Yee, V. | bioRxiv Science

bioRxiv2020

Complimentary action of structured and unstructured domains of epsin supports clathrin-mediated endocytosis at high tension

Membrane tension plays an inhibitory role in clathrin-mediated endocytosis (CME) by impeding the transition of flat plasma membrane to hemispherical clathrin-coated structures (CCSs). Membrane tension also impedes the transition of hemispherical domes to omegashaped CCSs, a necessary step before their internalization via dynamin-mediated membrane scission. However, CME is not completely halted in cells under high tension conditions. Here we find that epsin, a membrane bending protein which inserts its N-terminus H0 helix into lipid bilayer, supports flat-to-dome transition and increases the stability of CCSs at high tension. This discovery is supported by molecular dynamic simulation of the epsin N-terminal homology (ENTH) domain that becomes more structured when embedded in a lipid bilayer. In addition, epsin has an intrinsically disordered protein (IDP) C-terminus domain which induces membrane curvature via steric repulsion. Insertion of H0 helix into lipid bilayer is not sufficient for stable epsin recruitment as deleting the IDP domain in epsin renders it cytosolic. Epsins binding to adaptor protein 2 and clathrin is critical for epsins association with CCSs under high tension conditions, supporting the importance of multivalent interactions in CCSs. Together, our results support a model where the ENTH and IDP domains of epsin have complementary roles to ensure CME initiation and CCS maturation are unimpeded under high tension environments.

cell biology↗

bioRxiv2019

Discovery of a redox-thiol switch regulating cellular energy metabolism

Previously, we reported that increased synthesis of the gas hydrogen sulfide (H2S) during the Integrated Stress Response (ISR) induced proteome-wide cysteine-sulfhydration with the predominant modified pathway being enzymes of cellular energy metabolism (Gao, et al. 2015). Using pancreatic beta cells and quantitative proteomics in this study, we identified a Redox Thiol Switch from S-glutathionylation to S-sulfhydration and we named it, RTSGS. About half of the identified proteins are involved in energy metabolism, and one novel target was the mitochondrial phosphoenolpyruvate carboxykinase 2 (PCK2) whose catalytic Cys306was targeted by both modifications. The enzymatic activity of PCK2 was inhibited by S-glutathionylation, and this inhibition was largely reversed by S-sulfhydration. S-sulfhydration also reversed the S-glutathionylation-mediated inhibition of glucose flux, indicating a broad metabolic significance. We propose that a Redox Thiol Switch from S-glutathionylation to S-sulfhydration is a key mechanism to fine tune cellular energy metabolism in response to different levels of oxidative stress.

biochemistry↗