Biology subjects

Ziegler, A. R.

Publications and source records attributed to Ziegler, A. R..

5 recordsLinked to original sources

bioRxiv2026

Temporal N-terminomics analysis reveals proteome remodeling following brensocatib-mediated cathepsin C inhibition in promyeloblast cells

Cathepsin C (CatC) is known to activate neutrophil serine proteases (NSPs) involved in innate immune function although its broader impact on cellular proteolytic networks remains poorly defined. Here, we characterized the proteolytic landscape of HL-60 neutrophil progenitor cells following treatment with the CatC inhibitor brensocatib. Activity-based probes confirmed sustained inhibition of CatC by brensocatib, accompanied by progressive suppression of downstream elastase-like protease activation over 16 h, 72 h, and 7 days. An enrichment-free N-terminomics workflow was used to compare control and brensocatib-treated HL-60 cells. Following prolonged CatC inhibition, NSPs were reduced in abundance, while lysosomal cathepsins and endogenous protease inhibitors increased. These findings are consistent with remodeling of the protease-antiprotease network. Cleavage site analysis identified a pronounced, time-dependent NSP-associated P1 cleavage signature enriched for Val, Thr, Ala, Ile, and Cys in control cells that was progressively lost following brensocatib treatment. We also identified established and candidate CatC-dependent cleavage events, together with proteolytic adaptations that emerged in the absence of CatC activity. Collectively, these findings demonstrate that CatC inhibition extends beyond suppression of canonical NSP activation to progressive remodeling of the broader protease-antiprotease network, providing new mechanistic insight into the cellular consequences of therapeutic CatC inhibition.

biochemistry↗

bioRxiv2025

PAC/SP3 on-bead carboxyl derivatization allows combined C- and N-terminomics

On-bead single-pot solid-phase enhanced sample preparation, SP3, also known as Protein Aggregation Capture (PAC), is a robust, high-throughput, and widely utilized approach for proteomic sample preparation. Recent studies have highlighted PAC/SP3 as an ideal platform for chemoproteomics, allowing chemical labelling by minimizing sample loss and improving recovery of derivatized peptides. In this work, we establish an on-bead PAC/SP3 protein-level amine and carboxyl derivatization approach to facilitate C-terminal focused proteomics. We demonstrate that on-bead protein derivatization of carboxyl groups can be achieved using ethanolamine, (2-aminoethyl)trimethylammonium (AETMA), and (carboxymethyl)trimethylammonium (Girards reagent T, GT) via EDC/HOBt coupling, enabling the labelling of protein C-termini. Using a prokaryotic model system, Acinetobacter baumannii, we demonstrate that AETMA and ethanolamine labelling each enables the identification of unique protein C-terminal peptides, with AETMA improving the identification of C-terminal peptides lacking basic residues. Finally, we apply this approach to interrogate both N- and C-termini in response to etoposide-induced apoptosis within Jurkat cells, demonstrating that combined N- and C-terminomics is achievable using on-bead derivatization, yet provides modest coverage of the C-terminome in its current form. Overall, this work establishes bead-based carboxyl group derivatization as a viable platform to enable future C-terminomics method development.

biochemistry↗

bioRxiv2025

Probing the activity of cysteine cathepsins in inflammatory bowel diseases

Cathepsin S is a cysteine protease that has been implicated in inflammatory bowel diseases (IBD) for its ability to promote visceral pain. Given its pro-inflammatory roles, we hypothesized that cathepsin S would drive other symptoms associated with IBD. Using activity-based probes, we investigated cysteine cathepsin activation in human and murine colitis. We observed a significant increase in fecal cathepsin S in patients with ulcerative colitis compared to healthy controls, while cathepsin S in mucosal biopsies was unchanged. Mice with experimental colitis exhibited a modest increase in mucosal activity of both cathepsin S and X compared to naive mice. Luminal secretion of cathepsin S was dramatically increased upon colitis induction, although differences between mouse colonies were observed. To investigate the contribution of cathepsin S and cathepsin X to colitis, we induced colitis in cathepsin-deficient mice. Cathepsin X-deficient mice exhibited no clear differences in disease indicators compared to wild-type mice. While cathepsin S-deficient mice exhibited less rectal bleeding, less splenomegaly and marginally improved histological scores, weight loss, diarrhea, colon shortening, and myeloperoxidase activity were not significantly different from wild-type mice. To determine whether pharmacologic inhibition of cathepsin S activity would ameliorate symptoms of colitis, a reversible inhibitor LY3000328 was administered to mice at the initiation of colitis. LY3000328 provoked a clear upregulation of cathepsin S and L activity in the mucosa, most likely through a compensatory mechanism. This increase in protease activity was associated with exacerbated histological scores and splenomegaly. Collectively, these results suggest that cathepsin S, but not cathepsin X, may contribute to some of the symptoms of experimental colitis. While cathepsin S has potential to be a therapeutic target in colitis, improved strategies to sustain its inhibition are required in future.

pathology↗

bioRxiv2025

Legumain drives processing of cathepsins and nuclear localisation of cathepsin L

Lysosomal proteases such as the cathepsin family and the asparaginyl endopeptidase, legumain, govern vital processes to maintain cellular proteostasis, and their dysregulation contributes to diverse pathologies. Recent studies have reported extra-lysosomal localisation of these proteases, especially in the nucleus, cytoplasm, and extracellularly, yet their function is not completely understood. To examine the relationship between legumain and cathepsins, we assessed the activity and expression of cathepsins in wild-type and legumain-deficient (LGMN-/-) cells using chemical activity-based probes and immunoblots. Processing of cathepsins (CTS) L, V, B, and D from the single-chain to the two-chain form was abrogated in the absence of legumain, with some cell type- and species-specific variation observed. This processing was dependent on legumain activity, although the mechanism remains unclear since recombinant legumain does not appear to directly cleave cathepsins in vitro. In cell types where CTSL exists in the nucleus preferentially in its double chain form, loss of legumain led to a reduction in nuclear CTSL levels. To understand the potential role of these lysosomal proteases in the nucleus, we applied our newly refined chemical N-terminomics pipeline, No-enrichment Identification of Cleavage Events (NICE). This analysis revealed widespread changes in both protein abundance and proteolysis, including putative nuclear substrates of CTSL and legumain, that primarily suggest roles in cell proliferation, cell cycle regulation, inflammation, and ribosomal biogenesis. Overall, this study builds on our understanding of the relationship between legumain and cathepsins and provides the first systematic characterisation of lysosomal protease substrates in the nucleus. Our results offer valuable insight into the potential extra-lysosomal roles of these critical proteases.

biochemistry↗

bioRxiv2023

FAIMS-enabled N-terminomics analysis reveals novel legumain substrates in murine spleen

Aberrant levels of the asparaginyl endopeptidase legumain have been linked to inflammation, neurodegeneration and cancer, yet our understanding of this protease is incomplete. Systematic attempts to identify legumain substrates have previously been confined to in vitro studies, which fail to mirror physiological conditions and obscure biologically relevant cleavage events. Using high-field asymmetric waveform ion mobility spectrometry (FAIMS), we developed a sensitive and streamlined approach for proteome and N-terminome analyses in a single analytical method without the need for N-termini enrichment. Compared to unfractionated proteomic analysis, we demonstrate FAIMS fractionation improves neo-N- termini identification by >2.5 fold, resulting in identification of >2,882 unique neo-N-termini from limited sample amounts. Within murine spleens, this approach identifies 6,366 proteins and 2,528 unique neo-N-termini, with 235 cleavage events enriched in wild-type compared to legumain-deficient spleens. Among these, 119 neo-N-termini arose from asparaginyl endopeptidase activities, representing novel putative physiological legumain substrates. The direct cleavage of selected substrates by legumain was confirmed using in vitro assays, providing support for the existence of physiologically relevant extra-lysosomal legumain activity. Combined, these data shed critical light on the functions of legumain and demonstrates the utility of FAIMS as an accessible method to improve depth and quality of N- terminomics studies.

biochemistry↗