Biochemical Tools for Tracking Proteolysis

Abstract

All living organisms depend on tightly regulated cellular networks to control biological functions. Proteolysis is an important irreversible post-translational modification that regulates most, if not all, cellular processes. Proteases are a large family of enzymes that perform hydrolysis of protein substrates, leading to protein activation or degradation. The 473 known and 90 putative human proteases are divided into 5 main mechanistic groups: metalloproteases, serine proteases, cysteine proteases, threonine proteases, and aspartic acid proteases. Proteases are fundamental to all biological systems, and when dysregulated they profoundly influence disease progression. Inhibiting proteases has led to effective therapies for viral infections, cardiovascular disorders, and blood coagulation just to name a few. Between 5 and 10% of all pharmaceutical targets are proteases, despite limited knowledge about their biological roles. More than 50% of all human proteases have no known substrates. We present here a comprehensive list of all current known human proteases. We also present current and novel biochemical tools to characterize protease functions in vitro, in vivo, and ex vivo. These tools make it achievable to define both beneficial and detrimental activities of proteases in health and disease.

Proteomics Analysis of Tears and Saliva From Sjogren’s Syndrome Patients

“…Interestingly, upregulation of several cysteine protease inhibitors in the saliva of healthy controls included cystatin-D, cystatin-S and cystatin-SN, indicating a possible regulation and inhibition of cathepsin activity ( Balbín et al, 1994 ). Further studies to characterize these proteases enriched in SS tears and saliva using N-terminomics could identify their potential substrates, which could help find new drug targets in SS ( Dufour, 2015 ; Mainoli et al, 2020 ; Longxiang Wang, Kimberly Main, Henry; Wang, Olivier Julien, 2021 ). Our data present new proteases, metabolic and cellular signaling pathways that are elevated in SS and could lead to unbiased investigations of new regulators of SS pathogenesis.…”

Prioritized single-cell proteomics reveals molecular and functional polarization across primary macrophages

“…To avoid products of proteolysis that may occur during sample preparation, we focused only on proteolytic products present in the macrophages prior to trypsin digestion. These products were identified in discovery bulk samples in which amine groups were covalently labeled prior to trypsin digestion as commonly performed 30, 31 . The proteolytic products were matched to annotated proteolytic products in the MEROPS database 26 and analyzed by pSCoPE in single cells.…”

Matrix Metalloproteinases: From Molecular Mechanisms to Physiology, Pathophysiology, and Pharmacology

“…MMPs belong to the superfamily of metzincin proteases, which is a family of multidomain zinc-dependent endopeptidases that can be grouped into six families: the astacins, the adamalysins (a disintegrins and metalloproteinases, or ADAMs), and the ADAMs with thrombospondin motif (ADAMTSs), the pappalysins, the serralysins, and the MMPs (Sternlicht and Werb, 2001;Sela-Passwell et al, 2011b;Tokito and Jougasaki, 2016;Wang et al, 2021). The metzincins received this denomination due to a characteristic feature in their structure, a methionine (Met) residue at the active site and the use of zinc in the enzymatic reaction.…”

“…Pharmacology of Matrix Metalloproteinases mechanistic classes of catalysis: metallo, serine, cysteine, aspartic acid, and threonine proteases (Puente et al, 2003;Wang et al, 2021) (Fig. 1); see the MEROPS database (https://www.ebi.ac.uk/merops/) for the lists of all known proteases (Rawlings et al, 2010).…”