Biology subjects

Antosiewicz, A.

Publications and source records attributed to Antosiewicz, A..

3 recordsLinked to original sources

bioRxiv2024

Mechanism of catalytic apparatus of human chitotriosidase-1 and its dual inactivation mode by the first-in-class OATD-01 inhibitor.

Despite extensive research over the past three decades, there are still uncertainties regarding the catalytic mechanism of human chitotriosidase-1. To fill the gap, we reanalysed the structural information available for this enzyme. Based on the existing and new experimental data, complemented by multi-scale simulations, we modelled the full-length structure of human chitotriosidase-1 and proposed the general model of its catalytic mechanism. We have elucidated the catalytic role of the four highly conserved structural motifs present in glycoside hydrolases 18 family and demonstrated the impact of ions on achieving optimal catalytic conditions. Furthermore, we have identified distinct mechanical motions within the catalytic domain that collectively facilitate the catalysis. Finally, we demonstrate how subtle dynamical changes observed within the active site upon binding of the OATD-01 inhibitor correspond to long-range effects that are transmitted across enzyme subunits, leading to profound biological consequences.

biophysics↗

bioRxiv2024

Depletion of essential GroEL protein in Escherichia coli using Clp-Interacting Peptidic Protein Erasers (CLIPPERs)

New, universal tools for targeted protein degradation in bacteria can help to accelerate protein function studies and antimicrobial research. We have developed a new method for degrading bacterial proteins using plasmid-encoded degrader peptides which deliver target proteins for degradation by a highly conserved ClpXP protease. We demonstrated the mode of action of the degraders on a challenging essential target, GroEL. The studies in bacteria were complemented by in vitro binding and structural studies. Expression of degrader peptides resulted in a temperature-dependent growth inhibition and depletion of GroEL levels over time. The reduction of GroEL levels was accompanied by dramatic proteome alterations. The presented method offers a new alternative approach for regulating protein levels in bacteria without genomic modifications or tag fusions. Our studies demonstrate that ClpXP is an attractive protease for the future use in bacterial targeted protein degradation.

molecular biology↗

bioRxiv2021

A uniform benchmark for testing ssrA-derived degrons in the Escherichia coli ClpXP pathway

The ssrA degron is commonly used in fusion proteins to control protein stability in bacteria or as an interaction module. These applications often rely on the modular activities of the ssrA tag in binding to the SspB adaptor and in engaging the ClpXP protease. However, a comparison of these activities for a substantial standard set of degron variants has not been conducted previously, which may hinder developments of new variants optimized exclusively for one application. Here, we strive to establish a benchmark that will facilitate the comparison of ssrA variants under uniform conditions. In our workflow, we included methods for expression and purification of ClpX, ClpP, SspB and eGFP-degrons, assays of ClpX ATPase activity, of eGFP-degron binding to SspB and for measuring eGFP-degron degradation in vitro and in vivo. Using uniform, precise and sensitive methods under the same conditions on a range of eGFP-degrons allowed us to determine subtle differences in their properties that can affect their potential applications. Our findings can serve as a reference and a resource for developing targeted protein degradation approaches. SUMMARYThis work lays standards for assays used to compare engagement of SspB and ClpXP by a set of ssrA-derived degrons that can be used to fine-tune tools for protein stability control.

biochemistry↗