Current Protein & Peptide Science

ISSN: 1389-2042

Current Protein and Peptide Science
Volume 6, Number 6, December 2005

Contents


Aspartic Proteinase Content of the Arabidopsis Genome Pp.493
Carlos Faro and Susannah Gal
[Abstract]


Mapping of the Active Site of Proteases in the 1960s and Rational Design of Inhibitors/Drugs in the 1990s Pp.501
I. Schechter
[Abstract]


Nepenthesin, a Unique Member of a Novel Subfamily of Aspartic Proteinases: Enzymatic and Structural Characteristics Pp.513
Kenji Takahashi, Senarath B. P. Athauda, Koji Matsumoto, Sanath Rajapakshe,Masayuki Kuribayashi,Masaki Kojima,Nobuko Kubomura-Yoshida, Akihiro Iwamatsu, Chiaki Shibata and Hideshi Inoue
[Abstract]


Computational Methods for Remote Homolog Identification Pp.527
Xiu-Feng Wan and Dong Xu
[Abstract]


Endothelin Receptors in Gastrointestinal Smooth Muscle Pp.547
Shih-Che Huang
[Abstract]


Naturally Occurring Polyamines: Interaction with Macromolecules Pp.559
Uriel Bachrach
[Abstract]


The RING-Finger Protein Haprin: Domains and Function in the Acrosome Reaction Pp.567
Kouichi Kitamura, Hiromitsu Tanaka and Yoshitake Nishimune
[Abstract]




Abstracts

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Aspartic Proteinase Content of the Arabidopsis Genome
Carlos Faro and Susannah Gal

The sequence of the Arabidopsis genome has given us information about one plant's complement of aspartic proteinases. Using an in silico analysis based on the homology to known aspartic proteinase genes, we have uncovered 51 sequences that potentially encode these enzymes. This is substantial more than the number predicted for other eukaryotic systems. We have grouped the deduced amino acid sequences into 3 classes - typical plant aspartic proteinase, nucellin-like and atypical aspartic proteinase sequences-, depending on their putative domain organizations and their active site sequence motifs. Searching databases has revealed cDNAs or ESTs for nearly 90% of these genes. Sequence analysis using software that detects targeting signals indicates most of the predicted proteins have the expected localization in the secretory system although several of these are membrane bound. The analysis also predicts 8 chloroplast localized proteins and 2 mitochondria-localized aspartic proteinase-like proteins. The wide variety of structures and subcellular locations implies multiple functions for aspartic proteinases in plants.


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Mapping of the Active Site of Proteases in the 1960s and Rational Design of Inhibitors/Drugs in the 1990s
I. Schechter

For several decades the specificity of proteases has been presented as an active site divided into subsites, using the nomenclature of Schechter & Berger from 1967 (S1, S2…for subsites of the active site; P1, P2…for residues of the substrate occupying the corresponding subsites). At early stages of the research (1960s) it was realized that the size of the active site was larger than expected and important interactions occur in regions remote from the catalytic site. Since the active site was found to be large it was divided into subsites, and a procedure to map it up was developed. The map pro-vides information on the size of the active site (number of subsites), the properties of each subsite (free energy of ligand binding, nature of binding forces, etc.), and it enables rational design of new substrates and inhibitors. Already in 1968 inhibitors with binding constants ten thousand fold higher than available inhibitors, were prepared. The model of a large active site was initially met with strong opposition. Before long, however, predictions of the model (size of the active site, interactions in subsites remote from the catalytic site) were confirmed by X-ray crystallography (1970). During the 1990s proteolytic enzymes received renewed attention in biology and medicine, they became therapeutic targets, and protease inhibitors were successfully applied in the treatment of AIDS and hypertension. The model of large active site divided into subsites, proposed 38 years ago, stood the test of time. This model is still in use in basic research to evaluate enzyme activity, and in pharmaceutical research for the development of inhibitors/drugs.


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Nepenthesin, a Unique Member of a Novel Subfamily of Aspartic Proteinases: Enzymatic and Structural Characteristics
Kenji Takahashi, Senarath B. P. Athauda, Koji Matsumoto, Sanath Rajapakshe, Masayuki Kuribayashi, Masaki Kojima, Nobuko Kubomura-Yoshida, Akihiro Iwamatsu, Chiaki Shibata and Hideshi Inoue

Carnivorous plants are known to secrete acid proteinases to digest prey, mainly insects, for nitrogen uptake. In our recent study, we have purified, for the first time, to homogeneity two acid proteinases, nepenthesin I (Nep I) and ne-penthesin II (Nep II) from the pitcher fluid of Nepenthes distillatoria and investigated their enzymatic and structural char-acteristics. Both enzymes were optimally active at pH approx. 2.6 toward acid-denatured hemoglobin; the specificity of Nep I toward oxidized insulin B chain appears to be similar, but slightly wider than those of other aspartic proteinases (APs). At or below 50^oC both enzymes were remarkably stable; especially Nep I was extremely stable over a wide range of pH from 3 to 10 for over 30 days. This suggests an evolutionary adaptation of the enzymes to their specific habitat. We have also cloned the cDNAs and deduced the complete amino acid sequences of the precursors of Nep I and Nep II from the pitcher tissue of Nepenthes gracilis. Although the corresponding mature enzymes are homologous with ordinary pep-sin-type APs, both enzymes had a high content of cysteine residues (12 residues per molecule), which are assumed to form six unique disulfide bonds as suggested by computer modeling and are thought to contribute toward the remarkable stability of Neps. Moreover, the amino acid sequence identity of Neps with ordinary APs, including plant vacuolar APs, are remarkably low (approx. 20%), and phylogenetic comparison shows that Neps are distantly related to them to form a novel subfamily of APs with a high content of cysteine residues and a characteristic insertion, named ‘the Nep-type AP (NAP)-specific insertion’, including a large number of novel, orthologous plant APs emerging in the gene/protein data-bases.


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Computational Methods for Remote Homolog Identification
Xiu-Feng Wan and Dong Xu

As more and more protein sequences are available, homolog identification becomes increasingly important for functional, structural, and evolutional studies of proteins. Many homologous proteins were separated a very long time ago in their evolutionary history and thus their sequences share low sequence identity. These remote homologs have become a research focus in bioinformatics over the past decade, and some significant advances have been achieved. In this paper, we provide a comprehensive review on computational techniques used in remote homolog identification based on different methods, including sequence-sequence comparison, and sequence-structure comparison, and structure-structure com-parison. Other miscellaneous approaches are also summarized. Pointers to the online resources of these methods and their related databases are provided. Comparisons among different methods in terms of their technical approaches, their strengths, and limitations are followed. Studies on proteins in SARS-CoV are shown as an example for remote homolog identification application.


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Endothelin Receptors in Gastrointestinal Smooth Muscle
Shih-Che Huang

Endothelins (ETs) are a family of peptides with 21-amino-acid residues. ET-1 was identified as a potent vasoconstrictor produced by vascular endothelial cells. Three distinct isoforms of ET, i.e. ET-1, ET-2 and ET-3, have been found to exist in a variety of tissues. ET was later found to cause contraction as well as relaxation of smooth muscle in many physiologic systems. In the gastrointestinal tract, ET causes contraction and/or relaxation of the esophagus, stomach, ileum and colon. In the hepatobiliary system, ET causes contraction of the portal vein, hepatic stellate cells, gallbladder and common bile duct. In mammalian species, two classes of ET receptors, ET_A and ET_B, have been cloned. ET_A receptors have higher affinities for ET-1 and ET-2 than ET-3, while ET_B receptors have the same affinities for ET-1, ET-2 and ET-3. In the gastrointestinal system, ET causes smooth muscle contraction through interaction with ET_A receptors, ET_B receptors or both ET_A and ET_B receptors, depending on the tissues and species. In addition to contraction, ET causes smooth muscle relaxation through interaction with ET_A receptors or ET_B receptors. At the present time, there are no studies showing that ET causes smooth muscle relaxation through interaction with both ET_A and ET_B subtypes. ET induces contraction in most of the non-sphincter muscle except the fundus of the stomach. On the other hand, ET causes relaxation and contraction in the lower esophageal and internal anal sphincters. ET may play an important role in the control of human gastrointestinal motility and portal vein pressure.


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Naturally Occurring Polyamines: Interaction with Macromolecules
Uriel Bachrach

The naturally occurring polyamines, spermine [NH₂(CH₂)₃NH(CH₂)₄NH(CH₂)₃NH₂] and spermidine [NH₂(CH₂)₃NH(CH₂)₄NH₂], as well as the diamine putrescine [NH₂(CH₂)₄NH₂], are widely spread in nature. They occur in plants, micro-organisms and animal tissues and fulfil many important physiological functions. Due to their cationic nature they interact with negatively charged macromolecules such nucleic acids, phospholipids and proteins. This ionic interaction, which is reversible, leads to the stabilization of DNA, tRNA, membranes and some proteins. Early studies dem-onstrated that polyamines stimulate the growth of pro- and eukaryotic cells and that they play an important role in car-cinogenesis and in malignant transformation processes. As a result of these studies various inhibitors of polyamine bio-synthesis have been synthesized and are used to combat cancer and parasitic diseases (e.g., African sleeping sickness).


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The RING-Finger Protein Haprin: Domains and Function in the Acrosome Reaction
Kouichi Kitamura,Hiromitsu Tanaka, and Yoshitake Nishimune

The RBCC (RING finger, B-box type zinc finger, coiled-coil domain) motif family contains a large number of proteins implicated in many cellular processes, including vesicle exocytosis. The acrosome reaction, the sperm exocytotic event that is required for fertilization, involves essentially the same process of intracellular membrane fusions as vesicular exocytosis in somatic cells. We have previously isolated a haploid-germ-cell-specific gene designated haprin, which en-codes a RBCC motif protein that plays a role in the acrosome reaction of sperm by mediating protein complex formation via the RBCC motif. In this review, we describe the potential role of Haprin in the molecular mechanisms of acrosome re-action, as compared with some other RBCC proteins. The conserved structure and localization of the Haprin protein in human and mouse suggest an indispensable role for Haprin in the functioning of mammalian sperm.