Abstract
Cathepsin S is a lysosomal cysteine protease highly expressed in immune cells such as dendritic cells, B cells and macrophages. Its functions include extracellular matrix breakdown and cleavage of cell adhesion molecules to facilitate immune cell motility, as well as cleavage of the invariant chain during maturation of major histocompatibility complex II. The identification of these diverse specific functions has brought the challenge of delineating cathepsin S activity with great spatial precision, relative to related enzymes and substrates. Here, the development of a potent and highly selective two-step activity-based probe for cathepsin S and the application in multicolor bio-orthogonal correlative light-electron microscopy is presented. LHVS, which has been reported as a selective inhibitor of cathepsin S with nanomolar potency, formed the basis for our probe design. However, in competitive activity-based protein profiling experiments LHVS showed significant cross-reactivity toward Cat L. Introduction of an azide group in the P2 position expanded the selectivity window for cathepsin S, but rendered the probe undetectable, as demonstrated in bio-orthogonal competitive activity-based protein profiling. Incorporation of an additional azide handle for click chemistry on the solvent-exposed P1 position allowed for selective labeling of cathepsin S. This highlights the influence of click handle positioning on probe efficacy. This probe was utilized in multicolor bio-orthogonal confocal and correlative light-electron microscopy to investigate the localization of cathepsin S activity at an ultrastructural level in bone marrow-derived dendritic cells. The tools developed in this study will aid the characterization of the variety of functions of cathepsin S throughout biology.
Highlights
Cathepsin S (Cat S) is a lysosomal cysteine endopeptidase from the papain-superfamily and is an important mediator of adaptive immunity (Jakos et al, 2019)
Cat S is mainly expressed in immune cells such as B cells, macrophages and dendritic cells (DCs) and to a lesser extent in some epithelial cells (Gupta et al, 2008)
leucine homophenyl vinyl sulfone (LHVS) derivative 2 was synthesized using a modified procedure of the vinylsulfone synthesis described by Van der Linden and colleagues
Summary
Cathepsin S (Cat S) is a lysosomal cysteine endopeptidase from the papain-superfamily and is an important mediator of adaptive immunity (Jakos et al, 2019). Cat S is responsible for extracellular matrix (ECM) breakdown, e.g., elastin, fibronectin, collagen I, II and IV, and cleavage of cell adhesion molecules, such as JAM-B, ALCAM and L1CAM among many others (Fonovicand Turk, 2014; Sevenich et al, 2014; Soboticet al., 2015). This assists immune cell motility and function, Cat S overexpression by tumor and tumor-associated cells, in turn facilitates tumor growth, invasion and metastasis that can even surpass the blood brain barrier (Wang et al, 2006; Sevenich et al, 2014). Inhibition of these processes by small molecule inhibitors reduces cancer malignancy and improves survival in mouse models (Sevenich et al, 2014; Olson and Joyce, 2015; McDowell et al, 2020)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.