Evolution in the structure and function of carboxyl proteases

Summary A model for the structure and function of extracellular carboxyl (acid) proteases can be established from three amino acid sequences and four crystal structures of these enzymes. The carboxyl proteases from gastric and fungal origins are very homologous in both primary and tertiary structures. The molecules consist of about 320 residues organized with a secondary structure which is primarily comprised of -strands and very similar tertiary structures. An apparent binding cleft, which can accommodate a substrate with about eight amino acid residues, contains near its midpoint the active center residues Asp-215, Asp-32, and Ser-35. These three residues are hydrogen bonded to each other.An intracellular carboxyl protease, cathepsin D, is very homologous to the extracellular enzymes in N-terminal amino acid sequence and primary structure location of active center residues. The tertiary structure of cathepsin D is probably similar, as well. However, cathepsin D contains a unique hydrophobic tail made up of about 100 residues added on the C-terminal side. Cathepsin D precursor is over 100,000 daltons in molecular weights, as contrasted to the gastric carboxyl protease zymogens, which are about 40,000 daltons.     

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.     

Evolution of the placenta during the early radiation of placental mammals

The chorioallantoic placenta is an organ of gaseous exchange that exhibits a high degree of structural diversity. One factor determining oxygen transfer across the placenta, the diffusion distance, is in part dependent on the number of cell layers separating maternal from fetal blood. This interhaemal barrier occurs in three principal variants. The focus of this review is on determining how the barrier evolved in placental mammals. The analysis was based on current knowledge of placental structure, as far as possible using ultrastructural data, and on current views about the evolution of placental mammals, derived from molecular phylogenetics. We show that epitheliochorial placentation, the least invasive type, is a derived state and discuss factors that may have determined its evolution with reference to conflict theory, as applied to the allocation of resources between mother and fetus. It is not yet possible to determine which of the two more invasive types of placentation occurred in the last common ancestor of crown placentals. Depending on tree topology and taxon sampling, the result achieved is either endotheliochorial, haemochorial or unresolved. Finally we discuss other factors important to placental gas exchange and point to physiological variables that might become amenable to phylogenetic analysis.     

Evolution of placental specializations in viviparous African and South American lizards

Phylogenetic information offers an important resource in analyses of reproductive diversity, including interpretations of fetal membrane evolution. In this paper, we draw upon ongoing studies of South American and African lizards to consider the value of combining phylogenetic and reproductive evidence in the construction of evolutionary interpretations. South American lizards of the genus Mabuya exhibit several reproductive specializations that are convergent on those of eutherian mammals, including viviparity, long gestation periods, ovulation of tiny eggs, and placental supply of the nutrients for development. Studies of placental morphology and development indicate that New World Mabuya share several other derived features, including chorionic areolae and a "Type IV" epitheliochorial placenta with a villous, mesometrial placentome. Some characteristics of these lizards are shared by two African skinks, M. ivensii and Eumecia anchietae, including minuscule eggs, placentotrophy, an absorptive chorioallantois, and features of the yolk sac. Available evidence is consistent with two explanations: (1) placentotrophy originated in Africa, predating a trans-Atlantic colonization by Mabuya of the New World; and (2) placentotrophy arose two or three separate times among these closely related skinks. As illustrated by analysis of these animals, not only can data on fetal membrane morphology yield phylogenetic information, but phylogenetic evidence in turn provides a valuable way to reconstruct the evolution of fetal membranes in a biogeographic context. When appropriately interpreted, morphological and phylogenetic evidence can be combined to yield robust evolutionary conclusions that avoid the pitfalls of circular reasoning.     

Detergent proteases

Over the past 20 years, the development of subtilisins as typical detergent proteases has employed all the tools of enzyme technology, resulting in a constant flow of new and improved enzymes. The number of molecules identified and characterized, however, is in clear opposition to the number of molecules that are entering the market. Will the next-generation detergent proteases be based on new backbones different from subtilisins, or will the use of all available technologies (rational design, directed evolution and exploitation of natural diversity) yield improved subtilisins, ending the current era dominated by high alkaline subtilisins? These questions will have to be answered not only by the performance of the molecules themselves, but also by their yield in fermentation and their compatibility with existing production technologies.     

Cytochemistry of membrane proteases

Summary Membrane proteases that are detectable by cytochemical means are the classified exopeptidases, aminopeptidases A and M (or N), -glutamyl transpeptidase (which also acts as transferase), dipeptidyl peptidase IV and the endopeptidase, enteropeptidase (also known as enterokinase). Not yet classified are the possible expeptidase, tripeptidyl peptidase and endopeptidases I (Ala-endopeptidase) and II (Arg-endopeptidase). All these membrane proteases can be investigated with either chromogenic or fluorogenic procedures using synthetic peptide substrates. The most useful substrates are 4-methoxy-2-naphthylamine amino acids and peptides for cytochemical localizations at the light and electron microscope levels, for cytophotometric quantification and the study of membrane protease isoenzymes after analytical isoelectric focusing. Amino acid or peptide derivatives of naphthylamine AS can be recommended for light microscopical localization and cytofluorometric quantification, and 7-amino-4-methylcoumarin and 7-amino-4-trifluoromethylcoumarin amino acids and peptides for the development of enzyme bands after isoelectric focusing. Cytochemistry reveals the heterogeneity in the distribution and species differences of membrane proteases in adult cells, tissues and organs and during development. It also reveals some common localizations, such as in small intestinal enterocytes and proximal tubule cells. The species and organ differences are substantiated and extended considerably by isoelectric focusing in combination with methods for the cytochemical detection of proteases. In addition, continuous cytophotometry or cytofluorometry (section and cultured cell biochemistry) allows the kinetic characteristics, initial reaction rates and maximum activities of all membrane proteases to be determined.The physiological functions of the endopeptidases and exopeptidases are still a matter of debate. However, from cytochemical inhibition studies with natural peptide substrates, e.g. peptide hormones, there is increasing evidence that the proteases detected with synthetic peptides play a decisive role in many physiological circumstances, e.g. in endocrine regulation mechanisms or the regulation of blood pressure. In this respect, capillary endothelium-linked surface membrane proteases may be especially important.     

Immobilization of milk-clotting proteases

Traditionally, cheese manufacturing is a batch process and current practice is to use a milk-clotting enzyme in a soluble form. Immobilization of proteases for milk coagulation has received renewed interest and potential applications have recently been reported. Use of immobilized proteases would permit renneting of milk as a continuous process. In addition, it should be possible to recover and re-use the enzyme for coagulation of further batches of milk. This review elaborates on the recent developments in the area of immobilized proteases and their application in cheese-making.Paper No. 7334 through the Experiment Station, G.B. Pant University of Agriculture & Technology, Pantnagar-263 145, India.     

Sulphydryl plant proteases

Plant proteases, characterized by the reduced sulphydryl group required for catalysis, can be produced from tropical plants. Crude or partially refined mixtures, including minor quantities of other enzymes, are used in a variety of food industries. Enzymes extracted from edible fruit should prove acceptable. In terms of value or volume papain is the major enzyme used, though stem bromelain and ficin also appear in commerce. Other enzymes such as fruit bromelain or calotropain are either less well known or not commercially available. Meat tenderization and beer chill-proofing, mostly with papain, are reviewed and the effects of plant proteases on flavour in foods noted. Fish waste and oilseed processing, especially to produce proteins or polypeptides with functional properties, are amongst the currently minor or potential uses considered. Immobilization on a solid support merits further investigation. Development has been slow because of problems arising from the macromolecular nature of the substrate, namely protein, which is actually attacked in foods. Competition from microbial and other enzymes necessitates more attention being given to production methods if usage is to be maintained or increased.     

Chloroplast Deg proteases

For some chloroplast proteases ATP binding and hydrolysis is not necessary for their catalytic activity, most probably because even strongly unfolded substrates may penetrate their catalytic chamber. Deg1, 2, 5 and 8 are the best known of Arabidopsis thaliana ATP- independent chloroplast proteases, encoded by orthologues of genes coding for DegP, DegQ and DegS proteases of Escherichia coli. Current awareness in the area of structure and functions of chloroplast Degs is much more limited vs the one about their bacterial counterparts. Deg5 and Deg8 form a catalytic heterododecamer which is loosely attached to luminal side of thylakoid membrane. The complex catalyses--supported by Deg1 and one of FtsH proteases--the degradation of PsbA damaged due to plant exposition to elevated irradiance and thus these protease are of key importance for the plants' sensitivity to photoinhibition. Deg2 role in the disposal of damaged PsbA has not been elucidated. Recombinant Deg1 may degrade PsbO and plastocyanin in vitro but it is not clear whether this reaction is performed in vivo as well.     

Transmembrane segment proteases

Summary Transmembrane segment proteases comprise a novel class of proteases that cleave substrates within hydrophobic membrane-spanning segments. They cleave in parts of proteins that upon first glance should be protected by the hydrophobic environment of the lipid bilayer. At present, no such protease has been isolated and biochemically characterized. They are defined according to the appearance of the respective cleavage products. All trans-membrane segment proteases seem to participate in a regulated two-step proteolytic process that plays a central role in cellular regulation or is part of a protein degradation pathway.Abbreviations -APP -amyloid precursor protein - S1P site-1 protease - S2P site-2 protease - SPase signal peptidase - SPPase signal peptide peptidase - SREBP sterol regulatory element-binding protein - SCAP SREBP cleavage-activating protein     

Interaction of human placental ribonuclease with placental ribonuclease inhibitor

The interactions of human placental ribonuclease inhibitor (PRI) with bovine pancreatic ribonuclease (RNase) A and human angiogenin, a plasma protein that induces blood vessel formation, have been characterized in detail in earlier studies. However, studies on the interaction of PRI with the RNase(s) indigenous to placenta have not been performed previously, nor have any placental RNases been identified. In the present work, the major human placental RNase (PR) was purified to homogeneity by a five-step procedure and was obtained in a yield of 110 micrograms/kg of tissue. The placental content of angiogenin was also examined and was found to be at least 10-fold lower than that of PR. On the basis of its amino acid composition, amino-terminal sequence, and catalytic properties, PR appears to be identical with an RNase previously isolated from eosinophils (eosinophil-derived neurotoxin), liver, and urine. The apparent second-order rate constant of association for the PR.PRI complex, measured by examining the competition between PR and angiogenin for PRI, is 1.9 X 10(8) M-1 s-1. The rate constant for dissociation of the complex, determined by HPLC measurement of the rate of release of PR from its complex with PRI in the presence of a scavenger for free PRI, is 1.8 X 10(-7) s-1. Thus the Ki value for the PR.PRI complex is 9 X 10(-16) M, similar to that obtained with angiogenin, and 40-fold lower than that measured with RNase A. Complex formation causes a small red shift in the protein fluorescence emission spectrum, with no significant change in overall intensity. The fluorescence quantum yield of PR and the Stern-Volmer constant for fluorescence quenching by acrylamide are both high, possibly due to the presence of an unusual posttranslationally modified tryptophan residue at position 7 in the primary sequence.     

Digestive proteases of blood-feeding nematodes

Blood-feeding parasites employ a battery of proteolytic enzymes to digest the contents of their bloodmeal. Host haemoglobin is a major substrate for these proteases and, therefore, a driving force in the evolution of parasite-derived proteolytic enzymes. This review will focus on the digestive proteases of the major blood-feeding nematodes - hookworms (Ancylostoma spp. and Necator americanus) and the ruminant parasite, Haemonchus contortus - but also compares and contrasts these proteases with recent findings from schistosomes and malaria parasites. Haematophagous nematodes express proteases of different mechanistic classes in their intestines, many of which have proven or putative roles in degradation of haemoglobin and other proteins involved in nutrition. Moreover, the fine specificity of the relationships between digestive proteases and their substrate proteins provides a new molecular paradigm for understanding host-parasite co-evolution. Numerous laboratories are actively investigating these molecules as antiparasite vaccine targets.     

Extracellular proteases of Staphylococcus spp

Bacterial proteases secreted into an infected host may exhibit a wide range of pathogenic potentials. Staphylococci, in particular Staphylococcus aureus, are known to produce several extracellular proteases, including serine-, cysteine- and metalloenzymes. Their insensitivity to most human plasma protease inhibitors and, even more, the ability to inactivate some of these make the proteases potentially harmful. Indeed, several recent studies have shown that staphylococcal proteases are able to interact with the host defense mechanisms and tissue components as well as to modify other pathogen-derived virulence factors. A tight, cell density-dependent control of proteolytic activity expression, similar to that of the well-defined virulence determinants, further suggests the role of staphylococcal proteases in the infection process. Consistently, alterations in coordinated expression of extracellular proteins markedly diminished the virulence. However, despite these data and the fact that a strain deficient in sspABC operon coding for serine (sspA) and cysteine (sspB) proteases was highly attenuated in virulence in the animal infection model, it was impossible to unambiguously demonstrate the importance of any particular protease as a virulence factor. Therefore, it can be assumed that the orchestrated expression and interaction of a variety of extracellular and cell surface proteins rather than any particular one is responsible for the staphylococcal pathogenicity and that the proteases apparently play an important role in this complex process. Such redundant mechanism is very well suited for promoting the survival of staphylococci under diverse environmental conditions encountered in the infected host.     

Structural principles of intramembrane proteases

Intramembrane proteases are present in most organisms, and are used by cells to send signal across membranes, to activate growth factors, and to accomplish many other tasks that are beyond the capability of their soluble cousins. These enzymes specialize in cleaving peptide bonds that are normally embedded in cell membranes. They contain multiple membrane-spanning segments, and their catalytic residues are often found within these hydrophobic domains. In the past year, a number of important papers have been published that began to address the structural features of these membrane proteins by X-ray crystallography, electron microscopy, and biochemical methods, including the first report of an intramembrane protease crystal structure, that of Escherichia coli GlpG. Taken together, these studies started to reveal patterns of how intramembrane proteases are constructed, how waters are supplied to the membrane-embedded active site, and how membrane protein substrates interact with them.     

The AbetaCs of Abeta-cleaving proteases

The amyloid beta-protein (Abeta), which accumulates abnormally in Alzheimer disease (AD), is degraded by a diverse set of proteolytic enzymes. Abeta-cleaving proteases, largely ignored until only recently, are now known to play a pivotal role in the regulation of cerebral Abeta levels and amyloid plaque formation in animal models, and accumulating evidence suggests that defective Abeta proteolysis may be operative in many AD cases. This review summarizes the growing body of evidence supporting the involvement of specific Abeta-cleaving proteases in the etiology and potential treatment of AD. Recognition of the importance of Abeta degradation to the overall economy of Abeta has revised our thinking about the mechanistic basis of AD pathogenesis and identified a novel class of enzymes that may serve as both therapeutic targets and therapeutic agents.     

Cysteine proteases of malaria parasites

A number of cysteine proteases of malaria parasites have been described, and many more putative cysteine proteases are suggested by analysis of the Plasmodium falciparum genome sequence. Studies with protease inhibitors have suggested roles for cysteine proteases in hemoglobin hydrolysis, erythrocyte rupture, and erythrocyte invasion by erythrocytic malaria parasites. The best characterised Plasmodium cysteine proteases are the falcipains, a family of papain-family (clan CA) enzymes. Falcipain-2 and falcipain-3 are hemoglobinases that appear to hydrolyse host erythrocyte hemoglobin in the parasite food vacuole. This function was recently confirmed for falcipain-2, with the demonstration that disruption of the falcipain-2 gene led to a transient block in hemoglobin hydrolysis. A role for falcipain-1 in erythrocyte invasion was recently suggested, but disruption of the falcipain-1 gene did not alter parasite development. Other papain-family proteases predicted by the genome sequence include dipeptidyl peptidases, a calpain homolog, and serine-repeat antigens. The serine-repeat antigens have cysteine protease motifs, but in some the active site Cys is replaced by a Ser. One of these proteins, SERA-5, was recently shown to have serine protease activity. As SERA-5 and some other serine-repeat antigens localise to the parasitophorous vacuole in mature parasites, they may play a role in erythrocyte rupture. The P. falciparum genome sequence also predicts more distantly related (clan CD and CE) cysteine proteases, but biochemical characterisation of these proteins has not been done. New drugs for malaria are greatly needed, and cysteine proteases may provide useful new drug targets. Cysteine protease inhibitors have demonstrated potent antimalarial effects, and the optimisation and testing of falcipain inhibitor antimalarials is underway.