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Proceedings of the National Academy of... Feb 2022Understanding the molecular consequences of mutations in proteins is essential to map genotypes to phenotypes and interpret the increasing wealth of genomic data. While...
Understanding the molecular consequences of mutations in proteins is essential to map genotypes to phenotypes and interpret the increasing wealth of genomic data. While mutations are known to disrupt protein structure and function, their potential to create new structures and localization phenotypes has not yet been mapped to a sequence space. To map this relationship, we employed two homo-oligomeric protein complexes in which the internal symmetry exacerbates the impact of mutations. We mutagenized three surface residues of each complex and monitored the mutations' effect on localization and assembly phenotypes in yeast cells. While surface mutations are classically viewed as benign, our analysis of several hundred mutants revealed they often trigger three main phenotypes in these proteins: nuclear localization, the formation of puncta, and fibers. Strikingly, more than 50% of random mutants induced one of these phenotypes in both complexes. Analyzing the mutant's sequences showed that surface stickiness and net charge are two key physicochemical properties associated with these changes. In one complex, more than 60% of mutants self-assembled into fibers. Such a high frequency is explained by negative design: charged residues shield the complex from self-interacting with copies of itself, and the sole removal of the charges induces its supramolecular self-assembly. A subsequent analysis of several other complexes targeted with alanine mutations suggested that such negative design is common. These results highlight that minimal perturbations in protein surfaces' physicochemical properties can frequently drive assembly and localization changes in a cellular context.
Topics: Genotype; Mutation; Phenotype; Proteins
PubMed: 35078932
DOI: 10.1073/pnas.2101117119 -
Genetics Sep 2020Natural environments are seldom static and therefore it is important to ask how a population adapts in a changing environment. We consider a finite, diploid population...
Natural environments are seldom static and therefore it is important to ask how a population adapts in a changing environment. We consider a finite, diploid population evolving in a periodically changing environment and study how the fixation probability of a rare mutant depends on its dominance coefficient and the rate of environmental change. We find that, in slowly changing environments, the effect of dominance is the same as in the static environment, that is, if a mutant is beneficial (deleterious) when it appears, it is more (less) likely to fix if it is dominant. But, in fast changing environments, the effect of dominance can be different from that in the static environment and is determined by the mutant's fitness at the time of appearance as well as that in the time-averaged environment. We find that, in a rapidly varying environment that is neutral on average, an initially beneficial (deleterious) mutant that arises while selection is decreasing (increasing) has a fixation probability lower (higher) than that for a neutral mutant as a result of which the recessive (dominant) mutant is favored. If the environment is beneficial (deleterious) on average but the mutant is deleterious (beneficial) when it appears in the population, the dominant (recessive) mutant is favored in a fast changing environment. We also find that, when recurrent mutations occur, dominance does not have a strong influence on evolutionary dynamics.
Topics: Adaptation, Physiological; Diploidy; Environment; Evolution, Molecular; Genes, Dominant; Genetic Fitness; Models, Genetic; Selection, Genetic
PubMed: 32723776
DOI: 10.1534/genetics.120.303519 -
Journal of Virology Jul 2022Multigene family (MGF) gene products are increasingly reported to be implicated in African swine fever virus (ASFV) virulence and attenuation of host defenses, among...
Multigene family (MGF) gene products are increasingly reported to be implicated in African swine fever virus (ASFV) virulence and attenuation of host defenses, among which the MGF360-9L and MGF505-7R gene products are characterized by convergent but distinct mechanisms of immune evasion. Herein, a recombinant ASFV mutant, ASFV-Δ9L/Δ7R, bearing combinational deletions of MGF360-9L and MGF505-7R, was constructed from the highly virulent ASFV strain CN/GS/2018 of genotype II that is currently circulating in China. Pigs inoculated intramuscularly with 10 50% hemadsorption doses (HAD) of the mutant remained clinically healthy without any serious side effects. Importantly, in a virulence challenge, all four within-pen contact pigs demonstrated clinical signs and pathological findings consistent with ASF. In contrast, vaccinated pigs (5/6) were protected and clinical indicators tended to be normal, accompanied by extensive tissue repairs. Similar to most viral infections, innate immunity and both humoral and cellular immune responses appeared to be vital for protection. Notably, transcriptome sequencing (RNA-seq) and quantitative PCR (qPCR) analysis revealed a regulatory function of the mutant in dramatic and sustained expression of type I/III interferons and inflammatory and innate immune genes . Furthermore, infection with the mutant elicited an early and robust p30-specific IgG response, which coincided and was strongly correlated with the protective efficacy. Analysis of the cellular response revealed a strong ASFV-specific interferon gamma (IFN-γ) response and immunostaining of CD4 T cells coupled with a high level of CD163 macrophage infiltration in spleens of vaccinated pigs. Our study identifies a new mechanism of immunological regulation by ASFV MGFs that rationalizes the design of live attenuated vaccine for implementation of improved control strategies to eradicate ASFV. Currently, the deficiency in commercially available vaccines or therapeutic options against African swine fever constitutes a matter of major concern in the swine industry globally. Here, we report the design and construction of a recombinant ASFV mutant harboring combinational deletions of interferon inhibitors MGF360-9L and MGF505-7R based on a genotype II ASFV CN/GS/2018 strain currently circulating in China. The mutant was completely attenuated when inoculated at a high dose of 10 HAD. In the virulence challenge with homologous virus, sterile immunity was achieved, demonstrating the mutant's potential as a promising vaccine candidate. This sufficiency of effectiveness supports the claim that this live attenuated virus may be a viable vaccine option with which to fight ASF.
Topics: African Swine Fever; African Swine Fever Virus; Animals; Gene Deletion; Interferon Type I; Swine; Vaccines, Attenuated; Viral Vaccines
PubMed: 35867564
DOI: 10.1128/jvi.00329-22 -
International Journal of Molecular... Nov 2022Water shortages caused by climate change seriously threaten the survival and production of plants and are also one of the major environmental pressures faced by plants....
Water shortages caused by climate change seriously threaten the survival and production of plants and are also one of the major environmental pressures faced by plants. DORN1 was the first identified purinoceptor for the plant response to extracellular ATP. It has been established that DORN1 could play key roles in a series of biological activities in plants. However, the biological roles of DORN1 and the mechanism remain unclear under drought stress conditions in plants. Here, DORN1 was targeted for knockout by using the CRISPR/Cas 9 system. It was found that the loss function of DORN1 resulted in a significant decrease in the effective quantum yield of PSII [Y(II)], the photochemical quenching coefficient (qP), and the rate of photosynthetic electron transport through PSII (ETR), which reflected plants' photochemical efficiency. Whereas Y values showed obvious enhancement under drought stress conditions. Further experimental results showed that the Y, q, and ETR, which reflect plants' photochemical efficiency, increased significantly with CaCl treatment. These results indicated that the drought tolerance of the mutant was decreased, and the exogenous application of calcium ions could effectively promote the drought tolerance of the mutant. Transpiration loss controlled by stomata is closely related to drought tolerance, further, we examined the transpirational water loss in and found that it was greater than wild-type (WT). Besides, the mutant's stomatal aperture significantly increased compared with the WT and the stomata of mutant plants tend to close after CaCl treatment. Taken together, our results show that DORN1 plays a key role in drought stress tolerance in plants, which may depend on calcium and calcium-related signaling pathways.
Topics: Droughts; Calcium; Calcium Chloride; Photosynthesis; Water
PubMed: 36430696
DOI: 10.3390/ijms232214213 -
Cell Cycle (Georgetown, Tex.) Jun 2021Doxorubicin induces both DNA damage and metabolic interference. How these effects interact to modulate cellular toxicity is not completely understood but important given...
Doxorubicin induces both DNA damage and metabolic interference. How these effects interact to modulate cellular toxicity is not completely understood but important given the widespread use of doxorubicin in cancer treatment. This study tests the hypothesis that cell cycle arrest and survival are affected by distinct mitochondrial activities during doxorubicin exposure.Parental and mutant strains deficient in selected genes with mitochondrial function were treated with doxorubicin and assayed for changes in proliferation rates, cell survival and cell cycle arrest kinetics. Mitochondrial DNA content was estimated using quantitative PCR. Mitochondrial function was assessed by measuring oxygen consumption with and without an uncoupler.Parental cells growing in a non-fermentable carbon source medium and mutants lacking mitochondria and grown in glucose medium both show abrupt cell cycle and proliferation arrest during doxorubicin exposure compared to parental cells grown in glucose. Mitochondrial DNA increases during doxorubicin exposure in and in human breast cancer cells. Yeast strains deficient in TCA cycle activity or electron transport both show more abrupt cell cycle arrest than parental cells when exposed to doxorubicin. Concurrent treatment with the mitochondrial uncoupler dinitrophenol facilitates cell cycle progression and proliferation during doxorubicin exposure.Doxorubicin exposure induces mitochondrial DNA synthesis with TCA cycle and oxidative phosphorylation activity having opposing effects on cell proliferation, survival and cell cycle kinetics. TCA cycle activity provides biosynthetic substrates to support cell cycle progression and cell proliferation while electron transport and oxidative phosphorylation facilitate cell cycle arrest and possibly increased cytotoxicity.
Topics: Antibiotics, Antineoplastic; Cell Cycle; Cell Proliferation; DNA, Mitochondrial; Doxorubicin; Electron Transport; Humans; MCF-7 Cells; Mitochondria; Oxygen Consumption; Saccharomyces cerevisiae
PubMed: 33978554
DOI: 10.1080/15384101.2021.1919839 -
Biochemistry and Biophysics Reports Sep 2022V. fluvialis is an emerging foodborne pathogen and could cause cholera-like gastroenteritis syndrome and poses a potential threat to public health. VflT6SS2 is a...
V. fluvialis is an emerging foodborne pathogen and could cause cholera-like gastroenteritis syndrome and poses a potential threat to public health. VflT6SS2 is a functionally active type VI secretion system (T6SS) in which confers bactericidal activity. VflT6SS2 is composed of one major cluster and three - orphan clusters. Previously, we identified two quorum sensing (QS) systems CqsA/LuxS-HapR and VfqI-VfqR in and demonstrated that the former regulates VflT6SS2. However, whether VfqI-VfqR QS regulates VflT6SS2 is unknown. In this study, we showed that the mRNA abundances of VflT6SS2 2 (), 2 () and 2 () were all significantly decreased in VfqI or/and VfqR deletion mutant(s). Consistently, Hcp expression/secretion was reduced too in these mutants. Complementation assay with VfqR mutant further confirmed that the reduced Hcp expression/secretion and impaired antibacterial virulence are restored by introducing VfqR-expressing plasmid. Reporter fusion analyses revealed that VfqR modulates the promoter activities of VflT6SS2. Bioinformatical prediction and further reporter fusion assay in supported that VfqR acts as a transcriptional factor to bind and regulate the gene expression of the VflT6SS2 major cluster. However, VfqR seems to promote transcription of (2) in the orphan clusters through elevating the expression of which is encoded by the VflT6SS2 major cluster. Additionally, we found that the regulation intensity of VfqR on VflT6SS2 is weaker than that of HapR. In conclusion, our current study disclosed that in , VfqI-VfqR circuit upregulates the expression and function of VflT6SS2 by directly or indirectly activating its transcription. These findings will enhance our understanding of the complicated regulatory network between QS and T6SS in .
PubMed: 35669988
DOI: 10.1016/j.bbrep.2022.101282 -
Microbiology Spectrum Feb 2022Bacteriophage-mediated transduction of bacterial DNA is a major route of horizontal gene transfer in the human pathogen, Staphylococcus aureus. Transduction involves the...
Bacteriophage-mediated transduction of bacterial DNA is a major route of horizontal gene transfer in the human pathogen, Staphylococcus aureus. Transduction involves the packaging of bacterial DNA by viruses and enables the transmission of virulence and resistance genes between cells. To learn more about transduction in S. aureus, we searched a transposon mutant library for genes and mutations that enhanced transfer mediated by the temperate phage, ϕ11. Using a novel screening strategy, we performed multiple rounds of transduction of transposon mutant pools selecting for an antibiotic resistance marker within the transposon element. When determining the locations of transferred mutations, we found that the screen had selected for just 1 or 2 transposon mutant(s) within each pool of 96 mutants. Subsequent analysis showed that the position of the transposon, rather than the inactivation of bacterial genes, was responsible for the phenotype. Interestingly, from multiple rounds, we identified a pattern of transduction that encompassed mobile genetic elements as well as chromosomal regions both upstream and downstream of the phage integration site. The latter was confirmed by DNA sequencing of purified phage lysates. Importantly, transduction frequencies were lower for phage lysates obtained by phage infection rather than induction. Our results confirmed previous reports of lateral transduction of bacterial DNA downstream of the integrated phage but also indicated a novel form of specialized transduction of DNA upstream of the phage. These findings illustrated the complexity of transduction processes and increased our understanding of the mechanisms by which phages transfer bacterial DNA. Horizontal transfer of DNA between bacterial cells contributes to the spread of virulence and antibiotic resistance genes in human pathogens. For Staphylococcus aureus, bacterial viruses play a major role in facilitating the horizontal transfer. These viruses, termed bacteriophages, can transfer bacterial DNA between cells by a process known as transduction, which despite its importance is only poorly characterized. Here, we employed a transposon mutant library to investigate transduction in S. aureus. We showed that the genomic location of bacterial DNA relative to where bacteriophages integrated into that bacterial genome affected how frequently that DNA was transduced. Based on serial transduction of transposon mutant pools and direct sequencing of bacterial DNA in bacteriophage particles, we demonstrated both lateral and specialized transduction. The use of mutant libraries to investigate the genomic patterns of bacterial DNA transferred between cells could help us understand how horizontal transfer influences virulence and resistance development.
Topics: DNA, Bacterial; Gene Transfer, Horizontal; Interspersed Repetitive Sequences; Staphylococcus Phages; Staphylococcus aureus; Transduction, Genetic
PubMed: 35138167
DOI: 10.1128/spectrum.02423-21 -
Probiotics and Antimicrobial Proteins Feb 2023The COVID-19 pandemic caused by a novel coronavirus (SARS-CoV-2) is a serious health concern in the twenty-first century for scientists, health workers, and all humans....
The COVID-19 pandemic caused by a novel coronavirus (SARS-CoV-2) is a serious health concern in the twenty-first century for scientists, health workers, and all humans. The absence of specific biotherapeutics requires new strategies to prevent the spread and prophylaxis of the novel virus and its variants. The SARS-CoV-2 virus shows pathogenesis by entering the host cells via spike protein and Angiotensin-Converting Enzyme 2 receptor protein. Thus, the present study aims to compute the binding energies between a wide range of bacteriocins with receptor-binding domain (RBD) on spike proteins of wild type (WT) and beta variant (lineage B.1.351). Molecular docking analyses were performed to evaluate binding energies. Upon achieving the best bio-peptides with the highest docking scores, further molecular dynamics (MD) simulations were performed to validate the structure and interaction stability. Protein-protein docking of the chosen 22 biopeptides with WT-RBD showed docking scores lower than -7.9 kcal/mol. Pediocin PA-1 and salivaricin P showed the lowest (best) docking scores of - 12 kcal/mol. Pediocin PA-1, salivaricin B, and salivaricin P showed a remarkable increase in the double mutant's predicted binding affinity with -13.8 kcal/mol, -13.0 kcal/mol, and -12.5 kcal/mol, respectively. Also, a better predicted binding affinity of pediocin PA-1 and salivaricin B against triple mutant was observed compared to the WT. Thus, pediocin PA-1 binds stronger to mutants of the RBD, particularly to double and triple mutants. Salivaricin B showed a better predicted binding affinity towards triple mutant compared to WT, showing that it might be another bacteriocin with potential activity against the SARS-CoV-2 beta variant. Overall, pediocin PA-1, salivaricin P, and salivaricin B are the most promising candidates for inhibiting SARS-CoV-2 (including lineage B.1.351) entrance into the human cells. These bacteriocins derived from lactic acid bacteria hold promising potential for paving an alternative way for treatment and prophylaxis of WT and beta variants.
Topics: Humans; Bacteriocins; SARS-CoV-2; Lactobacillales; COVID-19; Molecular Docking Simulation; Pandemics
PubMed: 34837166
DOI: 10.1007/s12602-021-09879-0 -
Journal of Pharmacy & Pharmaceutical... 2018Liver fatty acid binding protein (FABP1) is a cytoplasmic polypeptide that transports substrates throughout the cytosol and functions as an antioxidant. A common...
PURPOSE
Liver fatty acid binding protein (FABP1) is a cytoplasmic polypeptide that transports substrates throughout the cytosol and functions as an antioxidant. A common polymorphic variant, FABP1 T94A has a minor allele frequency of 26-38%, 8.3±1.9% homozygous in the human population. The purpose of this study was to mutate and isolate recombinant rat FABP1 to the T94A variant to evaluate the mutant's antioxidant activity using in vitro studies.
METHODS
Site-directed mutagenesis was used to generate a mutation in rat cDNA within a pGEX-6p-2 vector. This plasmid was transformed into competent cells and cultured for expression of FABP1 T94A mutant. The mutated protein was purified using GSTrap Fastflow columns within an ÄKTA FPLC system. A 2,7-dichlorofluorescein (DCF) assay was used to screen the T94A variant antioxidant activity. Additionally, Thiobarbituric Acid Reactive Substances (TBARS) assay was used in determining T94A mutant antioxidant activity in hydrophilic and lipophilic environments through the use of the azo compounds AAPH and MeO-AMVN, respectively and in the presence and absence of the long-chain fatty acid palmitate and α-bromo palmitate.
RESULTS
Although the FABP1 T94A (20 μM) mutant significantly reduced DCF fluorescence compared to control (no protein; P< 0.001), there were no significant difference when compared to the wild-type (WT) FABP1. T94A was able to diminish the formation of malondialdehyde (MDA) in both lipophilic and hydrophilic systems. There were significant differences between T94A mutant and WT FABP1 at concentrations 1 and 10 μM (P< 0.05) in the hydrophilic milieu, however, this was not seen at 20 μM and also not seen in the lipophilic milieu at all concentrations. When T94A was pre-incubated with the long-chain fatty acids palmitate or α -bromo palmitate, MDA formation was decreased in both lipid peroxidation systems. There were no statistical differences between the WT FABP1 and T94A bound with fatty acids in both lipid peroxidation systems, however, there was a slight statistical difference when the T94A and WT FABP1 bound α-Br-PA in the AAPH lipid peroxidation system only.
CONCLUSIONS
The T94A has antioxidant activity in both hydrophilic and lipophilic environments. The T94A variant of FABP1 does not have a loss of function in regard to acting as an antioxidant but the extent of function may be influenced by ligand binding. We conclude that populations having the minor T94A allele frequency would have similar ROS scavenging potential as those with nascent FABP1.
Topics: Animals; Antioxidants; Fatty Acid-Binding Proteins; Fluoresceins; Fluorescent Dyes; Mutagenesis, Site-Directed; Rats; Recombinant Proteins
PubMed: 30407907
DOI: 10.18433/jpps30246 -
Current Protocols in Molecular Biology Apr 2014The lagging annotation of bacterial genomes and the inherent genetic complexity of many phenotypes is hindering the discovery of new drug targets and the development of... (Review)
Review
The lagging annotation of bacterial genomes and the inherent genetic complexity of many phenotypes is hindering the discovery of new drug targets and the development of new antimicrobial agents and vaccines. This unit presents Tn-seq, a method that has made it possible to quantitatively determine fitness for most genes in a microorganism and to screen for quantitative genetic interactions on a genome-wide scale and in a high-throughput fashion. Tn-seq can thus direct studies on the annotation of genes and untangle complex phenotypes. The method is based on the construction of a saturated transposon insertion library. After library selection, changes in the frequency of each insertion mutant are determined by sequencing flanking regions en masse. These changes are used to calculate each mutant's fitness. The method was originally developed for the Gram-positive bacterium Streptococcus pneumoniae, a causative agent of pneumonia and meningitis, but has now been applied to several different microbial species.
Topics: DNA, Bacterial; Genome, Bacterial; High-Throughput Nucleotide Sequencing; Sequence Analysis, DNA; Streptococcus pneumoniae
PubMed: 24733243
DOI: 10.1002/0471142727.mb0716s106