipocitos. La inhibición de la colesterogénesis en estas células produjo un descenso en la actividad de la vía de ERK1/2, que afectó a su vez, la fosforilación en Thr188 del factor C/EBPß, necesaria para que se una al DNA y active la expresión de los factores de transcripción PPAR¿ y C/EBP¿, cuya expresión disminuye al inhibir la colesterogénesis. Por otra parte, la inhibición de la síntesis de colesterol a distintos niveles provocó una desestructuración de los ?lipid rafts? en células 3T3-L1 que condujo a una menor capacidad del receptor de la insulina para activarse y fosforilar proteínas efectoras como Akt. Es posible que la menor fosforilación de ERK1/2, y en consecuencia de C/EBPß venga provocada por una desestabilización de los ?lipid rafts? y una menor funcionalidad del receptor de insulina en células 3T3-L1 tratadas con los inhibidores de la colesterogénesis, que conduce a su incapacidad para diferenciarse a adipocitos antes los estímulos adecuados. Por otra parte, el haloperidol, antipsicótico típico, se ha relacionado con un bloqueo de la acción de la ?7-reductasa. Comprobamos que el haloperidol inhibiá, además la acción de la ?8,7-isomerasa y la ?14-reductasa. Nos planteamos pues, valorar sus efectos sobre la estructura y funcionalidad de los ?lipid rafts? en células SH-SY5Y de neuroblastoma como posible causa de algunos de los efectos secundarios que provoca. En este sentido, el haloperidol a la dosis de 10 y 50 ?M alteró la composición de los ?lipid rafts? y la funcionalidad de la señalización mediada por insulina y somatostatina, afectando en este último caso a la actividad de la adenilato ciclasa y provocando aumentos en los niveles de AMPc. Finalmente, y en cuanto al papel regulador del colesterol en el ciclo celular, observamos que la inhibición de la colesterogénesis provocó un descenso en la actividad del promotor de la ciclina B1 en células MOLT-4 de linfoblastoma, hecho que concuerda con la parada en la fase de G2/M del ciclo celular en estas células cuando la biosíntesis de colesterol se encuentra inhibida. Además, encontramos la presencia de un elemento en la región promotora del gen de la ciclina B1 capaz de responder a la disminución de los niveles de colesterol celular

Decoding protein methylation function with thermal stability analysis

Methylation; ProteomicsMetilació; ProteòmicaMetilación; ProteómicaProtein methylation is an important modification beyond epigenetics. However, systems analyses of protein methylation lag behind compared to other modifications. Recently, thermal stability analyses have been developed which provide a proxy of a protein functional status. Here, we show that molecular and functional events closely linked to protein methylation can be revealed by the analysis of thermal stability. Using mouse embryonic stem cells as a model, we show that Prmt5 regulates mRNA binding proteins that are enriched in intrinsically disordered regions and involved in liquid-liquid phase separation mechanisms, including the formation of stress granules. Moreover, we reveal a non-canonical function of Ezh2 in mitotic chromosomes and the perichromosomal layer, and identify Mki67 as a putative Ezh2 substrate. Our approach provides an opportunity to systematically explore protein methylation function and represents a rich resource for understanding its role in pluripotency.We thank all members of the CNIO Proteomics Unit for discussions, the CNIO Flow Cytometry Unit for flow cytometry support, Cyan Lynch for sharing reagents and Ana Martinez-Val for support with data analysis. This work was supported by SAF2016-74962-R (MINECO) and the European Union Horizon 2020 program INFRAIA project EPIC-XS (project 823839). The CNIO Proteomics Unit belongs to ProteoRed, PRB3- ISCIII, supported by grant PT17/0019/0005. J.M. is supported by the Ikerbasque Programme, Basque Foundation for Science. O.F-C. is supported by grants from the Spanish Ministry of Science, Innovation and Universities (PID2021-128722OB-I00, co-financed with European FEDER funds) and the Spanish Association Against Cancer (AECC; PROYE20101FERN). M.M. lab was supported by grants from MINECO (PID2021-128726 and PDC2022-133408-I00), and Comunidad de Madrid (Y2020/BIO-6519 and S2022/BMD-7437)

An allosteric switch between the activation loop and a c-terminal palindromic phosphomotif controls c-Src function.

Autophosphorylation controls the transition between discrete functional and conformational states in protein kinases, yet the structural and molecular determinants underlying this fundamental process remain unclear. Here we show that c-terminal Tyr 530 is a de facto c-Src autophosphorylation site with slow time-resolution kinetics and a strong intermolecular component. On the contrary, activation-loop Tyr 419 undergoes faster kinetics and a cis-to-trans phosphorylation switch that controls c-terminal Tyr 530 autophosphorylation, enzyme specificity, and strikingly, c-Src non-catalytic function as a substrate. In line with this, we visualize by X-ray crystallography a snapshot of Tyr 530 intermolecular autophosphorylation. In an asymmetric arrangement of both catalytic domains, a c-terminal palindromic phospho-motif flanking Tyr 530 on the substrate molecule engages the G-loop of the active kinase adopting a position ready for entry into the catalytic cleft. Perturbation of the phosphomotif accounts for c-Src dysfunction as indicated by viral and colorectal cancer (CRC)-associated c-terminal deleted variants.Weshow that c-terminal residues 531 to 536 are required for c-Src Tyr 530 autophosphorylation, and such a detrimental effect is caused by the substrate molecule inhibiting allosterically the active kinase. Our work reveals a crosstalk between the activation and c-terminal segments that control the allosteric interplay between substrateand enzyme-acting kinases during autophosphorylation.post-print5137 K

Atg4 proteolytic activity can be inhibited by Atg1 phosphorylation

The biogenesis of autophagosomes depends on the conjugation of Atg8-like proteins with phosphatidylethanolamine. Atg8 processing by the cysteine protease Atg4 is required for its covalent linkage to phosphatidylethanolamine, but it is also necessary for Atg8 deconjugation from this lipid to release it from membranes. How these two cleavage steps are coordinated is unknown. Here we show that phosphorylation by Atg1 inhibits Atg4 function, an event that appears to exclusively occur at the site of autophagosome biogenesis. These results are consistent with a model where the Atg8-phosphatidylethanolamine pool essential for autophagosome formation is protected at least in part by Atg4 phosphorylation by Atg1 while newly synthesized cytoplasmic Atg8 remains susceptible to constitutive Atg4 processing

Conserved Atg8 recognition sites mediate Atg4 association with autophagosomal membranes and Atg8 deconjugation

Deconjugation of the Atg8/LC3 protein family members from phosphatidylethanolamine (PE) by Atg4 proteases is essential for autophagy progression, but how this event is regulated remains to be understood. Here, we show that yeast Atg4 is recruited onto autophagosomal membranes by direct binding to Atg8 via two evolutionarily conserved Atg8 recognition sites, a classical LC3-interacting region (LIR) at the C-terminus of the protein and a novel motif at the N-terminus. Although both sites are important for Atg4-Atg8 interaction in vivo, only the new N-terminal motif, close to the catalytic center, plays a key role in Atg4 recruitment to autophagosomal membranes and specific Atg8 deconjugation. We thus propose a model where Atg4 activity on autophagosomal membranes depends on the cooperative action of at least two sites within Atg4, in which one functions as a constitutive Atg8 binding module, while the other has a preference toward PE-bound Atg8

Conserved Atg8 recognition sites mediate Atg4 association with autophagosomal membranes and Atg8 deconjugation

Deconjugation of the Atg8/LC3 protein family members from phosphatidylethanolamine (PE) by Atg4 proteases is essential for autophagy progression, but how this event is regulated remains to be understood. Here, we show that yeast Atg4 is recruited onto autophagosomal membranes by direct binding to Atg8 via two evolutionarily conserved Atg8 recognition sites, a classical LC3-interacting region (LIR) at the C-terminus of the protein and a novel motif at the N-terminus. Although both sites are important for Atg4-Atg8 interaction in vivo, only the new N-terminal motif, close to the catalytic center, plays a key role in Atg4 recruitment to autophagosomal membranes and specific Atg8 deconjugation. We thus propose a model where Atg4 activity on autophagosomal membranes depends on the cooperative action of at least two sites within Atg4, in which one functions as a constitutive Atg8 binding module, while the other has a preference toward PE-bound Atg8