https://www.cell.com/cell/pdf/0092-8674(88)90462-X.pdf

Immunochemical identification of the serine protease inhibitor α1-antichymotrypsin in the brain amyloid deposits of Alzheimer's disease

Cysteine proteases have traditionally been viewed as lysosomal mediators of terminal protein degradation. However, recent findings refute this limited view and suggest a more expanded role for cysteine proteases in human biology. Several newly discovered members of this enzyme class are regulated proteases with limited tissue expression, which implies specific roles in cellular physiology. These roles appear to include apoptosis, MHC class II immune responses, prohormone processing, and extracellular matrix remodeling important to bone development. The ability of macrophages and other cells to mobilize elastolytic cysteine proteases to their surfaces under specialized conditions may also lead to accelerated collagen and elastin degradation at sites of inflammation in diseases such as atherosclerosis and emphysema. The development of inhibitors of specific cysteine proteases promises to provide new drugs for modifying immunity, osteoporosis, and chronic inflammation.

http://www3.uah.es/farmamol/Public/AnnReviews/PDF/Physiology/cysteine_proteases.pdf?origin=publication_detail

Emerging roles for cysteine proteases in human biology

Kinetics of association of serine proteinases with native and oxidized alpha-1-proteinase inhibitor and alpha-1-antichymotrypsin.

Polymorphonuclear neutrophils are the first cells recruited to inflammatory sites and form the earliest line of defense against invading microorganisms. Neutrophil elastase, proteinase 3, and cathepsin G are three hematopoietic serine proteases stored in large quantities in neutrophil cytoplasmic azurophilic granules. They act in combination with reactive oxygen species to help degrade engulfed microorganisms inside phagolysosomes. These proteases are also externalized in an active form during neutrophil activation at inflammatory sites, thus contributing to the regulation of inflammatory and immune responses. As multifunctional proteases, they also play a regulatory role in noninfectious inflammatory diseases. Mutations in the ELA2/ELANE gene, encoding neutrophil elastase, are the cause of human congenital neutropenia. Neutrophil membrane-bound proteinase 3 serves as an autoantigen in Wegener granulomatosis, a systemic autoimmune vasculitis. All three proteases are affected by mutations of the gene (CTSC) encoding dipeptidyl peptidase I, a protease required for activation of their proform before storage in cytoplasmic granules. Mutations of CTSC cause Papillon-Lefevre syndrome. Because of their roles in host defense and disease, elastase, proteinase 3, and cathepsin G are of interest as potential therapeutic targets. In this review, we describe the physicochemical functions of these proteases, toward a goal of better delineating their role in human diseases and identifying new therapeutic strategies based on the modulation of their bioavailability and activity. We also describe how nonhuman primate experimental models could assist with testing the efficacy of proposed therapeutic strategies.

Neutrophil Elastase, Proteinase 3, and Cathepsin G as Therapeutic Targets in Human Diseases

Colonization or emergence of microbial pathogens may result in tissue destruction by activation of one or more of five distinct host degradative pathways (matrix metalloproteinase pathway, plasminogen-dependent pathway, phagocytic pathway, PMN-serine proteinase pathway and osteoclastic bone resorption) or by direct cleavage of extracellular matrix constituents by microbial proteinases. Activation of endogenous destructive pathways may be mediated by immune responses resulting in expression of degradative cellular phenotypes among both immigrant and resident cell populations. In addition, expression of degradative phenotypes may be triggered by direct influences on host cells of microbial products (LPS, enzymes, toxins). A body of evidence suggests that each of these mechanisms involves local production of proinflammatory cytokines and growth factors. The matrix metalloproteinase pathway is centrally involved in dissolution of all unmineralized connective tissues and perhaps in resorption of bone as well. The matrix metalloproteinase family consists of nine or more genetically distinct Zn++ endopeptidases which collectively cleave all of the constituents of the extracellular matrix. Recent studies have uncovered many essential elements of a complex, but still incomplete, regulatory network that governs tissue destruction. Proinflammatory cytokines and growth factors induce signalling pathways several of which are dependent on protein kinase C and result in transient expression of the transcription factors c-jun and c-fos. Initiation of transcription of most matrix metalloproteinase genes requires binding of the transcription factor AP-1 (c-jun/c-fos) to a specific promoter sequence but attainment of maximal transcription rates is dependent on interaction with other promoter elements as well. Several matrix metalloproteinases have been detected in crevicular fluids and tissues of inflamed human gingiva as have the proinflammatory cytokines (IL-1 and TNF-alpha) which regulate their transcription. Although the mere presence of enzymes and cytokines does not necessarily impart function per se, these observations suggest that some level of spatial or temporal linkage exists between metalloproteinase/cytokine expression and gingival inflammation.

Role of cytokines and inflammatory mediators in tissue destruction

Human granulocytic elastases have been purified by a two-step procedure involving affinity chromatography of crude extracts of leukocytic granules on Sepharose-Trasylol, followed by ion-exchange chromatography on CM-cellulose to resolve the isoelastases. All of these enzymes were found to be glycoproteins with the carbohydrate content of the major form being composed essentially of only neutral sugars. The molecular weight of this form was found to be near 30 000 daltons with the other forms being slightly higher. Preliminary structural analyses indicate that all of the elastase isozymes have identical NH2-terminal sequences suggesting that the differences in mobility of the four proteins are not due to different degrees of activation from a common zymogen but, more likely, from minor changes in carbohydrate content. Human granulocytic elastases are less active on ligament elastin than porcine pancreatic elastase, but both are inhibited by synthetic elastase active-site directed low molecular weight compounds (Tuhy, P. M., and Powers, J. C. (1975), FEBS Lett. 50, 359) as well as by plasma alpha-1-proteinase inhibitor (formerly called alpha-1-antitrypsin). In the latter case a stable complex with mol wt of 78 000 daltons is formed indicating the formation of a 1:1 complex.

Human leukocyte granule elastase: rapid isolation and characterization

The interaction of human plasma alpha-1-antichymotrypsin with serine proteinases from different tissues has been investigated. The protein was found to form stable complexes with pancreatic chymotrypsin, leukocyte cathepsin G, and mast cell chymotrypsin. No inhibition of pancreatic trypsin or leukocyte elastase could be demonstrated. With mixtures containing both alpha-1-antichymotrypsin and alpha-1-proteinase inhibitor, it was found that the former preferentially inactivated leukocyte cathepsin G, while the latter showed a strong preference for pancreatic chymotrypsin. However, leukocyte elastase was specifically inactivated by alpha-1-proteinase inhibitor even in 1:1 mixtures with chymotrypsin. All of these results taken together suggest that one of the primary functions of alpha-1-antichymotrypsin is to inactivate leukocyte cathepsin G, while alpha-1-proteinase inhibitor controls the activity of other serine proteinases, particularly leukocyte elastase.

Human alpha-1-antichymotrypsin: interaction with chymotrypsin-like proteinases.

Two of the major enzymes present in an released from neutrophil granulocytes are the endoproteinases elastase and cathepsin G. While the former is believed to be one of the major causative agents responsible for tissue destruction in emphysema and rheumatoid arthritis, little is known about the function of cathepsin G. We have recently developed simple procedures for isolating the isoenzymes of each type of proteinase as well as for their specific controlling plasma inhibitors. We have also prepared synthetic substrates and inhibitor analogues. Some sequence studies have been initiated and the results indicate homology of these enzymes not only with each other and with the pancreatic proteinases but also between cathepsin G and proteolytic enzymes present in muscle and mast cell tissue. Significantly, both types of enzyme can degrade the structural protein myosin, as well as elastin and proteoglycan. However, their relative importance in muscle protein turnover or muscle disease has not yet been clarified.

Robert J. Baugh

Papers

Human leukocyte granule elastase: rapid isolation and characterization

Human alpha-1-antichymotrypsin: interaction with chymotrypsin-like proteinases.

Human leucocyte elastase and cathepsin G: structural and functional characteristics.