Biology subjects

Delgado, J. M.

Publications and source records attributed to Delgado, J. M..

3 recordsLinked to original sources

bioRxiv2023

The ER membrane protein complex governs lysosomal turnover of a mitochondrial tail-anchored protein, BNIP3, to restrict mitophagy

Lysosomal degradation of autophagy receptors is a common proxy for selective autophagy. However, we find that two established mitophagy receptors, BNIP3 and BNIP3L/NIX, violate this assumption. Rather, BNIP3 and NIX are constitutively delivered to lysosomes in an autophagy-independent manner. This alternative lysosomal delivery of BNIP3 accounts for nearly all of its lysosome-mediated degradation, even upon mitophagy induction. To identify how BNIP3, a tail-anchored protein in the outer mitochondrial membrane, is delivered to lysosomes, we performed a genome-wide CRISPR screen for factors influencing BNIP3 flux. By this approach, we revealed both known modifiers of BNIP3 stability as well as a pronounced reliance on endolysosomal components, including the ER membrane protein complex (EMC). Importantly, the endolysosomal system regulates BNIP3 alongside, but independent of, the ubiquitin-proteosome system (UPS). Perturbation of either mechanism is sufficient to modulate BNIP3-associated mitophagy and affect underlying cellular physiology. In short, while BNIP3 can be cleared by parallel and partially compensatory quality control pathways, non-autophagic lysosomal degradation of BNIP3 is a strong post-translational modifier of BNIP3 function. More broadly, these data reveal an unanticipated connection between mitophagy and TA protein quality control, wherein the endolysosomal system provides a critical axis for regulating cellular metabolism. Moreover, these findings extend recent models for tail-anchored protein quality control and install endosomal trafficking and lysosomal degradation in the canon of pathways that ensure tight regulation of endogenous TA protein localization.

cell biology↗

bioRxiv2022

Phosphoprotein Phosphatase Activity Positively Regulates Oligomeric Pyrin to Trigger Inflammasome Assembly in Response to Bacterial Effectors and Toxins that Inactivate RhoA in Macrophages

Pyrin is a pattern-recognition receptor in phagocytes that triggers capase-1 inflammasome assembly in response to bacterial toxins and effectors that inactivate RhoA. Pyrin contains oligomerization domains and is negatively regulated by phosphorylation of two residues, S205 and S241 (murine) or S208 and S242 (human), via the kinases PKN1/2, which are activated by RhoA. Familial Mediterranean Fever (FMF) is caused by phagocyte production of pyrin gain of function variants, which have a lower threshold for inflammasome assembly upon RhoA-PKN axis inhibition. Inactivation of the RhoA-PKN axis removes negative regulation but a phosphoprotein phosphatase (PPP) is needed to positively regulate pyrin. No PPP that dephosphorylates pyrin has been identified, oligomerization of murine pyrin has not been studied, and the phosphorylation status of oligomeric pyrin is unknown. We used murine macrophages and FMF patients monocytes combined with the use of bacterial agonists and chemical inhibitors, native PAGE, phospho-specific antibodies and siRNA knockdowns to determine if a PPP positively regulates oligomeric pyrin. Results with broadly-specific inhibitors indicate that PPP activity is required to dephosphorylate murine and human pyrin in wild type or FMF patients phagocytes. Findings from native PAGE show that murine pyrin forms oligomers that are phosphorylated on S205 prior to RhoA inactivation. Inhibitors cause reduced mobility of murine pyrin on native PAGE and hyperphosphorylation of S242 in human pyrin, suggesting a PPP constitutively counterbalances PKN to keep the second site hypophosphorylated. Data from siRNA knockdown experiments implicate PP2A in dephosphorylation of S205 and positive regulation of pyrin in response to RhoA inactivation. Key pointsMurine pyrin is oligomeric and phosphorylated on S205 prior to inflammasome assembly PPP activity positively regulates pyrin inflammasome assembly in mice and humans The alpha and beta subunits of PP2A dephosphorylate murine pyrin S205 in macrophages

immunology↗

bioRxiv2020

Molecular basis for SARS-CoV-2 spike affinity for human ACE2 receptor

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused substantially more infections, deaths, and economic disruptions than the 2002-2003 SARS-CoV. The key to understanding SARS-CoV-2s higher infectivity may lie in its host receptor recognition mechanism. This is because experiments show that the human ACE2 protein, which serves as the primary receptor for both CoVs, binds to CoV-2s spike protein 5-20 fold stronger than SARS-CoVs spike protein. The molecular basis for this difference in binding affinity, however, remains unexplained and, in fact, a comparison of X-ray structures leads to an opposite proposition. To gain insight, we use all-atom molecular dynamics simulations. Free energy calculations indicate that CoV-2s higher affinity is due primarily to differences in specific spike residues that are local to the spike-ACE2 interface, although there are allosteric effects in binding. Comparative analysis of equilibrium simulations reveals that while both CoV and CoV-2 spike-ACE2 complexes have similar interfacial topologies, CoV-2s spike protein engages in greater numbers, combinatorics and probabilities of hydrogen bonds and salt bridges with ACE2. We attribute CoV-2s higher affinity to these differences in polar contacts, and these findings also highlight the importance of thermal structural fluctuations in spike-ACE2 complexation. We anticipate that these findings will also inform the design of spike-ACE2 peptide blockers that, like in the cases of HIV and Influenza, can serve as antivirals.

biophysics↗