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PubMedDate not supplied

The utility of Ki-ras mutation analysis in the cytologic diagnosis of pancreatobiliary neoplasma.

PURPOSE: Mutations involved in neoplastic progression may be able to serve as markers for the presence of small numbers of neoplastic cells that would otherwise escape detection in diagnostic assays. Previous retrospective studies have suggested that the sensitivity of the cytologic diagnosis of pancreatic and biliary tract carcinomas is improved when analysis includes Ki-ras exon 1, which is commonly mutated in these neoplasms. We report our experience with the systematic prospective application of Ki-ras gene analysis to the evaluation of fine-needle aspirates and brushings from the pancreatobiliary tract. MATERIALS AND METHODS: Between September 1996 and April 1999, 75 pancreatic fine-needle aspirates and common bile duct brushings submitted for routine cytologic diagnosis were also evaluated for mutations in Ki-ras exon 1 by polymerase chain reaction/single-strand conformation polymorphism analysis. After routine preparation of the specimens, residual material was used for molecular analysis. Results are compared with the morphologic diagnosis and available clinical information. RESULTS: Single-strand conformation polymorphism mutation patterns in Ki-ras were detected in 22 of the 70 consecutive clinical specimens with adequate DNA and at least 6 months of available clinical follow-up. Sensitivity, specificity, and positive predictive value for the presence of concurrent or subsequent pancreatobiliary carcinoma were 33%, 97%, and 93%, respectively, for definitive cytologic diagnosis alone, and 53%, 97%, and 95% for positive Ki-ras single-strand conformation polymorphism mutation pattern alone. If definitive positive cytology or atypical/suspicious cytology with a positive Ki-ras single-strand conformation polymorphism mutation pattern is used, sensitivity is 55%, specificity is 97%, and positive predictive value is 96% for the presence of pancreatobiliary carcinoma. DISCUSSION: Results support the routine use of Ki-ras mutational analysis to increase the sensitivity of the cytologic evaluation of pancreatobiliary fine-needle aspirates and common bile duct brushings with atypical or suspicious morphology without compromising specificity.

Adenocarcinoma↗
PubMed1996

Mutation analysis of the RET proto-oncogene in Dutch families with MEN 2A, MEN 2B and FMTC: two novel mutations and one de novo mutation for MEN 2A.

Hereditary C-cell carcinoma is encountered in multiple endocrine neoplasia type 2A (MEN 2A), MEN 2B, and familial medullary thyroid carcinoma (FMTC). Mutations of the RET proto-oncogene are associated with all three diseases. To obtain an insight into the molecular heterogeneity of MEN 2 syndromes and FMTC in the Netherlands, probands of 20 MEN 2A families, two FMTC families, and seven MEN 2B families were analyzed by the polymerase chain reaction (PCR), DNA sequencing, and restriction enzyme digestion for abnormalities in the RET proto-oncogene. RET mutations were found in all cases. All MEN 2A families had a mutation involving one of five cysteine codons in exons 10 and 11 of RET. Two novel dinucleotide mutations and a de novo mutation were found. Both FMTC families had a mutation of the Cys at codon 618. All MEN 2B probands carried a Met to Thr mutation in exon 16. All mutations could be confirmed by restriction enzyme digestion of PCR amplicons. Identification of the RET mutation in the Dutch population with hereditary C-cell carcinoma facilitates genetic testing for families or individuals at risk for MEN 2A, FMTC, and MEN 2B.

Amino Acid Sequence↗
PubMed1996

Spliced leader RNA of trypanosomes: in vivo mutational analysis reveals extensive and distinct requirements for trans splicing and cap4 formation.

In trypanosomes mRNAs are generated through trans splicing. The spliced leader (SL) RNA, which donates the 5'-terminal mini-exon to each of the protein coding exons, plays a central role in the trans splicing process. We have established in vivo assays to study in detail trans splicing, cap4 modification, and RNP assembly of the SL RNA in the trypanosomatid species Leptomonas seymouri. First, we found that extensive sequences within the mini-exon are required for SL RNA function in vivo, although a conserved length of 39 nt is not essential. In contrast, the intron sequence appears to be surprisingly tolerant to mutation; only the stem-loop II structure is indispensable. The asymmetry of the sequence requirements in the stem I region suggests that this domain may exist in different functional conformations. Second, distinct mini-exon sequences outside the modification site are important for efficient cap4 formation. Third, all SL RNA mutations tested allowed core RNP assembly, suggesting flexible requirements for core protein binding. In sum, the results of our mutational analysis provide evidence for a discrete domain structure of the SL RNA and help to explain the strong phylogenetic conservation of the mini-exon sequence and of the overall SL RNA secondary structure; they also suggest that there may be certain differences between trans splicing in nematodes and trypanosomes. This approach provides a basis for studying RNA-RNA interactions in the trans spliceosome.

Animals↗
PubMed2002

Mutational analysis identifies a short atypical membrane attachment sequence (KYWFYR) within caveolin-1.

Caveolae are vesicular invaginations of the plasma membrane. Their formation is strictly dependent on the expression of the caveolin coat proteins. During transit to the plasma membrane, approximately 15 monomers of caveolin-1 assemble into a multivalent homo-oligomer. Caveolae are most likely generated through the subsequent interaction of these caveolin homo-oligomers with one another, with sphingolipids, and with cholesterol. Membrane association of caveolin-1 is critical to this process and is facilitated by an atypical N-terminal membrane attachment domain (residues 82-101), termed N-MAD. To better understand the membrane attachment function of N-MAD, we performed a detailed mutational analysis of the 20 amino acid N-MAD peptide sequence fused to the C-terminus of the soluble reporter green fluorescent protein (GFP). Removal of the distal six residues (KYWFYR) within N-MAD prevents membrane attachment in cells as assessed by hypotonic lysis, detergent solubility, carbonate extraction, and fluorescence microscopy. These six residues (KYWFYR) are sufficient to confer membrane attachment to GFP, an otherwise soluble protein. Both the central aromatic and flanking basic residues in this sequence are required for membrane attachment, as the sequence YWFY does not confer membrane affinity to GFP. Although the KYWFYR sequence within N-MAD facilitates membrane association, we show that the entire N-MAD sequence is required for targeting to lipid rafts/caveolae.

Amino Acid Sequence↗
PubMed1993

Assembly of the U2 small nuclear ribonucleoprotein from Trypanosoma brucei. A mutational analysis.

trans-Splicing in trypanosomes requires the functions of U2 and U4/U6 small nuclear (sn) RNPs. We have analyzed protein binding and assembly of the Trypanosoma brucei U2 snRNP, using specific antibodies against U2 snRNP proteins and in vitro reconstitution assays of U2 deletion derivatives and human-trypanosome hybrid RNAs. Stable binding of both the U2-specific 40-kDa and the common proteins requires only the 3'-terminal domain (stem-loop IIb, single-stranded region, and stem-loop IV), with loop IV providing the critical sequence determinant; stem-loop IV suffices for binding of the 40 kDa-protein, but not of the common proteins; surprisingly, the sequence of the "Sm-analogous" single-stranded region between stem-loops IIb and IV is not essential for protein binding. Our mutational analysis further indicates that interactions between common and specific proteins play an important role in the assembly of a stable core complex. Finally, a partially assembled U2 RNP complex could be identified as a kinetic intermediate of U2 snRNP assembly. We propose a model of the domain structure and assembly of the trans-spliceosomal U2 snRNP, which deviates in several aspects from that of the cis-spliceosomal U2 snRNP; these differences may be related to the trans-splicing-specific functions of the trypanosomal U2 snRNP.

Animals↗
PubMed1994

Mutational analysis and molecular modeling of the N-terminal kringle-containing domain of hepatocyte growth factor identifies amino acid side chains important for interaction with the c-Met receptor.

The hepatocyte growth factor receptor (HGFr) transduces a wide range of biological signals, including mitogenesis, motogenesis and morphogenesis. We recently localized a region within the N-terminal 175 amino acids of hepatocyte growth factor (HGF), termed HGF/NK1, that is necessary and sufficient for binding to the HGFr. HGF/NK1 contains an as-yet structurally undefined N-terminal region followed by the first of four HGF kringles. We have used a combination of molecular modeling and mutagenesis to dissect the function of this region of HGF. Two mutation-sensitive patches on the proposed surface regions of HGF kringle one (K1) were identified. The first patch consists of residues E159, S161, E195 and R197, all of which are predicted to be close to each other in the tertiary structure of K1. The second patch, lying on the opposite side of the kringle, consists of residues D171 and Q173. Mutational analysis of the N-terminal region of HGF identified residue D117 which also appeared to influence receptor binding. We also investigated the properties of a naturally occurring HGF variant (delta 5-HGF) that arises from an alternatively spliced transcript and therefore lacks five residues within K1. Our data suggest that in wild-type HGF, F162 is crucial in maintaining the hydrophobic core of the kringle. In delta 5-HGF, the loss of this residue is compensated for by a functional substitution of F162 with Y167, which is predicted to occupy the delta 5-HGF K1 core. Comparison of the models of wild-type and delta 5 kringles reveals that the positions of the presumed receptor binding determinants remain unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Heterogeneity in Li-Fraumeni families: p53 mutation analysis and immunohistochemical staining.

We have screened two families for constitutional TP53 mutations, one family with Li-Fraumeni syndrome and the other with features of this syndrome. We report a germline mutation in exon 7 of the TP53 gene in the family with "Li-Fraumeni-like" syndrome. The mutation occurred at codon 245 and causes a Gly-Ser amino acid change. It was inherited by both affected and unaffected subjects. Malignant tumours from all members of this family showed strong positive nuclear immunohistochemical staining with antibodies CM-1 and DO1, directed against TP53. In contrast, no constitutional TP53 mutations were found in a "classic" Li-Fraumeni family. In this family positive staining was seen in both malignant and normal tissues. These results support previous findings that variants of the Li-Fraumeni syndrome exist since not all LFS families carry TP53 germline mutations. Secondly, immunohistochemical positivity is not synonymous with an underlying mutation and is therefore inadequate as an exclusive diagnostic marker.

Adolescent↗
PubMed2003

Site-directed mutational analysis of the novel catalytic domains of alpha-aminoadipate reductase (Lys2p) from Candida albicans.

The alpha-aminoadipate reductase, a novel enzyme in the alpha-aminoadipic acid pathway for the biosynthesis of lysine in fungi, catalyzes the conversion of alpha-aminoadipic acid to alpha-aminoadipic-delta-semialdehyde in the presence of ATP, NADPH and MgCl(2). This reaction requires two distinct gene products, Lys2p and Lys5p. In the presence of CoA, Lys5p posttranslationally activates Lys2p for the alpha-aminoadipate reductase activity. Sequence alignments indicate the presence of all functional domains required for the activation, adenylation, dehydrogenation and alpha-aminoadipic acid binding in the Lys2p. In this report we present the results of site-directed mutational analysis of the conserved amino acid residues in the catalytic domains of Lys2p from the pathogenic yeast Candida albicans. Mutants were generated in the LYS2 sequence of pCaLYS2SEI by PCR mutagenesis and expressed in E. coli BL21 cells. Recombinant mutants and the wild-type Lys2p were analyzed for their alpha-aminoadipate reductase activity. Substitution of threonine 416, glycine 418, serine 419, and lysine 424 of the adenylation domain (TXGSXXXXK, residues 416-424) resulted in a significant reduction in alpha-aminoadipate reductase activity compared to the unmutagenized Lys2p control. Similarly replacement of glycine 978, threonine 980, glycine 981, phenylalanine 982, leucine 983 and glycine 984 of the NADPH binding domain (GXTGFLG, residues 978-984) caused a drastic decrease in alpha-aminoadipate reductase activity. Finally, substitution of histidine 460, aspartic acid 461, proline 462, isoleucine 463, glutamine 464, arginine 465, and aspartic acid 466 of the putative alpha-aminoadipic acid binding domain (HDPIQRD, residues 460-466) resulted in a highly reduced alpha-aminoadipate reductase activity. These results confirm the hypothesis that specific amino acid residues in highly conserved catalytic domains of Lys2p are essential for the alpha-aminoadipate reductase activity.

Adenosine Triphosphate↗
PubMed2006

Insight in eukaryotic ABC transporter function by mutation analysis.

With regard to structure-function relations of ATP-binding cassette (ABC) transporters several intriguing questions are in the spotlight of active research: Why do functional ABC transporters possess two ATP binding and hydrolysis domains together with two ABC signatures and to what extent are the individual nucleotide-binding domains independent or interacting? Where is the substrate-binding site and how is ATP hydrolysis functionally coupled to the transport process itself? Although much progress has been made in the elucidation of the three-dimensional structures of ABC transporters in the last years by several crystallographic studies including novel models for the nucleotide hydrolysis and translocation catalysis, site-directed mutagenesis as well as the identification of natural mutations is still a major tool to evaluate effects of individual amino acids on the overall function of ABC transporters. Apart from alterations in characteristic sequence such as Walker A, Walker B and the ABC signature other parts of ABC proteins were subject to detailed mutagenesis studies including the substrate-binding site or the regulatory domain of CFTR. In this review, we will give a detailed overview of the mutation analysis reported for selected ABC transporters of the ABCB and ABCC subfamilies, namely HsCFTR/ABCC7, HsSUR/ABCC8,9, HsMRP1/ABCC1, HsMRP2/ABCC2, ScYCF1 and P-glycoprotein (Pgp)/MDR1/ABCB1 and their effects on the function of each protein.

ATP-Binding Cassette Transporters↗
PubMed2002

Abietadiene synthase catalysis: mutational analysis of a prenyl diphosphate ionization-initiated cyclization and rearrangement.

Abietadiene synthase catalyzes the committed step in resin acid biosynthesis, forming a mixture of abietadiene double-bond isomers by two sequential, mechanistically distinct cyclizations at separate active sites. The first reaction, protonation-initiated cyclization, converts the universal diterpene precursor geranylgeranyl diphosphate to the stable bicyclic intermediate copalyl diphosphate. In the second, magnesium ion-dependent reaction, diphosphate ester ionization-initiated cyclization generates the tricyclic perhydrophenanthrene-type backbone and is coupled, by intramolecular proton transfer within a transient pimarenyl intermediate, to a 1,2-methyl migration that generates the C13 isopropyl group characteristic of the abietane structure. Alternative deprotonations of the terminal abietenyl carbocation provide a mixture of abietadiene, levopimaradiene, and neoabietadiene, and this product profile varies as a function of pH. Mutational analysis of amino acids at the active site of a modeled structure has identified residues critical for catalysis, as well as several that play roles in specifying product formation, apparently by ligation of a magnesium ion cofactor. These results strongly suggest that choice between alternatives for deprotonation of the abietenyl intermediate depends more on the positioning effects of the carbocation-diphosphate anion reaction partners than on the pKa of multiple participating bases. In one extreme case, mutant N765A is unable to mediate the intramolecular proton transfer and aborts the reaction, without catalyzing 1,2-methyl migration, to produce only sandaracopimaradiene, thereby providing supporting evidence for the corresponding stereochemistry of the cryptic pimarenyl intermediate of the reaction pathway.

Amino Acid Motifs↗
PubMed2000

Mutation analysis of the 5' noncoding regulatory region of the BCL-6 gene in non-Hodgkin lymphoma: evidence for recurrent mutations and intraclonal heterogeneity.

The BCL-6 proto-oncogene is involved in the genesis of non-Hodgkin lymphoma (NHL). Rearrangements due to chromosomal translocations and somatic mutations of the 5' noncoding regulatory region of the BCL-6 gene are potential mechanisms for altering its expression in NHL. To further elucidate the nature of the somatic mutations in the regulatory region of this gene, we have studied 10 healthy donors and 11 NHL biopsy samples by extensive molecular cloning and sequencing. In addition, we analyzed the BCL-6 genes of tumor and nontumor cells from 2 of the cases. The germ line sequence of this region was defined, which differs in 7 positions from that previously reported. In addition, 1 polymorphic variation at position 397(G or C) was identified. Deletions, insertions, and repeated substitution mutations were detected among the molecular isolates in 8 tumor specimens, with a mutational incidence ranging from 1.3 x 10(-3) to 1.3 x 10(-2)/bp (base pair). A total of 20 distinct substitution mutations, 1 insertion and 3 deletions were observed. One of these deletion mutations and 2 of the substitutions were observed in more than 1 tumor specimen from different individuals. In 3 tumor samples, identical mutations affecting both alleles were observed. These findings suggest the presence of mutational hot spots and hot specific events, a finding supported by our compilation of previously published data. In 6 samples, the nucleotide sequences showed evidence of intraclonal heterogeneity, consistent with a stepwise ongoing mutational process affecting the BCL-6 gene in the tumor cells. These mutations accumulating in the regulatory region of the BCL-6 gene could play a role in lymphoma progression and in the transformation of follicular lymphomas to more aggressive large cell lymphomas. (Blood. 2000;95:1400-1405)

Base Sequence↗
PubMed2006

Mutational analysis of the HER2 gene in lung tumors from Caucasian patients: mutations are mainly present in adenocarcinomas with bronchioloalveolar features.

Activating mutations in the tyrosine kinase domain of the HER2 gene have recently been reported in lung adenocarcinomas, mainly in East Asian patients. Our study was devised to evaluate the prevalence and nature of HER2 mutations in lung adenocarcinomas from Caucasian patients. The mutational status of the HER2 gene was evaluated in 403 lung adenocarcinomas by PCR-single strand conformation polymorphism analysis and direct sequencing of Exons 19 and 20. We found HER2 mutations in 9 (2.2%) cases. Seven (78%) of the mutations were in frame duplications/insertions at codons 776-779 (YVMA), the other 2 were base substitutions resulting in aminoacid changes. The hotspot mutation at bases 776-779 was previously found to be the most frequent HER2 mutation in Asiatic patients. The distribution of mutations was significantly different between conventional lung adenocarcinomas (CLAs) and lung adenocarcinomas with bronchioloalveolar features (ABAFs). Seven (6.2%) of 113 ABAFs and 2 (0.7%) of 290 CLA were mutated (p = 0.0025). In addition, the frequency of HER2 mutations was slightly higher in females (4.1%) than in males (1.8%) and in never smokers (3.1%) than in smokers (1.9%), but differences were not statistically significant. This series of tumors was also investigated for EGFR and K-ras mutations. EGFR mutations were observed in 43 (10.7%) cases, and K-ras mutations in 110 (27.3%) cases. EGFR, HER2 and K-ras mutations were found to be mutually exclusive events. The presence of HER2 mutations in a subset of patients with lung adenocarcinoma raise hope to treat these patients with HER2 specific kinase inhibitors.

Adenocarcinoma↗
PubMed2000

Phenotypic features, androgen receptor binding, and mutational analysis in 278 clinical cases reported as androgen insensitivity syndrome.

Androgen insensitivity syndrome (AIS) is the most common single entity that results in male under-masculinization, but large cohort studies of AIS have rarely been performed. Over the last decade, nationwide cooperation between pediatric endocrinologists in the United Kingdom has allowed the creation of a database of cases of intersex and ambiguous genitalia where detailed clinical information on every notified case has been collected via a questionnaire. Among the 816 entries recorded by January 1999, there were 105 clinically diagnosed cases of complete AIS (CAIS) and 173 cases of partial AIS (PAIS). A masculinization score was devised by scoring the external phenotype, and a score of 12 represented normal masculinization. Androgen receptor (AR) binding was determined by studying binding capacity (Bmax) and receptor affinity (K(d)), and cases were classified as either zero, abnormal, or normal binding. Mutation screening of all eight exons of the AR gene was performed by single-strand conformational polymorphism analysis, followed by direct DNA sequencing. All cases of PAIS presented within the first month of birth. The median age at presentation of children with CAIS was 1 yr (P10,P90: 0.1,10.4). The testes were palpable in the labioscrotal folds or the inguinal region in 77% and 41% of cases of CAIS and PAIS, respectively. There was marked overlap between the masculinization score of those children with PAIS reared as girls [2.5(P10,P90:1, 6)] and those reared as boys [3(P10,P90:2, 7.5)]. Gonadectomy was performed prepubertally in 66% and postpubertally in 29% of the cases of CAIS. The median age of the latter group was older at 14 yr (P10,P90:0.1,18). No cases of malignancy or carcinoma in situ were reported in the 121 cases of AIS where histology results were available. Biochemical endocrine investigations were reported to have been performed in a greater number of cases of PAIS than CAIS (98% vs. 48%). AR binding was abnormal in 44 of 51 (86%) and 40 of 113 (35%) cases of CAIS and PAIS, respectively. Zero binding was encountered in 29 of 43 (67%) and 1 of 55 (2%) cases of CAIS and PAIS, respectively. Mutational analysis of the AR gene, performed in 102 index cases was positive in 57 of 69 (83%) cases of CAIS and 12 of 43 (28%) cases of PAIS. In 24 of these cases, the mutation identified was novel. The mutations in PAIS cases were all missense, whereas in CAIS the mutations were more diverse. AR binding was only normal in 3 of 69 mutation-positive cases. In the PAIS group, mutation-positive cases had a significantly higher Kd and Bmax compared to the mutation negative cases. The clinical diagnosis of AIS can be confirmed in a significant number of cases by a combination of androgen-binding studies and mutational analysis. There is some correlation between the phenotypic features and the abnormalities discovered on mutational analysis of the AR gene, but there is a need to improve this further by developing optimal bioassays of AR function. The phenotypic heterogeneity among clinically diagnosed cases of AIS emphasizes the need for appropriate comprehensive evaluation of male under-masculinization.

Amino Acid Sequence↗
PubMed2005

Mutation analysis of HNF-4 binding sites in the human glucose-6-phosphatase promoter.

HNF-4, a member of the nuclear receptor superfamily, binds to HNF-4 response elements (HRE), consisting of a direct repeat of the hexameric half-sites spaced by 1 nt (direct repeat 1) and activates a number of genes, which play central roles in fatty acids and glucose metabolism. Glucose-6-phosphatase (G6Pase) catalyzes the terminal step in the gluconeogenic and glycogenolytic pathways. A previous study has shown that HNF-4 binds to two DR1s in the regions A (located between -266 and -234) and B (located between -306 and -274) on the human G6Pase promoter. We found that the region B contains the one more DR1 element, composed of the two half-sites, designated half-sites a and b, the latter of which overlaps with the previously identified DR1 consisting of two half-sites, designated half-sites b and c. In this study, electrophoretic mobility shift assay (EMSA) using point mutations in each half-site a, b, or c indicated that HNF-4 binds to the combination of half-sites a and b, but not to half-sites b and c. Furthermore, mutational analysis demonstrated that, in the context of the human G6Pase promoter, the half-sites a and b, but not the half-sites b and c, are required for the stimulatory effect of HNF-4. These results suggested that the DR1 element containing the half-sites a and b is a functional HRE that mediates the induction of hG6Pase promoter activity by HNF-4.

Base Sequence↗
PubMed2001

Mutational analysis of the PTEN gene in endometrial carcinoma and hyperplasia.

To determine the potential role of PTEN in the process of endometrial carcinogenesis, we examined a series of endometrial carcinoma and hyperplasia of the uterine corpus for the presence of a PTEN mutation. The entire coding region of the gene was screened for the presence of mutations by single-strand conformation polymorphism analysis, and mutations were confirmed by sequencing. We detected mutations in 14 of 57 endometrial carcinomas (13 of 50 endometrioid adenocarcinomas and 1 of 7 nonendometrioid adenocarcinomas) and 7 of 73 endometrial hyperplasias (1 of 24 simple hyperplasias without atypia, none of 16 complex hyperplasias without atypia, and 6 of 33 complex hyperplasias with atypia). Most (88%) mutations were clustered in exons 5, 7, and 8. Of the 24 mutations detected in 21 cases, 12 were frameshifts, 9 were nonsense, 2 were missense, and 1 was a silent mutation. Patients with a PTEN mutation had a better prognosis than those with no PTEN mutation. The presence of PTEN mutations in hyperplasia suggests that PTEN inactivation may occur as an initiating event in endometrial carcinogenesis and is involved in the development of cytologic atypia in hyperplasia.

Carcinoma↗
PubMed1998

Crystal structure and mutational analysis of the Escherichia coli putrescine receptor. Structural basis for substrate specificity.

PotF protein is a periplasmic substrate-binding protein of the putrescine transport system in Escherichia coli. We have determined the crystal structure of PotF protein in complex with the substrate at 2.3-A resolution. The PotF molecule has dimensions of 54 x 42 x 30 A and consists of two similar globular domains. The PotF structure is reminiscent of other periplasmic receptors with a highest structural homology to another polyamine-binding protein, PotD. Putrescine is tightly bound in the deep cleft between the two domains of PotF through 12 hydrogen bonds and 36 van der Waals interactions. The comparison of the PotF structure with that of PotD provides the insight into the differences in the specificity between the two proteins. The PotF structure, in combination with the mutational analysis, revealed the residues crucial for putrescine binding (Trp-37, Ser-85, Glu-185, Trp-244, Asp-247, and Asp-278) and the importance of water molecules for putrescine recognition.

Base Sequence↗
PubMed1999

Mutational analysis of glycosylation, membrane translocation, and cell surface expression of the hepatitis E virus ORF2 protein.

Hepatitis E virus (HEV) is the etiological agent for viral hepatitis type E, which is a major problem in the developing world. Because HEV cannot be cultured in vitro, very little information exists on the mechanisms of HEV gene expression and genome replication. HEV is a positive-strand RNA virus with three potential open reading frames (ORFs), one of which (ORF2) is postulated to encode the major viral capsid protein (pORF2). We earlier showed (S. Jameel, M. Zafrullah, M. H. Ozdener, and S. K. Panda, J. Virol. 70:207-216, 1996) pORF2 to be a approximately 88-kDa glycoprotein, carrying N-linked glycans and a potential endoplasmic reticulum (ER)-directing signal at its N terminus. Treatment with the drugs brefeldin A and monensin suggest that the protein may accumulate within the ER. Based on mutational analysis, we demonstrate Asn-310 to be the major site of N-glycan addition. In COS-1 cell expression and in vitro translation experiments, we confirm the ER-translocating nature of the pORF2 N-terminal hydrophobic sequence and show that the protein is cotranslationally, but not posttranslationally, translocated across the ER membrane. Earlier, we had also demonstrated cell surface localization of a fraction of the COS-1 cell-expressed pORF2. Using glycosylation- and translocation-defective mutants of pORF2, we now show that while transit of pORF2 into the ER is necessary for its cell surface expression, glycosylation of the protein is not required for such localization. These results may offer clues to the mechanisms of gene expression and capsid assembly in HEV.

Amino Acid Sequence↗
PubMed2004

Mutational analysis of peptidyl carrier protein and acyl carrier protein synthase unveils residues involved in protein-protein recognition.

4'-Phosphopantetheinyl transferases (PPTases) are essential for the production of fatty acids by fatty acid synthases (primary metabolism) and natural products by nonribosomal peptide synthetases and polyketide synthases (secondary metabolism). These systems contain carrier proteins (CPs) for the covalent binding of reaction intermediates during synthesis. PPTases transfer the 4'-phosphopantetheine moiety from coenzyme A (CoA) onto conserved serine residues of the apo-CPs to convert them to their functionally active holo form. In bacteria, two types of PPTases exist that are evolutionary related but differ in their substrate spectrum. Acyl carrier protein synthases (AcpSs) recognize CPs from primary metabolism, whereas Sfp- (surfactin production-) type PPTases have a preference for CPs of secondary metabolism. Previous investigations showed that a peptidyl carrier protein (PCP) of secondary metabolism can be altered to serve as substrate for AcpS. We demonstrate here that a single mutation in PCP suffices for the modification of this CP by AcpS, and we have identified by mutational analysis several other PCP residues and two AcpS residues involved in substrate discrimination by this PPTase. These altered PCPs were still capable of serving their designated function in NRPS modules, and selective use of AcpS or Sfp leads to production of two different products by a trimodular NRPS.

Bacterial Proteins↗