Proteolytic Cleavage—Mechanisms, Function, and “Omic” Approaches for a Near-Ubiquitous Posttranslational Modification

Abstract

Proteases enzymatically hydrolyze peptide bonds in substrate proteins, resulting in a widespread, irreversible posttranslational modification of the protein's structure and biological function. Often regarded as a mere degradative mechanism in destruction of proteins or turnover in maintaining physiological homeostasis, recent research in the field of degradomics has led to the recognition of two main yet unexpected concepts. First, that targeted, limited proteolytic cleavage events by a wide repertoire of proteases are pivotal regulators of most, if not all, physiological and pathological processes. Second, an unexpected in vivo abundance of stable cleaved proteins revealed pervasive, functionally relevant protein processing in normal and diseased tissue-from 40 to 70% of proteins also occur in vivo as distinct stable proteoforms with undocumented N- or C-termini, meaning these proteoforms are stable functional cleavage products, most with unknown functional implications. In this Review, we discuss the structural biology aspects and mechanisms of catalysis by different protease classes. We also provide an overview of biological pathways that utilize specific proteolytic cleavage as a precision control mechanism in protein quality control, stability, localization, and maturation, as well as proteolytic cleavage as a mediator in signaling pathways. Lastly, we provide a comprehensive overview of analytical methods and approaches to study activity and substrates of proteolytic enzymes in relevant biological models, both historical and focusing on state of the art proteomics techniques in the field of degradomics research.

Multiple Architectures and Mechanisms of Latency in Metallopeptidase Zymogens

“…The zymogens of the MMP family were the first to be structurally analyzed within the metzincin clan of MPs (Table 1; 87 ). MMPs are mostly secreted enzymes and broadly process extracellular matrix components, but they also shed membrane-bound substrates and activate other peptidases through limited proteolysis 8,88 . They are involved in chronic inflammatory diseases and degrade basement membrane components during several pathologies, including cancer and metastasis processes 7 .…”

PDX models reflect the proteome landscape of pediatric acute lymphoblastic leukemia but divert in select pathways

“…We recently developed a new enrichment procedure (High-efficiency Undecanal-based N Termini Enrichment, HUNTER) to extensively profile the N terminome of minimal disease samples 24,25 . In addition to profiling proteolytic proteoforms, we and others have shown that the N terminome provides a reliable repertoire to verify the existence of truncated proteins, including stable fragments that are established biomarkers 26 and identify their functional relevance in biological processes such as apoptosis 25,[27][28][29] .…”

Spatial Distribution of Endogenous Tissue Protease Activity in Gastric Carcinoma Mapped by MALDI Mass Spectrometry Imaging

“…Our MSI and quantitative proteomics results reveal differential distribution of protease activity -with strongest activity being observed in mouse tumor tissue, suggesting the general applicability of the workflow in animal pharmacology and clinical studies. Proteases control cell and tissue protein homeostasis (1). They influence cell proliferation, tissue morphogenesis and remodeling, and they are therefore associated with many pathological conditions including cancer.…”

“…In the past, various optical methods including (near infrared; NIR) fluorogenic and bioluminescence-based substrate-or activity reporter probes have been used to visualize protease activity in vitro, in cultured cells and in vivo (5,6). Analytical probes typically enable fluorescence quenching, fluorescence resonance energy transfer (FRET) 1 , and other optical readouts. Whereas several probes have been described for cell studies and in vivo molecular imaging (7), direct visualization and biochemical investigation of protease activity in tissue sections remains an underexplored field of research.…”