Biology subjects

Scavone, F.

Publications and source records attributed to Scavone, F..

3 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↗

bioRxiv2023

RPL26/uL24 UFMylation is essential for ribosome-associated quality control at the endoplasmic reticulum

Ribosomes that stall while translating cytosolic proteins are incapacitated by incomplete nascent chains, termed "arrest peptides" (APs) that are destroyed by the ubiquitin proteasome system (UPS) via a process known as the ribosome-associated quality control (RQC) pathway. By contrast, APs on ribosomes that stall while translocating secretory proteins into the endoplasmic reticulum (ER-APs) are shielded from cytosol by the ER membrane and the tightly sealed ribosome-translocon junction (RTJ). How this junction is breached to enable access of cytosolic UPS machinery and 26S proteasomes to translocon- and ribosome-obstructing ER-APs is not known. Here, we show that UPS and RQC-dependent degradation of ER-APs strictly requires conjugation of the ubiquitin-like (Ubl) protein UFM1 to 60S ribosomal subunits at the RTJ. Therefore, UFMylation of translocon-bound 60S subunits modulates the RTJ to promote access of proteasomes and RQC machinery to ER-APs. Significance StatementUFM1 is a ubiquitin-like protein that is selectively conjugated to the large (60S) subunit of ribosomes bound to the endoplasmic reticulum (ER), but the specific biological function of this modification is unclear. Here, we show that UFMylation facilitates proteasome-mediated degradation of arrest polypeptides (APs) which are generated following splitting of ribosomes that stall during co-translational translocation of secretory proteins into the ER. We propose that UFMylation weakens the tightly sealed ribosome-translocon junction, thereby allowing the cytosolic ubiquitin-proteasome and ribosome-associated quality control machineries to access ER-APs.

cell biology↗

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↗