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PubMed2004

Crystal structure of Mycobacterium tuberculosis catalase-peroxidase.

The Mycobacterium tuberculosis catalase-peroxidase is a multifunctional heme-dependent enzyme that activates the core anti-tuberculosis drug isoniazid. Numerous studies have been undertaken to elucidate the enzyme-dependent mechanism of isoniazid activation, and it is well documented that mutations that reduce activity or inactivate the catalase-peroxidase lead to increased levels of isoniazid resistance in M. tuberculosis. Interpretation of the catalytic activities and the effects of mutations upon the action of the enzyme to date have been limited due to the lack of a three-dimensional structure for this enzyme. In order to provide a more accurate model of the three-dimensional structure of the M. tuberculosis catalase-peroxidase, we have crystallized the enzyme and now report its crystal structure refined to 2.4-A resolution. The structure reveals new information about dimer assembly and provides information about the location of residues that may play a role in catalysis including candidates for protein-based radical formation. Modeling and computational studies suggest that the binding site for isoniazid is located near the delta-meso heme edge rather than in a surface loop structure as currently proposed. The availability of a crystal structure for the M. tuberculosis catalase-peroxidase also permits structural and functional effects of mutations implicated in causing elevated levels of isoniazid resistance in clinical isolates to be interpreted with improved confidence.

Amino Acid Sequence↗
PubMed1977

Regulation of yeast mitochondrial DNA synthesis. I. Analysis of a mutant conditionally deficient in mitochondrial DNA metabolism.

A single nuclear gene mutation has been isolated from strain 123.1C of Saccharomyces cerevisiae which is conditionally deficient in mitochondrial DNA metabolism. Growth of the haploid in media containing dextrose, a repressing carbon source, at 36 degrees C causes the rapid cessation of mitochondrial DNA synthesis as analyzed by radioactive 3H-adenine incorporation into mitochondrial DNA. These cells continue to grow and divide giving rise to neutral petites which are devoid of mitochondrial DNA as measured by radioactive incorporation of 3H-adenine at the permissive temperature. Growth of the haploid cells in media containing glycerol, a non-repressing carbon source, at 36 degrees C does not prevent mitochondrial DNA synthesis, however, the population of cells becomes partially petite. When such petites are analyzed, they are found to be suppressive and to contain mitochondrial DNA as measured in the manner described above. The action of this mutated gene appears to involve the sunthetic aspects of mitochondrial DNA metabolism, as haploid cells prelabeled in dextrose media with 3H-adenine show no loss or degradation of mitochondrial DNA at the restrictive temperature of 36 degrees C.

Cold Temperature↗
PubMed1982

Trans-dominant mutation affecting restriction and modification in Escherichia coli K-12.

We have analysed the mechanism of action of a ts mutation in E. coli, which has an effect on the expression of the restriction and modification phenotype. The frequencies of recombinants obtained in transduction experiments support the idea that the temperature sensitive mutation is located outside the hsd operon in the gene denoted hsd.X. Complementation experiments demonstrated the trans-dominant nature of the temperature sensitive mutation. The possible role of the hsd.X product in the formation of EcoR.K and EcoM.K complexes and their interaction with the recognition site on the DNA is discussed.

Bacteriophage lambda↗
PubMed1991

A dominant negative mutation suppresses the function of normal epidermal growth factor receptors by heterodimerization.

Recent studies provide evidence that defective receptors can function as a dominant negative mutation suppressing the action of wild-type receptors. This causes various diminished responses in cell culture and developmental disorders in murine embryogenesis. Here, we describe a model system and a potential mechanism underlying the dominant suppressing response caused by defective epidermal growth factor (EGF) receptors. We used cultured 3T3 cells coexpressing human wild-type receptors and an inactive deletion mutant lacking most of the cytoplasmic domain. When expressed alone, EGF was able to stimulate the dimerization of either wild-type or mutant receptors in living cells as revealed by chemical covalent cross-linking experiments. In response to EGF, heterodimers and homodimers of wild-type and mutant receptors were observed in cells coexpressing both receptor species. However, only homodimers of wild-type EGF receptors underwent EGF-induced tyrosine autophosphorylation in living cells. These results indicate that the integrity of both receptor moieties within receptor dimers is essential for kinase activation and autophosphorylation. Moreover, the presence of mutant receptors in cells expressing wild-type receptors diminished the number of high-affinity binding sites for EGF, reduced the rate of receptor endocytosis and degradation, and diminished biological signalling via EGF receptors. We propose that heterodimerization with defective EGF receptors functions as a dominant negative mutation suppressing the activation and response of normal receptors by formation of unproductive heterodimers.

Animals↗
PubMed2006

Structures of wild-type and mutant human spermidine/spermine N1-acetyltransferase, a potential therapeutic drug target.

Spermidine/spermine N1-acetyltransferase (SSAT) is a key enzyme in the control of polyamine levels in human cells, as acetylation of spermidine and spermine triggers export or degradation. Increased intracellular polyamine levels accompany several types of cancers as well as other human diseases, and compounds that affect the expression, activity, or stability of SSAT are being explored as potential therapeutic drugs. We have expressed human SSAT from the cloned cDNA in Escherichia coli and have determined high-resolution structures of wild-type and mutant SSAT, as the free dimer and in binary and ternary complexes with CoA, acetyl-CoA (AcCoA), spermine, and the inhibitor N1,N11bis-(ethyl)-norspermine (BE-3-3-3). These structures show details of binding sites for cofactor, substrates, and inhibitor and provide a framework to understand enzymatic activity, mutations, and the action of potential drugs. Two dimer conformations were observed: a symmetric form with two open surface channels capable of binding substrate or cofactor, and an asymmetric form in which only one of the surface channels appears capable of binding and acetylating polyamines. SSAT was found to self-acetylate lysine-26 in the presence of AcCoA and absence of substrate, a reaction apparently catalzyed by AcCoA bound in the second channel of the asymmetric dimer. These unexpected and intriguing complexities seem likely to have some as yet undefined role in regulating SSAT activity or stability as a part of polyamine homeostasis. Sequence signatures group SSAT with proteins that appear to have thialysine Nepsilon-acetyltransferase activity.

Acetyl Coenzyme A↗
PubMed2000

The use of frailty models in genetic studies: application to the relationship between end-stage renal failure and mutation type in Alport syndrome. European Community Alport Syndrome Concerted Action Group (ECASCA).

BACKGROUND: Alport syndrome (AS) is a severe hereditary disease usually transmitted as an X dominant trait and involving a mutation of the COL4A5 gene. It leads to end-stage renal failure (ESRF), but this progression is heterogeneous. Mutations of the COL4A5 gene have been characterised in numerous families using molecular biology. Our objective was to evaluate the interfamilial heterogeneity of the disease and to study relationships between mutation types and progression to ESRF in the European Community Alport Syndrome Concerted Action group (ECASCA) registry database. METHODS: We used the frailty model framework. Frailty models have been developed to analyse censored data with non-independent observations. Random effects are introduced in a Cox proportional regression model to take into account the intracluster correlations. In this study, ESRF is considered a censored event and the intrafamilial correlations are taken into account in the frailty models. RESULTS: These approaches allow us to demonstrate the existence of an interfamilial heterogeneity; the role of the mutation type explains the interfamilial variability. In particular, the results suggest that some mutation types are associated with a higher risk of ESRF for males. CONCLUSIONS: This study shows the importance of characterising the mutation at the molecular level in genetic studies, to understand the relationship between genotype and phenotype. The frailty models constitute an attractive approach in this context, when the phenotype is characterised by a censored end-point.

Alleles↗
PubMed2025

Detection and Characterization of RB1 Mosaicism in Patients With Retinoblastoma Receiving cfDNA Test.

IMPORTANCE: Plasma cell-free DNA (cfDNA) testing is increasingly used for disease diagnosis and monitoring in retinoblastoma, with RB1 allele fraction in cfDNA actively corresponding to disease status and treatment response. However, while RB1 mosaicism has been reported in retinoblastoma, its clinical implications and potential impact on cfDNA testing remain unclear. OBJECTIVES: To identify RB1 mosaicism using paired plasma and buffy coat (containing lymphocytes, monocytes, granulocytes, and platelets) DNA testing, and to characterize the implications of RB1 mosaicism on cfDNA testing outcomes. DESIGN, SETTING, AND PARTICIPANTS: In this cross-sectional study, participants with retinoblastoma underwent testing with MSK-ACCESS (Memorial Sloan Kettering-Analysis of Circulating cfDNA to Examine Somatic Status), a clinical assay that combines plasma cfDNA and buffy coat genomic DNA sequencing, enabling the detection and differentiation of somatic, heterozygous, and mosaic variants, between July 2020 and April 2024 at the Memorial Sloan Kettering Cancer Center. Mosaic findings from MSK-ACCESS were correlated with those from a subgroup of patients who concurrently underwent testing using the MSK-IMPACT germline assay. Data analysis was performed from April to September 2024. EXPOSURE: RB1 mosaicism in retinoblastoma. MAIN OUTCOMES AND MEASURES: The RB1 variant allele fractions in cfDNA and buffy coat genomic DNA were used to detect RB1 mosaicism. RESULTS: A total of 136 consecutive patients with retinoblastoma (median age at diagnosis, 1.0 year [IQR, 0.4-1.7 years]; 74 [54.4%] female; 67 with bilateral disease and 69 with unilateral disease) who underwent testing with the MSK-ACCESS assay were included. RB1 mosaicism was identified in buffy coat DNA from 20 patients (14.7%), with consistent results detected in all 11 participants tested concurrently by the MSK-IMPACT (Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets) germline assay. Four participants with RB1 mosaicism previously tested negative for germline RB1 variants by external laboratories. Compared with heterozygous participants, participants with RB1 mosaicism had a lower risk of developing bilateral disease (91.7% vs 55.0%, respectively; difference, 36.7% [95% CI, 13.8%-59.6%]; P = .002). In cfDNA, the mosaicism variant was detected both before and after treatment, with variant allele fraction initially decreasing after treatment but then stabilizing at levels consistent with mosaicism, despite the absence of clinical disease. CONCLUSIONS AND RELEVANCE: The accurate detection and quantification of RB1 mosaicism are crucial. RB1 mosaicism should be considered when RB1 variants persist in cfDNA after treatment without evidence of disease; failure to do so may lead to false-positive results and overtreatment in patients with RB1 mosaicism. Identifying RB1 mosaicism may improve patient counseling, inform treatment decisions, and enhance surveillance efforts.

Humans↗
PubMed2007

Phenotypic variation among brothers with the McLeod neuroacanthocytosis syndrome.

McLeod syndrome is an X-linked multisystem disorder affecting red blood cells, the peripheral and central nervous systems, and skeletal and cardiac muscle. No clear correlations of the clinical findings with the genotype of XK mutations have yet been uncovered. Here, we report the clinical features and progression in 10 affected brothers from 4 families with McLeod syndrome. There is significant variation in clinical presentation within families, including in causes of morbidity and mortality. This phenotypic variation, despite shared mutations, suggests the action of disease-modifying factors that may explain some of the difficulties with genotype-phenotype correlation in McLeod syndrome.

Adult↗
PubMedDate not supplied

Bcl-2 mediates chemoresistance in matched pairs of primary E(mu)-myc lymphomas in vivo.

The oncoprotein Bcl-2 is a potent survival factor antagonizing p53-dependent and -independent apoptotic cell death. Although many anticancer agents are known to engage apoptotic pathways, the clinical impact of Bcl-2 on treatment outcome remains controversial. Since it might be difficult to assess the contribution of a single gene to treatment response in patient material due to technical considerations, we sought to address Bcl-2's role in a mouse model of primary lymphomas treated at their natural site. Driven by the E(mu)-enhancer controlled c-myc transgene, primary B cell lymphomas arise in this model by several months of age and resemble closely typical clinical and histopathological features of human non-Hodgkin lymphomas. We introduced either bcl-2 or a control construct into identical samples of freshly isolated E(mu)-myc lymphomas by retroviral gene transfer in order to obtain matched pairs of primary lymphomas differing only in their Bcl-2 status. While no Bcl-2-mediated effect was detectable in clonogenic survival assays in vitro, treatment of the genetically modified lymphoma pairs propagated in nontransgenic recipient mice revealed Bcl-2's impact on drug sensitivity in vivo. Bcl-2 efficiently blocked short- and long-term drug-mediated cell death in vivo. In a comparison of 15 matched pairs of primary lymphomas, the bcl-2 transduced sample never achieved longer remission periods than the control counterpart and most of the Bcl-2 overexpressing lymphomas failed to respond at all. We conclude that-when assessed in the physiological environmental context-MBcl-2 contributes to chemoresistance of B cell lymphomas in vivo. This model, able to test any other candidate gene, will be particularly useful to study the implications of specific mutations for drug action in vivo.

Animals↗
PubMed2002

Mechanism of ruminant placental lactogen action: molecular and in vivo studies.

Ruminant placental lactogens (PLs) are structurally related to prolactins (PRLs) and growth hormones (GHs) and are secreted by placentae. Ruminant PLs are unusual in their capacity to bind and activate PRL and GH receptors (Rs) from other species. The present minireview summarizes several works showing that unlike in heterologous species (rat, rabbit, human), in homologous (ruminant) species, PLs act by activating PRLRs or by heterodimerizing GHRs and PRLRs, and suggests that this may be the main mechanism of PL action in vivo. Mutations impairing the ability of ovine (o)PL or bovine (b)PL to form complexes with PRLRs (but not with GHRs) do not cause loss of biological activity, because the transient existence of the homodimeric complex is still sufficient to initiate the signal transduction; however, mutants do lose their ability to activate homologous PRLRs. To explain this difference, we proposed a novel term-minimal time of homodimer persistence-which assumes that to initiate the signal transduction, a "minimal time" of homodimer existence is required for transphosphorylation of associated JAK2s. In interactions between ruminant PLs and homologous PRLRs, this minimal time is met through the interaction with homologous PRLRs, which has a shorter half-life than with heterologous PRLRs. Thus oPL or bPL are active in cells possessing both homologous and heterologous PRLRs. Mutations of PLs decrease the affinity, shortening the "time of homodimer persistence." In heterologous interactions, the minimal time is still sufficient to initiate the biological activity, whereas in homologous interactions, which in any case are weaker, further destabilization of the complex shortens its persistence below the minimal time, causing loss of biological activity.

Animals↗
PubMed1992

Acquisition of regional and cellular identities in the developing zebrafish nervous system.

In the past year, several new techniques have been used with great success in the study of nervous system development in the zebrafish. Perhaps the most exciting results have come from experiments in which single identified cells or small groups of cells have been transplanted between embryos in order to examine cell determination and the site of action of genetic mutations.

Animals↗
PubMed2003

The E box motif CAGGTG enhances somatic hypermutation without enhancing transcription.

The frequency of somatic hypermutations of an Ig kappa transgene with an artificial test insert, RS, is at least 4-fold higher than that of three related transgenes. The four transgenes differ only in the sequence of a 96 bp insert within the variable region. RS is hypermutable over the total 625 nucleotides of the variable/joining region. The RS insert contains two CAGGTG sequences, potential binding sites for basic helix-loop-helix proteins. Changing CAGGTG to AAGGTG reduces the mutability to that of the non-RS transgenes without altering the mutation pattern. The CAGGTG motif enhances somatic hypermutation without enhancing transcription. A DNA probe containing the two CAGGTG sites, but not AAGGTG, binds E47 and gives rise to two specific EMSA bands with nuclear extracts from mutating cells. Possible actions of this enhancer of somatic hypermutation are discussed.

Animals↗
PubMed1975

On some theoretical studies on gene differentiation in natural populations.

Different mathematical approaches to study the extent of genetic variation of natural populations are reviewed. The modern understanding of the gene structure permits new interpretations of existing concepts like fixation or inbreeding. A more recent measure of genic divergence, which at molecular level is designed to measure net codon differences is also seen to be related with gene diversity in a substructed population. It is argued that such variations are produced and preserved possibly by simultaneous action of migration, mutation, selection, and random genetic drift. At the present moment it is very difficult to isolate out the effect of each factor because of varying degrees of variation at the different gene sites and between different sets of populations.

Biological Evolution↗
PubMed2005

Mode of binding of methyl acceptor substrates to the adrenaline-synthesizing enzyme phenylethanolamine N-methyltransferase: implications for catalysis.

Here we report three crystal structure complexes of human phenylethanolamine N-methyltransferase (PNMT), one bound with a substrate that incorporates a flexible ethanolamine side chain (p-octopamine), a second bound with a semirigid analogue substrate [cis-(1R,2S)-2-amino-1-tetralol, cis-(1R,2S)-AT], and a third with trans-(1S,2S)-2-amino-1-tetralol [trans-(1S,2S)-AT] that acts as an inhibitor of PNMT rather than a substrate. A water-mediated interaction between the critical beta-hydroxyl of the flexible ethanolamine group of p-octopamine and an acidic residue, Asp267, is likely to play a key role in positioning the side chain correctly for methylation to occur at the amine. A second interaction with Glu219 may play a lesser role. Catalysis likely occurs via deprotonation of the amine through the action of Glu185; mutation of this residue significantly reduced the kcat without affecting the Km. The mode of binding of cis-(1R,2S)-AT supports the notion that this substrate is a conformationally restrained analogue of flexible PNMT substrates, in that it forms interactions with the enzyme similar to those observed for p-octopamine. By contrast, trans-(1S,2S)-AT, an inhibitor rather than a substrate, binds in an orientation that is flipped by 180 degrees compared with cis-(1R,2S)-AT. A consequence of this flipped binding mode is that the interactions between the hydroxyl and Asp267 and Glu219 are lost. However, the amines of inhibitor trans-(1S,2S)-AT and substrate cis-(1R,2S)-AT are both within methyl transfer distance of the cofactor. These results suggest that PNMT catalyzes transfer of methyl to ligand amines only when "anchor" interactions, such as those identified for the beta-hydroxyls of p-octopamine and cis-AT, are present.

Amino Acid Substitution↗
PubMed1995

Variants of the melanocyte-stimulating hormone receptor gene are associated with red hair and fair skin in humans.

Melanin pigmentation protects the skin from the damaging effects of ultraviolet radiation (UVR). There are two types of melanin, the red phaeomelanin and the black eumelanin, both of which are present in human skin. Eumelanin is photoprotective whereas phaeomelanin, because of its potential to generate free radicals in response to UVR, may contribute to UV-induced skin damage. Individuals with red hair have a predominance of phaeomelain in hair and skin and/or a reduced ability to produce eumelanin, which may explain why they fail to tan and are at risk from UVR. In mammals the relative proportions of phaeomelanin and eumelanin are regulated by melanocyte stimulating hormone (MSH), which acts via its receptor (MC1R), on melanocytes, to increase the synthesis of eumelanin and the product of the agouti locus which antagonises this action. In mice, mutations at either the MC1R gene or agouti affect the pattern of melanogenesis resulting in changes in coat colour. We now report the presence of MC1R gene sequence variants in humans. These were found in over 80% of individuals with red hair and/or fair skin that tans poorly but in fewer than 20% of individuals with brown or black hair and in less than 4% of those who showed a good tanning response. Our findings suggest that in humans, as in other mammals, the MC1R is a control point in the regulation of pigmentation phenotype and, more importantly, that variations in this protein are associated with a poor tanning response.

Base Sequence↗
PubMed2000

Tryptophan scanning mutagenesis in TM2 of the GABA(A) receptor alpha subunit: effects on channel gating and regulation by ethanol.

1. Each residue in the second transmembrane segment (TM2) of the human GABA(A) receptor alpha(2) subunit was individually mutated to tryptophan. The wild-type or mutant alpha(2) subunits were expressed with the wild-type human GABA(A) receptor beta(2) subunit in Xenopus oocytes, and the effects of these mutations were investigated using two-electrode voltage-clamp recording. 2. Four mutations (V257W, T262W, T265W and S270W) produced receptors which were active in the absence of agonist, and this spontaneous open channel activity was blocked by both picrotoxin and bicuculline, except in the alpha(2)(V257W)beta(2) mutant receptor, which was not sensitive to picrotoxin. 3. Six mutations (V257W, V260W, T262W, T267W, S270W and A273W) enhanced the agonist sensitivity of the receptor, by 10 - 100 times compared with the wild-type alpha(2)beta(2) receptor. Other mutations (T261W, V263W, L269W, I271W and S272W) had little or no effect on the apparent affinity of the receptor to GABA. Eight of the tryptophan mutations (R255, T256, F258, G259, L264, T265, M266 or T268) resulted in undetectable GABA-induced currents. 4. The S270W mutation eliminated potentiation of GABA by ethanol, whereas T261W markedly increased the action of ethanol. The T262W mutation produced direct activation (10% of maximal GABA response) by ethanol in the absence of GABA, while other mutations did not alter the action of ethanol significantly. 5. These results are consistent with a unique role for S270 in the action of ethanol within the TM2 region, and with models of GABA(A) receptor channel function, in which specific residues within TM2 are critical for the regulation of channel gating (S270, L264), while other residues (L269, I271 and S272) have little effect on these functions and may be non-critical structural residues.

Amino Acid Sequence↗
PubMed2003

A two-locus gene conversion model with selection and its application to the human RHCE and RHD genes.

A two-locus gene conversion model with selection is developed. Under the joint action of selection, mutation, gene conversion, recombination, and random genetic drift, approximate formulas for the expectations of the moments of allele frequencies and the expected amounts of variation within and between two loci are obtained by a diffusion method assuming relatively strong selection. It is shown that the pattern of allelic variation is mainly determined by the balance between gene conversion and selection, because these two mechanisms act in opposite directions. As an application of the theoretical results, the human RHCE and RHD genes are considered. The very high level of amino acid divergence between the two genes is observed only in a short region around exon 7. It is known that exon 7 encodes amino acids that characterize the difference between the RHCE and RHD antigens. The observed pattern of DNA variation in this region is consistent with the selection model developed in this article, suggesting that strong selection might be working to maintain the RHCE/RHD antigen variation in the two-locus system. The selection intensity is estimated on the basis of the theoretical result.

Alleles↗
PubMed2002

Early stages of energy transduction by myosin: roles of Arg in switch I, of Glu in switch II, and of the salt-bridge between them.

On the basis of the crystallographic snapshots of Rayment and his collaborators [Fisher, A. J., Smith, C. A., Thoden, J. B., Smith, R., Sutoh, K., Holden, H. M., & Rayment, I. (1995) Biochemistry 34, 8960-8972], we have understood some basic principles about the early stages of myosin catalysis, namely, ATP is drawn into the active site, over which the cleft closes. Catalyzed hydrolysis occurs, and the first product (orthophosphate) is released from the backdoor of the cleft. In the cleft-closing process, the active site incidentally signals its movement to a particular remote tryptophan residue, Trp-512. In this work, we expand on some of these ideas to rationalize the behavior of a mutated system in action. From the behavior of recombinant myosin systems in which Arg-247 and Glu-470 were substituted in several ways, we draw the conclusions that (i) the force between Arg-247 and gamma-phosphate of ATP may assist in closing the cleft, and incidentally in signaling to the remote Trp, and (ii) in catalysis, Glu-470 is involved in holding the lytic H(2)O (w(1)). We also propose that w(1) and also a second water, w(2), enter into a structure that bridges Glu-470 and the gamma-phosphate of bound ATP, and at the same time positions w(1) for its in-line hydrolytic attack.

Actins↗