Proteolysis to Identify Protease Substrates: Cleave to Decipher

Proteolysis is an irreversible post‐translational modification process, characterized by highly precise yet stable cleavage of proteins. Downstream events in signaling processes are reliant on proteolysis triggered by the protease activity. Studies indicate that abnormal proteolytic activity may lead to the manifestation of diseased conditions. Therefore, characterization of proteases may provide clues to understand their role in fundamental cellular processes like cellular growth, differentiation, apoptosis, and survival. The relevance of proteases and their substrates as clinical targets are being studied. Understanding the mechanism of proteolytic activity, the identity, and the role of repertoire of its substrates in a physiological pathway has opened avenues for novel drug designing. However, only a limited knowledge of protease substrates is currently available. In this review, the authors recapitulate the library screening, proteomics, and bioinformatics based approaches that have been employed for the identification of protease substrates.     

Profiling protease activities by dynamic proteomics workflows

Proteases play prominent roles in many physiological processes and the pathogenesis of various diseases, which makes them interesting drug targets. To fully understand the functional role of proteases in these processes, it is necessary to characterize the target specificity of the enzymes, identify endogenous substrates and cleavage products as well as protease activators and inhibitors. The complexity of these proteolytic networks presents a considerable analytic challenge. To comprehensively characterize these systems, quantitative methods that capture the spatial and temporal distributions of the network members are needed. Recently, activity-based workflows have come to the forefront to tackle the dynamic aspects of proteolytic processing networks in vitro, ex vivo and in vivo. In this review, we will discuss how mass spectrometry-based approaches can be used to gain new insights into protease biology by determining substrate specificities, profiling the activity-states of proteases, monitoring proteolysis in vivo, measuring reaction kinetics and defining in vitro and in vivo proteolytic events. In addition, examples of future aspects of protease research that go beyond mass spectrometry-based applications are given.     

Proteases in autophagy

Autophagy is a process involved in the proteolytic degradation of cellular macromolecules in lysosomes, which requires the activity of proteases, enzymes that hydrolyse peptide bonds and play a critical role in the initiation and execution of autophagy. Importantly, proteases also inhibit autophagy in certain cases. The initial steps of macroautophagy depend on the proteolytic processing of a particular protein, Atg8, by a cysteine protease, Atg4. This processing step is essential for conjugation of Atg8 with phosphatidylethanolamine and, subsequently, autophagosome formation. Lysosomal hydrolases, known as cathepsins, can be divided into several groups based on the catalitic residue in the active site, namely, cysteine, serine and aspartic cathepsins, which catalyse the cleavage of peptide bonds of autophagy substrates and, together with other factors, dispose of the autophagic flux. Whilst most cathepsins degrade autophagosomal content, some, such as cathepsin L, also degrade lysosomal membrane components, GABARAP-II and LC3-II. In contrast, cathepsin A, a serine protease, is involved in inhibition of chaperon-mediated autophagy through proteolytic processing of LAMP-2A. In addition, other families of calcium-dependent non-lysosomal cysteine proteases, such as calpains, and cysteine aspartate-specific proteases, such as caspases, may cleave autophagy-related proteins, negatively influencing the execution of autophagic processes. Here we discuss the current state of knowledge concerning protein degradation by autophagy and outline the role of proteases in autophagic processes. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.     

Proteases in autophagy

Autophagy is a process involved in the proteolytic degradation of cellular macromolecules in lysosomes, which requires the activity of proteases, enzymes that hydrolyse peptide bonds and play a critical role in the initiation and execution of autophagy. Importantly, proteases also inhibit autophagy in certain cases. The initial steps of macroautophagy depend on the proteolytic processing of a particular protein, Atg8, by a cysteine protease, Atg4. This processing step is essential for conjugation of Atg8 with phosphatidylethanolamine and, subsequently, autophagosome formation. Lysosomal hydrolases, known as cathepsins, can be divided into several groups based on the catalitic residue in the active site, namely, cysteine, serine and aspartic cathepsins, which catalyse the cleavage of peptide bonds of autophagy substrates and, together with other factors, dispose of the autophagic flux. Whilst most cathepsins degrade autophagosomal content, some, such as cathepsin L, also degrade lysosomal membrane components, GABARAP-II and LC3-II. In contrast, cathepsin A, a serine protease, is involved in inhibition of chaperon-mediated autophagy through proteolytic processing of LAMP-2A. In addition, other families of calcium-dependent non-lysosomal cysteine proteases, such as calpains, and cysteine aspartate-specific proteases, such as caspases, may cleave autophagy-related proteins, negatively influencing the execution of autophagic processes. Here we discuss the current state of knowledge concerning protein degradation by autophagy and outline the role of proteases in autophagic processes. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.     

Proteases released from Xenopus laevis eggs at activation and their role in envelope conversion

During fertilization of the Xenopus laevis egg, the egg envelope is converted so that further sperm contact with the egg is prevented. In this study two envelope conversion reactions were investigated, envelope hardening and limited hydrolysis of two structurally related envelope glycoproteins. Both of these reactions were shown to be sensitive to protease inhibitors. In an attempt to identify egg proteases involved in envelope conversion, the medium around activated dejellied eggs was collected and analyzed. The exudate was able to convert isolated envelopes and, when the exudate was analyzed using peptide substrates, two major activities were found, one with a preference for cleavage after argininyl peptide bonds and one with a preference for phenylalaninyl peptide bonds. Analysis of exudate using SDS-polyacrylamide gel electrophoresis with gelatin cast into the gel showed two bands of proteolytic activity, one at Mr 45,000 that was identified as the trypsin-like activity and one at Mr 30,000 that was identified as the chymotrypsin-like activity. When cortical granule exocytosis was suppressed using ammonium chloride, release of the two exudate proteases was also suppressed. Studies of the envelope conversion reactions using protease inhibitors indicated that the chymotrysin-like protease was involved in envelope conversion once it had been activated by the trypsin-like protease.     

Discovery of chemokine substrates for matrix metalloproteinases by exosite scanning: a new tool for degradomics

Increasingly it is being recognized that matrix metalloproteinases (MMPs) are important processing enzymes that regulate cellular behaviour and immune cell function by selective proteolysis of cell surface receptors and adhesion molecules, cytokines and growth factors. These functions will likely prove to be as important in vivo as the proposed roles of MMPs in pathological matrix degradation. To screen for new protease substrates we have reported a novel 'exosite scanning' strategy that utilizes protease substrate-binding exosite domains as yeast two-hybrid baits. We discovered that the chemokine monocyte chemoattractant protein-3 (MCP-3) binds the hemopexin C domain of gelatinase A (MMP-2) leading to its efficient cleavage, converting an agonist to a potent receptor antagonist. We have now found that other MMPs cleave MCP-1, MCP-2, MCP-3, MCP-4, SDF-lalpha and SDF-1beta indicating that the intersection between the chemokine and MMP families is broad with important implications for the control of inflammatory and immune processes. Use of engineered substrates with altered exosite binding affinities further revealed the power of exosites in dictating proteolytic specificity - either directing cleavage of non-preferred sites or in other cases virtually eliminating proteolysis of readily accessible scissile bonds. Hence, bioinformatic searches for protease substrates based on scissile bond preference will only reveal a subset of substrates unless the influence of exosites is considered.     

Consensus statement understanding health and malnutrition through a systems approach: the ENOUGH program for early life

Nutrition research, like most biomedical disciplines, adopted and often uses experimental approaches based on Beadle and Tatum’s one gene—one polypeptide hypothesis, thereby reducing biological processes to single reactions or pathways. Systems thinking is needed to understand the complexity of health and disease processes requiring measurements of physiological processes, as well as environmental and social factors, which may alter the expression of genetic information. Analysis of physiological processes with omics technologies to assess systems’ responses has only become available over the past decade and remains costly. Studies of environmental and social conditions known to alter health are often not connected to biomedical research. While these facts are widely accepted, developing and conducting comprehensive research programs for health are often beyond financial and human resources of single research groups. We propose a new research program on essential nutrients for optimal underpinning of growth and health (ENOUGH) that will use systems approaches with more comprehensive measurements and biostatistical analysis of the many biological and environmental factors that influence undernutrition. Creating a knowledge base for nutrition and health is a necessary first step toward developing solutions targeted to different populations in diverse social and physical environments for the two billion undernourished people in developed and developing economies.     

Effect of nutritional conditions on extracellular protease production by the haloalkaliphilic archaeon Natrialba magadii

AIMS: The effect of various nitrogen sources and nutritional starvation was examined on the production of an extracellular protease secreted by the haloalkaliphilic archaeon Natrialba magadii. METHODS AND RESULTS: Cell growth and proteolytic activity were measured in cells grown with different nitrogen sources. Proteolytic activity was produced in complex and easily metabolized nitrogen sources such as yeast extract, casein and casamino acids; meanwhile, ammonium repressed enzyme production. The time course and amount of protease accumulated showed an inverse correlation with growth rate and nutrient concentration. Starvation did not induce extracellular protease production. CONCLUSION: The accumulation of Nab. magadii extracellular protease is stimulated by nutrient limitation and slow growth rate indicating that it is probably induced in response to a deficit in the energetic status of the cells. Nutritional starvation did not induce protease accumulation suggesting that de novo synthesis of this protease and/or factor/s necessary for its activation are required. This enzyme may be regulated by nitrogen catabolite repression and it does not require protein substrates for induction. SIGNIFICANCE AND IMPACT OF THE STUDY: These results contribute to the basic knowledge on protease regulation in haloalkaliphilic archaea and will help to optimize the production of this extremozyme for biotechnological applications such as protease-catalysed peptide synthesis.     

Influence of enzymatic specificity on the behavior of ephemeral gels

Ephemeral gels, called Enzgels, successively undergo sol-gel and then gel-sol transition under the action of two antagonistic enzymes, transglutaminase and protease. Molecular and macroscopic properties of Enzgels are directly dependent on the enzymatic activities and their ratios. This work studies the characteristics of Enzgels according to the specificity of three different proteases: thermolysin, trypsin, and collagenase. The experiments are conducted using three types of gelatin networks, one created only by triple helices, one only by covalent bonds, and the last network by both triple helices and covalent bonds. Rheology and polarimetry measurements show that the evolution of Enzgels is directly dependent on the specificity of the protease used. Moreover, gelatin network conformation has different influences according to this proteolytic specificity. Collagenase is not very sensitive to gelatin conformation, whereas trypsin is very limited by the presence of covalent bonds. This study considerably expands the knowledge of Enzgel properties.     

Structure of the pig pancreatic GP-2: role of intramolecular disulfides in the resistance to proteolysis

GP-2 is the major membrane glycoprotein characteristic of the pancreatic zymogen granule membrane. When granules are lysed in the presence of DTT, GP-2 becomes completely and specifically degraded. This proteolysis was reproducible with the same characteristics in the purified granule membrane. The protease was purified from this source using hydrophobic interaction chromatography. The proteolytic activity was identified as a 29-kDa protein because, in a reconstituted system containing both the purified GP-2 and the 29-kDa protein, the proteolytic degradation of GP-2 was sensitive to the same spectrum and concentrations of inhibitors or reducing agents as in the membrane. The activity was characteristic of a serine protease. It was also shown that GP-2 only becomes sensitive to proteolytic digestion when its disulfide bonds are reduced, and that DTT does not activate the protease. Seven intramolecular disulfide bonds were identified on GP-2. All of them are located in a 65-kDa tryptic fragment that is very resistant to exogenous proteases under nonreducing conditions. Because of the quite specific degradation of GP-2 under reducing conditions, we believe that the 29-kDa protease must be closely associated with GP-2 on the membrane. This protease could be responsible, in part, for the solubilization of the GP-2 from the membrane into the zymogen granule content and its resulting secretion by the pancreas.     

ATP-dependent Clp Proteases in Photosynthetic Organisms— A Cut Above the Rest!

Proteases are critical regulatory factors for many metabolic cellular processes as well as being vital for degrading proteins damaged during environmental stresses. Many of those responsible for targeted protein degradation require the hydrolysis of ATP, and one class that has attracted much attention recently are the Clp proteases. They are among the best characterized proteases to date, and were the first shown to rely on an ATPase regulatory subunit possessing molecular chaperone activity, which functions both within the proteolytic complex and independently. A range of Clp proteins has been identified from many different bacteria and eukaryotes, with by far the greatest number and diversity of forms in oxygenic photobionts such as cyanobacteria and higher plants. Functionally, Clp proteins have also evolved into one of the more critical proteolytic enzymes within photobionts, and it is now somewhat of a paradox that we currently know least about Clp protease functions in the photosynthetic organisms, where they have their most important roles. This discrepancy is now being addressed, with studies on Clp protein in cyanobacteria and, in an increasing number, in higher plants.     

Proteolytic activities and proteases of plant chloroplasts

A concise overview on the current knowledge of the proteolytic activities in chloroplasts is presented, with an emphasis on the proteolytic events associated with thylakoid membranes. The Dl reaction centre protein of photosystem II undergoes rapid light-dependent turnover and chlorophyll a/b -binding proteins are effectively degraded upon acclimation of plants to higher irradiances. Insights into the partially characterized proteolytic systems in each case will be presented, but the proteases involved still remain unknown. It can be envisaged, however, that the proteolysis is probably an as highly regulated phenomenon as the various steps during biosynthesis of the photosynthetic multiprotein complexes. From the protease point of view, more progress has recently been made in characterization of processing proteases involved in protein import into chloroplasts and in C-terminal processing of the Dl protein. Moreover, there are an increasing number of proteases in chloroplasts which have been discovered and identified as bacterial homologues. These include a Clp-type protease, a homologue of the bacterial protease FtsH and the cyanobacterial PcrA protease, all of which have a specific location in the chloroplast but their definite physiological substrates are still missing. Attempts are made to bring together the recent progress in the identification of proteases and characterisation of proteolytic events in chloroplasts.     

Pituitary endopeptidases

This review summarizes our knowledge of pituitary endopeptidases. Emphasis has been placed on well-characterized enzymes and their potential roles in proteolytic processes of the pituitary. Because of space limitations, degradation of biologically active peptide by crude preparations has generally not been discussed. Only a few proteolytic enzymes are at present adequately characterized, and knowledge of their physiological function in vivo is insufficient. Among the many functions of proteolytic enzymes, those that are specific for the pituitary as an endocrine gland are of primary interest. Such functions include inactivation of neuropeptides and factors that control the secretory function of the pituitary, processing of precursors destined for secretion, selective cleavage of prohormones into active fragments, and degradation of inactive fragments. While some of the enzymes described here, such as cathepsin D, could be expected to have primarily a degradative function, others could potentially be involved in hormonal metabolism, since they exhibit trypsin-like, chymotrypsin-like, and dipeptidyl carboxypeptidase-like activities, all potentially useful in hormonal conversions. Data suggestive of the presence in the pituitary of enzymes involved in removal of the 'signal sequence', and enzymes involved in hormone processing by cleavage of bonds after a pair of basic residues and in the subsequent removal of these residues by a carboxypeptidase B-like activity have been published. None of these enzymes, however, has been isolated or purified to a degree that would allow determination of its specificity, mechanisms of action, physicochemical properties, and susceptibility to specific inhibitors. Questions that remain unresolved ask whether differences in the processing pathways in various anatomical parts of the pituitary are due to the presence of proteases with different specificities, or to different disposition of these enzymes, and factors, such as conformation of the substrate and its secondary modification, for example by glycosylation or phosphorylation. Proof of a functional involvement of a protease in hormonal processing should include demonstration that inhibition of activity results in inhibition of processing in the intact cell. Specific inhibitors of processing enzymes could potentially be used to modulate pituitary function, and thus have pharmacological interest. Although there are few answers to the above problems at present, the questions are well defined, and it can be expected that the rapidly expanding research on pituitary proteases will soon provide some of the answers.     

Ulp1-SUMO crystal structure and genetic analysis reveal conserved interactions and a regulatory element essential for cell growth in yeast

Modification of cellular proteins by the ubiquitin-like protein SUMO is essential for nuclear processes and cell cycle progression in yeast. The Ulp1 protease catalyzes two essential functions in the SUMO pathway: (1) processing of full-length SUMO to its mature form and (2) deconjugation of SUMO from targeted proteins. Selective reduction of the proteolytic reaction produced a covalent thiohemiacetal transition state complex between a Ulp1 C-terminal fragment and its cellular substrate Smt3, the yeast SUMO homolog. The Ulp1-Smt3 crystal structure and functional testing of elements within the conserved interface elucidate determinants of SUMO recognition, processing, and deconjugation. Genetic analysis guided by the structure further reveals a regulatory element N-terminal to the proteolytic domain that is required for cell growth in yeast.     

Local proteolytic activity in tumor cell invasion and metastasis

Proteolytic cleavage of extracellular matrix (ECM) is a critical regulator of many physiological and pathological events. It affects fundamental processes such as cell growth, differentiation, apoptosis and migration. Most proteases are produced as inactive proenzymes that undergo proteolytic cleavage for activation. Proteolytic activity is additionally modified by endogenous inhibitors. Mechanisms that localize and concentrate protease activity in the pericellular microenvironment of cells are prerequisites for processes like angiogenesis, bone development, inflammation and tumor cell invasion. Methods that enable real-time, high-resolution imaging and precise quantification of local proteolytic activity in vitro and in vivo remain major challenges. These methods will play an important role in the understanding of basic principles e.g. in cancer cell invasion, the identification of new therapeutical targets and hence drug design. This review highlights mechanisms and functions of local proteolytic activity with special emphasis on tumor cell invasion and metastasis, and focuses on techniques for the investigation of this process.     

The primary structure and structural characteristics of Achromobacter lyticus protease I, a lysine-specific serine protease

The complete amino acid sequence of Achromobacter lyticus protease I (EC 3.4.21.50), which specifically hydrolyzes lysyl peptide bonds, has been established. This has been achieved by sequence analysis of the reduced and S-carboxymethylated protease and of peptides obtained by enzymatic digestion with Achromobacter protease I itself and Staphylococcus aureus V8 protease and by chemical cleavage with cyanogen bromide. The protease consists of 268 residues with three disulfide bonds, which have been assigned to Cys6-Cys216, Cys12-Cys80, and Cys36-Cys58. Comparison of the amino acid sequence of Achromobacter protease and other serine proteases of bacterial and mammalian origins has revealed that Achromobacter protease I is a mammalian-type serine protease of which the catalytic triad comprises His57, Asp113, and Ser194. It has also been shown that the protease has 9- and 26-residue extensions of the peptide chain at the N and C termini, respectively, and overall sequence homology is as low as 20% with bovine trypsin. The presence of a disulfide bridge between the N-terminal extension Cys6 and Cys216 close to the putative active site in the C-terminal region is thought to be responsible for the generation of maximal proteolytic function in the pH range 8.5-10.7 and enhanced stability to denaturation.     

The matriptase-prostasin proteolytic cascade in epithelial development and pathology

The type II transmembrane serine protease matriptase has an essential role in the integrity and function of multiple epithelial tissues. In the epidermis, matriptase activates the glycosylphosphatidylinositol (GPI) anchored membrane serine protease prostasin to initiate a proteolytic cascade that is required for the development of the stratum corneum barrier function. Accordingly, mice deficient for matriptase phenocopy mice deficient for epidermal prostasin and present with impaired corneocyte differentiation, imparied lipid matrix formation, loss of profilaggrin processing and loss of tight junction formation and function. Together, these defects lead to a compromised epidermal barrier and result in fatal dehydration during the neonatal period. Proteolytic activity of the matriptase-prostasin cascade is regulated in the epidermis via inhibition by the Kunitz-type serine protease inhibitor hepatocyte growth factor activator inhibitor-1 (HAI-1). Importantly, targeted post-natal ablation of matriptase in mice perturbs the function of multiple adult tissues, indicating an ongoing requirement for matriptase proteolysis in the maintenance of diverse types of epithelia. Impaired matriptase proteolytic activity has been linked to human Autosomal Recessive Icthyosis with Hypotrichosis (ARIH), whereas aberrant matriptase activity has been implicated in Netherton’s Syndrome. This review will summarize information pertaining to the role of matriptase in epithelial biology and will discuss recent advancements in our understanding of how matriptase activity is regulated and the down-stream effectors of matriptase proteolysis.     

Proteolytic enzymes, past and present

William Beaumont's pioneering research on gastric secretion has been germinal in the discovery of proteolytic enzymes and the elucidation of their chemical structure, physiological roles, and biochemical evolution. Although the mammalian digestive enzymes, notably those of gastric and pancreatic origin, have been among the best characterized, of even greater interest and complexity are those that fulfill regulatory functions by limiting their action on specific peptide bonds in target protein substrates. The difference between digestive and regulatory proteases can best be understood by considering their evolutionary relationships on the basis of the organization of both their genes and the proteins themselves. An analysis of representative members of protease families, notably the mammalian serine proteases, suggests that they are the products of processes of recombination of gene segments that give rise to functionally and structurally distinct domains. The evolutionary variability introduced by combinations of domains appears to be far more restricted than if each protein molecule were the product of a single and unique evolutionary event.