Current insights into protease dynamics in human epithelial disease and barrier function

Abstract

The fundamental importance of proteolytic enzymes to the biology of all living organisms is manifestly demonstrated by genomic analyses which demonstrate that genes encoding proteases and their inhibitors account for over 2 % of the coding sequences of most genomes sequenced to date. For example, over 500 protease genes have been annotated in the human genome. This extraordinary diversity reflects the wide range of biological functions performed by this family of enzymes: in addition to relatively non-specific degradative functions, characterized for example by digestive enzymes of the intestine, proteases also perform the selective and limited cleavage of a myriad of protein substrates which defines their subsequent biological functions and properties. In so doing, protease-dependent peptide bond hydrolysis ranks among the most significant of all posttranslational modification systems and underpins the processes of development, tissue homeostasis, turnover and remodelling, immune protection and healing. Given their central role in health, it is unsurprising that imbalances in protease activity are also intimately associated with both inherited and acquired human conditions. Proteases are generally classified on the basis of their mechanism of peptide bond cleavage. Four major families are well known. In the case of serine and cysteine enzymes, the hydroxyl and thiol residues respectively act as the nucleophilic groups in catalyzing hydrolytic peptide bond cleavage. Metalloenzymes, utilize a metal ion, frequently Zn, at their active site to activate a molecule of water to act as the nucleophile attack group. In aspartyl, sometimes referred to as acidic, proteases a similar mechanism of action to the metallo-enzymes is proposed in which a molecule of water is co-ordinated in the active site between two conserved aspartic acid residues, one of which causes activation of the water by removal of a proton and formation of the nucleophilic group required for peptide bond cleavage. In addition to these well known major catalytic classes, a number of additional mechanisms of peptide bond cleavage are now recognized leading to an expanded classification system which includes three additional classes. A subset of acidic proteases was originally identified based on their resistance to pepstatin, a broad spectrum inhibitor of proteases active at acidic pH. The unusual active sites of this pepstatin-insensitive subset proved difficult to characterise, but it is now recognized that some members of the subset were actually misclassified serine proteases, but with an acidic pH optimum, whilst the remainder represented a new class of protease containing glutamic acid/glutamine in their active site rather than aspartic acid. A further catalytic type of protease was identified following the elucidation of the crystallographic structure of the yeast proteasome which demonstrated the presence of an N-terminal threonine at the reactive centre fulfilling a similar nucleophilic role to the serine residue in the active sites of serine proteases. Once again this helped explain an inhibitor resistance phenomenon in that the proteasome was well known to be insensitive to inhibition by a wide range of standard protease inhibitors. The final and most recent addition to the list of classes is unusual in that members of this group are not proteases at all: they do not achieve peptide bond through hydrolysis, but instead act through the specific and unusual chemistry of asparagine. An asparagine residue at the active site is able to act as a nucleophile and cause autolytic cleavage of the peptide chain. In this instance the asparagine residue is destroyed and therefore the active site functions only once. The reader is referred to the MEROPS database (http://merops.sanger.ac.uk) which provides an excellent on M. A. Curtis :D. P. Kelsell (*) Blizard Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, Whitechapel, London E1 2AT, UK e-mail: d.p.kelsell@qmul.ac.uk