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International Journal of Molecular... Jun 2024The compound 15-deacetylcalonectrin (15-deCAL) is a common pathway intermediate in the biosynthesis of trichothecenes. This tricyclic intermediate is metabolized to...
The compound 15-deacetylcalonectrin (15-deCAL) is a common pathway intermediate in the biosynthesis of trichothecenes. This tricyclic intermediate is metabolized to calonectrin (CAL) by trichothecene 15--acetyltransferase encoded by . Unlike other trichothecene pathway gene mutants, the Δ mutant produces lower amounts of the knocked-out enzyme's substrate 15-deCAL, and instead, accumulates higher quantities of earlier bicyclic intermediate and shunt metabolites. Furthermore, evolutionary studies suggest that may play a role in shaping the chemotypes of trichothecene-producing strains. To better understand the functional role of Tri3p in biosynthesis and evolution, we aimed to develop a method to produce 15-deCAL by using transgenic strains derived from a trichothecene overproducer. Unfortunately, introducing mutant , encoding a catalytically impaired but structurally intact acetylase, did not improve the low 15-deCAL production level of the Δ deletion strain, and the bicyclic products continued to accumulate as the major metabolites of the active-site mutant. These findings are discussed in light of the enzyme responsible for 15-deCAL production in trichothecene biosynthesis machinery. To efficiently produce 15-deCAL, we tested an alternative strategy of using a CAL-overproducing transformant. By feeding a crude CAL extract to a strain that was isolated in this study and capable of specifically deacetylating C-15 acetyl, 15-deCAL was efficiently recovered. The substrate produced in this manner can be used for kinetic investigations of this enzyme and its possible role in chemotype diversification.
Topics: Fusarium; Trichothecenes; Mutation; Acetyltransferases; Fungal Proteins; Biosynthetic Pathways
PubMed: 38928120
DOI: 10.3390/ijms25126414 -
International Journal of Molecular... Jun 2024In our prior investigations, we elucidated the role of the tryptophan-to-tyrosine substitution at the 61st position in the nonstructural protein NSsW61Y in diminishing...
The Effect of Tryptophan-to-Tyrosine Mutation at Position 61 of the Nonstructural Protein of Severe Fever with Thrombocytopenia Syndrome Virus on Viral Replication through Autophagosome Modulation.
In our prior investigations, we elucidated the role of the tryptophan-to-tyrosine substitution at the 61st position in the nonstructural protein NSsW61Y in diminishing the interaction between nonstructural proteins (NSs) and nucleoprotein (NP), impeding viral replication. In this study, we focused on the involvement of NSs in replication via the modulation of autophagosomes. Initially, we examined the impact of NP expression levels, a marker for replication, upon the infection of HeLa cells with severe fever thrombocytopenia syndrome virus (SFTSV), with or without the inhibition of NP binding. Western blot analysis revealed a reduction in NP levels in NSsW61Y-expressing conditions. Furthermore, the expression levels of the canonical autophagosome markers p62 and LC3 decreased in HeLa cells expressing NSsW61Y, revealing the involvement of individual viral proteins on autophagy. Subsequent experiments confirmed that NSsW61Y perturbs autophagy flux, as evidenced by reduced levels of LC3B and p62 upon treatment with chloroquine, an inhibitor of autophagosome-lysosome fusion. LysoTracker staining demonstrated a decrease in lysosomes in cells expressing the NS mutant compared to those expressing wild-type NS. We further explored the mTOR-associated regulatory pathway, a key regulator affected by NS mutant expression. The observed inhibition of replication could be linked to conformational changes in the NSs, impairing their binding to NP and altering mTOR regulation, a crucial upstream signaling component in autophagy. These findings illuminate the intricate interplay between NSsW61Y and the suppression of host autophagy machinery, which is crucial for the generation of autophagosomes to facilitate viral replication.
Topics: Humans; Viral Nonstructural Proteins; Virus Replication; Autophagosomes; HeLa Cells; Phlebovirus; Autophagy; Tyrosine; Tryptophan; TOR Serine-Threonine Kinases; Mutation; Amino Acid Substitution; Severe Fever with Thrombocytopenia Syndrome; Lysosomes; Nucleoproteins
PubMed: 38928101
DOI: 10.3390/ijms25126394 -
International Journal of Molecular... Jun 2024We analyzed the thermal stability of the HPr protein through the site-directed point mutation Lys62 replaced by Ala residue using molecular dynamics simulations at five...
We analyzed the thermal stability of the HPr protein through the site-directed point mutation Lys62 replaced by Ala residue using molecular dynamics simulations at five different temperatures: 298, 333, 362, 400, and 450 K, for periods of 1 μs and in triplicate. The results from the mutant thermophilic HPrm protein were compared with those of the wild-type thermophilic HPr protein and the mesophilic HPr protein. Structural and molecular interaction analyses show that proteins lose stability as temperature increases. Mutant and wild-type proteins behave similarly up to 362 K. However, at 400 K the mutant protein shows greater structural instability, losing more buried hydrogen bonds and exposing more of its non-polar residues to the solvent. Therefore, in this study, we confirmed that the salt bridge network of the Glu3-Lys62-Glu36 triad, made up of the Glu3-Lys62 and Glu36-Lys62 ion pairs, provides thermal stability to the thermophilic HPr protein.
Topics: Molecular Dynamics Simulation; Protein Stability; Hydrogen Bonding; Temperature; Mutation; Bacterial Proteins; Amino Acid Substitution; Protein Conformation; Mutagenesis, Site-Directed
PubMed: 38928023
DOI: 10.3390/ijms25126316 -
International Journal of Molecular... Jun 2024In yeast , there are two translation termination factors, eRF1 (Sup45) and eRF3 (Sup35), which are essential for viability. Previous studies have revealed that presence...
In yeast , there are two translation termination factors, eRF1 (Sup45) and eRF3 (Sup35), which are essential for viability. Previous studies have revealed that presence of nonsense mutations in these genes leads to amplification of mutant alleles ( and ), which appears to be necessary for the viability of such cells. However, the mechanism of this phenomenon remained unclear. In this study, we used RNA-Seq and proteome analysis to reveal the complete set of gene expression changes that occur during cellular adaptation to the introduction of the nonsense allele. Our analysis demonstrated significant changes in the transcription of genes that control the cell cycle: decreases in the expression of genes of the anaphase promoting complex APC/C (, ) and their activator , and increases in the expression of the transcription factor , the main cell cycle kinase , and cyclins that induce DNA biosynthesis. We propose a model according to which yeast adaptation to nonsense mutations in the translation termination factor genes occurs as a result of a delayed cell cycle progression beyond the G2-M stage, which leads to an extension of the S and G2 phases and an increase in the number of copies of the mutant allele.
Topics: Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Codon, Nonsense; Gene Expression Regulation, Fungal; Peptide Termination Factors; Adaptation, Physiological; Cell Cycle
PubMed: 38928012
DOI: 10.3390/ijms25126308 -
International Journal of Molecular... Jun 2024Oxidative stress represents a critical facet of the array of abiotic stresses affecting crop growth and yield. In this paper, we investigated the potential differences...
Oxidative stress represents a critical facet of the array of abiotic stresses affecting crop growth and yield. In this paper, we investigated the potential differences in the functions of two highly homologous Arabidopsis DSS1 proteins in terms of maintaining genome integrity and response to oxidative stress. In the context of homologous recombination (HR), it was shown that overexpressing AtDSS1(I) using a functional complementation test increases the resistance of the Δ mutant of to genotoxic agents. This indicates its conserved role in DNA repair via HR. To investigate the global transcriptome changes occurring in plant mutant lines, gene expression analysis was conducted using Illumina RNA sequencing technology. Individual RNA libraries were constructed from three total RNA samples isolated from , , and wild-type (WT) plants under hydrogen peroxide-induced stress. RNA-Seq data analysis and real-time PCR identification revealed major changes in gene expression between mutant lines and WT, while the and mutant lines exhibited analogous transcription profiles. The Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed significantly enriched metabolic pathways. Notably, genes associated with HR were upregulated in mutants compared to the WT. Otherwise, genes of the metabolic pathway responsible for the synthesis of secondary metabolites were downregulated in both mutant lines. These findings highlight the importance of understanding the molecular mechanisms of plant responses to oxidative stress.
Topics: Oxidative Stress; Arabidopsis; Seedlings; Transcriptome; Arabidopsis Proteins; Gene Expression Regulation, Plant; Gene Knockout Techniques; Gene Expression Profiling; Mutation; Hydrogen Peroxide
PubMed: 38927997
DOI: 10.3390/ijms25126291 -
Genes Jun 2024Retinitis pigmentosa (RP) is a heterogeneous inherited retinal disorder. Mutations in cause autosomal recessive (AR) RP. We aimed to characterize the genotype,...
Retinitis pigmentosa (RP) is a heterogeneous inherited retinal disorder. Mutations in cause autosomal recessive (AR) RP. We aimed to characterize the genotype, expression pattern, and phenotype in a large cohort of cases. Sanger and whole exome sequencing were used to identify the variants. Medical records were reviewed and analyzed. Thirty-one patients with biallelic mutations were identified: 28 homozygous for c.226C>T (p.R76*), 2 compound heterozygous for p.R76* and c.3G>A (p.M1?), and one homozygous for c.247C>T (p.R83*). c.226C>T is a founder mutation among patients of Jewish descent. The clinical parameters were less severe in compared to and cases. RT-PCR analysis in fibroblast cells revealed the presence of four different transcripts in both WT and mutant samples with a lower percentage of the WT transcript in patients. Sequence analysis identified an exonic sequence enhancer (ESE) that includes the c.226 position which is affected by the mutation. mutations are an uncommon cause of IRD worldwide but are not rare among Ashkenazi Jews. Our data indicate that p.R76* affect an ESE which in turn results in the pronounced skipping of exon 3. Therefore, RNA-based therapies might show low efficacy since the mutant transcripts are spliced.
Topics: Humans; Retinitis Pigmentosa; Female; Male; Mutation; Adult; Jews; Exome Sequencing; Pedigree; Eye Proteins; Phenotype; Middle Aged; Adolescent
PubMed: 38927740
DOI: 10.3390/genes15060804 -
Genes Jun 2024Many enzymes in the Raetz pathway for lipid A biosynthesis in are essential. A homologous protein Pa1792|LpxH in is known to complement the loss of LpxH in ....
Many enzymes in the Raetz pathway for lipid A biosynthesis in are essential. A homologous protein Pa1792|LpxH in is known to complement the loss of LpxH in . Genome-wide transposon-insertion sequencing analysis indicates that is essential in . However, genetic analysis of in has not been carried out, partly because the conditional alleles of essential genes are not readily constructed. In this study, we first constructed a plasmid-based temperature-sensitive mutant or in PAO1. Spot-plating assay indicated that was lethal at a restrictive temperature, confirming its essentiality for growth. Microscopic analysis revealed that exhibited an oval-shaped morphology, suggesting that was required for rod-shape formation. SDS-PAGE and Western blotting analysis showed that failed to synthesize lipid A, consistent with its function in lipid A biosynthesis. Strong expression of but not the non-homologous isoenzyme or impeded growth and caused cell lysis, implying that -specific cofactors were required for this toxic effect in . Together, our results demonstrate that is essential for lipid A biosynthesis, rod-shaped growth, and viability in . We propose that this plasmid-based conditional allele is a useful tool for the genetic study of essential genes in .
Topics: Pseudomonas aeruginosa; Plasmids; Bacterial Proteins; Temperature; Mutation; Lipid A; Escherichia coli
PubMed: 38927720
DOI: 10.3390/genes15060784 -
Genes Jun 2024is a LIM-homeodomain transcription factor that affects body size in mammals by regulating the secretion of pituitary hormones. Akita, Shiba Inu, and Mame Shiba Inu dogs...
is a LIM-homeodomain transcription factor that affects body size in mammals by regulating the secretion of pituitary hormones. Akita, Shiba Inu, and Mame Shiba Inu dogs are Japanese native dog breeds that have different body sizes. To determine whether plays a role in the differing body sizes of these three dog breeds, we sequenced the gene in the three breeds, which led to the identification of an SNP in codon 280 (S280N) associated with body size. The allele frequency at this SNP differed significantly between the large Akita and the two kinds of smaller Shiba dogs. To validate the function of this SNP on body size, we introduced this change into the gene of mice. Homozygous mutant mice (S279N) were found to have significantly increased body lengths and weights compared to heterozygous mutant (S279N) and wild-type (S279N) mice several weeks after weaning. These results demonstrate that a nonsynonymous substitution in plays an important role in regulating body size in mammals.
Topics: Animals; LIM-Homeodomain Proteins; Transcription Factors; Mice; Body Size; Dogs; Polymorphism, Single Nucleotide; Gene Frequency; Male; Female
PubMed: 38927675
DOI: 10.3390/genes15060739 -
Genes May 2024Grain filling is critical for determining yield and quality, raising the question of whether central coordinators exist to facilitate the uptake and storage of various...
Grain filling is critical for determining yield and quality, raising the question of whether central coordinators exist to facilitate the uptake and storage of various substances from maternal to filial tissues. The duplicate NAC transcription factors ZmNAC128 and ZmNAC130 could potentially serve as central coordinators. By analyzing differentially expressed genes from mutants across different genetic backgrounds and growing years, we identified 243 highly and differentially expressed genes (hdEGs) as the core target genes. These 243 hdEGs were associated with storage metabolism and transporters. ZmNAC128 and ZmNAC130 play vital roles in storage metabolism, and this study revealed two additional starch metabolism-related genes, and , as their direct targets. A key finding of this study was the inclusion of 17 transporter genes within the 243 hdEGs, with significant alterations in the levels of more than 10 elements/substances in mutant kernels. Among them, six out of the nine upregulated transporter genes were linked to the transport of heavy metals and metalloids (HMMs), which was consistent with the enrichment of cadmium, lead, and arsenic observed in mutant kernels. Interestingly, the levels of Mg and Zn, minerals important to biofortification efforts, were reduced in mutant kernels. In addition to their direct involvement in sugar transport, ZmNAC128 and ZmNAC130 also activate the expression of the endosperm-preferential nitrogen and phosphate transporters and . This coordinated regulation limits the intake of HMMs, enhances biofortification, and facilitates the uptake and storage of essential nutrients.
Topics: Zea mays; Gene Expression Regulation, Plant; Plant Proteins; Transcription Factors; Seeds; Edible Grain; Nutrients
PubMed: 38927600
DOI: 10.3390/genes15060663 -
Biomolecules May 2024The p53 protein is the master regulator of cellular integrity, primarily due to its tumor-suppressing functions. Approximately half of all human cancers carry mutations... (Review)
Review
The p53 protein is the master regulator of cellular integrity, primarily due to its tumor-suppressing functions. Approximately half of all human cancers carry mutations in the TP53 gene, which not only abrogate the tumor-suppressive functions but also confer p53 mutant proteins with oncogenic potential. The latter is achieved through so-called gain-of-function (GOF) mutations that promote cancer progression, metastasis, and therapy resistance by deregulating transcriptional networks, signaling pathways, metabolism, immune surveillance, and cellular compositions of the microenvironment. Despite recent progress in understanding the complexity of mutp53 in neoplastic development, the exact mechanisms of how mutp53 contributes to cancer development and how they escape proteasomal and lysosomal degradation remain only partially understood. In this review, we address recent findings in the field of oncogenic functions of mutp53 specifically regarding, but not limited to, its implications in metabolic pathways, the secretome of cancer cells, the cancer microenvironment, and the regulating scenarios of the aberrant proteasomal degradation. By analyzing proteasomal and lysosomal protein degradation, as well as its connection with autophagy, we propose new therapeutical approaches that aim to destabilize mutp53 proteins and deactivate its oncogenic functions, thereby providing a fundamental basis for further investigation and rational treatment approaches for TP53-mutated cancers.
Topics: Humans; Tumor Suppressor Protein p53; Neoplasms; Tumor Microenvironment; Proteolysis; Proteasome Endopeptidase Complex; Autophagy; Animals; Mutation; Lysosomes; Carcinogenesis
PubMed: 38927053
DOI: 10.3390/biom14060649