Golgi/granule processing of peptide hormone and neuropeptide precursors: a minireview

Proteolytic processing of precursor proteins is a phylogenetically ancient and widely used mechanism for producing biologically active peptides. Proteolytic cleavage of proproteins begins only after transport to the Golgi apparatus has been completed and in most systems may continue for many hours within newly formed secretory vesicles as these are stored in the cytosol or transported along axons to more peripheral sites of release. Paired basic residues are required for efficient proteolysis in most precursors, suggesting that a small number of specialized tryptic proteases exist that have great site selectivity but can process many sites within the same precursor or in different precursors within the same cell, or in different cells or tissues. Cleavage-site choice may be strongly influenced by other factors, such as secondary and tertiary structure, but definitive structural information on precursor proteins is lacking. Modifications such as glycosylation, phosphorylation, and sulfation also are Golgi associated but are not known to influence proteolytic processing patterns. Golgi/granule processing also rarely occurs at sites other than pairs of basic amino acids, including single basic residues ( trypsinlike ), Leu-Ala, Leu-Ser, or Tyr-Ala bonds ( chymotrysinlike ) as well as other specialized nontryptic cleavages, suggesting that mixtures of proteases coexist in the Golgi/granule system. Cathepsin B-like thiol proteases, or their precursors, have been implicated as the major processing endopeptidases in several systems. Carboxypeptidase B-like enzymes also have been identified in secretion granules in several tissues and appear to be metalloenzymes similar in mechanism to the pancreatic carboxypeptidases, but with a lower pH optimum. The role of the Golgi apparatus in sorting newly formed secreted products from lysosomal hydrolases may have permitted the development in evolution of an intimate relationship between certain of the lysosomal degradative enzymes, such as cathepsin B or its precursors, and the Golgi/granule processing systems. The sequestration of the proteolytic products of precursors within secretion granules leads to the coordinate discharge of highly complex mixtures of peptides having related or overlapping biological activities. The cosecretion of nonfunctional peptide " leftovers ," such as the proinsulin C-peptide, can serve as useful markers of secretion or cellular localization, as well as of evolutionary relation ships. Errors in cleavage due to point mutations in precursors have been identified in several systems, leading to the accumulation of incorrectly processed materials in the circulation. These and/or defects (ABSTRACT TRUNCATED AT 400 WORDS)     

Unconventional serine proteases: variations on the catalytic Ser/His/Asp triad configuration

Serine proteases comprise nearly one-third of all known proteases identified to date and play crucial roles in a wide variety of cellular as well as extracellular functions, including the process of blood clotting, protein digestion, cell signaling, inflammation, and protein processing. Their hallmark is that they contain the so-called "classical" catalytic Ser/His/Asp triad. Although the classical serine proteases are the most widespread in nature, there exist a variety of "nonclassical" serine proteases where variations to the catalytic triad are observed. Such variations include the triads Ser/His/Glu, Ser/His/His, and Ser/Glu/Asp, and include the dyads Ser/Lys and Ser/His. Other variations are seen with certain serine and threonine peptidases of the Ntn hydrolase superfamily that carry out catalysis with a single active site residue. This work discusses the structure and function of these novel serine proteases and threonine proteases and how their catalytic machinery differs from the prototypic serine protease class.     

植物类Caspase及其调控细胞程序性死亡的研究进展

植物细胞程序性死亡（PCD）在植物生长发育和逆境适应中发挥重要作用。半胱氨酸蛋白酶（caspase）调控动物PcD的启动、执行及信号转导。通过人工合成底物、动物caspase抑制剂等方法已证实在植物中存在类caspase,可分为metacas．pases、VPEs（vacuolar processing enzymes）和saspases等。本文综述了植物类caspase的种类、结构、定位、功能及其调控PCD的研究进展,提出植物PCD中类caspase作用的调控途径,为深入研究植物PCD提供参考。     

Salicylic acid influences the protease activity and posttranslation modifications of the secreted peptides in the moss Physcomitrella patens

Plant secretome comprises dozens of secreted proteins. However, little is known about the composition of the whole secreted peptide pools and the proteases responsible for the generation of the peptide pools. The majority of studies focus on target detection and characterization of specific plant peptide hormones. In this study, we performed a comprehensive analysis of the whole extracellular peptidome, using moss Physcomitrella patens as a model. Hundreds of modified and unmodified endogenous peptides that originated from functional and nonfunctional protein precursors were identified. The plant proteases responsible for shaping the pool of endogenous peptides were predicted. Salicylic acid (SA) influenced peptide production in the secretome. The proteasome activity was altered upon SA treatment, thereby influencing the composition of the peptide pools. These results shed more light on the role of proteases and posttranslational modification in the “active management” of the extracellular peptide pool in response to stress conditions. It also identifies a list of potential peptide hormones in the moss secretome for further analysis.     

Physiological roles of protein inhibitors

Protein inhibitors of proteases are widespread in nature. They are found in many sources, such as seeds, blood, eggs, and in many types of cells and tissues. Many physiological roles have been attributed to the protein inhibitors. Germination, wound healing, blood clotting, angiogenesis, etc., are some of the normal physiological activities in which protein inhibitors of proteases are involved. Lung destruction during emphysema and cartilage breakdown due to inflammation are two of the pathological conditions in which deficiency of protein inhibitors contributes to tissue breakdown by proteases.     

Protease propeptide structures,mechanisms of activation,and functions

Abstract

Proteases are a diverse group of hydrolytic enzymes, ranging from single-domain catalytic molecules to sophisticated multi-functional macromolecules. Human proteases are divided into five mechanistic classes: aspartate, cysteine, metallo, serine and threonine proteases, based on the catalytic mechanism of hydrolysis. As a protective mechanism against uncontrolled proteolysis, proteases are often produced and secreted as inactive precursors, called zymogens, containing inhibitory N-terminal propeptides. Protease propeptide structures vary considerably in length, ranging from dipeptides and propeptides of about 10 amino acids to complex multifunctional prodomains with hundreds of residues. Interestingly, sequence analysis of the different protease domains has demonstrated that propeptide sequences present higher heterogeneity compared with their catalytic domains. Therefore, we suggest that protease inhibition targeting propeptides might be more specific and have less off-target effects than classical inhibitors. The roles of propeptides, besides keeping protease latency, include correct folding of proteases, compartmentalization, liganding, and functional modulation. Changes in the propeptide sequence, thus, have a tremendous impact on the cognate enzymes. Small modifications of the propeptide sequences modulate the activity of the enzymes, which may be useful as a therapeutic strategy. This review provides an overview of known human proteases, with a focus on the role of their propeptides. We review propeptide functions, activation mechanisms, and possible therapeutic applications.     

Sequence of an epidermal growth factor-binding protein

A cDNA clone has been isolated that corresponds to the entire translated region of the mRNA coding for the epidermal growth factor-binding protein type B. The complete nucleotide sequence and the predicted amino acid sequence of the protein were elucidated. The protein sequence was compared to some related serine proteases. This comparison supports the notion that several serine proteases suggested to be involved in the processing of precursors to polypeptide hormones and growth factors are closely related to each other. It appears that these proteases are descendants of a common ancestral gene and thus form a distinct subfamily among the serine proteases.     

Caspase-dependant activation of chymotrypsin-like proteases mediates nuclear events during Jurkat T cell apoptosis

Apoptosis involves a cascade of biochemical and morphological changes resulting in the systematic disintegration of the cell. Caspases are central mediators of this process. Supporting and primary roles for serine proteases as pro-apoptotic mediators have also been highlighted. Evidence for such roles comes largely from the use of pharmacological inhibitors; as a consequence information regarding their apoptotic function and biochemical properties has been limited. Here, we circumvented limitations associated with traditional serine protease inhibitors through use of a fluorescently labelled inhibitor of serine proteases (FLISP) that allowed for analysis of the specificity, regulation and positioning of apoptotic serine proteases within a classical apoptotic cascade. We demonstrate that staurosporine triggers a caspase-dependant induction of chymotrypsin-like activity in the nucleus of apoptotic Jurkat T cells. We show that serine protease activity is required for the generation of late stage nuclear events including condensation, fragmentation and DNA degradation. Furthermore, we reveal caspase-dependant activation of two chymotrypsin-like protein species that we hypothesize mediate cell death-associated nuclear events.     

The role of protease activity in ErbB biology

Proteases are now recognized as having an active role in a variety of processes aside from their recognized metabolic role in protein degradation. Within the ErbB system of ligands and receptors, proteases are known to be necessary for the generation of soluble ligands from transmembrane precursors and for the processing of the ErbB4 receptor, such that its intracellular domain is translocated to the nucleus. There are two protease activities involved in the events: proteases that cleave within the ectodomain of ligand (or receptor) and proteases that cleave the substrate within the transmembrane domain. The former are the ADAM proteases and the latter are the γ-secretase complex and the rhomboid proteases. This review discusses the roles of each of these protease systems within the ErbB system.     

Evaluation of the presence of aspartic proteases from Centaurea calcitrapa during seed germination

Aspartic proteinases are present in a variety of organisms including plants. Common features of aspartic proteases include an active site cleft that contains two catalytic aspartic residues, acid pH optima for enzymatic activity, inhibition by pepstatin A. Plant aspartic proteinases occur in seeds and may be involved in the processing of storage proteins. Many of them have been purified and characterized. The presence of aspartic proteases in seeds of Centaurea calcitrapa during germination was investigated by measuring the activity on enzyme extracts. The aspartic proteases are present mainly in the beginning of seed germination suggesting that they could initiate the degradation of protein reserves in germinating seeds.

These proteases were purified by salt precipitation followed by anion-exchange chromatography. Purified aspartic proteases have an optimal pH between 3.5 and 4.5, using FTC-hemoglobin as substrate and an optimal temperature at 52 °C. The ability of seed extracts for milk clotting was tested and the clotting time that was achieved is in the same range found for flower extracts appropriated for special cheeses in which weak clotting agents are required.     

Purification and properties of proteases produced byalternaria alternata (Fr.) Keissl,regulation of production by fructose

A strain ofAlternaria alternata (Fr.) Keissl, when grown on wheat bran Czapek Dox medium was found to secrete one neutral and two alkaline proteases. The purified enzymes were found to be endo peptidases, the alkaline proteases being serine proteases and neutral proteases being cysteine proteases. Fructose when added to the culture medium was found to give rise to a new neutral protease at the expense of the neutral protease produced in the absence of fructose and was also found to enhance the production of alkaline proteases. It also appears that fructose modifies the alkaline proteases with respect to some characteristics such asV _max, Ea etc. Sodium dodecyl sulphate Polyacrylamide gel electrophoresis indicated a significantly altered protein profile in fructose supplemented medium.     

Growth factor-binding proteases in the murine submaxillary gland: isolation of a cDNA clone

The submaxillary gland of the adult male mouse contains a number of serine proteases, several of which are involved in the proteolytic processing of precursors to growth factors and other biologically active polypeptides. Here we report the isolation and identification of a cDNA clone corresponding to one of the proteases, the type B of the epidermal growth factor-binding protein. A pronounced sequence homology was found between the predicted activation peptide of this protease and the NH2-terminal extension of the nerve growth factor alpha subunit, suggesting that the latter protein has an uncleaved activation peptide attached to its NH2 terminus.     

An overview of Bacillus proteases: from production to application

Proteases have a broad range of applications in industrial processes and products and are representative of most worldwide enzyme sales. The genus Bacillus is probably the most important bacterial source of proteases and is capable of producing high yields of neutral and alkaline proteolytic enzymes with remarkable properties, such as high stability towards extreme temperatures, pH, organic solvents, detergents and oxidizing compounds. Therefore, several strategies have been developed for the cost-effective production of Bacillus proteases, including optimization of the fermentation parameters. Moreover, there are many studies on the use of low-cost substrates for submerged and solid state fermentation. Other alternatives include genetic tools such as protein engineering in order to obtain more active and stable proteases and strain engineering to better secrete recombinant proteases from Bacillus through homologous and heterologous protein expression. There has been extensive research on proteases because of the broad number of applications for these enzymes, such as in detergent formulations for the removal of blood stains from fabrics, production of bioactive peptides, food processing, enantioselective reactions, and dehairing of skins. Moreover, many commercial proteases have been characterized and purified from different Bacillus species. Therefore, this review highlights the production, purification, characterization, and application of proteases from a number of Bacillus species.     

Leucoagaricus gongylophorus uses leaf-cutting ants to vector proteolytic enzymes towards new plant substrate

The mutualism between leaf-cutting ants and their fungal symbionts revolves around processing and inoculation of fresh leaf pulp in underground fungus gardens, mediated by ant fecal fluid deposited on the newly added plant substrate. As herbivorous feeding often implies that growth is nitrogen limited, we cloned and sequenced six fungal proteases found in the fecal fluid of the leaf-cutting ant Acromyrmex echinatior and identified them as two metalloendoproteases, two serine proteases and two aspartic proteases. The metalloendoproteases and serine proteases showed significant activity in fecal fluid at pH values of 5–7, but the aspartic proteases were inactive across a pH range of 3–10. Protease activity disappeared when the ants were kept on a sugar water diet without fungus. Relative to normal mycelium, both metalloendoproteases, both serine proteases and one aspartic protease were upregulated in the gongylidia, specialized hyphal tips whose only known function is to provide food to the ants. These combined results indicate that the enzymes are derived from the ingested fungal tissues. We infer that the five proteases are likely to accelerate protein extraction from plant cells in the leaf pulp that the ants add to the fungus garden, but regulatory functions such as activation of proenzymes are also possible, particularly for the aspartic proteases that were present but without showing activity. The proteases had high sequence similarities to proteolytic enzymes of phytopathogenic fungi, consistent with previous indications of convergent evolution of decomposition enzymes in attine ant fungal symbionts and phytopathogenic fungi.     

Processing of mitochondrial presequences

Mitochondrial proteins are synthesized as precursor proteins on either cytosolic or mitochondrial ribosomes. The synthesized precursors from both translation origins possess targeting signals that guide the protein to its final destination in one of the four subcompartments of the organelle. The majority of nuclear-encoded mitochondrial precursors and also mitochondrial-encoded preproteins have an N-terminal presequence that serves as a targeting sequence. Specific presequence peptidases that are found in the matrix, inner membrane and intermembrane space of mitochondria proteolytically remove the signal sequence upon import or sorting. Besides the classical presequence peptidases MPP, IMP and Oct1, several novel proteases have recently been described to possess precursor processing activity, and analysis of their functional relevance revealed a tight connection between precursor processing, mitochondrial dynamics and protein quality control. This article is part of a Special Issue entitled: Mitochondrial Gene Expression.     

Intestinal proteolytic profile changes during larval development of Anticarsia gemmatalis caterpillars

Soybean is one of most consumed and produced grains in the world, and Anticarsia gemmatalis is a pest that causes great damage to this crop due to severe defoliation during its larval phase. Plants have mechanisms that lead to the inhibition of proteases in the intestine of these herbivores, hampering their development. Understanding this complex protease inhibitor is important for pest control. The objective of this study was to evaluate the enzymatic profiles of the intestinal proteases of the soybean caterpillar at different instars. For this, the proteolytic profile of the gut in the third, fourth, and fifth instars were analyzed. Irreversible inhibitors of proteases were separately incubated with A. gemmatalis enzyme extracts at the third, fourth, and fifth instar to assess the contribution of these proteases to total proteolytic activity. The enzymatic extracts were also evaluated with specific substrates to confirm changes in the specific activities of trypsin-like, chymotrypsin-like, and cysteine proteases at different instars. The results showed that the protease profile of A. gemmatalis gut changes throughout its larval development. The activity of cysteine proteases was more intense in the first instar. On the contrary, the serine proteases showed major activities in the late stages of the larval phase. Zymogram analysis and protein identification by liquid chromatography–mass spectrometry indicated serine protease as the main protease class expressed in the fifth instar. These results may shift the focus from the rational development of the protease inhibitor to A. gemmatalis and other Lepidoptera, as the expression of major proteases is not constant.     

Intracellular proteolysis: Signals of selective protein degradation

Selective proteolysis is one of the mechanisms for the maintenance of cell homeostasis via rapid degradation of defective polypeptides and certain short-lived regulatory proteins. In prokaryotic cells, high-molecular-mass oligomeric ATP-dependent proteases are responsible for selective protein degradation. In eukaryotes, most polypeptides are attacked by the multicatalytic 26S proteasome, and the degradation of the majority of substrates involves their preliminary modification with the protein ubiquitin. The proteins undergoing the selective proteolysis often contain specific degradation signals necessary for their recognition by the corresponding proteases. This article is dedicated to the 25th Anniversary of the journal Bioorganicheskaya Khimiya     

Elastases and elastokines: elastin degradation and its significance in health and disease

Abstract

Elastin is an important protein of the extracellular matrix of higher vertebrates, which confers elasticity and resilience to various tissues and organs including lungs, skin, large blood vessels and ligaments. Owing to its unique structure, extensive cross-linking and durability, it does not undergo significant turnover in healthy tissues and has a half-life of more than 70?years. Elastin is not only a structural protein, influencing the architecture and biomechanical properties of the extracellular matrix, but also plays a vital role in various physiological processes. Bioactive elastin peptides termed elastokines – in particular those of the GXXPG motif – occur as a result of proteolytic degradation of elastin and its non-cross-linked precursor tropoelastin and display several biological activities. For instance, they promote angiogenesis or stimulate cell adhesion, chemotaxis, proliferation, protease activation and apoptosis. Elastin-degrading enzymes such as matrix metalloproteinases, serine proteases and cysteine proteases slowly damage elastin over the lifetime of an organism. The destruction of elastin and the biological processes triggered by elastokines favor the development and progression of various pathological conditions including emphysema, chronic obstructive pulmonary disease, atherosclerosis, metabolic syndrome and cancer. This review gives an overview on types of human elastases and their action on human elastin, including the formation, structure and biological activities of elastokines and their role in common biological processes and severe pathological conditions.     

Peptidases and proteases of Escherichia coli and Salmonella typhimurium

A number of peptidases and proteases have been identified in Escherichia coli. Although their specific physiological roles are often not known, some of them have been shown to be involved in: the maturation of nascent polypeptide chains; the maturation of protein precursors; the signal peptide processing of exported proteins; the degradation of abnormal proteins; the use of small peptides as nutrients; the degradation of colicins; viral morphogenesis; the inactivation of some regulatory proteins for which a limited lifetime is a physiological necessity. Some of these enzymes act in concert to carry out specific functions. At present, twelve peptidases and seventeen proteases have been characterized. The specificity for only a few of them is known. The possible roles and the properties of these enzymes are discussed in this review.     

Proteolytic cleavage of staphylococcal exoproteins analyzed by two-dimensional gel electrophoresis

Extracellular proteases of Staphylococcus aureus are emerging as potential virulence factors that are relevant to the pathogenicity of staphylococcal infections. These proteases may also be involved in the proteolytic cleavage of other exoproteins released from this organism. To define the target exoproteins and their sites of cleavage by proteases, high-resolution two-dimensional polyacrylamide gel electrophoresis followed by N-terminal amino acid sequencing of exoprotein spots was performed. Two to three hundred exoprotein spots were detected at the early-stationary phase of cultures of S. aureus NCTC8325, and then at the late-stationary stage most of these high molecular protein spots became invisible due to further proteolytic degradation. As the result of N-terminal analysis, lipase, triacylglycerol lipase, orf619 protein and orf388 protein were detected as multiple spots at the early-stationary phase. We found that these exoproteins were cleaved at 3, 7, 4 and 4 different sites, respectively, by proteases. According to the M.W. and pI of each peptide spot obtained from the gel and their matches with calculated values in addition to their N-terminal sequences, we showed that the positions of putative peptides resulted from proteolytic cleavage of these proteins.