Gel-based Protease Proteomics for Identifying the Novel Calpain Substrates in Dopaminergic Neuronal Cell

Calpains are a family of calcium-dependent cysteine proteases that are ubiquitously expressed in mammals and play critical roles in neuronal death by catalyzing substrate proteolysis. Here, we developed two-dimensional gel electrophoresis-based protease proteomics to identify putative calpain substrates. To accomplish this, cellular lysates from neuronal cells were first separated by pI, and the immobilized sample on a gel strip was incubated with a recombinant calpain and separated by molecular weight. Among 25 altered protein spots that were differentially expressed by at least 2-fold, we confirmed that arsenical pump-driving ATPase, optineurin, and peripherin were cleaved by calpain using in vitro and in vivo cleavage assays. Furthermore, we found that all of these substrates were cleaved in MN9D cells treated with either ionomycin or 1-methyl-4-phenylpyridinium, both of which cause a calcium-mediated calpain activation. Their cleavage was blocked by calcium chelator or calpain inhibitors. In addition, calpain-mediated cleavage of these substrates and its inhibition by calpeptin were confirmed in a middle cerebral artery occlusion model of cerebral ischemia, as well as a stereotaxic brain injection model of Parkinson disease. Transient overexpression of each protein was shown to attenuate 1-methyl-4-phenylpyridinium-induced cell death, indicating that these substrates may confer protection of varying magnitudes against dopaminergic injury. Taken together, the data indicate that our protease proteomic method has the potential to be applicable for identifying proteolytic substrates affected by diverse proteases. Moreover, the results described here will help us decipher the molecular mechanisms underlying the progression of neurodegenerative disorders where protease activation is critically involved.     

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.     

N- and C-terminal degradomics: new approaches to reveal biological roles for plant proteases from substrate identification

Proteolysis is an irreversible post-translational modification that regulates many intra- and intercellular processes, including essential go/no-go decisions during cell proliferation, development and cell death. Hundreds of protease-coding genes have been identified in plants, but few have been linked to specific substrates. Conversely, proteolytic processes are frequently observed in plant biology but rarely have they been ascribed to specific proteases. In mammalian systems, unbiased system-wide proteomics analyses of protease activities have recently been tremendously successful in the identification of protease substrate repertoires, also known as substrate degradomes. Knowledge of the substrate degradome is key to understand the role of proteases in vivo. Quantitative shotgun proteomic studies have been successful in identifying protease substrates, but while simple to perform they are biased toward abundant proteins and do not reveal precise cleavage sites. Current degradomics techniques overcome these limitations by focusing on the information-rich amino- and carboxy-terminal peptides of the original mature proteins and the protease-generated neo-termini. Targeted quantitative analysis of protein termini identifies precise cleavage sites in protease substrates with exquisite sensitivity and dynamic range in in vitro and in vivo systems. This review provides an overview of state-of-the-art methods for enrichment of protein terminal peptides, and their application to protease research. These emerging degradomics techniques promise to clarify the elusive biological roles of proteases and proteolysis in plants.     

Evaluation of a D-amino-acid-containing fluorescence resonance energy transfer peptide library for profiling prokaryotic proteases

Bacterial proteases play an important role in a broad spectrum of processes, including colonization, proliferation, and virulence. In this respect, bacterial proteases are potential biomarkers for bacterial diagnosis and targets for novel therapeutic protease inhibitors. To investigate these potential functions, the authors designed and used a protease substrate fluorescence resonance energy transfer (FRET) library comprising 115 short d- and l-amino-acid-containing fluorogenic substrates as a tool to generate proteolytic profiles for a wide range of bacteria. Bacterial specificity of the d-amino acid substrates was confirmed using enzymes isolated from both eukaryotic and prokaryotic organisms. Interestingly, bacterial proteases that are known to be involved in housekeeping and nutrition, but not in virulence, were able to degrade substrates in which a d-amino acid was present. Using our FRET peptide library and culture supernatants from a total of 60 different bacterial species revealed novel, bacteria-specific, proteolytic profiles, although in-species variation was observed for Pseudomonas aeruginosa, Porphyromonas gingivalis, and Staphylococcus aureus. Overall, the specific characteristic of our substrate peptide library makes it a rapid tool to high-throughput screen for novel substrates to detect bacterial proteolytic activity.     

Towards the control of intracellular protein turnover: mitochondrial Lon protease inhibitors versus proteasome inhibitors

Cellular protein homeostasis results from the combination of protein biogenesis processes and protein quality control mechanisms, which contribute to the functional state of cells under normal and stress conditions. Proteolysis constitutes the final step by which short-lived, misfolded and damaged intracellular proteins are eliminated. Protein turnover and oxidatively modified protein degradation are mainly achieved by the proteasome in the cytosol and nucleus of eukaryotic cells while several ATP-dependent proteases including the matrix protease Lon take part in the mitochondrial protein degradation. Moreover, Lon protease seems to play a major role in the elimination of oxidatively modified proteins in the mitochondrial matrix. Specific inhibitors are commonly used to assess cellular functions of proteolytic systems as well as to identify their protein substrates. Here, we present and discuss known proteasome and Lon protease inhibitors. To date, very few inhibitors of Lon have been described and no specific inhibitors of this protease are available. The current knowledge on both catalytic mechanisms and inhibitors of these two proteases is first described and attempts to define specific non-peptidic inhibitors of the human Lon protease are presented.     

Proteomic techniques and activity-based probes for the system-wide study of proteolysis

Proteolysis constitutes a major post-translational modification but specificity and substrate selectivity of numerous proteases have remained elusive. In this review, we highlight how advanced techniques in the areas of proteomics and activity-based probes can be used to investigate i) protease active site specificity; ii) protease in vivo substrates; iii) protease contribution to proteome homeostasis and composition; and iv) detection and localization of active proteases. Peptide libraries together with genetical or biochemical selection have traditionally been used for active site profiling of proteases. These are now complemented by proteome-derived peptide libraries that simultaneously determine prime and non-prime specificity and characterize subsite cooperativity. Cell-contextual discovery of protease substrates is rendered possible by techniques that isolate and quantitate protein termini. Here, a novel approach termed Terminal Amine Isotopic Labeling of Substrates (TAILS) provides an integrated platform for substrate discovery and appropriate statistical evaluation of terminal peptide identification and quantification. Proteolytically generated carboxy-termini can now also be analyzed on a proteome-wide level. Proteolytic regulation of proteome composition is monitored by quantitative proteomic approaches employing stable isotope coding or label free quantification. Activity-based probes specifically recognize active proteases. In proteomic screens, they can be used to detect and quantitate proteolytic activity while their application in cellular histology allows to locate proteolytic activity in situ. Activity-based probes – especially in conjunction with positron emission tomography – are also promising tools to monitor proteolytic activities on an organism-wide basis with a focus on in vivo tumor imaging. Together, this array of methodological possibilities enables unveiling physiological protease substrate repertoires and defining protease function in the cellular- and organism-wide context.     

Using peptide libraries to identify optimal cleavage motifs for proteolytic enzymes

Proteases and peptidases are involved in a vast array of fundamental cellular processes, including cell growth, survival, motility, death, and differentiation, and can be important players in multicellular systems such as angiogenesis, inflammation, and immunity. Though long considered to be essentially digestive enzymes that mediate complete degradation of their substrates, many proteases are now known to be highly site specific. Knowledge of the cleavage site motif for a protease or peptidase can be useful in the design of substrates and inhibitors for the enzyme, and can also provide insight into its biological function through the identification and characterization of its protein substrates. Here, we describe in detail methodology that allows for the rapid and general determination of optimal recognition sequences for proteolytic enzymes.     

Cystatins, thyropins and inhibitors homologous to propeptides of cysteine proteases

Protein inhibitors of proteolytic enzymes play an important role in regulating the activity of endogenous proteases and in host defense mechanisms against pathogens preventing the deleterious effects of exogenous proteases. In recent years a great interest in protein inhibitors of cysteine proteases has increased due to the extensive growth of knowledge about the contribution of cysteine proteases to pathological processes associated with many human diseases, as well as due to prospects for treatment of these disorders which may arise from the thorough understanding of their inhibitory mechanisms. This paper reviews the most important aspects of three families of cysteine protease inhibitors: cystatins, thyropins and inhibitors homologous to propeptides of cysteine proteases. Special attention is given to structural bases of the interactions between the inhibitors and their target enzymes. The paper presents a general characterization of the families according to the MEROPS classification of protease inhibitors, pointing out new members.     

Catalytic properties of the caspases

Caspase stands for cysteine-dependent aspartate specific protease, and is a term coined to define proteases related to interleukin 1beta converting enzyme and CED-3.1 Thus their enzymatic properties are governed by a dominant specificity for substrates containing Asp, and by the use of a Cys side-chain for catalyzing peptide bond cleavage. The use of a Cys side chain as a nucleophile during peptide bond hydrolysis is common to several protease families. However, the primary specificity for Asp turns out to be very rare among protease families throughout biotic kingdoms. Of all known mammalian proteases only the caspase activator granzyme B, a serine protease, has the same primary specificity. In addition to this unusual primary specificity, caspases are remarkable in that certain of their zymogens have intrinsic proteolytic activity. This latter property is essential to trigger the proteolytic pathways that lead to apoptosis. Here we review the known enzymatic properties of the caspases and their zymogens within the broad context of structure:mechanism:activity relationships of proteases in general.     

Substrate recognition by AAA+ ATPases: distinct substrate binding modes in ATP-dependent protease Yme1 of the mitochondrial intermembrane space

The energy-dependent proteolysis of cellular proteins is mediated by conserved proteolytic AAA(+) complexes. Two such machines, the m- and i-AAA proteases, are present in the mitochondrial inner membrane. They exert chaperone-like properties and specifically degrade nonnative membrane proteins. However, molecular mechanisms of substrate engagement by AAA proteases remained elusive. Here, we define initial steps of substrate recognition and identify two distinct substrate binding sites in the i-AAA protease subunit Yme1. Misfolded polypeptides are recognized by conserved helices in proteolytic and AAA domains. Structural modeling reveals a lattice-like arrangement of these helices at the surface of hexameric AAA protease ring complexes. While helices within the AAA domain apparently play a general role for substrate binding, the requirement for binding to surface-exposed helices within the proteolytic domain is determined by the folding and membrane association of substrates. Moreover, an assembly factor of cytochrome c oxidase, Cox20, serves as a substrate-specific cofactor during proteolysis and modulates the initial interaction of nonassembled Cox2 with the protease. Our findings therefore reveal the existence of alternative substrate recognition pathways within AAA proteases and shed new light on molecular mechanisms ensuring the specificity of proteolysis by energy-dependent proteases.     

Purification and characterization of proteolytic enzymes from normal and opaque-2Zea mays L. developing endosperms

Purification and characterization of proteases from developing normal maize endosperm and high lysine opaque-2 maize endosperm have been carried out with a view to understand their role in storage protein modification. At day 15, normal maize endosperm had two types of proteolytic enzymes, namely, protease I and protease II, while at day 25 protease n disappeared and in place protease III appeared. However, in opaque-2 maize endosperm at both the stages only one type of enzyme (protease I) was present. These proteases had many properties in common-optimum pH and temperature were respectively, 5.7and 40°C; their activity was inhibited to the extent of 75 –93 % by p-chloromercuribenzoate; trypsin inhibitor inhibited the activity more at early stages of endosperm development; all proteases cleaved synthetic substrates p-tosyl-L-arginine methylesler and N-benzoyl-L-tyrosine ethyl ester and poly-L-glutamic acid. TheKm values of day 15 and 25 normal maize endosperm proteases ranged from 2.73–3.30, while for opaque-2 maize endosperm protease I it was 3.33 mg azocasein per ml assay medium. These enzymes, however, differed with respect to proteolytic activity towards poly-L-lysine. Only normal maize endosperm protease III at day 25 followed by protease II at day 15 showed high activity towards this homopolypeptide suggesting thereby their role in determining the quality of normal maize endosperm protein. Part of Ph.D. thesis submitted by the first author     

Proteases involved in long-term potentiation

Much attention has been paid to proteases involved in long-term potentiation (LTP). Calpains, Ca-dependent cysteine proteases, have first been demonstrated to be the mediator of LTP by the proteolytic cleavage of fodrin, which allows glutamate receptors located deep in the postsynaptic membrane to move to the surface. It is now generally considered that calpain activation is necessary for LTP formation in the cleavage of substrates such as protein kinase Czeta, NMDA receptors, and the glutamate receptor-interacting protein. Recent studies have shown that serine proteases such as tissue-type plasminogen activator (tPA), thrombin, and neuropsin are involved in LTP. tPA contributes to LTP by both receptor-mediated activation of cAMP-dependent protein kinase and the cleavage of NMDA receptors. Thrombin induces a proteolytic activation of PAR-1, resulting in activation of protein kinase C, which reduces the voltage-dependent Mg2+ blockade of NMDA receptor-channels. On the other hand, neuropsin may act as a regulatory molecule in LTP via its proteolytic degradation of extracellular matrix protein such as fibronectin. In addition to such neuronal proteases, proteases secreted from microglia such as tPA may also contribute to LTP. The enzymatic activity of each protease is strictly regulated by endogenous inhibitors and other factors in the brain. Once activated, proteases can irreversibly cleave peptide bonds. After cleavage, some substrates are inactivated and others are activated to gain new functions. Therefore, the issue to identify substrates for each protease is very important to understand the molecular basis of LTP.     

Membrane protein turnover by the m-AAA protease in mitochondria depends on the transmembrane domains of its subunits

AAA proteases are membrane-bound ATP-dependent proteases that are present in eubacteria, mitochondria and chloroplasts and that can degrade membrane proteins. Recent evidence suggests dislocation of membrane-embedded substrates for proteolysis to occur in a hydrophilic environment; however, next to nothing is known about the mechanism of this process. Here, we have analysed the role of the membrane-spanning domains of Yta10 and Yta12, which are conserved subunits of the hetero-oligomeric m-AAA protease in the mitochondria of Saccharomyces cerevisiae. We demonstrate that the m-AAA protease retains proteolytic activity after deletion of the transmembrane segments of either Yta10 or Yta12. Although the mutant m-AAA protease is still capable of processing cytochrome c peroxidase and degrading a peripheral membrane protein, proteolysis of integral membrane proteins is impaired. We therefore propose that transmembrane segments of m-AAA protease subunits have a direct role in the dislocation of membrane-embedded 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.     

Silencing barley cystatins HvCPI‐2 and HvCPI‐4 specifically modifies leaf responses to drought stress

Protein breakdown and mobilization are some of the major metabolic features associated with abiotic stresses, essential for nutrient recycling and plant survival. Genetic manipulation of protease and/or protease inhibitors may contribute to modulate proteolytic processes and plant responses. The expression analysis of the whole cystatin family, inhibitors of C1A cysteine proteases, after water deprivation in barley leaves highlighted the involvement of Icy‐2 and Icy‐4 cystatin genes. Artificial microRNA lines independently silencing the two drought‐induced cystatins were generated to assess their function in planta. Phenotype alterations at the final stages of the plant life cycle are represented by the stay‐green phenotype of silenced cystatin 2 lines. Besides, the enhanced tolerance to drought and differential responses to water deprivation at the initial growing stages are observed. The mutual compensating expression of Icy‐2 and Icy‐4 genes in the silencing lines pointed to their cooperative role. Proteolytic patterns by silencing these cystatins were concomitant with modifications in the expression of potential target proteases, in particular, HvPap‐1, HvPap‐12, and HvPap‐16 C1A proteases. Metabolomics analysis lines also revealed specific modifications in the accumulation of several metabolites. These findings support the use of plants with altered proteolytic regulation in crop improvement in the face of climate change.     

The serine protease HhoA from Synechocystis sp. strain PCC 6803: substrate specificity and formation of a hexameric complex are regulated by the PDZ domain

Enzymes of the ATP-independent Deg serine endopeptidase family are very flexible with regard to their substrate specificity. Some family members cleave only one substrate, while others act as general proteases on unfolded substrates. The proteolytic activity of Deg proteases is regulated by PDZ protein interaction domains. Here we characterized the HhoA protease from Synechocystis sp. strain PCC 6803 in vitro using several recombinant protein constructs. The proteolytic activity of HhoA was found to increase with temperature and basic pH and was stimulated by the addition of Mg(2+) or Ca(2+). We found that the single PDZ domain of HhoA played a critical role in regulating protease activity and in the assembly of a hexameric complex. Deletion of the PDZ domain strongly reduced proteolysis of a sterically challenging resorufin-labeled casein substrate, but unlabeled beta-casein was still degraded. Reconstitution of the purified HhoA with total membrane proteins isolated from Synechocystis sp. wild-type strain PCC 6803 and a DeltahhoA mutant resulted in specific degradation of selected proteins at elevated temperatures. We concluded that a single PDZ domain of HhoA plays a critical role in defining the protease activity and oligomerization state, combining the functions that are attributed to two PDZ domains in the homologous DegP protease from Escherichia coli. Based on this first enzymatic study of a Deg protease from cyanobacteria, we propose a general role for HhoA in the quality control of extracytoplasmic proteins, including membrane proteins, in Synechocystis sp. strain PCC 6803.     

Effects of protease treatment on growth,morphology, adhesion,and cell surface proteins of secondary chick embryo fibroblasts

Several proteolytic enzymes have been studied with regard to their ability to induce DNA synthesis and cell proliferation in resting chick embryo fibroblasts. Of the enzymes examined, thrombin, bromelin, and trypsin exhibit potent mitogenic activity, elastase has significant but less marked activity, whereas thermolysin, papain, and α-protease are inactive. The enzymes were also tested for their ability to induce morphological change or to remove two iodinatable proteins of 250,000 and 205,000 daltons. Although the larger protein is removed by some but not all of the proteases examined, every protease tested removed the smaller cell surface protein. The ability of proteases to stimulate cell growth could not be correlated directly with removal of either of these cell surface proteins; however, loss of the smaller protein does correlate with the reduction of both cytoplasmic spreading and cell-cell interactions observed after protease treatment. A secondary, later event of migration of cells into clumps is observed in those instances when protease treatment did not result in a loss of the 250K protein. A role for each of these proteins in the processes of cellular adhesion is discussed.