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Unraveling Proteases' Diverse Roles

Apr 14，2025

Proteases have been called biology’s version of Swiss army knives, able to cut long sequences of proteins into fragments. A protease is an enzyme that breaks the long, chainlike molecules of proteins so they can be digested. This process is called proteolysis, and it turns protein molecules into shorter fragments, called peptides, and eventually into their components, called amino acids. We need a steady supply of amino acids for proper growth and repair.

Proteases: Multifunctional Enzymes

Proteases are ubiquitous in biosystems where they have diverse roles in the biochemical, physiological, and regulatory aspects of cells and organisms. Proteases represent the largest segment of the industrial enzyme market where they are used in detergents, in food processing, in leather and fabric upgrading, as catalysts in organic synthesis, and as therapeutics. Microbial protease overproducing strains have been developed by conventional screening, mutation/selection strategies and genetic engineering, and wholly new enzymes, with altered specificity or stability, have been designed through techniques such as site-directed mutagenesis and directed evolution. Complete sequencing of the genomes of key Bacillus and Aspergillus workhorse extracellular enzyme producers and other species of interest has contributed to enhanced production yields of indigenous proteases as well as to production of heterologous proteases. With annual protease sales of about $1.5–1.8 billion, proteases account for 60% of the total enzyme market. Detergent proteases, with an annual market of about $1 billion account for the largest protease application segment. Subtilisin Carlsberg and related subtilisin serine proteases represent the first generation of detergent proteases with pH optima of 9–10.[1]

The second generation, having higher pH optima (10–11) and greater temperature stability, is produced from alkalophilic strains including Bacillusclausii and B. halodurans. The third generation consists of detergent proteases whose active sites and/or stability have been modified by protein engineering. The principal applications of proteases in food processing are in brewing, cereal mashing, and beer haze clarification, in the coagulation step in cheese making, in altering the viscoelastic properties of dough in baking and in production of protein hydrolysates. In organic synthesis, proteases have application in synthesis and/or hydrolysis of peptide, ester, and amide bonds involving carboxylic acids and are effective tools for resolution of pairs of enantiomers. Proteases have applications in nutrition as digestive aids and in therapy in thrombosis and cancer treatment. Hyperproteolytic endogenous activity may play significant roles in abnormal physiological functioning as well as in microbial and viral pathophysiological conditions and this has created substantial momentum for development of protease inhibitors as therapeutic agents against disease-causing proteases.

Multifunctional Enzymes in Life and Disease

Proteases likely arose at the earliest stages of protein evolution as simple destructive enzymes necessary for protein catabolism and the generation of amino acids in primitive organisms. For many years, studies on proteases focused on their original roles as blunt aggressors associated with protein demolition. However, the realization that, beyond these nonspecific degradative functions, proteases act as sharp scissors and catalyze highly specific reactions of proteolytic processing, producing new protein products, inaugurated a new era in protease research. The current success of research in this group of ancient enzymes derives mainly from the large collection of findings demonstrating their relevance in the control of multiple biological processes in all living organisms. Thus, proteases regulate the fate, localization, and activity of many proteins, modulate protein-protein interactions, create new bioactive molecules, contribute to the processing of cellular information, and generate, transduce, and amplify molecular signals. As a direct result of these multiple actions, proteases influence DNA replication and transcription, cell proliferation and differentiation, tissue morphogenesis and remodeling, heat shock and unfolded protein responses, angiogenesis, neurogenesis, ovulation, fertilization, wound repair, stem cell mobilization, hemostasis, blood coagulation, inflammation, immunity, autophagy, senescence, necrosis, and apoptosis.[2]

At the beginning of the post-genome era, a large body of information is available about the composition and organization of proteolytic systems in many living organisms. These global genomic views have revealed that the protease landscape is vast and quite unexplored. Therefore, it is very likely that the size of the different degradomes will grow in the near future, as new enzymes with unusual structural designs and catalytic mechanisms are identified and characterized. The recent finding of two novel and evolutionarily conserved cysteine proteases called UfSP1 and UfSP2 represents an example of experimental work that has led to the unmasking of “hidden proteases” that had remained invisible to homology-based screening methods. Many newly identified proteases remain as in silico predictions without experimental evidence for enzymatic activity. A major challenge for the future will be to demonstrate enzymatic properties for these predicted proteases. The comparative genomic studies have also provided interesting information about conservation, neofunctionalization, and subfunctionalization events in the protease field. Thus, the lineage-specific expansion of reproductive proteases in rodents may help to explain some of the pronounced reproductive differences between mammalian species, whereas changes in immune-related proteases may reflect evolutionary diversification of host defense mechanisms in response to new environmental conditions.

Proteases as therapeutics

Proteases are an expanding class of drugs that hold great promise. The U.S. FDA (Food and Drug Administration) has approved 12 protease therapies, and a number of next generation or completely new proteases are in clinical development. Although they are a well-recognized class of targets for inhibitors, they have not typically been considered as a drug class despite their application in the clinic over the last several decades; initially as plasma fractions and later as purified products. Although the predominant use of proteases has been in treating cardiovascular disease, they are also emerging as useful agents in the treatment of sepsis, digestive disorders, inflammation, cystic fibrosis, retinal disorders, psoriasis and other diseases. In the present review, we outline the history of proteases as therapeutics, provide an overview of their current clinical application, and describe several approaches to improve and expand their clinical application.[3]

Sequencing of the human genome revealed that more than 2% of our genes encode proteases, suggesting that these enzymes possess functions more complex than the simple digestive role that they are often assumed to play. For example, proteases regulate growth factors, cytokines, chemokines and cellular receptors, both through activation and inactivation leading to downstream intracellular signalling and gene regulation. For most proteases, is it unclear how many physiologically relevant substrates they have, how active a given protease is within particular tissues in the human body, and how these characteristics differ in disease. Up-regulation of proteolysis is associated commonly with different types of cancer and is linked to tumour metastasis, invasion and growth. Dysregulated proteolysis is also a feature of various inflammatory, and other, diseases. Prior to the use of high-throughput proteomic analyses it was assumed that most human proteases had thousands of substrates, but now a more accurate view is that most of them have 100 or fewer substrates, which are profoundly determined by spatial and temporal factors.

The therapeutic use of proteases over the past several decades has provided clinical results that clearly suggest a bright future for their expanded use. When administered in their active form, proteases can have a biological half-life on the scale of minutes and this can be extended by several hours using one of several approaches. Protease engineering has been, and will continue to be, used successfully to modify their properties. The therapeutic benefits of protease drugs need not solely arise from their primary proteolytic functions and they can be applied in situations where they are not normally involved. Importantly, proteases can be administered in conjunction with conventional small molecule therapies.