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Results for “mutational profiling”

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PubMed2001

Can an optimization/scoring procedure in ligand-protein docking be employed to probe drug-resistant mutations in proteins?

A simple ligand-protein structural optimization and binding evaluation procedure has been routinely used in high-speed ligand-protein docking studies. In this work, we examine whether such an optimization/scoring procedure is useful in indicating possible drug-resistant mutations in proteins. Crystal structures of three wild-type enzymes (HIV-1 protease, HIV-1 reverse transcriptase, and Mycobacterium tuberculosis H37Rv enoyl-ACP reductase) complexed to a variety of inhibitors are studied. Mutations are introduced into these structures by using the molecular modeling software, SYBYL. Structural optimization and scoring of a mutant complex is conducted by a procedure similar to that used in a recent docking study (Wang et al., 1999). The computed results are compared with observed drug resistance data and the profile of nonresistant mutations. Most mutations studied show an energy change in the same direction as those indicated by observed resistance data. 50% of the polar to polar or nonpolar to nonpolar mutations are found to correlate qualitatively with observed drug resistance data. Van der Waals interactions account for most of these changes, which is in agreement with conclusions from structural studies. Substantially larger deviations are found between computed results and observed data for most polar to nonpolar or nonpolar to polar mutations, which result from deficiency in modelling and scoring ligand-protein interactions in our procedure. Our results suggest that an optimization/docking scoring procedure is useful for qualitatively probing polar to polar or nonpolar to nonpolar resistant mutations in addition to its application in screening active compounds. More accurate description of ligand-protein interactions and the use of methods such as free energy perturbation and Poisson-Boltzmann may be needed to further improve the quality of prediction.

Computer Simulation↗

PubMed1998

Characterization of monoclonal antibodies to calpain 3 and protein expression in muscle from patients with limb-girdle muscular dystrophy type 2A.

Monoclonal antibodies were raised to two regions of calpain 3 (muscle-specific calcium-activated neutral protease), which is the product of the gene that is defective in limb-girdle muscular dystrophy type 2A. The antibodies produced characteristic patterns of bands on Western blots: normal calpain 3 protein was represented by bands at 94 kd, plus additional fragments at approximately 60 or 30 kd, according to the antibody used. Specificity was confirmed by the loss of all bands in patients with null gene mutations. The "normal" profile of bands was observed in muscle from 33 control subjects and 70 disease-control patients. Calpain 3 protein was found to be extremely stable in fresh human muscle, with full-size protein being detected 8 hours after the muscle had been removed. Blots of muscle from nine limb-girdle muscular dystrophy type 2A patients with defined mutations showed variation in protein expression, with seven showing a clear reduction in the abundance of protein detected. No simple relationship was found between the abundance and clinical severity. Two patients showed normal expression of the full-size 94 kd band accompanied by a clear reduction in the smaller fragments. This pattern was also observed in one patient with an undefined form of limb-girdle dystrophy. These results indicate that immunodiagnosis is feasible, but caution will need to be exercised with the interpretation of near-normal protein profiles.

Adult↗

PubMed2026

Gastroenteropancreatic Neuroendocrine Carcinoma (GEP-NEC): An Aggressive Disease Course and Limitations for Personalized Oncology.

Neuroendocrine carcinoma (NEC) is a rare, aggressive malignancy with limited treatment options and poor prognosis. We report a male patient diagnosed with a gastroenteropancreatic (GEP)-NEC with synchronous liver metastasis at the time of surgery who underwent a radical resection attempt. Despite radical-intent surgery followed by adjuvant carboplatin/etoposide, early recurrence developed with progression through multiple subsequent chemotherapy lines. During the treatment process, genetic profiling was performed twice to identify actionable genomic targets, with inclusion in the national IMPRESS study as a last resort. Comprehensive genomic profiling revealed TP53 mutation and RB1 loss but no actionable alterations. A patient-derived organoid (PDO) was successfully established from resected tumor tissue and retained key neuroendocrine and proliferative features, with partial genomic concordance to the primary tumor. Differences between the primary and subsequent PDO in variant allele frequencies suggest clonal selection during culture. Exploratory metabolomic profiling of tryptophan pathway metabolites in patient serum and PDO-culture media indicated tumor-associated metabolic alterations. We present clinical and translational efforts in difficult-to-treat NEC, illustrating both the translational challenges and the potential role of PDOs in advancing personalized treatment strategies for a cancer with very limited treatment options.

Gastroenteropancreatic neuroendocrine carcinoma↗

PubMed2004

Studies of the enzymic mechanism of Candida tenuis xylose reductase (AKR 2B5): X-ray structure and catalytic reaction profile for the H113A mutant.

Xylose reductase from the yeast Candida tenuis (CtXR) is a family 2 member of the aldo-keto reductase (AKR) superfamily of proteins and enzymes. Active site His-113 is conserved among AKRs, but a unified mechanism of how it affects catalytic activity is outstanding. We have replaced His-113 by alanine using site-directed mutagenesis, determined a 2.2 A structure of H113A mutant bound to NADP(+), and compared catalytic reaction profiles of NADH-dependent reduction of different aldehydes catalyzed by the wild type and the mutant. Deuterium kinetic isotope effects (KIEs) on k(cat) and k(cat)/K(m xylose) show that, relative to the wild type, the hydride transfer rate constant (k(7) approximately 0.16 s(-1)) has decreased about 1000-fold in H113A whereas xylose binding was not strongly affected. No solvent isotope effect was seen on k(cat) and k(cat)/K(m xylose) for H113A, suggesting that proton transfer has not become rate-limiting as a result of the mutation. The pH profiles of log(k(cat)/K(m xylose)) for the wild type and H113A decreased above apparent pK(a) values of 8.85 and 7.63, respectively. The DeltapK(a) of -1.2 pH units likely reflects a proximally disruptive character of the mutation, affecting the position of Asp-50. A steady-state kinetic analysis for H113A-catalyzed reduction of a homologous series of meta-substituted benzaldehyde derivatives was carried out, and quantitative structure-reactivity correlations were used to factor the observed kinetic substituent effect on k(cat) and k(cat)/K(m aldehyde) into an electronic effect and bonding effects (which are lacking in the wild type). Using the Hammett sigma scale, electronic parameter coefficients (rho) of +0.64 (k(cat)) and +0.78 (k(cat)/K(m aldehyde)) were calculated and clearly differ from rho(k(cat)/K(aldehyde)) and rho(k(cat)) values of +1.67 and approximately 0.0, respectively, for the wild-type enzyme. Hydride transfer rate constants of H113A, calculated from kinetic parameters and KIE data, display a substituent dependence not seen in the corresponding wild-type enzyme rate constants. An enzymic mechanism is proposed in which His-113, through a hydrogen bond from Nepsilon2 to aldehyde O1, assists in catalysis by optimizing the C=O bond charge separation and orbital alignment in the ternary complex.

Alanine↗

PubMed2006

pncA mutations in pyrazinamide-resistant Mycobacterium tuberculosis clinical isolates from the southeast region of Brazil.

OBJECTIVES: To investigate the presence of mutations in the pncA gene in 31 pyrazinamide-resistant Mycobacterium tuberculosis and 5 susceptible strains. MICs and pyrazinamidase (PZase) activity were also determined. METHODS: All 36 M. tuberculosis clinical isolates were genotyped by mycobacterial interspersed repetitive units (MIRUs) and most were also typed by spoligotyping. The MIC value necessary to inhibit 99% of the resistant mycobacterial isolates was determined by microplate Alamar Blue assay (MABA) and by Löwenstein-Jensen assay (LJA). The PZase activity was measured by pyrazinamide deamination to pyrazinoic acid and ammonia, and the entire pncA sequence including the 410 bp upstream from the start codon was determined by DNA sequencing of purified PCR products. RESULTS: Of the 31 isolates resistant to pyrazinamide, 26 (83.9%) showed at least one mutation in the pncA gene or in its putative regulatory region. Among the 22 different mutations detected in the pncA gene and in its regulatory region, 9 (40.9%) mutations (consisting of six substitutions, two insertions and one deletion) have not been described in previous studies. Three pyrazinamide-resistant isolates, confirmed by MIC varying from 800 to 1600 mg/L, carried the wild-type pncA sequence and retained PZase activity. CONCLUSIONS: These results contribute to the knowledge of the molecular mechanism of pyrazinamide resistance in Brazil and also expand the profile of pncA mutations worldwide. The MABA was successfully used to determine the MICs of pyrazinamide.

Amidohydrolases↗

PubMed2005

Detection of three common G6PD gene mutations in Chinese individuals by probe melting curves.

OBJECTIVE: In order to develop and validate an assay for rapid detection of three common G6PD gene mutations in Chinese individuals. METHODS: In this report we design two sets of primers and fluorescently labeled hybridization probes recognizing adjacent sequences in the amplicon; after annealing, the fluorophores were in resonance energy transfer, providing real-time monitoring of the amplication process. At the completion of PCR, fluorescence was monitored as the temperature increased through the Tm of the probe/product duplex, and a characteristic melting profile for each mutation was obtained. By using the fluorescence method and PCR/RE, a total of 57 samples obtained from two groups of G6PD-deficient individuals were studied. RESULT: A rapid method for detection of three common G6PD gene mutations in Chinese individuals by probe melting curves was developed. This method shows 100% accordance with the traditional method. CONCLUSION: This fluorescent melting curve analysis is a simple, rapid, and effective method for clinical diagnosis and screening of G6PD deficiency.

China↗

PubMed2007

New insight into BRAF mutations in cancer.

There has been much recent progress in our understanding of the role played by the RAS-RAF-MEK-ERK cascade in human cancer. RAS is an oncogene and this pathway is known to promote proliferation and malignant transformation. More recently, however, RAF has become the focus of attention, particularly in melanoma, where approximately 70% of cases carry mutations in the BRAF gene. The majority of the mutations in BRAF in cancer are activating, but rare mutants that cannot activate MEK have provided new insight into RAF signalling networks that exist in cancer and normal cells. Surprisingly, germline mutations in BRAF that occur in rare genetic syndromes have also recently been described. The induction of BRAF mutations in melanoma depends on the type of UV exposure that the skin receives, and some studies have suggested the existence of susceptibility loci that make it more likely that some individuals will acquire these mutations. Importantly, genetic profiling and microarray studies have provided insight into the spectrum of melanomas in which BRAF plays a role and also revealed intriguing new data that could be important for the diagnosis and treatment of human cancers.

Gene Regulatory Networks↗

PubMed1993

Dose-dependent ras mutation spectra in N-nitrosodiethylamine induced mouse liver tumors and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone induced mouse lung tumors.

In a previous study, the spectrum of H-ras mutations detected in B6C3F1 mouse liver tumors induced by 5, 50 or 150 mumol/kg body wt of N-nitrosodiethylamine (NDEA) was similar to that in spontaneous B6C3F1 mouse liver tumors, suggesting that activation of the H-ras gene in NDEA-induced mouse liver tumors may not be the direct result of the chemical interaction with the H-ras gene. In the present study, mutations in the H-ras oncogene from B6C3F1 mouse liver tumors induced by 5 or 50 mumol/kg body wt of NDEA were characterized by DNA amplification with polymerase chain reaction (PCR), single-strand conformation of polymorphism (SSCP) and direct sequence analysis. Twenty-one of 66 NDEA-induced B6C3F1 mouse liver tumors contained activated H-ras gene with 2 of 21 having a CG to AT transversion at the first base of codon 61, 17 of 21 having AT to GC transition and 2 of 21 having an AT to TA transversion at the second base of codon 61 in the H-ras gene. The predominant mutation, AT to GC transition (17/21, 81%) is consistent with the formation of O4-ethylthymine adduct, and is distinct from the predominant CG to AT transversion (50%) at the first base of codon 61 detected in H-ras gene from NDEA-induced B6C3F1 mouse liver tumors in a previous study by Stowers et al. Mutations in the K-ras oncogene from 59 A/J mouse lung tumors induced by 0.53 mmol/kg body wt of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were also characterized by using the above mentioned methods. Forty-six of 59 NNK-induced A/J mouse lung tumors contained activated K-ras genes. All 46 (100%) of the activated K-ras gene had GC to AT transitions at the second base of codon 12. The same mutation was observed in 70% (7/10) of the K-ras oncogene from A/J lung tumors induced by 4.8 mmol/kg body wt (given in 21 doses) of NNK. These data suggest that other factors in addition to genotoxic effect might be involved in the induction of rodent tumors by some carcinogens when given at higher doses. Therefore, further studies to compare the dose-dependent differences in the profile of ras mutations induced by chemical carcinogens may help to assess human cancer risk. Mutation(s) in exons 5-8 of the p53 gene was not found in these NDEA-induced mouse liver tumors and NNK-induced mouse lung tumors.

Animals↗

PubMed2005

Tissue-specific time courses of spontaneous mutation frequency and deviations in mutation pattern are observed in middle to late adulthood in Big Blue mice.

To better define the time course of spontaneous mutation frequency in middle to late adulthood of the mouse, measurements were made at 10, 14, 17, 23, 25, and 30 months of age in samples of adipose tissue, liver, cerebellum (90% neurons), and the male germline (95% germ cells). A total of 46 million plaque-forming units (pfus) were screened at the six time points and 1,450 circular blue plaques were harvested and sequenced. These data improve resolution and confirm the previously observed occurrence of at least two tissue-specific profiles of spontaneous mutation frequency (elevation with age in adipose tissue and liver, and constancy with age in neurons and male germ cells), a low mutation frequency in the male germline, and a mutation pattern unchanged with age within a tissue. These findings appear to extend to very old age (30 months). Additional findings include interanimal variation in spontaneous mutation frequency is larger in adipose tissues and liver compared with neurons and male germ cells, and subtle but significant differences in the mutation pattern among tissues, consistent with a minor effect of tissue-specific metabolism. The presumptive unaltered balance of DNA damage and repair with age in the male germline has evolutionary consequences. It is of particular interest given the controversy over whether or not increasing germline mutation frequency with paternal age underlies the reports associating older males with a higher incidence of some types of genetic disease. These most detailed measurements available to date regarding the time course of spontaneous mutation frequency and pattern in individual tissues help to constrain hypotheses regarding the role of mutational mechanisms in DNA repair and aging.

Adipose Tissue↗

PubMed2004

[Expression profile and prognostic significance of CD24, p53 and p21 in lymphomas. A tissue microarray study of over 600 non-Hodgkin lymphomas].

BACKGROUND AND OBJECTIVE: Tissue microarrays allow the simultanous, rapid and standardized in-situ analysis of up to 1000 different tissue samples on a single glass slide. We investigated the expression profil and the prognostic significance of CD24, p53 and p21 using a Non-Hodgkin-Lymphoma tissue microarray. METHODS: Over 600 well documented Non-Hodgkin-Lymphomas, consisting of 341 diffuse large B-cell lymphomas, 86 follicular lymphomas, 81 chronic lymphatic leukemias/small lymphocytic lymphomas, 51 primary mediastinal diffuse large B-cell lymphomas, 26 mantle cell lymphomas, 8 lymphoplasmocytic lymphomas, 8 T-cell-lymphomas NOS und 2 Burkitt lymphomas were brought into array format. The expression of CD24, p53 and p21 was analysed semiquantitatively by immunohistochemistry. The immunophenotype p53+/p21- (Deltap53) was used as a surrogat for p53 gene mutations. The expression profile was compared to clinical data and the overall survival in the subgroup of diffuse large B-cell lymphomas. RESULTS: 91% of the analyzed Non-Hodgkin-lymphomas (473 of 522 cases) showed CD24 positivity. CD24 Expression was not associated with survival. Deltap53 was found in thirteen percent of all lymphomas (70 of 539 cases), the subgroup of the diffuse large B-cell lymphomas demonstrated the highest Deltap53 (21%). In a multivariate cox regression analysis, a high international prognostic index and Deltap53 were independent markers of bad survival. CONCLUSION: The TMA-technology allows also in lymphoma research the simultanous, cost-effective and standardized analysis of large patient cohorts. Deltap53 revealed as an independent negative prognostic factor in diffuse large B-cell lymphomas. It can easily be determined in daily routine practice.

Adolescent↗

PubMed2006

Acute myeloid leukemia carrying cytoplasmic/mutated nucleophosmin (NPMc+ AML): biologic and clinical features.

The nucleophosmin (NPM1) gene encodes for a multifunctional nucleocytoplasmic shuttling protein that is localized mainly in the nucleolus. NPM1 mutations occur in 50% to 60% of adult acute myeloid leukemia with normal karyotype (AML-NK) and generate NPM mutants that localize aberrantly in the leukemic-cell cytoplasm, hence the term NPM-cytoplasmic positive (NPMc+ AML). Cytoplasmic NPM accumulation is caused by the concerted action of 2 alterations at mutant C-terminus, that is, changes of tryptophan(s) 288 and 290 (or only 290) and creation of an additional nuclear export signal (NES) motif. NPMc+ AML shows increased frequency in adults and females, wide morphologic spectrum, multilineage involvement, high frequency of FLT3-ITD, CD34 negativity, and a distinct gene-expression profile. Analysis of mutated NPM has important clinical and pathologic applications. Immunohistochemical detection of cytoplasmic NPM predicts NPM1 mutations and helps rationalize cytogenetic/molecular studies in AML. NPM1 mutations in absence of FLT3-ITD identify a prognostically favorable subgroup in the heterogeneous AML-NK category. Due to their frequency and stability, NPM1 mutations may become a new tool for monitoring minimal residual disease in AML-NK. Future studies should focus on clarifying how NPM mutants promote leukemia, integrating NPMc+ AML in the upcoming World Health Organization leukemia classification, and eventually developing specific antileukemic drugs.

Active Transport, Cell Nucleus↗

PubMed2002

The Liverpool Lung Project: a molecular epidemiological study of early lung cancer detection.

Lung cancer is the major cause of death from neoplastic disease in the world, and even with politically-motivated smoking cessation campaigns throughout Europe, the disease remains the major cause of death. The development of molecular epidemiological population-based research into early lung cancer detection, such as the Liverpool Lung Project (LLP), may provide a way forward. This is the first major molecular epidemiological study of detection of early lung cancer. The use of molecular epidemiological risk assessments prior to clinical diagnosis and markers of preclinical carcinogenesis in patients with a high risk of developing lung cancer will reduce the incidence of clinically-detectable lung cancer, given the appropriate intervention strategies. The aims are as follows: 1) to prepare a molecular genetic and epidemiological risk assessment model based on environmental exposures and genetic predisposition; 2) to develop an archive of specimens relating to at-risk individuals and those with lung cancer; 3) to redefine lung cancer based on molecular pathology using the fields of expression profiling, genetic instability and molecular cytogenetics; 4) to identify and assess novel markers of precarcinogenesis in high-risk populations; and 5) to facilitate the development of new treatment strategies (e.g. chemoprevention programmes and targeted drug therapies). The LLP has two components: 1) a case-controlled study of newly-diagnosed cases of lung cancer that will provide a baseline, risk assessment; and 2) a prospective cohort study to be carried out over a 10-yr period that will identify markers of preclinical carcinogenesis. In-depth interviews are carried out using structured and semi-structured questionnaires. Sputum, blood and tumour specimens are collected and will be assessed for specific molecular markers (e.g. genetic instability, mutation and expression profiling, and methylation status). Conclusions from The Liverpool Lung Project will be based around molecular-epidemiological and genotyping risk assessment models, as well as redefining the disease, and ultimately contributing to the development of new early lung cancer detection and treatment strategies.

Age Factors↗

PubMed2005

Large-scale screens for cDNAs with in vivo activity.

The pace of biological investigation has been greatly accelerated by technologies that permit comprehensive inventory of gene expression under diverse conditions. We have been developing and using approaches to understand gene function that are based on functional assessments of gene activity, using automated expression cloning followed by gene knockdown using shRNA or by gene knockout. Some recent developments in expression screens and methods to expedite the formation of targeted mutations in mice are discussed. A resource for large scale quantitative profiling of mRNA abundance is also described.

Animals↗

PubMed2000

Immunohistochemical detection of mismatch repair gene proteins as a useful tool for the identification of colorectal carcinoma with the mutator phenotype.

There are two well-defined pathways for colorectal carcinogenesis, the suppressor and the mutator pathways. The latter is characteristic of hereditary non-polyposis colorectal cancer (HNPCC), but can also be found in a subset of sporadic colorectal cancer (SCC) possessing distinctive clinical and pathological features, namely early age of onset, location in the right colon, poor differentiation, and a predominant mucinous component. This mutator pathway results from inactivation of mismatch repair (MMR) genes, namely MSH2 and MLH1. The aim of this study was to ascertain if abnormal MMR protein gene expression is a good indicator for identifying tumours from the mutator pathway. Seventy-six cases of SCC were studied by immunohistochemistry using two monoclonal mouse antibodies that react against MSH2 and MLH1 protein gene products. Immunoexpression was assessed both in tumour and in non-neoplastic, adjacent and distant mucosa. Microsatellite instability (MSI) was detected by evaluating the length of poly(CA) repeated sequences at seven loci, or by the detection of small unstable alleles in a poly(A) repeat - BAT-26. Except for BAT-26, in which only tumour DNA was used, MSI analysis was performed in both tumour and normal mucosal DNA. MSI was classified as high (MSI-H), low (MSI-L) or stable (MSS). Abnormal protein expression was found in 9/76 (12%) tumours. Immunohistochemistry for hmlh1 and hmsh2 detected 75% of MSI-H. There was also a highly significant correlation between the observed immunoexpression and several clinical and pathological characteristics described as the phenotypic profile of the mutator pathway, such as right-sided location (p=0.003), mucin production (p=0.008), and a peritumoural lymphoid infiltrate (p=0.009). Non-neoplastic adjacent mucosa showed normal hMSH2 expression in all cases, but in ten cases there was no hMLH1 expression in this transitional mucosa, which is known to display an alterated mucin pattern and a high proliferative rate. These results demonstrated a good correlation between hMLH1 and hMSH2 gene immunoexpression and the clinico-pathological features characteristic of the mutator phenotype and support the use of this method as a rapid and efficient way to detect tumours arising from this pathway.

Adult↗

PubMed1992

Evaluation of genotoxicity, pathological lesions, and cell proliferation in livers of rats and mice treated with furan.

Preliminary results from the National Toxicology Program (NTP) bioassays of furan given by gavage indicate the induction of hepatocellular carcinomas in male F-344 rats and in both sexes of B6C3F1 mice, and cholangiocarcinomas in both sexes of rats. To assess the genotoxicity of furan, chemically induced unscheduled DNA synthesis was evaluated in the in vivo hepatocyte DNA repair assay. Furan did not induce unscheduled DNA synthesis in hepatocytes isolated after single gavage treatment of male F-344 rats (5, 30, and 100 mg/kg) or male B6C3F1 mice (10, 50, 100, and 200 mg/kg). Furan induced cytotoxicity and enhanced cell proliferation were evaluated in livers of rats and mice as events that also might give rise to mutations and/or drive tumor formation. The labeling index (LI, percentage of hepatocyte nuclei in S-phase) was measured histoautoradiographically following a single gavage administration of furan (30 mg/kg, male rats; 50 mg/kg, male mice) followed by an injection of 3H-thymidine 2 hr prior to sacrifice. Hepatocellular necrosis and a sharp increase in LI (23.9 for mice and 17.8 for rats vs. less than 0.5 for controls) was observed 48 hr after treatment with furan, indicative of restorative cell proliferation secondary to cytotoxicity. Hepatocyte proliferation was evaluated also at the highest NTP bioassay dose (15 mg/kg/day for mice and 8 mg/kg/day for rats, 5 days/week) by labeling with 3H-thymidine administered via a 6 day osmotic pump implanted subcutaneously. Necrosis and inflammation were observed along the subcapsular visceral surface of the left or caudate liver lobes, likely due to diffusion of furan directly through the stomach to the liver. After 6 weeks of furan administration, male and female rats, but not mice, exhibited bile duct hyperplasia as well as metaplasia in the areas of fibrosis along the subcapsular visceral surface of the left or caudate liver lobes. The fold increase in hepatocyte LI in treated animals relative to the combined controls measured at weeks 1, 3, and 6 ranged from 39 to 5 for male mice, 18 to 51 for male rats, and 12 to 19 for female rats. Taken together, these data suggest that mechanisms other than direct DNA-reactivity might explain the profile of oncogene mutations observed in the mouse liver tumors, including selective promotion of different subpopulations of preneoplastic cells and/or mutational events secondary to sustained cell proliferation or inflammation. The extensive amount of furan-induced cell proliferation subsequent to cytotoxicity likely had a significant impact on tumor development, and such data should be considered in risk evaluations for this compound.

Animals↗

PubMed2004

Conservation and covariance in PH domain sequences: physicochemical profile and information theoretical analysis of XLA-causing mutations in the Btk PH domain.

Mutations that cause X-linked agammaglobulinemia (XLA) appear throughout the Bruton tyrosine kinase (Btk) sequence, including the pleckstrin homology (PH) domain. To analyze the basis of this disease with respect to protein structure, we studied the relationships between PH domain sequences and structures by comparing sequence-based profiles of physicochemical properties and solvent accessibility profiles. The diversity of the distribution of amino acids was measured by calculating entropies for sequences containing mutations at different positions in multiple sequence alignments. Mutual information was calculated to quantify positional covariation. Eight conserved extrema were apparent in all profiles. The majority of the XLA disease-causing mutations in the Btk PH domain were found at positions having significant mutual information, indicating that there are covariant constraints for both structure and function. Together with additional structural analyses, all the XLA mutations that were analyzed could be explained at the molecular level. The method developed here is applicable to the design of mutations for protein engineering.

Agammaglobulinemia↗

PubMed1998

Substitution of leucine 8 in the simian immunodeficiency virus matrix protein impairs particle formation without affecting N-myristylation of the Gag precursor.

In the late stages of replication of the simian immunodeficiency viruses (SIV), the matrix protein (MA) plays a central role in the transport of Pr55gag to the plasma membrane, assembly of virus particles, and incorporation of the envelope glycoprotein into particles. Targeting of Pr55gag to the plasma membrane is mediated by two motifs within the MA protein: the N-terminal myristate and a cluster of positively charged amino acids. In this report, we characterized the assembly phenotype of an SIV Gag mutant (L8Q) carrying the single amino acid substitution of glutamine for leucine at position 8 in the MA domain. The hydropathic profile of the mutated MA protein indicated that the L8Q amino acid change disrupts the hydrophobic character of the region comprising the first 10 residues of the protein. Expression of mutant L8Q Gag protein in CV-1 cells, by means of the vaccinia virus vector system, resulted in efficient synthesis and N-myristylation of Pr55gag. However, this mutation severely impaired particle production, as inferred from both biochemical and electron microscopy analyses. Cellular fractionation assays revealed that in cells expressing mutant L8Q, the proportion of cytosol-associated Pr55gag was significantly increased compared to that observed upon expression of wild-type Gag. Furthermore, mutant L8Q Gag partitioned onto cytosol and membrane fractions in a manner similar to nonmyristylated Gag polyprotein. Taken together, these results indicate that the L8Q mutation reduces the membrane-binding capacity of the Gag precursor. It is therefore likely that in the SIV MA, in addition to the N-myristate group, the hydrophobicity of the neighboring region is important for efficient association of Pr55gag with the plasma membrane.

Amino Acid Sequence↗

PubMed2002

The little difference: in vivo analysis of pheromone discrimination in Schizophyllum commune.

The B mating type of Schizophyllum commune is defined by a multi-specific pheromone/receptor system. The interaction of pheromone receptors and their ligands, encoded by the Balpha locus, triggers sexual development. The receptors belong to the family of G protein-coupled seven-transmembrane-domain receptors, while the ligands are small lipopeptide pheromones. A productive interaction is only possible between molecules derived from different specificities. There is no induction of sexual development by pheromones of self-specificity. Since there are nine versions of different specificity for pheromones and receptors in Balpha, this system can be used to study multi-ligand discrimination. We investigated pheromone discrimination using chimeric receptor molecules and the influence of single point mutations on activation profiles of the receptor.

Base Sequence↗