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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 -
Frontiers in Plant Science 2023Cuticular wax is a characteristic feature of land plants that provides protection against both biotic and abiotic stresses. In this study, a glossy mutant lacking an...
Cuticular wax is a characteristic feature of land plants that provides protection against both biotic and abiotic stresses. In this study, a glossy mutant lacking an epicuticular wax layer was identified in the γ-irradiated M mutant population of the onion cultivar Bhima Super. The inheritance of the mutant's glossy phenotype was determined to be recessive and single locus. Scanning electron microscopy analysis showed poor accumulation of wax crystals in the glossy mutant, concentrated near the stomata. The plant height, number of leaves per plant, and stomatal parameters of the mutant were similar to the wild-type. RNA-seq was used to comprehend the expression variations of waxy cuticle-related genes in the glossy mutant and its wild-type waxy cultivars. Differential gene expression analysis of the RNA-seq data revealed that the genes involved in wax biosynthesis, such as , and , were downregulated by 2.72, 1.74, 2.59 and 2.12-fold, respectively, in the glossy mutant respectively. The expression patterns of these four unigenes were validated using semi-quantitative RT-PCR. The glossy mutant displayed a substantial 3.5-fold reduction in cuticular wax load compared to the wild-type due to the significant downregulation of these wax biosynthesis genes. These findings represent early advancements in understanding the molecular mechanisms of wax biosynthesis in onions. Furthermore, they provide a foundation for utilizing the glossy mutant trait in breeding programmes to enhance stress and pest resilience.
PubMed: 37680361
DOI: 10.3389/fpls.2023.1245308 -
Journal of Experimental & Clinical... Jul 2019We reported previously that phenethyl isothiocyanate (PEITC), a dietary compound, can reactivate p53 mutant in vitro and in SK-BR-3 (p53) breast xenograft model...
BACKGROUND
We reported previously that phenethyl isothiocyanate (PEITC), a dietary compound, can reactivate p53 mutant in vitro and in SK-BR-3 (p53) breast xenograft model resulting in tumor inhibition. Because of the diversity of human cancers with p53 mutations, these findings raise important questions whether this mechanism operates in different cancer types with same or different p53 mutations. In this study, we investigated whether PEITC recuses mutant p53 in prostate cancer cells harboring different types of p53 mutants, structural and contact, in vitro and in vivo.
METHODS
Cell proliferation, cell apoptosis and cell cycle arrest assays were performed to examine the effects of PEITC on prostate cancer cell lines with p53 mutation(s), wild-type p53, p53 null or normal prostate cells in vitro. Western blot analysis was used to monitor the expression levels of p53 protein, activation of ATM and upregulation of canonical p53 targets. Immunoprecipitation, subcellular protein fraction and qRT-PCR was performed to determine change in conformation and restoration of transactivation functions/ inhibition of gain-of-function (GOF) activities to p53 mutant(s). Mice xenograft models were established to evaluate the antitumor efficacy of PEITC and PEITC-induced reactivation of p53 mutant(s) in vivo. Immunohistochemistry of xenograft tumor tissues was performed to determine effects of PEITC on expression of Ki67 and mutant p53 in vivo.
RESULTS
We demonstrated that PEITC inhibits the growth of prostate cancer cells with different "hotspot" p53 mutations (structural and contact), however, preferentially towards structural mutants. PEITC inhibits proliferation and induces apoptosis by rescuing mutant p53 in p53 contact (VCaP) and p53 structural (LAPC-4) mutant cells with differential potency. We further showed that PEITC inhibits the growth of DU145 cells that co-express p53 (structural) and p53 (contact) mutants by targeting p53 mutant selectively, but not p53. The mutant p53 restored by PEITC induces apoptosis in DU145 cells by activating canonical p53 targets, delaying cells in G1 phase and phosphorylating ATM. Importantly, PEITC reactivated p53 and p53 mutants in LAPC-4 and DU145 prostate xenograft models, respectively, resulting in significant tumor inhibition.
CONCLUSION
Our studies provide the first evidence that PEITC's anti-cancer activity is cancer cell type-independent, but p53 mutant-type dependent.
Topics: Animals; Anticarcinogenic Agents; Ataxia Telangiectasia Mutated Proteins; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Humans; Isothiocyanates; Male; Mice; Mutation; Phosphorylation; Prostatic Neoplasms; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays
PubMed: 31307507
DOI: 10.1186/s13046-019-1267-z -
Nature Methods Oct 2009Biological pathways are structured in complex networks of interacting genes. Solving the architecture of such networks may provide valuable information, such as how...
Biological pathways are structured in complex networks of interacting genes. Solving the architecture of such networks may provide valuable information, such as how microorganisms cause disease. Here we present a method (Tn-seq) for accurately determining quantitative genetic interactions on a genome-wide scale in microorganisms. Tn-seq is based on the assembly of a saturated Mariner transposon insertion library. After library selection, changes in frequency of each insertion mutant are determined by sequencing the flanking regions en masse. These changes are used to calculate each mutant's fitness. Using this approach, we determined fitness for each gene of Streptococcus pneumoniae, a causative agent of pneumonia and meningitis. A genome-wide screen for genetic interactions of five query genes identified both alleviating and aggravating interactions that could be divided into seven distinct categories. Owing to the wide activity of the Mariner transposon, Tn-seq has the potential to contribute to the exploration of complex pathways across many different species.
Topics: Chromosome Mapping; Computer Simulation; DNA, Bacterial; Models, Genetic; Proteome; Sequence Analysis, DNA; Signal Transduction; Streptococcus pneumoniae
PubMed: 19767758
DOI: 10.1038/nmeth.1377 -
Molecules (Basel, Switzerland) Dec 2004This report describes a new set of macromolecular descriptors of relevance to protein QSAR/QSPR studies, protein's quadratic indices. These descriptors are calculated... (Review)
Review
This report describes a new set of macromolecular descriptors of relevance to protein QSAR/QSPR studies, protein's quadratic indices. These descriptors are calculated from the macromolecular pseudograph's alpha-carbon atom adjacency matrix. A study of the protein stability effects for a complete set of alanine substitutions in Arc repressor illustrates this approach. Quantitative Structure-Stability Relationship (QSSR) models allow discriminating between near wild-type stability and reduced-stability A-mutants. A linear discriminant function gives rise to excellent discrimination between 85.4% (35/41)and 91.67% (11/12) of near wild-type stability/reduced stability mutants in training and test series, respectively. The model's overall predictability oscillates from 80.49 until 82.93, when n varies from 2 to 10 in leave-n-out cross validation procedures. This value stabilizes around 80.49% when n was > 6. Additionally, canonical regression analysis corroborates the statistical quality of the classification model (Rcanc = 0.72, p-level <0.0001). This analysis was also used to compute biological stability canonical scores for each Arc A-mutant. On the other hand, nonlinear piecewise regression model compares favorably with respect to linear regression one on predicting the melting temperature (tm)of the Arc A-mutants. The linear model explains almost 72% of the variance of the experimental tm (R = 0.85 and s = 5.64) and LOO press statistics evidenced its predictive ability (q2 = 0.55 and scv = 6.24). However, this linear regression model falls to resolve t(m) predictions of Arc A-mutants in external prediction series. Therefore, the use of nonlinear piecewise models was required. The tm values of A-mutants in training (R = 0.94) and test(R = 0.91) sets are calculated by piecewise model with a high degree of precision. A break-point value of 51.32 degrees C characterizes two mutants' clusters and coincides perfectly with the experimental scale. For this reason, we can use the linear discriminant analysis and piecewise models in combination to classify and predict the stability of the mutants' Arc homodimers. These models also permit the interpretation of the driving forces of such a folding process. The models include protein's quadratic indices accounting for hydrophobic (z1), bulk-steric (z2), and electronic (z3) features of the studied molecules. Preponderance of z1 and z3 over z2 indicates the higher importance of the hydrophobic and electronic side chain terms in the folding of the Arc dimer. In this sense, developed equations involve short-reaching (k < or = 3), middle- reaching (3 < k < or = 7) and far-reaching (k= 8 or greater) z1, 2, 3-protein's quadratic indices. This situation points to topologic/topographic protein's backbone interactions control of the stability profile of wild-type Arc and its A-mutants. Consequently, the present approach represents a novel and very promising way to mathematical research in biology sciences.
Topics: Alanine; Amino Acid Substitution; Animals; Computational Biology; Dimerization; Humans; Models, Molecular; Predictive Value of Tests; Protein Engineering; Protein Folding; Quantitative Structure-Activity Relationship; Repressor Proteins; Stereoisomerism; Viral Regulatory and Accessory Proteins
PubMed: 18007508
DOI: 10.3390/91201124 -
Current Genetics Apr 2007The PSO3 gene of Saccharomyces cerevisiae was molecularly cloned by complementing the cold-sensitivity phenotype of a pso3-1 mutant and was found to be allelic to RNR4,...
The PSO3 gene of Saccharomyces cerevisiae was molecularly cloned by complementing the cold-sensitivity phenotype of a pso3-1 mutant and was found to be allelic to RNR4, encoding one of the two DNA damage-inducible small subunits of the ribonucleotide reductase (RNR) complex. Compared to a rnr4Delta mutant that allows only very little mutation induction at very low doses of 254(nm) ultraviolet light (UVC), the pso3-1 mutant allele confers leakiness in that it permits some DNA damage-induced mutagenesis at low doses of UVC. Similarly, the pso3 mutant is slightly less sensitive to UVC than an rnr4Delta mutant. Cloning and sequencing of the RNR4 locus of the pso3-1 mutant revealed that its intermediate phenotype is attributable to a G --> A transition at nucleotide 352, leading to replacement of glycine by arginine [G118R] in the mutant's protein. Both RNR4 mutant alleles confer significantly less sensitivity to UVC than mutant alleles of non-UVC-mutable REV3, indicating that, apart from nucleotide excision repair, RAD6-dependent error-free DNA repair may still be functional. The phenotype of a strongly reduced UVC-induced mutagenesis for rnr4 mutant alleles has not yet been described; it suggests the importance of this gene for a fully functional RNR providing correct amounts of DNA precursor molecules, thereby, allowing translesion synthesis (error-prone) of UVC-damaged DNA. Stationary phase cells of the rnr4Delta mutant, but not of the original pso3-1 mutant, are swollen with a fourfold to eightfold increase in volume. The central role of RNR in DNA precursor metabolism and its complex regulation allow for several modes of suppression that may influence the phenotypes of RNR4 mutants, especially those containing the leaky pso3-1 mutant allele.
Topics: Alleles; Base Sequence; Cloning, Molecular; DNA Damage; DNA Primers; DNA Repair; DNA, Fungal; Genes, Fungal; Genetic Complementation Test; Models, Biological; Mutation; Phenotype; Ribonucleoside Diphosphate Reductase; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Ultraviolet Rays
PubMed: 17287963
DOI: 10.1007/s00294-007-0120-7 -
Canadian Journal of Microbiology Nov 2020Brewer's yeast has been widely used in the food industry, and the autolysates thereof are increasingly being studied for their valuable nutritional compositions. Yeast...
Brewer's yeast has been widely used in the food industry, and the autolysates thereof are increasingly being studied for their valuable nutritional compositions. Yeast autolysis is most affected by medium composition and temperature. In this study, a thermosensitive autolytic brewer's yeast P-510 was obtained with atmospheric and room temperature plasma mutagenesis plus 5-bromo-chloro-3-indolyl phosphate screening. The mutant rapidly autolyzed at 37 °C and the autolysates contained more active components and showed higher antioxidant activities compared with that of the parental strain, which indicated that the mutant's autolysates can potentially be used as functional food and nutritional ingredients. Transcriptomic analysis of the mutant and parental strains at 28 and 37 °C suggested that thermosensitive autolysis of P-510 was probably caused by mitochondrial disfunction, glycogen metabolic flux of glycolysis and pentose phosphate pathway disorder, as well as hexose transport inhibition. The results revealed the important role of mitochondrial metabolism and glycogen utilization regulation in heat stress response of yeast.
Topics: Antioxidants; Gene Expression Profiling; Heat-Shock Response; Mutagenesis; Mutation; Saccharomyces cerevisiae; Temperature
PubMed: 32619357
DOI: 10.1139/cjm-2019-0456 -
Plant Direct Jul 2022In nature, plants experience rapid changes in light intensity and quality throughout the day. To maximize growth, they have established molecular mechanisms to optimize...
In nature, plants experience rapid changes in light intensity and quality throughout the day. To maximize growth, they have established molecular mechanisms to optimize photosynthetic output while protecting components of the light-dependent reaction and CO fixation pathways. Plant phenotyping of mutant collections has become a powerful tool to unveil the genetic loci involved in environmental acclimation. Here, we describe the phenotyping of the transfer-DNA (T-DNA) insertion mutant line SALK_008491, previously known as . Growth in a fluctuating light regime caused a loss in growth rate accompanied by a spike in photosystem (PS) II damage and increased non-photochemical quenching (NPQ). Interestingly, an independent null allele did not recapitulate the NPQ phenotype. Through bulk sequencing of a backcrossed segregating F pool, we identified an ~14-kb large deletion on chromosome 3 (Chr3) in SALK_008491 affecting five genes upstream of . Besides , which encodes for a putative plastid Na/H antiporter, the stromal NAD-dependent D-3-phosphoglycerate dehydrogenase 3 () locus was eradicated. Although some changes in the SALK_008491 mutant's photosynthesis can be assigned to the loss of PGDH3, our follow-up studies employing respective single mutants and complementation with overlapping transformation-competent artificial chromosome (TAC) vectors reveal that the exacerbated fluctuating light sensitivity in SALK_008491 mutants result from the simultaneous loss of PGDH3 and NHD1. Altogether, the data obtained from this large deletion-carrying mutant provide new and unintuitive insights into the molecular mechanisms that function to protect the photosynthetic machinery. Moreover, our study renews calls for caution when setting up reverse genetic studies using T-DNA lines. Although second-site insertions, indels, and SNPs have been reported before, large deletion surrounding the insertion site causes yet another problem. Nevertheless, as shown through this research, such unpredictable genetic events following T-DNA mutagenesis can provide unintuitive insights that allow for understanding complex phenomena such as the plant acclimation to dynamic high light stress.
PubMed: 35875836
DOI: 10.1002/pld3.429 -
Cell Cycle (Georgetown, Tex.) Jan 2020mutations are found in 50% of all cancers and mutated status is considered poor for treatment. However, some mutations exhibit only partial loss-of-function (LOF),...
mutations are found in 50% of all cancers and mutated status is considered poor for treatment. However, some mutations exhibit only partial loss-of-function (LOF), meaning they retain residual transcriptional and non-transcriptional activities that are potentially beneficial for therapy. Earlier we have characterized a knock-in mouse model for the partial LOF mutant (p53RR). Reduced DNA binding cooperativity of this mutant led to the loss of p53-dependent apoptosis, while p53 functions in cell cycle control, senescence, metabolism, and antioxidant defense remained intact. Concomitantly, tumor suppression was evident but strongly compromised compared to wild-type mice. Here we used the mouse as a model to investigate whether residual functions of mutant p53 can be engaged to induce cell death, which is considered the most desirable outcome of tumor therapy. We made use of knock-out in developing embryos as a sensitive tool for detecting remaining p53 activities. Genetic ablation of led to embryonic lethality in homozygotes at days 9.5-11.5. This effect was not rescued by concomitant p21-knockout, indicating its independence of p21-mediated cell cycle arrest. Instead, immunohistochemical analysis showed widespread apoptosis in tissues of defective embryos accompanied by persistent accumulation of p53RR protein. This led to partial restoration of the mutant's proficiency in transcriptional induction of the pro-apoptotic genes (Puma) and . These data indicate that increased quantity can compensate for qualitative defects of p53 mutants and suggest that Mdm2-targeting (potentially in combination with other drugs) might be effective against cells bearing p53 partial LOF mutants.
Topics: Animals; Apoptosis; Cyclin-Dependent Kinase Inhibitor p21; Embryo Loss; Embryo, Mammalian; Gene Deletion; Homozygote; Mice; Mutant Proteins; Proto-Oncogene Proteins c-mdm2; Tumor Suppressor Protein p53
PubMed: 31749402
DOI: 10.1080/15384101.2019.1693748 -
IEEE Journal of Translational... 2021Mitochondrial [Formula: see text]-oxidation of fatty acids is the primary energy source for the heart and carried out by Hydroxy Acyl-CoA Dehydrogenase (HADH) encoded...
BACKGROUND
Mitochondrial [Formula: see text]-oxidation of fatty acids is the primary energy source for the heart and carried out by Hydroxy Acyl-CoA Dehydrogenase (HADH) encoded trifunctional protein. Mutations in the genes encoding mitochondrial proteins result in functionally defective protein complexes that contribute to energy deficiencies, excessive reactive oxygen species (ROS) production, and accumulation of damaged mitochondria. We hypothesize that a dramatic alternation in redox state and associated mitochondrial dysfunction is the underlying cause of Fatty Acid Oxidation (FAO) deficiency mutant, resulting in heart failure. Mitochondrial co-enzymes, NADH and FAD, are autofluorescent metabolic indices of cells when imaged, yield a quantitative assessment of the cells' redox status and, in turn, that of the tissue and organ.
METHOD
We utilized an optical cryo-imager to quantitively evaluate the three-dimensional distribution of mitochondrial redox state in newborn rats' hearts and kidneys. Redox ratio (RR) assessment shows that mitochondrial dysfunction is extreme and could contribute to severe heart problems and eventual heart failure in the mutants.
RESULTS
Three-dimensional redox ratio (NADH/FAD) rendering, and the volumetric mean value calculations confirmed significantly decreased cardiac RR in mutants by 31.90% and 12.32%, in renal mitochondrial RR compared to wild-type control. Further, histological assessment of newborn heart myocardial tissue indicated no significant difference in myocardial tissue architecture in both control and severe (HADHA) conditions.
CONCLUSION
These results demonstrate that optical imaging can accurately estimate the redox state changes in newborn rat organs. It is also apparent that the FAO mutant's heart tissue with a low redox ratio is probably more vulnerable to cumulative damages than kidneys and fails prematurely, contributing to sudden death.
Topics: Acyl-CoA Dehydrogenase; Animals; Animals, Newborn; Mitochondria; Myocardium; Oxidation-Reduction; Rats
PubMed: 34462673
DOI: 10.1109/JTEHM.2021.3104966