Evolution in the structure and function of aspartic proteases

Aspartic proteases (EC3.4.23) are a group of proteolytic enzymes of the pepsin family that share the same catalytic apparatus and usually function in acid solutions. This latter aspect limits the function of aspartic proteases to some specific locations in different organisms; thus the occurrence of aspartic proteases is less abundant than other groups of proteases, such as serine proteases. The best known sources of aspartic proteases are stomach (for pepsin, gastricsin, and chymosin), lysosomes (for cathepsins D and E), kidney (for renin), yeast granules, and fungi (for secreted proteases such as rhizopuspepsin, penicillopepsin, and endothiapepsin). These aspartic proteases have been extensively studied for their structure and function relationships and have been the topics of several reviews or monographs (Tang: Acid Proteases, Structure, Function and Biology. New York: Plenum Press, 1977; Tang: J Mol Cell Biochem 26:93-109, 1979; Kostka: Aspartic Proteinases and Their Inhibitors. Berlin: Walter de Gruyter, 1985). All mammalian aspartic proteases are synthesized as zymogens and are subsequently activated to active proteases. Although a zymogen for a fungal aspartic protease has not been found, the cDNA structure of rhizopuspepsin suggests the presence of a "pro" enzyme (Wong et al: Fed Proc 44:2725, 1985). It is probable that other fungal aspartic proteases are also synthesized as zymogens. It is the aim of this article to summarize the major models of structure-function relationships of aspartic proteases and their zymogens with emphasis on more recent findings. Attempts will also be made to relate these models to other aspartic proteases.     

半胱氨酸蛋白酶拟肽抑制剂设计新进展

半胱氨酸蛋白酶包括多种酶,这些酶在广泛的生命过程中发挥作用。人类正常的半胱氨酸蛋白酶表达失调,寄生虫、病毒的半胱氨酸蛋白酶表达与多种病理情况相关。对于这类疾病,抑制半胱氨酸蛋白酶是一个可行的药物治疗策略。当前这类药物设计的目标是3种结构不同的半胱氨酸蛋白酶,即木瓜蛋白酶家族、半胱氨酸-天冬氨基特异性蛋白酶家族(caspases)和小核糖核酸病毒科半胱氨酸蛋白酶抑制剂家族。本文综述了近年来有关半胱氨酸蛋白酶抑制剂的设计思路。     

Extracellular and membrane-bound proteases from Bacillus subtilis.

Bacillus subtilis YY88 synthesizes increased amounts of extracellular and membrane-bound proteases. More than 99% of the extracellular protease activity is accounted for by an alkaline serine protease and a neutral metalloprotease. An esterase having low protease activity accounts for less than 1% of the secreted protease. These enzymes were purified to homogeneity. Molecular weights of approximately 28,500 and 39,500 were determined for the alkaline and neutral proteases, respectively. The esterase had a molecular weight of approximately 35,000. Amino-terminal amino acid sequences were determined, and the actions of a number of inhibitors were examined. Membrane vesicles contained bound forms of alkaline and neutral proteases and a group of previously undetected proteases (M proteases). Membrane-bound proteases were extracted with Triton X-100. Membrane-bound alkaline and neutral proteases were indistinguishable from the extracellular enzymes by the criteria of molecular weight, immunoprecipitation, and sensitivity to inhibitors. The M protease fraction accounted for approximately 7% of the total activity in Triton X-100 extracts of membrane vesicles. The M protease fraction was partially fractionated into four species (M1 through M4) by ion-exchange chromatography. Immunoprecipitation and sensitivity to inhibitors distinguished membrane-bound alkaline and neutral proteases from M proteases. In contrast to alkaline and neutral proteases, proteases M2 and M3 exhibited exopeptidase activity.     

Commercial proteases: Present and future

This review presents a brief overview of the general categories of commercially used proteases, and critically surveys the successful strategies currently being used to improve the properties of proteases for various commercial purposes. We describe the broad application of proteases in laundry detergents, food processing, and the leather industry. The review also introduces the expanding development of proteases as a class of therapeutic agents, as well as highlighting recent progress in the field of protease engineering. The potential commercial applications of proteases are rapidly growing as recent technological advances are producing proteases with novel properties and substrate specificities.     

Digestive proteases during development of larvae of red palm weevil, Rhynchophorus ferrugineus (Olivier, 1790) (Coleoptera: Curculionidae).

The evolution of digestive proteases during larval development of Rhynchophorus ferrugineus (Olivier, 1790) has been studied. A progressive increase of protease activity has been found. The optimum pH for proteolytic activity against azocasein was determined. Caseinograms revealed an active complex of alkaline proteases from the early stages of the development. From the apparent molecular masses, three groups of proteases have been found - high molecular-mass proteases, medium molecular-mass proteases, and low molecular-mass proteases. Studies using specific protease inhibitors showed the major presence of serine proteases in gut extracts. The results obtained from larvae reared on different substrates have made possible a comparative assessment of the influence of diet on the development of the digestive enzymatic system. Larvae fed on an artificial diet showed a complete pattern of digestive proteases. Data suggest that this diet seems to be suitable for future research with this insect pest.     

Root proteases: reinforced links between nitrogen uptake and mobilization and drought tolerance

Integral subcellular and cellular functions ranging from gene expression, protein targeting and nutrient supply to cell differentiation and cell death require proteases. Plants have unique organelles such as chloroplasts composed of unique proteins that carry out the unique process of photosynthesis. Hence, along with proteases common across kingdoms, plants contain unique proteases. Improved knowledge on proteases can lead to a better understanding of plant development, differentiation and death. Because of their importance in multiple processes, plant proteases are actively studied. However, root proteases specifically are not as well studied. The associated rhizosphere, organic matter and/or inorganic matter make roots a difficult system. Yet recent research conclusively demonstrated the occurrence of endocytosis of proteins, peptides and even microbes by root cells, which, hitherto known for specialized pathogenesis or symbiosis, was unsuspected for nutrient uptake. These results reinforced the importance of root proteases in endocytosis or root exudate-mediated nutrient uptake. Rhizoplane, rhizosphere or in planta protease action on proteins, peptides and microbes generates sources of nitrogen, especially during abiotic stresses such as drought. This article highlights the recent research on root proteases for nitrogen uptake and the connection of the two to drought-tolerance mechanisms. Drought-induced proteases in rice roots, as known from rice expression databases, are discussed for future research on certain M50, Deg, FtsH, AMSH and deubiquitination proteases. The recent emphasis on linking drought and plant hydraulics to nutrient metabolism is illustrated and connected to the value of a systematic study of root proteases in crop improvement.     

Activity-based protein profiling reveals active serine proteases that drive malignancy of human ovarian clear cell carcinoma

Ovarian clear cell carcinoma (OCCC) is an understudied poor prognosis subtype of ovarian cancer lacking in effective targeted therapies. Efforts to define molecular drivers of OCCC malignancy may lead to new therapeutic targets and approaches. Among potential targets are secreted proteases, enzymes which in many cancers serve as key drivers of malignant progression. Here, we found that inhibitors of trypsin-like serine proteases suppressed malignant phenotypes of OCCC cell lines. To identify the proteases responsible for malignancy in OCCC, we employed activity-based protein profiling to directly analyze enzyme activity. We developed an activity-based probe featuring an arginine diphenylphosphonate warhead to detect active serine proteases of trypsin-like specificity and a biotin handle to facilitate affinity purification of labeled proteases. Using this probe, we identified active trypsin-like serine proteases within the complex proteomes secreted by OCCC cell lines, including two proteases in common, tissue plasminogen activator and urokinase-type plasminogen activator. Further interrogation of these proteases showed that both were involved in cancer cell invasion and proliferation of OCCC cells and were also detected in in vivo models of OCCC. We conclude the detection of tissue plasminogen activator and urokinase-type plasminogen activator as catalytically active proteases and significant drivers of the malignant phenotype may point to these enzymes as targets for new therapeutic strategies in OCCC. Our activity-based probe and profiling methodology will also serve as a valuable tool for detection of active trypsin-like serine proteases in models of other cancers and other diseases.     

The S8 serine, C1A cysteine and A1 aspartic protease families in Arabidopsis

The Arabidopsis thaliana genome has over 550 protease sequences representing all five catalytic types: serine, cysteine, aspartic acid, metallo and threonine (MEROPS peptidase database, http://merops.sanger.ac.uk/), which probably reflect a wide variety of as yet unidentified functions performed by plant proteases. Recent indications that the 26S proteasome, a T1 family-threonine protease, is a regulator of light and hormone responsive signal transduction highlight the potential of proteases to participate in many aspects of plant growth and development. Recent discoveries that proteases are required for stomatal distribution, embryo development and disease resistance point to wider roles for four additional multigene families that include some of the most frequently studied (yet poorly understood) plant proteases: the subtilisin-like, serine proteases (family S8), the papain-like, cysteine proteases (family C1A), the pepsin-like, aspartic proteases (family A1) and the plant matrixin, metalloproteases (family M10A). In this report, 54 subtilisin-like, 30 papain-like and 59 pepsin-like proteases from Arabidopsis, are compared with S8, C1A and A1 proteases known from other plant species at the functional, phylogenetic and gene structure levels. Examples of structural conservation between S8, C1A and A1 genes from rice, barley, tomato and soybean and those from Arabidopsis are noted, indicating that some common, essential plant protease roles were established before the divergence of monocots and eudicots. Numerous examples of tandem duplications of protease genes and evidence for a variety of restricted expression patterns suggest that a high degree of specialization exists among proteases within each family. We propose that comprehensive analysis of the functions of these genes in Arabidopsis will firmly establish serine, cysteine and aspartic proteases as regulators and effectors of a wide range of plant processes.     

Serine proteases in schistosomes and other trematodes

Trematodes, also known as flukes, are phylogenetically ancient parasitic organisms. Due to their importance as human and veterinary parasites, their proteins have been investigated extensively as drug and vaccine targets. Among those, proteases, as crucial enzymes for parasite survival, are considered candidate molecules for anti-parasitic interventions. Surprisingly however, trematode serine proteases, in comparison with other groups of proteases, are largely neglected. Genes encoding serine proteases have been identified in trematode genomes in significant abundance, but the biological roles and biochemical functions of these proteases are poorly understood. However, increasing volumes of genomic and proteomic studies, and accumulated experimental evidence, indicate that this class of proteases plays a substantial role in host–parasite interactions and parasite survival. Here, we discuss in detail serine proteases at genomic and protein levels, and their known or hypothetical functions.     

Evolution of Thrombin and Other Hemostatic Proteases by Survey of Protochordate, Hemichordate, and Echinoderm Genomes

Protochordate genomes enable a prevalence of hemostasis evolution. Broad searches were performed for homologs of human serine proteases of hemostasis on the genomes of Branchiostoma floridae, Saccoglossus kowalevskii, and Strongylocentrotus purpuratus. Sequences were analyzed by multiple bioinformatic tools. The survey revealed numerous homologous components. Amphioxus was rich in some serine proteases not accompanied by gamma-carboxyglutamic or kringle domains similar more to thrombin than to other coagulation factors. The serine proteases found in amphioxus exhibited the attributes similar to those of thrombin by phylogeny relationships, sequence conservation, gene synteny, spatial structure, and ligand docking. A few plasminogen- and plasminogen activators-like proteases with kringles were also present. Those serine proteases demonstrated the greatest proximity rather to plasminogen or plasminogen activators than to thrombin. Searching for homologs of serine protease hemostatic factors in acorn worm and sea urchin revealed several components similar to those found in amphioxus. Hypothetically, the common ancestor of chordates had three separate serine proteases that evolved independently into immunoglobulin-like and kringle proteases in lancelets, and prothrombin, plasminogen activators, and plasminogen in vertebrates. Ancestral proteases evolved in vertebrates into hemostasis factors after merging the proper N-terminal domains and duplications.     

Purification of active cysteine proteases by affinity chromatography with attached E-64 inhibitor

Cysteine proteases are implicated in many regulatory and degradative processes in animal and plant cells. Many of the proteases are strongly inhibited by an irreversible inhibitor, trans-(epoxysuccinyl)-l-leucylamino-4-guanidinobutane (E-64) from Aspergillus japonicus. Here we report a method for purification of cysteine proteases by affinity chromatography on E-64. Attachment of the inhibitor to thiopropyl Sepharose through its epoxy group resulted in the loss of its irreversible activity but did not affect the specificity of interaction or its capability to bind cysteine proteases. Papain that served as a model cysteine protease was fully active after elution. We also provide evidence for purification of active proteases from a mixture of extracellular fluid of Botrytis cinerea- and Trichoderma harzianum-inoculated bean plants. Since the proteases are eluted with urea after the column is washed with 1 M NaCl, this procedure may provide highly efficient purification.     

Visualization of proteases within a complex sample following their selective retention on immobilized bacitracin, a peptide antibiotic.

A method for the visualization of individual proteases within a complex biological sample is described. In a single chromatographic step, proteases can be separated from other biomolecules by selective binding to immobilized bacitracin, a peptide antibiotic. Following desorption, proteases may be separated by SDS-polyacrylamide gel electrophoresis. The application of this method is presented in the visualization of proteases secreted by the fungus Aspergillus niger.     

One- and two-dimensional SDS-PAGE zymography with quenched fluorogenic substrates provides identification of biological fluid proteases by direct mass spectrometry

We describe a simple and direct zymographic method for the detection of proteases using quenched fluorescent substrates. The proteases were separated using one- and two-dimensional electrophoresis, and the gel subsequently was incubated with the quenched fluorescent substrate. After a short incubation, the released fluorescence allowed the localization of the proteases directly using UV light. The protease spots could then be cut directly from the gel and processed for identification by mass spectrometry. This method could easily be used to develop or test whether a substrate is specific or not and also to detect the proteases that are able to cleave this substrate in a complex biological fluid. This also allowed direct identification of proteases without complex purification.     

The plant proteolytic machinery and its role in defence

The diverse roles of plant proteases in defence responses that are triggered by pathogens or pests are becoming clearer. Some proteases, such as papain in latex, execute the attack on the invading organism. Other proteases seem to be part of a signalling cascade, as indicated by protease inhibitor studies. Such a role has also been suggested for the recently discovered metacaspases and CDR1. Some proteases, such as RCR3, even act in perceiving the invader. These exciting recent reports are probably just the first examples of what lies beneath. More roles for plant proteases in defence, as well as the regulation and substrates of these enzymes, are waiting to be discovered.     

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。     

Identification and structural analysis of four serine proteases in a monotreme, the platypus, Ornithorhynchus anatinus

To study the emergence of the major subfamilies of serine proteases during vertebrate evolution, we present here the primary structure of four serine proteases expressed in the spleen of a monotreme, the platypus, Ornithorhynchus anatinus. Partial cDNA clones for four serine proteases were isolated by a PCR-based strategy. This strategy is based on the high level of sequence identity between various members of the large gene family of trypsin-related serine proteases, over two highly conserved regions, those of the histidine and the serine of the catalytic triad. The partial cDNA clones were used to isolate full-length or almost full-length cDNA clones for three of these proteases from a platypus spleen cDNA library. By phylogenetic analysis, these three clones were identified as being the platypus homologues of human coagulation factor X, neutrophil elastase, and a protease distantly related to the T-cell granzymes. The remaining partial clone was found to represent a close homologue of human complement factor D (adipsin). The isolation of these four clones shows that several of the major subfamilies of serine proteases had evolved as separate subfamilies long before the radiation of the major mammalian lineages of today, the monotremes, the marsupials, and the placental mammals. Upon comparison of the corresponding proteases of monotremes and eutherian mammals, the coagulation and complement proteases were shown to display a higher degree of conservation compared to the hematopoietic proteases N-elastase and the T-cell granzymes. This latter finding indicates a higher evolutionary pressure to maintain specific functions in the complement and coagulation enzymes compared to many of the hematopoietic serine proteases.     

Protease immobilization onto polyacrolein microspheres

Water-insoluble proteases were prepared by immobilizing papain and chymotrypsin onto the surface of polyacrolein microspheres with and without oligoglycines as spacer. The activity of immobilized proteases was found to be still high toward small ester substrates, but very low toward casein, a high-molecular-weight substrate. The relative activity of the immobilized proteases without spacer decreased gradually with the decreasing surface concentration of the immobilized proteases on the microspheres. On the contrary, the immobilized proteases with oligoglycine spacers gave an almost constant activity for the substrate hydrolysis within the surface concentration region studied and gave a much higher relative activity than those without any spacer. With the longer spacer, the immobilized enzymes showed a higher activity toward casein hydrolysis, whereas there was an optimum length for the spacer when hydrolysis was carried out toward the low-molecular-weight substrate. The thermal stability of the immobilized proteases was higher than that of the respective native proteases. The initial enzymatic activity of the immobilized proteases maintained almost unchanged without any elimination and inactivation of proteases, when the batch enzyme reaction was performed repeatedly, indicating the excellent durability.     

Signal peptide peptidase-like 2 proteases: Regulatory switches or proteasome of the membrane?

Signal peptide peptidase-like 2 (SPPL) proteases constitute a subfamily of SPP/SPPL intramembrane proteases which are homologues of the presenilins, the catalytic core of the γ-secretase complex. The three SPPL2 proteases SPPL2a, SPPL2b and SPPL2c proteolyse single-span, type II-oriented transmembrane proteins and/or tail-anchored proteins within their hydrophobic transmembrane segments. We review recent progress in defining substrate spectra and in vivo functions of these proteases. Characterisation of the respective knockout mice has implicated SPPL2 proteases in immune cell differentiation and function, prevention of atherosclerotic plaque development and spermatogenesis. Mechanisms how substrates are selected by these enzymes are still incompletely understood. We will discuss current views on how selective SPPL2-mediated cleavage is or whether these proteases may exhibit a generalised role in the turnover of membrane proteins. This has been suggested previously for the mechanistically related γ-secretase for which the term “proteasome of the membrane” has been coined based on its broad substrate spectrum. With regard to individual substrates, potential signalling functions of the resulting cytosolic cleavage fragments remain a controversial aspect. However, it has been clearly shown that SPPL2 proteases can influence cellular signalling and membrane trafficking by controlling levels of their membrane-bound substrate proteins which highlights these enzymes as regulatory switches. Based on this, regulatory mechanisms controlling activity of SPPL2 proteases would need to be postulated, which are just beginning to emerge. These different questions, which are relevant for other families of intramembrane proteases in a similar way, will be critically discussed based on the current state of knowledge.