Biology subjects

Pedowitz, N. J.

Publications and source records attributed to Pedowitz, N. J..

4 recordsLinked to original sources

bioRxiv2024

An mRNA Display Approach for Covalent Targeting of a Staphylococcus aureus Virulence Factor

Staphylococcus aureus (S. aureus) is an opportunistic human pathogen that causes over one million deaths around the world each year. We recently identified a family of serine hydrolases termed fluorophosphonate binding hydrolases (Fphs) that play important roles in lipid metabolism and colonization of a host. Because many of these enzymes are only expressed in Staphylococcus bacteria, they are valuable targets for diagnostics and therapeutics. Here we developed and screened highly diverse cyclic peptide libraries using mRNA display with a genetically encoded oxadiazolone (Ox) electrophile that was previously shown to potently and covalently inhibit multiple Fph enzymes. By performing multiple rounds of counter selections with WT and catalytic dead FphB, we were able to tune the selectivity of the resulting selected cyclic peptides containing the Ox residue towards the desired target. From our mRNA display hits, we developed potent and selective fluorescent probes that label the active site of FphB at single digit nanomolar concentrations in live S. aureus bacteria. Taken together, this work demonstrates the potential of using direct genetically encoded electrophiles for mRNA display of covalent binding ligands and identifies potent new probes for FphB that have the potential to be used for diagnostic and therapeutic applications.

microbiology↗

bioRxiv2023

Development of Oxadiazolone Activity-Based Probes Targeting FphE for Specific Detection of S. aureus Infections

Staphylococcus aureus is a major human pathogen responsible for a wide range of systemic infections. Since its propensity to form biofilms in vivo poses formidable challenges for both detection and treatment, tools that can be used to specifically image S. aureus biofilms are highly valuable for clinical management. Here we describe the development of oxadiazolone-based activity-based probes to target the S. aureus-specific serine hydrolase FphE. Because this enzyme lacks homologs in other bacteria, it is an ideal target for selective imaging of S. aureus infections. Using X-ray crystallography, direct cell labeling and mouse models of infection we demonstrate that oxadiazolone-based probes enable specific labeling of S. aureus bacteria through the direct covalent modification of the FphE active site serine. These results demonstrate the utility of the oxadizolone electrophile for activity-based probes (ABPs) and validate FphE as a target for development of imaging contrast agents for the rapid detection of S. aureus infections.

microbiology↗

bioRxiv2019

O-GlcNAcylation of small heat shock proteins enhances their anti-amyloid chaperone activity

A major role for the intracellular posttranslational modification O-GlcNAc appears to be the inhibition of protein aggregation. Most of the previous studies in this area have focused on O-GlcNAcylation of the amyloid-forming proteins themselves. Here, we use synthetic protein chemistry to discover that O-GlcNAc also activates the anti-amyloid activity of certain small heat shock proteins (sHSPs), a potentially more important modification event that can act broadly and substoichiometrically. More specifically, we find that O-GlcNAcylation increases the ability of sHSPs to block the amyloid formation of both -synuclein and A{beta}. Mechanistically, we show that O-GlcNAc near the sHSP IXI-domain prevents its ability to intramolecularly compete with substrate binding. Our results have important implications for neurodegenerative diseases associated with amyloid formation and potentially other areas of sHSP biology.

biochemistry↗

bioRxiv2019

MYPT1 O-GlcNAcylation controls the sensitivity of fibroblasts to sphingosine-1-phosphate mediated cellular contraction

Many intracellular proteins can be modified by N-acetylglucosamine, a posttranslational modification known as O-GlcNAc. Because this modification is found on serine and threonine side-chains, O-GlcNAc has the potential to dynamically regulate cellular signaling pathways through interplay with phosphorylation. Here, we discover and characterize one such example. First, we find that O-GlcNAc levels control the sensitivity of fibroblasts to actin contraction induced by the signaling lipid sphingosine-1-phosphate (S1P). In follow-up mechanistic investigations, we show that this O-GlcNAc dependence lies in the signaling pathway through the S1PR2 receptor and subsequent activation of the Rho and Rho kinase. This pathway typically culminates in the phosphorylation of myosin light chain (MLC), resulting in myosin activation and cellular contraction. We discovered that O-GlcNAc modification of the phosphatase subunit MYPT1 inhibits this pathway by blocking MYPT1 phosphorylation, maintaining its activity and causing the dephosphorylation of MLC. Therefore, MYTP1 O-GlcNAc levels function to regulate the sensitivity of cells to S1P-mediated cellular contraction. Finally, we demonstrate that O-GlcNAc levels alter the sensitivity of primary human dermal fibroblasts in a collagen matrix model of wound healing. Our findings have important implications for the role of O-GlcNAc in fibroblast motility and differentiation, particularly in diabetic wound healing, where increased levels of the modification may inhibit S1P-mediated healing phenotypes in fibroblasts.

cell biology↗