Biology subjects

Sheedy, C. J.

Publications and source records attributed to Sheedy, C. J..

3 recordsLinked to original sources

bioRxiv2025

CD47 prevents Rac-mediated phagocytosis through Vav1 dephosphorylation

CD47 is expressed by viable cells to protect against phagocytosis. CD47 is recognized by SIRP, an inhibitory receptor expressed by macrophages and other myeloid cells. Activated SIRP recruits SHP-1 and SHP-2 phosphatases but the inhibitory signaling cascade downstream of these phosphatases is not clear. In this study, we used time lapse imaging to measure how CD47 impacts the kinetics of phagocytosis. We found that targets with IgG antibodies were primarily phagocytosed through a Rac-based reaching mechanism. Targets also containing CD47 were only phagocytosed through a less frequent Rho-based sinking mechanism. Hyperactivating Rac2 eliminated the suppressive effect of CD47, suggesting that CD47 prevents activation of Rac and reaching phagocytosis. During IgG-mediated phagocytosis, the tyrosine kinase Syk phosphorylates the GEF Vav, which then activates the GTPase Rac to drive F-actin rearrangement and target internalization. CD47 inhibited Vav1 phosphorylation without impacting Vav1 recruitment to the phagocytic synapse or Syk phosphorylation. Macrophages expressing a hyperactive Vav1 were no longer sensitive to CD47. Together this data suggests that Vav1 is a key target of the CD47 signaling pathway.

cell biology↗

bioRxiv2024

PEX1G843D remains functional in peroxisome biogenesis but is rapidly degraded by the proteasome

The PEX1/PEX6 AAA-ATPase is required for the biogenesis and maintenance of peroxisomes. Mutations in HsPEX1 and HsPEX6 disrupt peroxisomal matrix protein import and are the leading cause of Peroxisome Biogenesis Disorders (PBDs). The most common disease-causing mutation in PEX1 is the HsPEX1G843D allele, which results in a reduction of peroxisomal protein import. Here we demonstrate that in vitro the homologous yeast mutant, ScPex1G700D, reduces the stability of Pex1s active D2 ATPase domain and impairs assembly with Pex6, but can still form an active AAA-ATPase motor. In vivo, ScPex1G700D exhibits only a slight defect in peroxisome import. We generated model human HsPEX1G843D cell lines and show that PEX1G843D is rapidly degraded by the proteasome, but that induced overexpression of PEX1G843D can restore peroxisome import. Additionally, we found that the G843D mutation reduces PEX1s affinity for PEX6, and that impaired assembly is sufficient to induce degradation of PEX1WT. Lastly, we found that fusing a deubiquitinase to PEX1G843D significantly hinders its degradation in mammalian cells. Altogether, our findings suggest a novel regulatory mechanism for PEX1/PEX6 hexamer assembly and highlight the potential of protein stabilization as a therapeutic strategy for PBDs arising from the G843D mutation and other PEX1 hypomorphs.

biochemistry↗

bioRxiv2024

A genome-wide screen links peroxisome regulation with Wnt signaling through RNF146 and tankyrase

Peroxisomes are membrane-bound organelles harboring metabolic enzymes. In humans, peroxisomes are required for normal development, yet the genes regulating peroxisome function remain unclear. We performed a genome-wide CRISPRi screen to identify novel factors involved in peroxisomal homeostasis. We found that inhibition of RNF146, an E3 ligase activated by poly(ADP-ribose), reduced the import of proteins into peroxisomes. RNF146-mediated loss of peroxisome import depended on the stabilization and activity of the poly(ADP-ribose) polymerase tankyrase, which binds the peroxisomal membrane protein PEX14. We propose that RNF146 and tankyrase regulate peroxisome import efficiency by PARsylation of proteins at the peroxisome membrane. Interestingly, we found that the loss of peroxisomes increased tankyrase and RNF146-dependent degradation of non-peroxisomal substrates, including the beta-catenin destruction complex component AXIN1, which was sufficient to alter the amplitude of beta-catenin transcription. Together, these observations not only suggest previously undescribed roles for RNF146 in peroxisomal regulation, but also a novel role in bridging peroxisome function with Wnt/beta-catenin signaling during development.

cell biology↗