scholarly journals Combination of Two Activating Mutations in One HOG1 Gene Forms Hyperactive Enzymes That Induce Growth Arrest

2003 ◽  
Vol 23 (14) ◽  
pp. 4826-4840 ◽  
Author(s):  
Gilad Yaakov ◽  
Michal Bell ◽  
Stefan Hohmann ◽  
David Engelberg
Keyword(s):  
Catalytic Activity ◽  
Growth Inhibition ◽  
Target Genes ◽  
Single Point ◽  
Expression System ◽  
Point Mutations ◽  
Intrinsic Activity ◽  
Single Point Mutation ◽  
Biological Potential ◽  
Mitogen Activated Protein

ABSTRACT Mitogen-activated protein kinases (MAPKs) play key roles in differentiation, growth, proliferation, and apoptosis. Although MAPKs have been extensively studied, the precise function, specific substrates, and target genes of each MAPK are not known. These issues could be addressed by sole activation of a given MAPK, e.g., through the use of constitutively active MAPK enzymes. We have recently reported the isolation of eight hyperactive mutants of the Saccharomyces cerevisiae MAPK Hog1, each of which bears a distinct single point mutation. These mutants acquired high intrinsic catalytic activity but did not impose the full biological potential of the Hog1 pathway. Here we describe our attempt to obtain a MAPK that is more active than the previous mutants both catalytically and biologically. We combined two different activating point mutations in the same gene and found that two of the resulting double mutants acquired unusual properties. These alleles, HOG1D170A,F318L and HOG1D170A,F318S , induced a severe growth inhibition and had to be studied through an inducible expression system. This growth inhibition correlated with very high spontaneous (in the absence of any stimulation) catalytic activity and strong induction of Hog1 target genes. Furthermore, analysis of the phosphorylation status of these active alleles shows that their acquired intrinsic activity is independent of either phospho-Thr174 or phospho-Tyr176. Through fluorescence-activated cell sorting analysis, we show that the effect on cell growth inhibition is not a result of cell death. This study provides the first example of a MAPK that is intrinsically activated by mutations and induces a strong biological effect.

scholarly journals Mutational Analysis of Quinolone-Resistant Determining Region gyrA and parC Genes in Quinolone-Resistant ESBL-Producing E. Coli

2021 ◽  
Vol 20 (3) ◽  
Author(s):  
Hairul Aini Hamzah ◽  
Rahmatullah Sirat ◽  
Mohammed Imad A. Mustafa Mahmud ◽  
Roesnita Baharudin
Keyword(s):  
Mutational Analysis ◽  
Single Point ◽  
Point Mutations ◽  
Multidrug Resistant ◽  
Quinolone Resistance ◽  
Resistant Bacteria ◽  
Single Point Mutation ◽  
Phenotypic Resistance ◽  
Drug Effectiveness ◽  
E Coli

 Introduction: Co-resistance to quinolones among extended spectrum β[1]lactamase (ESBL)-producing E. coli commonly occurs in clinical settings. Quinolones act on DNA gyrase and DNA topoisomerase enzymes, which are coded by gyrA and parC genes, thus any mutation to the genes may affect the drug effectiveness. The objective of the study was to characterize gyrA and parC genes in quinolone-resistant E. coli isolates and correlated the mutations with their phenotypic resistance. Materials and Methods: Thirty-two quinolone-resistant (QR) and six quinolone-sensitive (QS) ESBL-E. coli isolates were identified by antibiotic susceptibility and minimum inhibitory concentration tests. Bioinformatics analysis were conducted to study any mutations occurred in the genes and generate their codon compositions. Results: All the QR ESBL-E. coli isolates were identified as multidrug-resistant bacteria. A single point mutation in the quinolone resistance-determining region (QRDR) of gyrA, at codon 83, caused the substitution amino acid Ser83Leu. It is associated with a high level of resistance to nalidixic acid. However, double mutations Ser83Leu and Asp87Asn in the same region were significantly linked to higher levels of resistance to ciprofloxacin. Cumulative point mutations in gyrA and/or in parC were also correlated significantly (p<0.05) to increased resistance to ciprofloxacin. Conclusion: Together, the findings showed that the mutations in gyrA and parC genes handled the institution of intrinsic quinolone resistance in the ESBL-E. coli isolates. Thus, vigilant monitoring for emergence of new mutation in resistance genes may give an insight into dissemination of QR ESBL-E. coli in a particular region.

scholarly journals Single point mutation in tick-borne encephalitis virus prM protein induces a reduction of virus particle secretion

2004 ◽  
Vol 85 (10) ◽  
pp. 3049-3058 ◽  
Author(s):  
Kentarou Yoshii ◽  
Akihiro Konno ◽  
Akiko Goto ◽  
Junko Nio ◽  
Mayumi Obara ◽  
...  
Keyword(s):  
Point Mutation ◽  
Single Point ◽  
Expression System ◽  
Envelope Proteins ◽  
Viral Envelope ◽  
Single Point Mutation ◽  
Virus Envelope ◽  
Electron Microscopic ◽  
Transport Pathway ◽  
Tick Borne Encephalitis

Flaviviruses are assembled to bud into the lumen of the endoplasmic reticulum (ER) and are secreted through the vesicle transport pathway. Virus envelope proteins play important roles in this process. In this study, the effect of mutations in the envelope proteins of tick-borne encephalitis (TBE) virus on secretion of virus-like particles (VLPs), using a recombinant plasmid expression system was analysed. It was found that a single point mutation at position 63 in prM induces a reduction in secretion of VLPs. The mutation in prM did not affect the folding of the envelope proteins, and chaperone-like activity of prM was maintained. As observed by immunofluorescence microscopy, viral envelope proteins with the mutation in prM were scarce in the Golgi complex, and accumulated in the ER. Electron microscopic analysis of cells expressing the mutated prM revealed that many tubular structures were present in the lumen. The insertion of the prM mutation at aa 63 into the viral genome reduced the production of infectious virus particles. This data suggest that prM plays a crucial role in the virus budding process.

scholarly journals Autism-associated mutation inhibits protein kinase C-mediated neuroligin-4X enhancement of excitatory synapses

2015 ◽  
Vol 112 (8) ◽  
pp. 2551-2556 ◽  
Author(s):  
Michael A. Bemben ◽  
Quynh-Anh Nguyen ◽  
Tongguang Wang ◽  
Yan Li ◽  
Roger A. Nicoll ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Single Point ◽  
Point Mutations ◽  
Genetic Mutation ◽  
Autism Spectrum ◽  
Repetitive Behaviors ◽  
Single Point Mutation ◽  
Kinase C ◽  
Human Neurons

Autism spectrum disorders (ASDs) comprise a highly heritable, multifarious group of neurodevelopmental disorders, which are characterized by repetitive behaviors and impairments in social interactions. Point mutations have been identified in X-linked Neuroligin (NLGN) 3 and 4X genes in patients with ASDs and all of these reside in their extracellular domains except for a single point mutation in the cytoplasmic domain of NLGN4X in which an arginine is mutated to a cysteine (R704C). Here we show that endogenous NLGN4X is robustly phosphorylated by protein kinase C (PKC) at T707, and R704C completely eliminates T707 phosphorylation. Endogenous NLGN4X is intensely phosphorylated on T707 upon PKC stimulation in human neurons. Furthermore, a phospho-mimetic mutation at T707 has a profound effect on NLGN4X-mediated excitatory potentiation. Our results now establish an important interplay between a genetic mutation, a key posttranslational modification, and robust synaptic changes, which can provide insights into the synaptic dysfunction of ASDs.

scholarly journals Monitoring of Pyrethroid Resistance Allele Frequency in the Common Bed Bug (Cimex lectularius) in the Republic of Korea

2020 ◽  
Vol 58 (1) ◽  
pp. 99-102
Author(s):  
Susie Cho ◽  
Heung-Chul Kim ◽  
Sung-Tae Chong ◽  
Terry A. Klein ◽  
Deok Ho Kwon ◽  
...  
Keyword(s):  
Large Scale ◽  
Pyrethroid Resistance ◽  
Single Point ◽  
Point Mutations ◽  
Monitoring Program ◽  
Cimex Lectularius ◽  
Single Point Mutation ◽  
Resistance Allele ◽  
Bed Bugs ◽  
Bed Bug

Two-point mutations (V419L and L925I) on the voltage-sensitive sodium channel of bed bugs (<i>Cimex lectularius</i>) are known to confer pyrethroid resistance. To determine the status of pyrethroid resistance in bed bugs in Korea, resistance allele frequencies of bed bug strains collected from several US military installations in Korea and Mokpo, Jeollanamdo, from 2009-2019 were monitored using a quantitative sequencing. Most bed bugs were determined to have both of the point mutations except a few specimens, collected in 2009, 2012 and 2014, having only a single point mutation (L925I). No susceptible allele was observed in any of the bed bugs examined, suggesting that pyrethroid resistance in bed bug populations in Korea has reached a serious level. Large scale monitoring is required to increase our knowledge on the distribution and prevalence of pyrethroid resistance in bed bug populations in Korea. Based on present study, it is urgent to restrict the use of pyrethroids and to introduce effective alternative insecticides. A nation-wide monitoring program to determine the pyrethroid resistance level in bed bugs and to select alternative insecticides should be implemented.

scholarly journals Genetic perturbation enhances functional heterogeneity in alkaline phosphatase

2021 ◽  
Author(s):  
Morito Sakuma ◽  
Shingo Honda ◽  
Hiroshi Ueno ◽  
Kentaro Miyazaki ◽  
Nobuhiko Tokuriki ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Catalytic Activity ◽  
Single Molecule ◽  
Single Point ◽  
Point Mutations ◽  
Clear Correlation ◽  
Wild Type ◽  
Functional Heterogeneity ◽  
Genetic Perturbations ◽  
In The Wild

Enzymes inherently exhibit molecule-to-molecule heterogeneity in catalytic activity or function, which underlies the acquisition of new functions in evolutionary processes. However, correlations between the functional heterogeneity of an enzyme and its multi-functionality or promiscuity remain elusive. In addition, the modulation of functional heterogeneity upon genetic perturbation is currently unexplored. Here, we quantitatively analyzed functional heterogeneity in the wild-type and 69 single-point mutants of Escherichia coli alkaline phosphatase (AP) by employing single-molecule assay with a femtoliter reactor array device. Most mutant enzymes exhibited higher functional heterogeneity than the wild-type enzyme, irrespective of catalytic activity. These results indicated that the wild-type AP minimizes functional heterogeneity, and single-point mutations can significantly expand the span of functional heterogeneity in AP. Moreover, we identified a clear correlation between functional heterogeneity and promiscuous activities. These findings suggest that enzymes can acquire greater functional heterogeneity following marginal genetic perturbations that concomitantly promote catalytic promiscuity.

scholarly journals Selective disruption of synaptic BMP signaling by a Smad mutation adjacent to the highly conserved H2 helix

2019 ◽  
Author(s):  
Tho Huu Nguyen ◽  
Tae Hee Han ◽  
Stuart Newfeld ◽  
Mihaela Serpe
Keyword(s):  
Bone Morphogenetic Proteins ◽  
Target Genes ◽  
Normal Development ◽  
Single Point ◽  
Point Mutations ◽  
Bmp Signaling ◽  
Electrophysiological Properties ◽  
Molecular Features ◽  
Synaptic Junctions ◽  
And Function

ABSTRACTBone morphogenetic proteins (BMPs) shape normal development and function via canonical and non-canonical signaling pathways. When activating the canonical pathway, BMPs initiate signaling by binding to transmembrane receptors that phosphorylate pathway effectors, the Smad proteins, inducing their translocation into the nucleus and thus regulation of target genes. Phosphorylated Smads also accumulate at cellular junctions, but this non-canonical signaling modality remains less defined. We have recently reported that phosphorylated Smad (pMad in Drosophila) accumulates at synaptic junctions in complexes with genetically distinct composition and regulation. Here we examined a wide collection of Drosophila Mad alleles and searched for molecular features relevant to pMad accumulation at synaptic junctions. We found that strong Mad alleles generally disrupt both synaptic and nuclear pMad accumulation, whereas moderate Mad alleles have a wider range of phenotypes and could selectively impact different BMP signaling modalities. Interestingly, synaptic pMad appeared more sensitive to net reduction in Mad levels than nuclear pMad. Importantly, a previously uncharacterized allele, Mad8, showed markedly reduced synaptic pMad levels but only moderately diminished nuclear pMad signals. The postsynaptic composition and electrophysiological properties of Mad8 NMJs were similarly altered. Using biochemical approaches, we examined how single point mutations such as S359L, present in Mad8, could influence the Mad-receptor interface and we identified a key molecular determinant, the H2 helix. Our study highlights the biological relevance of the Smad-dependent, non-canonical BMP signaling and uncovers a highly conserved structural feature of Smads, critical for normal development and function.

Point mutations in the beta-subunit of cytochrome b558 leading to X- linked chronic granulomatous disease

Blood ◽  
1991 ◽  
Vol 77 (11) ◽  
pp. 2482-2487 ◽  
Author(s):  
BG Bolscher ◽  
M de Boer ◽  
A de Klein ◽  
RS Weening ◽  
D Roos
Keyword(s):  
Chronic Granulomatous Disease ◽  
Single Point ◽  
Genetic Defect ◽  
Point Mutations ◽  
Heme Iron ◽  
Beta Subunit ◽  
Stable Complex ◽  
Single Point Mutation ◽  
Granulomatous Disease ◽  
Cytochrome B558

The NADPH:O2 oxidoreductase of phagocytic leukocytes is an important enzyme for the bactericidal activity of these cells. Cytochrome b558 is a membrane component of this enzyme. In X-linked chronic granulomatous disease (Xb- CGD) the phagocytes are defective in the beta-subunit (gp91-phox) of this cytochrome. We have studied the genetic defect in a group of six X-linked CGD patients characterized by complete or partial loss of cytochrome b558 with the use of the polymerase chain reaction. All patients had a different single point mutation in the gp91-phox gene, indicating that the genetic defect in Xb- CGD is very heterogeneous. In one patient the mutation leads to a premature termination codon. In the other five cases these mutations predict incorporation of a different amino acid. The mutations were with one exception found in the N-terminal half of the protein, suggesting that this part of cytochrome b558 is important for the binding of the heme or for formation of a stable complex with p22-phox. Two histidyl residues were found that might be ligands of the heme iron.

scholarly journals mmCSM-AB: guiding rational antibody engineering through multiple point mutations

2020 ◽  
Vol 48 (W1) ◽  
pp. W125-W131 ◽  
Author(s):  
Yoochan Myung ◽  
Douglas E V Pires ◽  
David B Ascher
Keyword(s):  
Binding Affinity ◽  
Single Point ◽  
Point Mutations ◽  
Affinity Maturation ◽  
Antibody Engineering ◽  
Multiple Point ◽  
Single Point Mutation ◽  
Atomic Interaction ◽  
Therapeutic Class ◽  
Antibody Design

Abstract While antibodies are becoming an increasingly important therapeutic class, especially in personalized medicine, their development and optimization has been largely through experimental exploration. While there have been many efforts to develop computational tools to guide rational antibody engineering, most approaches are of limited accuracy when applied to antibody design, and have largely been limited to analysing a single point mutation at a time. To overcome this gap, we have curated a dataset of 242 experimentally determined changes in binding affinity upon multiple point mutations in antibody-target complexes (89 increasing and 153 decreasing binding affinity). Here, we have shown that by using our graph-based signatures and atomic interaction information, we can accurately analyse the consequence of multi-point mutations on antigen binding affinity. Our approach outperformed other available tools across cross-validation and two independent blind tests, achieving Pearson's correlations of up to 0.95. We have implemented our new approach, mmCSM-AB, as a web-server that can help guide the process of affinity maturation in antibody design. mmCSM-AB is freely available at http://biosig.unimelb.edu.au/mmcsm_ab/.

scholarly journals Mutations in Aspergillus fumigatus Resulting in Reduced Susceptibility to Posaconazole Appear To Be Restricted to a Single Amino Acid in the Cytochrome P450 14α-Demethylase

2003 ◽  
Vol 47 (2) ◽  
pp. 577-581 ◽  
Author(s):  
Paul A. Mann ◽  
Raulo M. Parmegiani ◽  
Shui-Qing Wei ◽  
Cara A. Mendrick ◽  
Xin Li ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Aspergillus Fumigatus ◽  
Single Point ◽  
Point Mutations ◽  
Drug Accumulation ◽  
Resistant Isolate ◽  
Single Amino Acid ◽  
Single Point Mutation ◽  
Genes Encoding ◽  
Moderately Resistant

ABSTRACT To better understand the molecular basis of posaconazole (POS) resistance in Aspergillus fumigatus, resistant laboratory isolates were selected. Spontaneous mutants arose at a frequency of 1 in 108 and fell into two susceptibility groups, moderately resistant and highly resistant. Azole resistance in A. fumigatus was previously associated with decreased drug accumulation. We therefore analyzed the mutants for changes in levels of transcripts of genes encoding efflux pumps (mdr1 and mdr2) and/or alterations in accumulation of [14C]POS. No changes in either pump expression or drug accumulation were detected. Similarly, there was no change in expression of cyp51A or cyp51B, which encode the presumed target site for POS, cytochrome P450 14α-demethylase. DNA sequencing revealed that each resistant isolate carried a single point mutation in residue 54 of cyp51A. Mutations at the same locus were identified in three clinical A. fumigatus isolates exhibiting reduced POS susceptibility but not in susceptible clinical strains. To verify that these mutations were responsible for the resistance phenotype, we introduced them into the chromosome of a POS-susceptible A. fumigatus strain under the control of the glyceraldehyde phosphate dehydrogenase promoter. The transformants exhibited reductions in susceptibility to POS comparable to those exhibited by the original mutants, confirming that point mutations in the cyp51A gene in A. fumigatus can confer reduced susceptibility to POS.

scholarly journals Assessing Protein-Drug Resistance Due to Mutations via a Rigidity Analysis in silico Approach

10.29007/7gnf ◽  
2020 ◽  
Author(s):  
Dylan Carpenter ◽  
Tess Thackray ◽  
Cecilia Kalthoff ◽  
Filip Jagodzinski
Keyword(s):  
Drug Resistance ◽  
Amino Acid ◽  
In Silico ◽  
Single Point ◽  
Point Mutations ◽  
Single Point Mutation ◽  
Protein Drug ◽  
Ligand Complex ◽  
Rigidity Analysis ◽  
The Impact

A mutation to the amino acid sequence of a protein can cause a biomolecule to be resistant to the intended effects of a drug. Assessing the changes of a drug’s efficacy in response to mutations via mutagenesis wet-lab experiments is prohibitively time consuming for even a single point mutation, let alone for all possible mutations. Existing approaches for inferring mutation-induced drug resistance are available, but all of them reason about mutations of residues at or very near the protein-drug interface. However, there are examples of mutations far away from the region where the ligand binds, but which nonetheless render a protein resistant to the effects of the drug. We present a proof-of-concept computational pipeline that generates in silico the set of all possible single point mutations in a protein-ligand complex. We assess drug resistance using a graph theoretic rigidity analysis approach. Unlike existing methods, we are able to assess the impact of mutations far away from the protein-drug interface. We introduce several visualizations for exploring how amino acid substitutions both near and far away from where the ligand interacts with a protein target have a stabilizing or destabilizing effect on the protein-drug complex. We discuss our analytical approach in the context of experimental data from the literature about clinically known protein-drug interactions.

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