Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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We have cloned a cDNA containing the entire coding sequence of a marsupial (the brushtail possum, Trichosurus vulpecula) zona pellucida protein (ZPB). The open reading frame of 1,581 nt is predicted to encode a ZPB polypeptide of 527 amino acids which contains 20 cysteine residues, 7 potential N-linked glycosylation sites, a potential N-terminal signal peptide and a potential C-terminal trans-membrane domain, preceded by a furin proteolytic processing signal. Sequence comparisons between possum ZPB and orthologous polypeptides from 7 eutherian species and from Xenopus laevis, reveal the existence of a high degree of sequence similarity, particularly in the central portion of the molecule. Cysteine residues are highly conserved, and all nine species possess potential N-terminal signal peptide sequences and C-terminal trans-membrane domains of approximately the same length. In situ hybridisation revealed that expression of ZPB was restricted to oocytes of primordial and primary follicles of adult possums; no expression was detected in the surrounding granulosa cells. The broad conservation of ZPB sequence, structure and expression over a wide range of mammalian species, revealed by our studies, makes it unlikely that these features account for the different properties of the marsupial and eutherian zona pellucidae.
Mol Reprod Dev 1999 Feb
PMID:Isolation and characterisation of a cDNA encoding a zona pellucida protein (ZPB) from the marsupial Trichosurus vulpecula (brushtail possum). 989 Jul 48

Free living amoeba, including pathogenic Acanthamoeba culbertsoni, are widely distributed in soil and fresh water. It has been found that cysteine proteinases are more active in pathogenic strains of amoeba whereas serine proteinases are found in both pathogenic and nonpathogenic strains. Cysteine proteinases thus play important roles in the pathogenesis of several parasitic infections and have been proposed as targets for the structure-based strategy of drug design. As the first step toward applying this strategy to design inhibitors as antiparasitic agents for A. culbertsoni, we isolated and sequenced the full length clone of a cysteine proteinase gene from A. culbertsoni by performing reverse transcription-polymerase chain reaction (RT-PCR) with degenerate oligonucleotide primers derived from conserved cysteine proteinase sequences. The 5' and the 3' regions of the cysteine proteinase gene were amplified using the PCR protocol for the rapid amplification of cDNA ends (RACE). It has an open reading frame of 1359 bp. The deduced amino acid sequence has the sequence homology with the cysteine proteinase genes of Paragonimus westermani metacercaria, Schistosoma mansoni, human cathepsin L and Fasciola hepatica, each by 45.3%, 45.9%, 57.9% and 50.8% respectively. Sequence analysis and alignment showed significant similarity to other eukaryotic cysteine proteinases, including the conservation of the cysteine, histidine, and asparagine residues that form the catalytic triad. A 1.5 kbp mRNA was detected on Northern blot analysis using full-length cysteine proteinase cDNA as a probe. The A. culbertsoni cysteine proteinase gene (AcCP2) was found to contain Ex3Rx3Wx2N at the proregion and also a proline/threonine-rich C-terminal extension. Therefore, it has cathepsin L-like characteristics. Phylogenetic analysis based on the amino acid sequences of cysteine proteinase indicated that AcCP2 was closely related with papaya, while it was remotely related with those of Schistosoma.
Mol Cells 1999 Oct 31
PMID:Cloning of a cysteine proteinase gene from Acanthamoeba culbertsoni. 1059 37

Melatonin is synthesized in pinealocytes of the pineal gland and in photoreceptors of the retina. Synthesis rate from serotonin to melatonin is controlled by the rapid and dramatic enzymatic increase in darkness of serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AA-NAT, EC 2.3.1.87) and hydroxyindole-O-methyltransferase (HIOMT, EC 2.1.1.4). The primary structure of these critical indoleamine enzymes is now known and the regulation of the enzyme catalysis can be examined. As a first step, the conserved cysteine (C) and histidine (H) residues were targeted for site-directed mutagenesis as potential amino acid residues involved in the N-acetylation reaction of AA-NAT. Our studies concluded that among 6 histidine (H) to alanine (A) mutations, three residues (H110A, H118A, H120A) within the AA-NAT protein showed little or no enzymatic activity, whereas the others (H28A, H70A, H125A) retained enzymatic activity, compared to the unaltered AA-NAT protein. Cysteine to alanine mutations, C37A and C177A, had no significant effect on the AA-NAT enzymatic activity; however, C61A had a four-fold increase in K(m) for acetyl CoA and an altered sensitivity to the thiol modification chemical, N-ethylmaleimide (NEM), implying that C61 may participate in the acetyl CoA binding. Further studies examined the AA-NAT enzyme regulation of the highly conserved carboxyl terminus. When 12 terminal amino acid residues were deleted systematically from the carboxyl terminus of the 205 amino acid residue AA-NAT protein, enzyme activity was retained. However, further residue deletion resulted in enzyme activity plummeting, implicating that the essential information either for the correct structural folding into an active enzyme form or for enzyme stability is in the 193 residues. To test the relative importance of the AA-NAT carboxyl terminal region, a single leucine (L) was altered to alanine (A) or proline (P). Both mutants, either L193A or L193P, had a marked decrease in AA-NAT enzymatic activity and a decrease in thermal stability, suggesting the leucine, in addition to the cysteine and histidine residues, is involved in either enzyme catalysis or stability. In light of the recently reported three-dimensional structure of AA-NAT (17,18), the site-directed mutagenesis data demonstrate experimentally the importance of essential amino acid residues for acetyl CoA binding and AA-NAT activation.
Brain Res Mol Brain Res 2000 Feb 22
PMID:Identification of specific histidine residues and the carboxyl terminus are essential for serotonin N-acetyltransferase enzymatic activity. 1068 40

We characterized the digestive proteinases of eight species of beetles to improve our understanding of the phylogenetic distribution of serine and cysteine proteinases. Serine proteinases function optimally under alkaline pH conditions, whereas cysteine proteinases require acidic pH. The phylogenetic distribution of cysteine proteinases suggests that they first appeared in an early cucujiform ancestor, however, data for some groups is patchy, and there has been speculation that they have been lost in at least one group, the long-horned beetles (Cerambycidae). The pattern we found supports the hypothesized origin of the proteinases and extends their distribution to an additional superfamily. In addition, we confirmed the presence of cysteine proteinases in some Curculionoidea. Cysteine proteinases were absent, however, from all three species of cerambycids surveyed, supporting the hypothesis that this group has reverted to the more ancestral serine (alkaline) digestive strategy. In four species we compared the pH optima for total proteolytic activity to the actual pH of the midgut and found the match between optimal and actual pH to be weaker in the cerambycids. These findings suggest that either a close correlation between midgut pH and the proteolytic pH optimum is not needed for adequate digestive efficiency, or that midgut pH is a more constrained digestive feature and there has been insufficient time for it to shift upwards to maximize serine proteinase activity.
Comp Biochem Physiol B Biochem Mol Biol 2000 Aug
PMID:Phylogenetic distribution of cysteine proteinases in beetles: evidence for an evolutionary shift to an alkaline digestive strategy in Cerambycidae. 1102 73

The Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) family of cytoadherent proteins has a central role in disease from malaria infection. This highly diverse gene family is involved in binding interactions between infected erythrocytes and host cells and is expressed in a clonally variant pattern at the erythrocyte surface. We describe by sequence analysis the structure and domain organization of 20 PfEMP1 from the GenBank database. Four domains comprise the majority of PfEMP1 extracellular sequence: the N-terminal segment (NTS) located at the amino terminus of all PfEMP1, the C2, the Cysteine-rich Interdomain Region (CIDR) and the Duffy Binding-like (DBL) domains. Previous work has shown that CIDR and DBL domains can possess adhesive properties. CIDR domains grouped as three distinct sequence classes (alpha, beta, and gamma) and DBL domains as five sequence classes (alpha, beta, gamma, delta, and epsilon). Consensus motifs are described for the different DBL and CIDR types. Whereas the number of DBL and CIDR domains vary between PfEMP1, PfEMP1 domain architecture is not random in that certain tandem domain associations--such as DBLalphaCIDRalpha, DBLdeltaCIDRbeta, and DBLbetaC2--are preferentially observed. This conservation may have functional significance for PfEMP1 folding, transport, or binding activity. Parasite binding phenotype appears to be a determinant of infected erythrocyte tissue tropism that contributes to parasite survival, transmission, and disease outcome. The sequence classification of DBL and CIDR types may have predictive value for identifying PfEMP1 domains with a particular binding property. This information might be used to develop interventions targeting parasite binding variants that cause disease.
Mol Biochem Parasitol 2000 Oct
PMID:Classification of adhesive domains in the Plasmodium falciparum erythrocyte membrane protein 1 family. 1107 Dec 84

KB-R7943 (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulfonate) is a potent and selective Na(+)/Ca(2+) exchange (NCX) inhibitor that is 3-fold more inhibitory to NCX3 than to NCX1 or NCX2. Here we searched for amino acid residues that may form the KB-R7943 receptor in the exchanger by analyzing the function of chimeras between NCX1 and NCX3 as well as of their site-directed mutants. We found that the highly conserved alpha-2 repeat of the exchanger is almost exclusively responsible for the difference in drug response of the isoforms. Such difference was mostly reproduced by single substitutions of residues in the alpha-2 repeat (V820G or Q826V in NCX1 and A809V or A809I in NCX3), suggesting their importance in drug sensitivity. Cysteine scanning mutagenesis of the alpha-2 repeat of NCX1 identified one residue (Gly833) that caused a large (> or = 30-fold) reduction in drug sensitivity. We found that the Gly-to-Thr substitution caused even larger reduction in drug sensitivity. Interestingly, extracellularly applied KB-R7943 at 0.8 microM markedly inhibited the whole-cell outward exchange current, whereas the drug applied intracellularly at 30 microM did not. These results suggest that KB-R7943 inhibits the exchanger from the external side in intact cells and that a region of the alpha-2 repeat of NCX1 containing Gly833 may participate in the formation of the drug receptor. Because we suggested previously that Gly833 is accessible from the inside of a cell, the results raised an interesting possibility that this residue may alter its position during Na(+)/Ca(2+) exchange in such a way that it becomes accessible to external drug.
Mol Pharmacol 2001 Mar
PMID:Structural domains influencing sensitivity to isothiourea derivative inhibitor KB-R7943 in cardiac Nna(+)/Ca(2+) exchanger. 1117 48

The substituted-cysteine accessibility method (SCAM) was applied to transmembrane span seven of the human A(1) adenosine receptor (hA(1)AR) to reveal a subset of amino acids that are exposed to the ligand-binding crevice. The SCAM approach involved a systematic probe of receptor structure by individual substitutions of residues K265 (7.30) to R296 (7.61) with cysteine. In most cases, hA(1)AR substituted-cysteine mutant membranes displayed antagonist dissociation binding constants that did not differ significantly from wild-type (WT). Radioligand binding assays were used to compare cell membranes that were treated with hydrophilic, sulfhydryl-specific methanethiosulfonate derivatives with control cell membranes. Position H278 was previously reported to be required for A(1)AR ligand binding; however, that report did not establish that H278 represents a contact point for ligands. Cysteine-substitution at H278 yields membrane preparations with greatly decreased receptor density compared with WT membranes from cells in the same transfection experiment. However, H278C membranes retain a measurable fraction of antagonist binding. This observation allows for the investigation of binding-crevice accessibility at position 278 and suggests that H278 may not be required for binding of antagonist ligands. Our data reveal the binding-crevice accessibility of residues T270 (7.35), A273 (7.38), I274 (7.39), T277 (7.42), H278 (7.43), N284 (7.49), and Y288 (7.53) in the hA(1)AR. These data are consistent with the high-resolution structure of bovine rhodopsin that features three alpha-helical turns in this region that are interrupted by an elongated, nonhelical structure from positions 7.43 to 7.48 in the primary amino acid sequence.
Mol Pharmacol 2001 May
PMID:Determination of amino acid residues that are accessible from the ligand binding crevice in the seventh transmembrane-spanning region of the human A(1) adenosine receptor. 1130 3

The 110-amino acid multidrug transporter from E. coli, EmrE, is a member of the family of MiniTexan or Smr drug transporters. EmrE can transport acriflavine, ethidium bromide, tetraphenylphosphonium (TPP+), benzalkonium and several other drugs with relatively high affinities. EmrE is an H+/drug antiporter, utilizing the proton electrochemical gradient generated across the bacterial cytoplasmic membrane by exchanging two protons with one substrate molecule. The EmrE multidrug transporter is unique in its small size and hydrophobic nature. Hydropathic analysis of the EmrE sequence predicts four alpha-helical transmembrane segments. This model is experimentally supported by FTIR studies that confirm the high alpha-helicity of the protein and by high-resolution heteronuclear NMR analysis of the protein structure. The TMS of EmrE are tightly packed in the membrane without any continuous aqueous domain, as was shown by Cysteine scanning experiments. These results suggest the existence of a hydrophobic pathway through which the substrates are translocated. EmrE is functional as a homo-oligomer as suggested by several lines of evidence, including co-reconstitution experiments of wild-type protein with inactive mutants in which negative dominance has been observed. EmrE has only one membrane embedded charged residue, Glu-14, that is conserved in more than fifty homologous proteins and it is a simple model system to study the role of carboxylic residues in ion-coupled transporters. We have used mutagenesis and chemical modification to show that Glu-14 is part of the substrate-binding site. Its role in proton binding and translocation was shown by a study of the effect of pH on ligand binding, uptake, efflux and exchange reactions. We conclude that Glu-14 is an essential part of a binding site, common to substrates and protons. The occupancy of this site is mutually exclusive and provides the basis of the simplest coupling of two fluxes. Because of some of its properties and its size, EmrE provides a unique system to understand mechanisms of substrate recognition and translocation.
J Mol Microbiol Biotechnol 2001 Apr
PMID:Precious things come in little packages. 1132 68

Fetal membranes overlying the cervix in patients prior to and during labour, and within the rupture tear after spontaneous delivery at term, exhibit altered morphology. In this study we report that in comparison to mid-zone fetal membranes biopsies, these regions are characterized by increased expression of the matricellular protein osteonectin or SPARC (Secreted Protein Acidic and Rich in Cysteine). In the reticular layer, the percentage of vimentin positive mesenchymal cells immunoreactive for osteonectin increased in these regions from 3-4% to 25-33% and represented a fraction of the alpha-smooth muscle actin positive myofibroblasts elevated in the same regions. In the fibroblastic layer, the percentage of osteonectin positive cells increased from 1-5% to 8-13%; however, these did not exhibit the same relationship to the alpha-smooth muscle actin positive myofibroblasts in this layer. In the cytotrophoblastic layer the percentage of cytotrophoblastic cells immunoreactive for osteonectin increased from 1% to 6-12%. Elevation of in-situ detectable mRNA was also observed in the same cellular populations in this region. The incidence of cells positive for osteonectin mRNA or protein in the reticular layer correlated with morphological changes. Osteonectin has been implicated in the regulation of extracellular matrix turnover, and its pattern of expression suggests a role in the regional connective tissue and cytotrophoblastic changes proposed to be involved in the cleavage and rupture of fetal membranes.
Mol Hum Reprod 2001 May
PMID:Regional and cellular localization of osteonectin/SPARC expression in connective tissue and cytotrophoblastic layers of human fetal membranes at term. 1133 70

Cysteine-proteinases from parasitic protozoa have been recently characterized as factors of virulence and pathogenicity in several human and veterinary diseases. In Chagas' disease, the chronic infection caused by Trypanosoma cruzi, structure-functional studies on cysteine proteases were thus far limited to the parasite's major isoform, a cathepsin L-like lysosomal protease designated as cruzipain, cruzain or GP57/51. Encoded by a large gene family, cruzipain is efficiently targeted by synthetic inhibitors, which prevent parasite intracellular growth and differentiation. We have previously demonstrated that the multicopy cruzipain gene family includes polymorphic sequences, which could encode functionally different isoforms. We report here a comparative kinetic study between cruzain, the archetype of the cruzipain family, and an isoform, termed cruzipain 2, which is expressed preferentially by the mammalian stages of T. cruzi. Heterologous expression of the catalytic domain of cruzipain 2 in Saccharomyces cerevisae yielded an enzyme that differs markedly from cruzain with respect to pH stability, substrate specificity and sensitivity to inhibition by natural and synthetic inhibitors of cysteine proteases. We suggest that the structural-functional diversification imparted by genetic polymorphism of cruzipain genes may have contributed to T. cruzi adaptation to vertebrate hosts.
Mol Biochem Parasitol 2001 Apr 25
PMID:Cysteine protease isoforms from Trypanosoma cruzi, cruzipain 2 and cruzain, present different substrate preference and susceptibility to inhibitors. 1135 12


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