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International Journal of Molecular... Jun 2024Cytokinesis in plant cells begins with the fusion of vesicles that transport cell wall materials to the center of the cell division plane, where the cell plate forms and...
Cytokinesis in plant cells begins with the fusion of vesicles that transport cell wall materials to the center of the cell division plane, where the cell plate forms and expands radially until it fuses with the parental cell wall. Vesicle fusion is facilitated by -SNARE complexes, with assistance from Sec1/Munc18 (SM) proteins. The SNARE protein KNOLLE and the SM protein KEULE are required for membrane fusion at the cell plate. Due to the crucial function of KEULE, all Arabidopsis () mutants identified to date are seedling lethal. Here, we identified the Arabidopsis () and mutants, which carry recessive, hypomorphic alleles of . Homozygous and plants are viable and fertile but have smaller rosettes and fewer leaves at bolting than the wild type. Their leaves are serrated, small, and wavy, with a complex venation pattern. The mutant leaves also develop necrotic patches and undergo premature senescence. RNA-seq revealed transcriptome changes likely leading to reduced cell wall integrity and an increase in the unfolded protein response. These findings shed light on the roles of KEULE in postembryonic development, particularly in the patterning of rosette leaves and leaf margins.
Topics: Arabidopsis; Arabidopsis Proteins; Gene Expression Regulation, Plant; Plant Leaves; Mutation; Cell Wall; Phenotype
PubMed: 38928373
DOI: 10.3390/ijms25126667 -
International Journal of Molecular... Jun 2024Myeloproliferative neoplasms (MPNs), namely, polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF), are clonal stem cell disorders...
Assessment of Total Antioxidant Capacity, 8-Hydroxy-2'-deoxy-guanosine, the Genetic Landscape, and Their Associations in -Negative Chronic and Blast Phase Myeloproliferative Neoplasms.
Myeloproliferative neoplasms (MPNs), namely, polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF), are clonal stem cell disorders defined by an excessive production of functionally mature and terminally differentiated myeloid cells. MPNs can transform into secondary acute myeloid leukemia (sAML/blast phase MPN) and are linked to alterations in the redox balance, i.e., elevated concentrations of reactive oxygen species and markers of oxidative stress (OS), and changes in antioxidant systems. We evaluated OS in 117 chronic phase MPNs and 21 sAML cases versus controls by measuring total antioxidant capacity (TAC) and 8-hydroxy-2'-deoxy-guanosine (8-OHdG) concentrations. TAC was higher in MPNs than controls ( = 0.03), particularly in ET ( = 0.04) and PMF ( = 0.01). -positive MPNs had higher TAC than controls ( = 0.002) and triple-negative MPNs ( = 0.01). PMF patients who had treatment expressed lower TAC than therapy-free subjects ( = 0.03). 8-OHdG concentrations were similar between controls and MPNs, controls and sAML, and MPNs and sAML. We noted associations between TAC and MPNs (OR = 1.82; = 0.05), i.e., ET (OR = 2.36; = 0.03) and PMF (OR = 2.11; = 0.03), but not sAML. 8-OHdG concentrations were not associated with MPNs (OR = 1.73; = 0.62) or sAML (OR = 1.89; = 0.49). In conclusion, we detected redox imbalances in MPNs based on disease subtype, driver mutations, and treatment history.
Topics: Humans; Male; Female; 8-Hydroxy-2'-Deoxyguanosine; Middle Aged; Aged; Myeloproliferative Disorders; Antioxidants; Adult; Oxidative Stress; Aged, 80 and over; Blast Crisis; Fusion Proteins, bcr-abl; Primary Myelofibrosis
PubMed: 38928358
DOI: 10.3390/ijms25126652 -
International Journal of Molecular... Jun 2024Our study investigates the genetic mechanisms underlying the spotted leaf phenotype in rice, focusing on the mutant. This mutant is characterized by persistent...
Our study investigates the genetic mechanisms underlying the spotted leaf phenotype in rice, focusing on the mutant. This mutant is characterized by persistent reddish-brown leaf spots from the seedling stage to maturity, leading to extensive leaf necrosis. Using map-based cloning, we localized the responsible locus to a 330 Kb region on chromosome 2. We identified , named , as the causative gene. A point mutation in , substituting valine for glutamic acid, was identified as the critical factor for the phenotype. Functional complementation and the generation of CRISPR/Cas9-mediated knockout lines in the IR64 background confirmed the central role of OsRPT5A in controlling this trait. The qPCR results from different parts of the rice plant revealed that is constitutively expressed across various tissues, with its subcellular localization unaffected by the mutation. Notably, we observed an abnormal accumulation of reactive oxygen species (ROS) in mutants by examining the physiological indexes of leaves, suggesting a disruption in the ROS system. Complementation studies indicated OsRPT5A's involvement in ROS homeostasis and catalase activity regulation. Moreover, the mutant exhibited enhanced resistance to pv. (), highlighting OsRPT5A's role in rice pathogen resistance mechanisms. Overall, our results suggest that OsRPT5A plays a critical role in regulating ROS homeostasis and enhancing pathogen resistance in rice.
Topics: Oryza; Plant Leaves; Plant Proteins; Chromosome Mapping; Xanthomonas; Plant Diseases; Reactive Oxygen Species; Disease Resistance; Mutation; Phenotype; Gene Expression Regulation, Plant
PubMed: 38928342
DOI: 10.3390/ijms25126637 -
International Journal of Molecular... Jun 2024Glucocerebrosidase (GCase) is a lysosomal enzyme that catalyzes the breakdown of glucosylceramide in the presence of its activator saposin C (SapC). SapC arises from the...
Glucocerebrosidase (GCase) is a lysosomal enzyme that catalyzes the breakdown of glucosylceramide in the presence of its activator saposin C (SapC). SapC arises from the proteolytical cleavage of prosaposin (encoded by gene), which gives rise to four saposins. GCase is targeted to the lysosomes by LIMP-2, encoded by gene. GCase deficiency causes Gaucher Disease (GD), which is mainly due to biallelic pathogenetic variants in the GCase-encoding gene, . However, impairment of GCase activity can be rarely caused by SapC or LIMP-2 deficiencies. We report a new case of LIMP-2 deficiency and a new case of SapC deficiency (missing all four saposins, PSAP deficiency), and measured common biomarkers of GD and GCase activity. Glucosylsphingosine and chitotriosidase activity in plasma were increased in GCase deficiencies caused by and mutations, whereas -linked deficiency showed only Glucosylsphingosine elevation. GCase activity was reduced in fibroblasts and leukocytes: the decrease was sharper in - and -mutant fibroblasts than -mutant ones; LIMP-2-deficient leukocytes displayed higher residual GCase activity than -mutant ones. Finally, we demonstrated that GCase mainly undergoes proteasomal degradation in LIMP-2-deficient fibroblasts and lysosomal degradation in PSAP-deficient fibroblasts. Thus, we analyzed the differential biochemical profile of GCase deficiencies due to the ultra-rare and biallelic pathogenic variants in comparison with the profile observed in -linked GCase deficiency.
Topics: Glucosylceramidase; Humans; Gaucher Disease; Saposins; Lysosomal Membrane Proteins; Receptors, Scavenger; Fibroblasts; Mutation; Lysosomes; Hexosaminidases; Male; Female
PubMed: 38928321
DOI: 10.3390/ijms25126615 -
International Journal of Molecular... Jun 2024Bacterial nitroreductase enzymes capable of activating imaging probes and prodrugs are valuable tools for gene-directed enzyme prodrug therapies and targeted cell...
Bacterial nitroreductase enzymes capable of activating imaging probes and prodrugs are valuable tools for gene-directed enzyme prodrug therapies and targeted cell ablation models. We recently engineered a nitroreductase ( NfsB F70A/F108Y) for the substantially enhanced reduction of the 5-nitroimidazole PET-capable probe, SN33623, which permits the theranostic imaging of vectors labeled with oxygen-insensitive bacterial nitroreductases. This mutant enzyme also shows improved activation of the DNA-alkylation prodrugs CB1954 and metronidazole. To elucidate the mechanism behind these enhancements, we resolved the crystal structure of the mutant enzyme to 1.98 Å and compared it to the wild-type enzyme. Structural analysis revealed an expanded substrate access channel and new hydrogen bonding interactions. Additionally, computational modeling of SN33623, CB1954, and metronidazole binding in the active sites of both the mutant and wild-type enzymes revealed key differences in substrate orientations and interactions, with improvements in activity being mirrored by reduced distances between the N5-H of isoalloxazine and the substrate nitro group oxygen in the mutant models. These findings deepen our understanding of nitroreductase substrate specificity and catalytic mechanisms and have potential implications for developing more effective theranostic imaging strategies in cancer treatment.
Topics: Nitroreductases; Nitroimidazoles; Metronidazole; Prodrugs; Escherichia coli Proteins; Positron-Emission Tomography; Escherichia coli; Catalytic Domain; Protein Engineering; Models, Molecular; Aziridines
PubMed: 38928299
DOI: 10.3390/ijms25126593 -
International Journal of Molecular... Jun 2024Rice prolamins are categorized into three groups by molecular size (10, 13, or 16 kDa), while the 13 kDa prolamins are assigned to four subgroups (Pro13a-I, Pro13a-II,...
Rice prolamins are categorized into three groups by molecular size (10, 13, or 16 kDa), while the 13 kDa prolamins are assigned to four subgroups (Pro13a-I, Pro13a-II, Pro13b-I, and Pro13b-II) based on cysteine residue content. Since lowering prolamin content in rice is essential to minimize indigestion and allergy risks, we generated four knockout lines using CRISPR-Cas9, which selectively reduced the expression of a specific subgroup of the 13 kDa prolamins. These four mutant rice lines also showed the compensatory expression of glutelins and non-targeted prolamins and were accompanied by low grain weight, altered starch content, and atypically-shaped starch granules and protein bodies. Transcriptome analysis identified 746 differentially expressed genes associated with 13 kDa prolamins during development. Correlation analysis revealed negative associations between genes in Pro13a-I and those in Pro13a-II and Pro13b-I/II subgroups. Furthermore, alterations in the transcription levels of 9 ER stress and 17 transcription factor genes were also observed in mutant rice lines with suppressed expression of 13 kDa prolamin. Our results provide profound insight into the functional role of 13 kDa rice prolamins in the regulatory mechanisms underlying rice seed development, suggesting their promising potential application to improve nutritional and immunological value.
Topics: Oryza; Prolamins; CRISPR-Cas Systems; Starch; Gene Editing; Gene Expression Regulation, Plant; Seed Storage Proteins; Seeds; Glutens; Plant Proteins; Gene Expression Profiling
PubMed: 38928285
DOI: 10.3390/ijms25126579 -
International Journal of Molecular... Jun 2024Plants photoreceptors perceive changes in light quality and intensity and thereby regulate plant vegetative growth and reproductive development. By screening a γ...
Plants photoreceptors perceive changes in light quality and intensity and thereby regulate plant vegetative growth and reproductive development. By screening a γ irradiation-induced mutant library of the soybean () cultivar "Dongsheng 7", we identified , a mutant with elongated nodes, yellowed leaves, decreased chlorophyll contents, altered photosynthetic performance, and early maturation. An analysis of bulked DNA and RNA data sampled from a population segregating for , using the BVF-IGV pipeline established in our laboratory, identified a 10 bp deletion in the first exon of the candidate gene . The causative mutation was verified by a variation analysis of over 500 genes in the candidate gene region and an association analysis, performed using two populations segregating for . () is a homolog of in , which encodes a PΦB synthase involved in the biosynthesis of phytochrome. A transcriptome analysis of using the Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed changes in multiple functional pathways, including photosynthesis, gibberellic acid (GA) signaling, and flowering time, which may explain the observed mutant phenotypes. Further studies on the function of GmHY2a and its homologs will help us to understand its profound regulatory effects on photosynthesis, photomorphogenesis, and flowering time.
Topics: Glycine max; Photosynthesis; Exons; Gene Expression Regulation, Plant; Hypocotyl; Sequence Deletion; Plant Proteins; Gibberellins; Gene Expression Profiling; Phenotype
PubMed: 38928189
DOI: 10.3390/ijms25126483 -
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