Judith S. Bond

Proteases: Multifunctional Enzymes in Life and Disease

Our view of proteases has come a long way since P. A. Levene reported his studies on “The Cleavage Products of Proteoses” in the first issue of The Journal of Biological Chemistry published October 1, 1905 (1). Today, after more than 100 years and 350,000 articles on these enzymes in the scientific literature, proteases remain at the cutting edge of biological research. 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 (2). 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 (3–11). 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. Consistent with these essential roles of proteases in cell behavior and survival and death of all organisms, alterations in proteolytic systems underlie multiple pathological conditions such as cancer, neurodegenerative disorders, and inflammatory and cardiovascular diseases. Accordingly, many proteases are a major focus of attention for the pharmaceutical industry as potential drug targets or as diagnostic and prognostic biomarkers (12). Proteases also play key roles in plants and contribute to the processing, maturation, or destruction of specific sets of proteins in response to developmental cues or to variations in environmental conditions (13). Likewise, many infectious microorganisms require proteases for replication or use proteases as virulence factors, which has facilitated the development of protease-targeted therapies for diseases of great relevance to human life such as AIDS (12). Finally, proteases are also important tools of the biotechnological industry because of their usefulness as biochemical reagents or in the manufacture of numerous products (e.g. Ref. 14). This outstanding diversity in protease functions directly results from the evolutionary invention of a multiplicity of enzymes that exhibit a variety of sizes and shapes. Thus, the architectural design of proteases ranges from small enzymes made up of simple catalytic units (∼20 kDa) to sophisticated protein-processing and degradation machines, like the proteasome and meprin metalloproteinase isoforms (0.7–6 MDa) (15). In terms of specificity, diversity is also a common rule. Thus, some proteases exhibit an exquisite specificity toward a unique peptide bond of a single protein (e.g. angiotensin-converting enzyme); however, most proteases are relatively nonspecific for substrates, and some are overtly promiscuous and target multiple substrates in an indiscriminate manner (e.g. proteinase K). Proteases also follow different strategies to establish their appropriate location in the cellular geography and, in most cases, operate in the context of complex networks comprising distinct proteases, substrates, cofactors, inhibitors, adaptors, receptors, and binding proteins, which provide an additional level of interest but also complexity to the study of proteolytic enzymes. This work aims at serving as a primer to a minireview series on proteases to be published in forthcoming issues of this Journal. This introductory article will focus on the discussion of the large and growing complexity of proteolytic enzymes present in all organisms, from bacteria to man. We will first show the results of comparative genomic analysis that have shed light on the real dimensions of the proteolytic space. The levels of protease complexity and mechanisms of protease regulation will then be addressed. Finally, we will discuss current frontiers and future perspectives in protease research.

Intracellular protein catabolism

Molecular Aspects of Proteinases and Inhibitors: The Metzincinsuperfamily of Zincpeptidases W. Bode, et al. Structure and Biosynthesis of Meprins P. Marchand, J.S. Bond Involvement of Tissue Inhibitor of Metalloproteinases (TIMPs) during Matrix Metalloproteinase Activation H. Nagase, et al. Protein Turnover in Health and Disease: Structural Aspects of Autophagy P.O. Seglen, et al. Mechanism of Autophagy in Permeabilized Hepatocytes M. Kadowaki, et al. Lysosomal Proteinolysis Based on Decreased Degradation of a Specific Protein, Mitochondrial ATP Synthetase Subunit C: Batten Disease J. Ezaki, et al. Functional Aspects of Proteolytic Systems: Endopeptidase 24.11 Neprilysin and Relatives: Twenty Years On A.J. Turner, et al. Function of Calpains: Possible Involvement in Myoblast Fusion M. Hayashi, et al. Ubiquitin and Proteasome Related Proteolysis: Protein and Gene Structures of 20S and 26S Proteasomes K. Tanaka, et al. The Proteasome and Protein Degradation in Yeast W. Hilt, et al. Pathological Aspects of Protein Turnover: Cellular Proteases Involved in the Pathogenicity of Human Immunodeficiency and Influenza Viruses H. Kido, et al. HIV Proteinase Mutations Leading to Reduced Inhibitor Susceptibility B. Korant, et al. 7 additional articles. Index.

Families of metalloendopeptidases and their relationships

Crystal structures available for four metalloendopeptidases have revealed zinc ligands for these enzymes. New sequence information has made it possible to compare the primary structures of the zinc‐binding site in metalloendopeptidases. A scheme based on the zinc‐binding site is proposed to classify metalloendopeptidases into five distinct families: thermolysin, astacin, serratia, matrixin, and snake venom metalloproteinases. Two histidines and one glutamate are zinc‐ligands in the thermolysin family. Three histidines and one tyrosine are zinc ligands in other four families, which are further distinguished by the identity of the residue following the third histidine and by the environment surrounding the tyrosine.

Meprin metalloprotease expression and regulation in kidney, intestine, urinary tract infections and cancer

Meprins are unique plasma membrane and secreted metalloproteinases that are highly regulated at the transcriptional and post‐translational levels. Meprin α and β subunits are abundantly expressed in kidney and intestinal epithelial cells, are secreted into the urinary tract and intestinal lumen, and are found in leukocytes and cancer cells under certain conditions. Their location and proteolytic activities indicate functions at the interface of the host and the external environment, and in trafficking of macrophages and metastases of cancer cells. These proteases can be detrimental when there is tissue damage or disruption, as in acute renal injury or intestinal inflammation, and there is evidence they are involved in movement of leukocytes and cancer cells to sites of infection or in metastasis, respectively.

Microbial-induced meprin β cleavage in MUC2 mucin and a functional CFTR channel are required to release anchored small intestinal mucus.

Significance Mucus with its major constituent, the gel-forming mucins, is important for protecting the host epithelium from bacteria. Under normal conditions, these mucin networks are constantly released into the small intestinal lumen. This release required a proteolytic cleavage in the mucin by the metalloprotease meprin β and was absent in germ-free animals but induced by bacteria. The small intestinal mucus in cystic fibrosis is also attached, not due to lack of enzyme, but rather that the mucin is not properly unfolded in the absence of a functional cystic fibrosis transmembrane conductance regulator channel and sufficient bicarbonate levels. Mucus can thus appear both attached and released as part of a system controlling bacterial removal. This new concept may lead to new ways for the treatment and therapy of cystic fibrosis. The mucus that covers and protects the epithelium of the intestine is built around its major structural component, the gel-forming MUC2 mucin. The gel-forming mucins have traditionally been assumed to be secreted as nonattached. The colon has a two-layered mucus system where the inner mucus is attached to the epithelium, whereas the small intestine normally has a nonattached mucus. However, the mucus of the small intestine of meprin β-deficient mice was now found to be attached. Meprin β is an endogenous zinc-dependent metalloprotease now shown to cleave the N-terminal region of the MUC2 mucin at two specific sites. When recombinant meprin β was added to the attached mucus of meprin β-deficient mice, the mucus was detached from the epithelium. Similar to meprin β-deficient mice, germ-free mice have attached mucus as they did not shed the membrane-anchored meprin β into the luminal mucus. The ileal mucus of cystic fibrosis (CF) mice with a nonfunctional cystic fibrosis transmembrane conductance regulator (CFTR) channel was recently shown to be attached to the epithelium. Addition of recombinant meprin β to CF mucus did not release the mucus, but further addition of bicarbonate rendered the CF mucus normal, suggesting that MUC2 unfolding exposed the meprin β cleavage sites. Mucus is thus secreted attached to the goblet cells and requires an enzyme, meprin β in the small intestine, to be detached and released into the intestinal lumen. This process regulates mucus properties, can be triggered by bacterial contact, and is nonfunctional in CF due to poor mucin unfolding.

The substrate degradome of meprin metalloproteases reveals an unexpected proteolytic link between meprin β and ADAM10

The in vivo roles of meprin metalloproteases in pathophysiological conditions remain elusive. Substrates define protease roles. Therefore, to identify natural substrates for human meprin α and β we employed TAILS (terminal amine isotopic labeling of substrates), a proteomics approach that enriches for N-terminal peptides of proteins and cleavage fragments. Of the 151 new extracellular substrates we identified, it was notable that ADAM10 (a disintegrin and metalloprotease domain-containing protein 10)—the constitutive α-secretase—is activated by meprin β through cleavage of the propeptide. To validate this cleavage event, we expressed recombinant proADAM10 and after preincubation with meprin β, this resulted in significantly elevated ADAM10 activity. Cellular expression in murine primary fibroblasts confirmed activation. Other novel substrates including extracellular matrix proteins, growth factors and inhibitors were validated by western analyses and enzyme activity assays with Edman sequencing confirming the exact cleavage sites identified by TAILS. Cleavages in vivo were confirmed by comparing wild-type and meprin−/− mice. Our finding of cystatin C, elafin and fetuin-A as substrates and natural inhibitors for meprins reveal new mechanisms in the regulation of protease activity important for understanding pathophysiological processes.

Deletion of the mouse meprin β metalloprotease gene diminishes the ability of leukocytes to disseminate through extracellular matrix

Meprins are metalloendopeptidases expressed by leukocytes in the lamina propria of the human inflamed bowel, that degrade extracellular matrix proteins in vitro implicating them in leukocyte transmigration events. The aims of these studies were to 1) examine the expression of meprins in the mouse mesenteric lymph node, 2) determine whether macrophages express meprins, and 3) determine whether deletion of the meprin β gene (Mep-1β) mitigated the ability of leukocytes to disseminate through extracellular matrix in vitro. These studies show that meprin α and β are expressed in leukocytes of the mouse mesenteric lymph node, and meprin α, but not β, decreased during intestinal inflammation. Deletion of Mep-1β gene decreased the ability of leukocytes to migrate through matrigel compared with wild-type leukocytes. Meprin β, but not α, was detected in cortical and medullary macrophages of the lymph node. Thus overall, meprin β is expressed by leukocytes in the draining lymph node of the intestine, regardless of the inflammatory status of the animal, and is likely to contribute to leukocyte transmigration events important to intestinal immune responses. Thus, the expression of meprins by leukocytes of the intestinal immune system may have important implications for diseases such as inflammatory bowel diseases, which are aggravated by leukocyte infiltration.

Metalloprotease Meprin β Generates Nontoxic N-terminal Amyloid Precursor Protein Fragments in Vivo

Identification of physiologically relevant substrates is still the most challenging part in protease research for understanding the biological activity of these enzymes. The zinc-dependent metalloprotease meprin β is known to be expressed in many tissues with functions in health and disease. Here, we demonstrate unique interactions between meprin β and the amyloid precursor protein (APP). Although APP is intensively studied as a ubiquitously expressed cell surface protein, which is involved in Alzheimer disease, its precise physiological role and relevance remain elusive. Based on a novel proteomics technique termed terminal amine isotopic labeling of substrates (TAILS), APP was identified as a substrate for meprin β. Processing of APP by meprin β was subsequently validated using in vitro and in vivo approaches. N-terminal APP fragments of about 11 and 20 kDa were found in human and mouse brain lysates but not in meprin β−/− mouse brain lysates. Although these APP fragments were in the range of those responsible for caspase-induced neurodegeneration, we did not detect cytotoxicity to primary neurons treated by these fragments. Our data demonstrate that meprin β is a physiologically relevant enzyme in APP processing.

Proteolysis and physiological regulation

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Prointerleukin-18 Is Activated by Meprin β in Vitro and in Vivo in Intestinal Inflammation

Interleukin-18 (IL-18), a pro-inflammatory cytokine, is a key factor in inflammatory bowel disease (IBD). Caspase-1 activates this cytokine, but other proteases are likely involved in maturation. Because meprin metalloproteinases have been implicated in IBD, the interaction of these proteases with proIL-18 was studied. The results demonstrate that the meprin β subunit of meprins A and B cleaves proIL-18 into a smaller 17-kDa product. The cleavage is at the Asn51–Asp52 bond, a site C-terminal to caspase-1 cleavage. The cleavage occurred in vitro with a Km of 1.3 μm and in Madin-Darby canine kidney cells transfected with meprin β when proIL-18 was added to the culture medium. The product of meprin B cleavage of proIL-18 activated NF-κB in EL-4 cells, indicating that it was biologically active. To determine the physiological significance of the interactions of meprins with proIL-18, an experimental model of IBD was produced by administering dextran sulfate sodium (DSS) to wild-type and meprin β knock-out (βKO) mice, and the serum levels of active IL-18 were determined. DSS-treated meprin βKO mice had lower levels of the active cytokine in the serum compared with wild-type mice. Furthermore, in meprin αKO mice, which express meprin β but not α, active IL-18 was elevated in the serum of DSS-treated mice compared with wild-type mice, indicating that the meprin isoforms have opposing effects on the IL-18 levels in vivo. This study identifies proIL-18 as a biologically important substrate for meprin β and implicates meprins in the modulation of inflammation.