Biology subjects

Godoy-Ruiz, R.

Publications and source records attributed to Godoy-Ruiz, R..

3 recordsLinked to original sources

bioRxiv2024

Mitophagosomes induced during EV-D68 infection promote viral nonlytic release

Enterovirus-D68 (EV-D68) is a plus-strand RNA virus that primarily causes infant respiratory infections. In rare pediatric cases, infection with EV-D68 has been associated with acute flaccid myelitis, a polio-like paralytic disease. We have previously demonstrated that EV-D68 induces nonselective autophagy for its benefit. Here, we demonstrate that EV-D68 induces mitophagy, the specific autophagic degradation of mitochondria. EV-D68 infection induces mitophagosome formation and several hallmarks of mitophagy, including mitochondrial fragmentation, mitochondrial membrane potential loss, and Parkin translocation to the mitochondria were observed in EV-D68 infected cells. The 3C protease of EV-D68 cleaves the mitochondrial fusion protein, mitofusin-2, near the C-terminal HR2 domain to induce mitochondrial fragmentation, and these fragmented mitochondria colocalized with double-stranded RNA (dsRNA), which labels viral RNA replication sites after peak viral RNA replication. Depleting components of mitophagy signaling specifically reduced EV-D68 release without impacting viral intracellular titers. Our results suggest that whereas the machinery of macroautophagy supports various stages of enterovirus replication, including viral genomic RNA replication and capsid maturation, mitophagy is the specific form of autophagy that regulates the nonlytic release of enteroviruses from cells. SignificanceEV-D68 is a respiratory pathogen and a significant cause of acute respiratory illness in infants, against which no prophylactic vaccines or antivirals exist. We have previously shown that EV-D68, like other enteroviruses, triggers and benefits from nonselective autophagy. However, it was unclear whether the virus induces selective autophagy. Here, we demonstrate that EV-D68 infection induces mitophagy and uses mitophagosomes to traffic virions to the surface of cells for nonlytic release. Our findings suggest that mitophagy may constitute the specific form of autophagy that facilitates the autophagosome-mediated nonlytic release of enteroviruses from infected cells and that targeting mitophagy could be an effective strategy to impede EV-D68 nonlytic release.

microbiology↗

bioRxiv2023

Endosomal activation of the Clostridioides difficile binary toxin is Ca2+-dependent

The C. difficile binary toxin (CDT) enters host cells via endosomal delivery like many other AB-type binary toxins. In this study, the cell-binding component of CDT, termed CDTb, was found to bind and form pores in lipid bilayers upon depleting free Ca2+ ion concentrations, and not by lowering pH, as found for other binary toxins (i.e., anthrax). Cryoelectron microscopy, nuclear magnetic resonance spectroscopy, surface plasmon resonance, electrochemical impedance spectroscopy, CDT toxicity studies, and site directed mutagenesis show that dissociation of Ca2+ from a single site in receptor binding domain 1 (RBD1) of CDTb is consistent with a molecular mechanism in which Ca2+ dissociation from RBD1 induces a "trigger" via conformational exchange that enables CDTb to bind and form pores in endosomal membrane bilayers as free Ca2+ concentrations decrease during CDT endosomal delivery. One-Sentence SummaryCa2+ dissociation and unfolding of a receptor binding domain in CDTb activates pore-formation by the C. difficile binary toxin.

biophysics↗

bioRxiv2020

Engineering protein-specific proteases: targeting active RAS

We describe the design, kinetic properties, and structures of engineered subtilisin proteases that degrade the active form of RAS by cleaving a conserved sequence in switch 2. RAS is a signaling protein that, when mutated, drives a third of human cancers. To generate high specificity for the RAS target sequence, the active site was modified to be dependent on a cofactor (imidazole or nitrite) and protease sub-sites were engineered to create a linkage between substrate and cofactor binding. Selective proteolysis of active RAS arises from a 2-step process wherein sub-site interactions promote productive binding of the cofactor, enabling cleavage. Proteases engineered in this way specifically cleave active RAS in vitro, deplete the level of RAS in a bacterial reporter system, and also degrade RAS in human cell culture. Although these proteases target active RAS, the underlying design principles are fundamental and will be adaptable to many target proteins.

biochemistry↗