Biology subjects

Cohen, R. E.

Publications and source records attributed to Cohen, R. E..

4 recordsLinked to original sources

bioRxiv2023

STING induces LUBAC-mediated synthesis of linear ubiquitin chains to stimulate innate immune signaling

STING activation by cyclic dinucleotides in mammals induces IRF3- and NF{kappa}B -mediated gene expression, and the lipidation of LC3B at Golgi-related membranes. While mechanisms of the IRF3 response are well understood, the mechanisms of NF{kappa}B activation mediated by STING remain unclear. We report that STING activation induces linear/M1-linked ubiquitin chain (M1-Ub) formation and recruitment of the LUBAC E3 ligase, HOIP, to LC3B-associated Golgi membranes where ubiquitin is also localized. Loss of HOIP prevents formation of M1-Ub ubiquitin chains and reduces STING-induced NF{kappa}B and IRF3-mediated signaling in human monocytic THP1 cells and mouse bone marrow derived macrophages, without affecting STING activation. STING-induced LC3B lipidation is not required for M1-Ub chain formation or the immune-related gene expression, however the recently reported function of STING to neutralize the pH of the Golgi may be involved. Thus, LUBAC synthesis of M1 ubiquitin chains mediates STING-induced innate immune signaling.

cell biology↗

bioRxiv2021

Branched ubiquitin chain binding and deubiquitination by UCH37 facilitate proteasome clearance of stress-induced inclusions

UCH37, also known as UCHL5, is a highly conserved deubiquitinating enzyme (DUB) that associates with the 26S proteasome. Recently it was reported that UCH37 activity is stimulated by branched ubiquitin chain architectures. To understand how UCH37 achieves its unique debranching specificity, we performed biochemical and NMR structural analyses and found that UCH37 is activated by contacts with the hydrophobic patches of both distal ubiquitins that emanate from a branched ubiquitin. In addition, RPN13, which recruits UCH37 to the proteasome, further enhances branched-chain specificity by restricting linear ubiquitin chains from having access to the UCH37 active site. In cultured human cells under conditions of proteolytic stress, we show that substrate clearance by the proteasome is promoted by both binding and deubiquitination of branched polyubiquitin by UCH37. Proteasomes containing UCH37(C88A), which is catalytically inactive, aberrantly retain polyubiquitinated species as well as the RAD23B substrate shuttle factor, suggesting a defect in recycling of the proteasome. These findings provide a foundation to understand how proteasome degradation of substrates modified by a unique ubiquitin chain architecture is aided by a DUB.

biochemistry↗

bioRxiv2019

High-affinity free ubiquitin sensors as quantitative probes of ubiquitin homeostasis and deubiquitination

Ubiquitin (Ub) conjugation is an essential post-translational modification that affects nearly all proteins in eukaryotes. The functions and mechanisms of ubiquitination are areas of extensive and ongoing study, and yet the dynamics and regulation of even free (i.e., unconjugated) Ub are poorly understood. A major impediment has been the lack of simple and robust techniques to quantify Ub levels in cells and to monitor Ub release from conjugates. Here we describe the development of avidity-based fluorescent sensors that address this need. The sensors bind specifically to free Ub, have Kd values down to 60 pM, and, in concert with a newly developed workflow, allow us to distinguish and quantify the pools of free, protein-conjugated, and thioesterified forms of Ub from cell lysates. Alternatively, free Ub in fixed cells can be visualized microscopically by staining with a sensor. Real-time assays using the sensors afford unprecedented flexibility and precision to measure deubiquitination of virtually any (poly)Ub conjugate.

biochemistry↗

bioRxiv2017

Methods for addressing the protein-protein interaction between histone deacetylase 6 and ubiquitin

Histone deacetylase 6 (HDAC6) is a cytoplasmic HDAC isoform able to remove acetyl groups from cellular substrates such as -tubulin. In addition to the two deacetylase domains, HDAC6 has a C-terminal zinc-finger ubiquitin (Ub)-binding domain (ZnF-UBP) able to recognize free Ub. HDAC6-Ub interaction is thought to function in regulating the elimination of misfolded proteins during stress response through the aggresome pathway. Small molecules inhibiting deacetylation by HDAC6 were shown to reduce aggresomes, but the interplay between HDAC6 catalytic activity and Ub-binding function is not fully understood. Here we describe two methods to measure the HDAC6-Ub interaction in vitro using full-length HDAC6. Both methods were effective for screening inhibitors of the HDAC6-Ub protein-protein interaction independently of the catalytic activity. Our results suggest a potential role for the HDAC6 deacetylase domains in modulating HDAC6-Ub interaction. This new aspect of HDAC6 regulation can be targeted to address the roles of HDAC6-Ub interaction in normal and disease conditions.

biochemistry↗