Biology subjects

Turk, B.

Publications and source records attributed to Turk, B..

3 recordsLinked to original sources

bioRxiv2025

Engineering cathepsin S selective chemical probes and antibody-drug conjugates through substrate profiling with unnatural amino acids

Cysteine cathepsins, particularly cathepsin S, are important regulators of proteolytic signaling in health and disease, including cancer progression and immune modulation. Despite their therapeutic relevance, selective chemical tools to study individual cathepsins remain limited due to overlapping substrate preferences. Here we present design of cathepsin S selective chemical probes and cathepsin S-cleavable antibody-drug conjugates (ADCs) through substrate profiling with unnatural amino acids. First, Hybrid Combinatorial Substrate Library (HyCoSuL) technology incorporating a broad spectrum of unnatural amino acids was applied to comprehensively investigate the substrate specificity of cathepsin S. This approach enabled the identification of highly selective tetrapeptide motifs that served as scaffolds for the design of optimized fluorogenic substrates, irreversible inhibitors, and fluorescent activity-based probes (ABPs). These tools demonstrated high selectivity toward cathepsin S over closely related family members in both biochemical and cellular settings. We then translated these findings to develop cathepsin S-activated ADCs, incorporating the optimized peptide motifs as protease-cleavable linkers for targeted payload release. Using these linkers, we further generated MMAE-based antibody-drug conjugates directed against HER-2 and the TROP-2 proteins, demonstrating cathepsin S-dependent cytotoxicity in HER-2-positive as well as HER-2-negative/TROP-2-positive breast cancer models. Finally, we employed anti-cathepsin S antibodies in combination with mass cytometry (CyTOF) to assess the spatial distribution of cathepsin S expression in breast cancer patient samples. By correlating cathepsin S levels with tumor-associated markers such as TROP-2 and HER-2, we identified potential co-expression patterns that support the rationale for personalized therapy using antibody-drug conjugates selectively activated by cathepsin S. This integrative approach provides a comprehensive platform for profiling cathepsin S activity at the molecular, cellular, and tissue levels, with broad implications for the development of precision therapeutics and diagnostic strategies in oncology.

cancer biology↗

bioRxiv2022

The Cul5 E3 Ligase Complex Is a Key Negative Feedback Regulator of TCR/IL2 Signaling and Anti-Tumor Activity in CD8+ T Cells

CD8+ T cells play an important role in tumor immune surveillance and control. Better understanding of the regulation of their anti-tumor actions and improving their cytotoxic function and persistence will help advancing cancer immunotherapies. Here, we report the development of a step-wise CRISPR knockout (KO) screening strategy under the selection of TGF-{beta}, a clinically relevant immunosuppressive pressure. The screen identifies Cul5 as a negative-feedback regulator of the core signaling pathways, differentiation, and persistence of CD8+ T cell. Cul5 KO in mouse CD8+ T cells significantly improves their tumor control ability in vitro and in vivo with significant proteomic alterations that generally enhance TCR and cytokine signaling, effector function, stemness, and survival of CD8+ T cell. Mechanistically, Cul5, whose protein content and active, neddylated form increase upon TCR-stimulation, interacts with SOCS-box-containing Pcmtd2 and negatively regulates TCR and IL2/STAT5 signaling by decreasing TCR and IL2 signaling molecules. Moreover, Cul5 KO in human CD8+ T cells phenocopies that in mouse CD8+ T cells. Furthermore, KO of CTLA4 that is markedly upregulated by Cul5 KO in mouse and human CD8+ cells further enhances anti-tumor effect of Cul5 KO, and a neddylation inhibitor enhances CD8 effector activities largely dependently of Cul5. These results together not only reveal a previously unknown negative-feedback regulatory mechanism for CD8+ T cells, but also have strong translational implications in cancer immunotherapy.

immunology↗

bioRxiv2019

The Activome: multiplexed probing of activity of proteolytic enzymes using mass cytometry-compatible activity-based probes (TOF-probes)

The activome can be considered as a subset of the proteome that contains enzymes in their catalytically active form and can be interrogated by using probes targeted towards individual specific enzymes. A subset of such enzymes are proteases that are frequently studied with activity-based probes, small inhibitors equipped with a detectable tag, commonly a fluorophore. Due to the spectral overlap of these commonly used fluorophores, simultaneous analysis becomes limited. To overcome this, we developed a series of protease-selective lanthanide-labeled probes compatible with mass cytometry. Using lanthanide-based tags instead of fluorophores gives us the ability to monitor the activity of multiple proteases in parallel. As proof of concept we developed a panel of cathepsin and legumain specific probes and showed that we were able to identify an activome of these proteases in two cell lines and peripheral blood mononuclear cells, providing a framework for the use of mass cytometry for multiplexed enzyme activity detection.

biochemistry↗