This editorial by Dr. Luke Wiseman of The Scripps Research Institute accompanies the ACS Chemical Biology Special Issue on Proteolytic Regulation of Cellular Physiology. Like a block of granite in the hands of a sculptor, cellular proteomes must undergo constant reshaping and remodeling to accomplish all of the intricate biochemical processes necessary for organismal survival. […]

Like a block of granite in the hands of a sculptor, cellular proteomes must undergo constant reshaping and remodeling to accomplish all of the intricate biochemical processes necessary for organismal survival. One of the primary molecular mechanisms responsible for this reshaping is mediated through the activity of proteases. Proteases primarily function through the catabolic processing of substrate proteins into smaller peptides and/or amino acids. Despite this common function, proteases can have wide-ranging effects on the cellular proteome. For example, quality control proteases utilize the power of ATP to promiscuously degrade damaged or misfolded proteins, preventing their potentially toxic intracellular accumulation. Alternatively, highly specific proteases such as the SUMO peptidases cleave at a specific peptide bond to allow selective regulation of specific biological functions. Thus, cellular proteases serve as both the chisel and the scalpel to sensitively remodel the proteome in response to the continually changing physiologic environment in all organisms, from bacteria to mammals.

The potential for proteases to influence proteomes through both promiscuous and specific activities has allowed evolution to integrate proteolytic activity into nearly every aspect of cellular physiology. Proteases are intricately involved in the regulation of all biological functions, including membrane remodeling, protection against pathologic insults, stress-responsive signaling, and apoptosis. Considering the central importance of proteases in regulating all these aspects of cellular physiology, it is not surprising that imbalances in protease activity are implicated in the onset and pathogenesis of etiologically diverse pathologic conditions including viral infection, cancer, and neurodegenerative disease. This has led to a significant effort to understand both the molecular mechanism by which proteases regulate cellular physiology and identify specific contributions of proteases in dictating organismal function in the context of both health and disease.

As a field, chemical biology has been and continues to be at the forefront of defining the physiologic and pathologic importance of proteases in diverse organisms. Advances in chemical biology led to the development of new proteomic approaches to define the activity of both promiscuous and specific proteases, revealing critical new insights into protease function. Chemical biologists have also leveraged our understanding of proteolytic mechanisms to develop new ligand-regulated strategies to sensitively regulate protein stability post-translationally, transforming our ability to probe the activity of different proteins with temporal or dosable resolution. Furthermore, the establishment of chemical, biological approaches such as activity-based protein profiling has allowed us to probe the mechanism of protease regulation and activity both in vitro and in vivo. Finally, the development of compounds that modulate the activity of specific proteases or proteolytic mechanisms has provided new opportunities to define the translational potential for pharmacologically targeting protein degradation in etiologically diverse diseases. Suffice to say, chemical biology and protease biology have become intricately integrated over the years to form a mutually self-sustaining experimental enterprise.

ACS Chemical Biology put together a Special Issue with a collection of articles highlighting the continued contributions of chemical biology in defining the importance of proteases in the regulation of cellular physiology in the context of health and disease. This includes both reviews and original articles describing recent technological advances, new experimental resources, and new discoveries in protease biology. Collectively, these articles provide a snapshot of the ongoing contributions chemical biologists are making in the field of protease biology. Furthermore, this issue serves to identify new challenges and opportunities for chemical biologists to continue to contribute to both our ever-growing understanding of protease regulation and the development of new pharmacologic approaches that target protease or proteolytic pathways for therapeutic intervention.

The ability to probe the N-termini of the cellular proteome through the application of N-terminal proteomics provides a broadly applicable experimental platform to identify new proteolytic activities and define substrate specificity for diverse proteases. In a review by Luo et al. included in this special issue, the methodology, biological potential, and future for these approaches are discussed, highlighting the central importance of N-terminal proteomics in protease biology. Underscoring this importance, an original article by Gordon et al. utilizes N-terminal proteomics to define the proteolytic remodeling associated with ulcerative colitis, revealing new insights into the contributions of host and microbial proteases in the dysregulated proteolysis associated with this disease. Since proteolytic processing results in the formation of both new N- and C-termini, following the formation of new C-termini within the proteome offers an alternative approach to follow proteolytic events in the context of health and disease.

The conjugation and removal of small proteins such as SUMO through post-translational mechanisms have emerged as a critical determinant in dictating the biological activity of multiple cellular pathways. In a review included in this special issue, Jia et al. describe the contributions of SUMO specific proteases (SENPs) in regulating SUMOylation and its downstream functional outputs, specifically focusing on the importance of chemical biological tools and approaches in studying this class of specific proteases.

Some of the most significant contributions of chemical biology to the study of proteases have resulted from the development of compounds that target specific proteases. The development of these compounds has provided new opportunities to probe the specific contributions of proteases in complex biological systems and define the therapeutic potential for targeting different proteases in the context of health and disease. In this issue, we include articles applying different experimental approaches describing the establishment and/or implementation of inhibitors of both bacterial and mammalian proteases. Using a peptide substrate library, Babin et al. assess inhibitors of the E. coli AAA+ ATP-dependent protease Lon, identifying new substrates and compounds that can be employed to probe the activity of this important protease. Alternatively, using a more structure driven approach, Solania et al. describe the identification of selective cell-permeable inhibitors of caspase-3, establishing a new tool to probe the importance of this protease in cellular function. Furthermore, using a combination of activity-based protein profiling and proteomics, Griswold et al. demonstrate that catalytic activity of the serine peptidase DPP9 functions to inhibit the activity of the CARD8 inflammasome, revealing new insights into the regulation of cell death induced through this complex.

Apart from the direct targeting of proteases, chemical biological approaches that ‘hijack’ proteolytic mechanisms have emerged as a promising therapeutic approach to post-translationally influence the stability of proteins in the context of diverse types of disease. One such strategy used heterobifunctional compounds, termed proteolysis targeted chimeric compounds (PROTACs), that bind to both a target protein and E3 ligases to promote the ubiquitination and subsequent degradation of a protein of interest. However, only a few E3 ligases have been liganded, limiting the available toolbox for PROTAC development. In this issue, Ward et al. describe the application of activity-based protein profiling to identify compounds that bind to the E3 ligase RNF4 and are suitable for targeted degradation applications. This demonstrates the potential for implementing activity-based protein profiling strategies to expand the toolbox available for targeted protein degradation applications.

Mitochondrial proteostasis and function are regulated by a network of proteases localized throughout the different mitochondrial environments defined by the double membrane architecture. In two reviews included in this issue, the importance of mitochondrial proteases in dictating different aspects of mitochondrial function is highlighted. In a comprehensive review by Sam et al., the involvement of mitochondrial proteases in regulating mitochondrial phospholipid synthesis is discussed, revealing critical links between proteolytic mechanisms and the composition of cellular membranes. Extending this, Batterbsy et al. describe the importance of mitochondrial proteases in linking mitochondrial translation to stress-responsive alterations in mitochondrial morphology and function, including discussion of the evolutionary relationship between mitochondrial ribosomal synthesis and regulation of mitochondria. These articles, together, begin to highlight the opportunity for chemical biology to contribute to defining the importance of mitochondrial proteases in regulating diverse functional aspects of this important organelle.

One class of proteases that present a unique set of challenges is intramembrane proteases (IMPs), due to their localization within cellular membranes. However, despite this, IMPs represent a critical class of proteases involved in diverse biological functions. Beard et al. directly address these challenges in a review included in this issue, specifically emphasizing the contributions of chemical, biological tools, and approaches in deconvoluting the functional implications of IMPs in cellular regulation.

Ultimately, a goal of many chemical biologists studying proteases is to define new therapeutic opportunities to intervene in human diseases such as cancer through the pharmacologic targeting of specific proteases. Two articles included in this issue describe the importance of proteases in cancer, with a specific focus on how pharmacologic targeting of proteolytic mechanisms could influence disease progression. In a review by Boudreau et al., the importance of pro-caspase 3 in oncogenic transformation is discussed, highlighting the potential for activating this pro-apoptotic protease as a new approach to intervene in diverse types of cancer. Furthermore, Wong et al. describe the dependencies of many cancer cells on the mitochondrial AAA+ protease CLPP, indicating a new potential therapeutic opportunity to mitigate tumorigenesis through the pharmacologic targeting of CLPP.

While pharmacologic targeting of proteases and proteolytic pathways offers promise for therapeutic development, many challenges are associated with targeting these types of proteins for disease intervention. These include challenges in developing highly selective modulators of a specific protease and the difficulty in identifying specific proteases that can be modulated in diseased cells without significantly impacting healthy cells. In an original article by Henes et al., the authors describe other types of challenges associated with developing therapeutic protease inhibitors against HIV-1 protease. They show that the accumulation of mutations on sites distal from the protease active site can reduce the affinity of Darunavir for the HIV-1 protease and ultimately enhance drug resistance. This highlights important considerations for developing therapeutic strategies to target viral or bacterial proteases.

The scope and breadth of the articles included in this special issue highlight the wide-ranging impact of chemical biologists in the study of proteases. As we continue developing and implementing new approaches to probe protease biology, discoveries will continue to be made regarding this remarkable class of proteins and their impact on regulating cellular physiology across different organisms and different environments. Moreover, as we learn more about protease function and biology, we will identify new opportunities to therapeutically manipulate proteases to treat a host of diseases, with chemical biologists leading the way.

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