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Journal of Agricultural and Food... Jul 2024Multiprotein bridging factor 1 (MBF1) is a very important transcription factor (TF) in plants, whose members influence numerous defense responses. Our study found that...
Multiprotein bridging factor 1 (MBF1) is a very important transcription factor (TF) in plants, whose members influence numerous defense responses. Our study found that MBF1c in Cucurbitaceae was highly conserved. expression was induced by temperature, salt stress, and abscisic acid (ABA) in cucumber. Overexpressed CsMBF1c enhanced the heat resistance of a cucumber, and the mutant showed decreased resistance to high temperatures (HTs). CsMBF1c played an important role in stabilizing the photosynthetic system of cucumber under HT, and its expression was significantly associated with heat-related TFs and genes related to protein processing in the endoplasmic reticulum (ER). Protein interaction showed that CsMBF1c interacted with dehydration-responsive element binding protein 2 (CsDREB2) and nuclear factor Y A1 (CsNFYA1). Overexpression of CsNFYA1 in improved the heat resistance. Transcriptional activation of CsNFYA1 was elevated by CsMBF1c. Therefore, plays an important regulatory role in cucumber's resistance to high temperatures.
PubMed: 38949485
DOI: 10.1021/acs.jafc.4c02398 -
Angewandte Chemie (International Ed. in... Jul 2024Aurothiomalate (AuTM) is an FDA-approved antiarthritic gold drug with unique anticancer properties. To enhance its anticancer activity, we prepared a bioconjugate with...
Aurothiomalate (AuTM) is an FDA-approved antiarthritic gold drug with unique anticancer properties. To enhance its anticancer activity, we prepared a bioconjugate with human apoferritin (HuHf) by attaching some AuTM moieties to surface protein residues. The reaction of apoferritin with excess AuTM yielded a single adduct, that was characterized by ESI MS and ICP-OES analysis, using three mutant ferritins and trypsinization experiments. The adduct contains ~3 gold atoms per ferritin subunit, arranged in a small cluster bound to Cys90 and Cys102. MD simulations provide a plausible structural model for the cluster. The adduct was evaluated for its pharmacological properties and was found to be significantly more cytotoxic than free AuTM against A2780 cancer cells mainly due to higher gold uptake. NMR-metabolomics showed that AuTM bound to HuHf and free AuTM induced qualitatively similar changes in treated cancer cells, indicating that the effects on cell metabolism are approximately the same, in agreement with independent biochemical experiments. In conclusion, we have demonstrated here that a molecularly precise bioconjugate formed between AuTM and HuHf exhibits anticancer properties far superior to the free drug, while retaining its key mechanistic features. Evidence is provided that human ferritin can serve as an excellent carrier for this metallodrug.
PubMed: 38949226
DOI: 10.1002/anie.202410791 -
BioRxiv : the Preprint Server For... Jun 2024About one-third of all human cancers encode abnormal RAS proteins locked in a constitutively activated state to drive malignant transformation and uncontrolled tumor...
About one-third of all human cancers encode abnormal RAS proteins locked in a constitutively activated state to drive malignant transformation and uncontrolled tumor growth. Despite progress in development of small molecules for treatment of mutant KRAS cancers, there is a need for a pan-RAS inhibitor that is effective against all RAS isoforms and variants and that avoids drug resistance. We have previously shown that the naturally occurring bacterial enzyme RAS/RAP1-specific endopeptidase (RRSP) is a potent RAS degrader that can be re-engineered as a biologic therapy to induce regression of colorectal, breast, and pancreatic tumors. Here, we have developed a strategy for in vivo expression of this RAS degrader via mRNA delivery using a synthetic nonviral gene delivery platform composed of the poly(ethylene glycol)--poly(propylene sulfide) (PEG--PPS) block copolymer conjugated to a dendritic cationic peptide (PPDP2). Using this strategy, PPDP2 is shown to deliver mRNA to both human and mouse pancreatic cells resulting in RRSP gene expression, activity, and loss of cell proliferation. Further, pancreatic tumors are reduced with residual tumors lacking detectable RAS and phosphorylated ERK. These data support that mRNA-loaded synthetic nanocarrier delivery of a RAS degrader can interrupt the RAS signaling system within pancreatic cancer cells while avoiding side effects during therapy.
PubMed: 38948803
DOI: 10.1101/2024.06.11.598439 -
BioRxiv : the Preprint Server For... Jun 2024Liprin-α1 is a widely expressed scaffolding protein responsible for regulating cellular processes such as focal adhesion, cell motility, and synaptic transmission....
Liprin-α1 is a widely expressed scaffolding protein responsible for regulating cellular processes such as focal adhesion, cell motility, and synaptic transmission. Liprin-α1 interacts with many proteins including ELKS, GIT1, liprin-β, and LAR-family receptor tyrosine protein phosphatase. Through these protein-protein interactions, liprin-α1 assembles large higher-order molecular complexes; however, the regulation of this complex assembly/disassembly is unknown. Liquid-liquid phase separation (LLPS) is a process that concentrates proteins within cellular nano-domains to facilitate efficient spatiotemporal signaling in response to signaling cascades. While there is no report that liprin-α1 spontaneously undergoes LLPS, we found that GFP-liprin-α1 expressed in HEK293 cells occasionally forms droplet-like condensates. MS-based interactomics identified Protein Phosphatase 2A (PP2A)/B56δ (PPP2R5D) trimers as specific interaction partners of liprin-α1 through a canonical Short Linear Interaction Motif (SLiM) in its N-terminal dimerization domain. Mutation of this SLiM nearly abolished PP2A interaction, and resulted in significantly increased LLPS. GFP-liprin-α1 showed significantly increased droplet formation in HEK293 cells devoid of B56δ (PPP2R5D knockout), suggesting that PPP2R5D/PP2A holoenzyme inhibits liprin-α1 LLPS. Guided by reported liprin-α1 Ser/Thr phosphorylation sites, we found liprin-α1 phospho-mimetic mutant at serine 763 (S763E) is sufficient to drive its LLPS. Domain mapping studies of liprin-α1 indicated that the intrinsically disordered region, the N-terminal dimerization domain, and the SAM domains are all necessary for liprin-α1 LLPS. Finally, expression of p.E420K, a human PPP2R5D variant causing Houge-Janssens Syndrome type 1 (also known as Jordan's Syndrome), significantly compromised suppression of liprin-α1 LLPS. Our work identified B56δ-PP2A holoenzyme as an inhibitor of liprin-α1 LLPS via regulation at multiple phosphorylation sites.
PubMed: 38948786
DOI: 10.1101/2024.06.18.599485 -
BioRxiv : the Preprint Server For... Jun 2024Oxidative protein folding in the endoplasmic reticulum (ER) is essential for all eukaryotic cells yet generates hydrogen peroxide (H2O2), a reactive oxygen species...
Oxidative protein folding in the endoplasmic reticulum (ER) is essential for all eukaryotic cells yet generates hydrogen peroxide (H2O2), a reactive oxygen species (ROS). The ER-transmembrane protein that provides reducing equivalents to ER and guards the cytosol for antioxidant defense remains unidentified. Here we combine AlphaFold2- based and functional reporter screens in to identify a previously uncharacterized and evolutionarily conserved protein ERGU-1 that fulfills these roles. Deleting ERGU-1 causes excessive H2O2 and transcriptional gene up- regulation through SKN-1, homolog of mammalian antioxidant master regulator NRF2. ERGU-1 deficiency also impairs organismal reproduction and behaviors. Both and human ERGU-1 proteins localize to ER membranes and form network reticulum structures. We name this system ER-GUARD, E ndoplasmic R eticulum Gu ardian A egis of R edox D efense. Human and homologs of ERGU-1 can rescue mutant phenotypes, demonstrating evolutionarily ancient and conserved functions. Together, our results reveal an ER-membrane-specific protein machinery and defense-net system ER-GUARD for peroxide detoxification and suggest a previously unknown but conserved pathway for antioxidant defense in animal cells.
PubMed: 38948723
DOI: 10.1101/2024.06.19.599784 -
Sichuan Da Xue Xue Bao. Yi Xue Ban =... May 2024Infertility affects approximately one-sixth of the people of childbearing age worldwide, causing not only economic burdens of treatment for families with fertility...
OBJECTIVE
Infertility affects approximately one-sixth of the people of childbearing age worldwide, causing not only economic burdens of treatment for families with fertility problems but also psychological stress for patients and presenting challenges to societal and economic development. Premature ovarian insufficiency (POI) refers to the loss of ovarian function in women before the age of 40 due to the depletion of follicles or decreased quality of remaining follicles, constituting a significant cause of female infertility. In recent years, with the help of the rapid development in genetic sequencing technology, it has been demonstrated that genetic factors play a crucial role in the onset of POI. Among the population suffering from POI, genetic studies have revealed that genes involved in processes such as meiosis, DNA damage repair, and mitosis account for approximately 37.4% of all pathogenic and potentially pathogenic genes identified. FA complementation group M () is a group of genes involved in the damage repair of DNA interstrand crosslinks (ICLs), including -. Abnormalities in the genes are associated with female infertility and gene knockout mice also exhibit phenotypes similar to those of POI. During the genetic screening of POI patients, this study identified a suspicious variant in . This study aims to explore the pathogenic mechanisms of the genes of the FA pathway and their variants in the development of POI. We hope to help shed light on potential diagnostic and therapeutic strategies for the affected individuals.
METHODS
One POI patient was included in the study. The inclusion criteria for POI patients were as follows: women under 40 years old exhibiting two or more instances of basal serum follicle-stimulating hormone levels>25 IU/L (with a minimum interval of 4 weeks inbetween tests), alongside clinical symptoms of menstrual disorders, normal chromosomal karyotype analysis results, and exclusion of other known diseases that can lead to ovarian dysfunction. We conducted whole-exome sequencing for the POI patient and identified pathogenic genes by classifying variants according to the standards and guidelines established by the American College of Medical Genetics and Genomics (ACMG). Subsequently, the identified variants were validated through Sanger sequencing and subjected to bioinformatics analysis. Plasmids containing wild-type and mutant genes were constructed and introduced into 293T cells. The 293T cells transfected with wild-type and mutant human plasmids and pEGFP-C1 empty vector plasmids were designated as the EGFP - group, the EGFP - group, and the EGFP group, respectively. To validate the production of truncated proteins, cell proteins were extracted 48 hours post-transfection from the three groups and confirmed using GFP antibody. In order to investigate the impact on DNA damage repair, immunofluorescence experiments were conducted 48 hours post-transfection in the EGFP WT group and the EGFP -MUT group to examine whether the variant affected FANCM's ability to localize on chromatin. Mitomycin C was used to induce ICLs damage in both the EGFP - group and the EGFP - group, which was followed by verification of its effect on ICLs damage repair using γ-H2AX antibody.
RESULTS
In a POI patient from a consanguineous family, we identified a homozygous variant in the gene, c.1152-1155del:p.Leu386Valfs*10. The patient presented with primary infertility, experiencing irregular menstruation since menarche at the age of 16. Hormonal evaluation revealed an FSH level of 26.79 IU/L and an anti-Müllerian hormone (AMH) level of 0.07 ng/mL. Vaginal ultrasound indicated unsatisfactory visualization of the ovaries on both sides and uterine dysplasia. The patient's parents were a consanguineous couple, with the mother having regular menstrual cycles. The patient had two sisters, one of whom passed away due to osteosarcoma, while the other exhibited irregular menstruation, had been diagnosed with ovarian insufficiency, and remained childless. Bioinformatics analysis revealed a deletion of four nucleotides (c.1152-1155del) in the exon 6 of the patient's gene. This variant resulted in a frameshift at codon 386, introducing a premature stop codon at codon 396, which ultimately led to the production of a truncated protein consisting of 395 amino acids. experiments demonstrated that this variant led to the production of a truncated FANCM protein of approximately 43 kDa and caused a defect in its nuclear localization, with the protein being present only in the cytoplasm. Following treatment with mitomycin C, there was a significant increase in γ-H2AX levels in 293T cells transfected with the mutant plasmid (<0.01), indicating a statistically significant impairment of DNA damage repair capability caused by this variant.
CONCLUSIONS
The homozygous variant in the gene, c.1152-1155del:p.Leu386Valfs*10, results in the production of a truncated FANCM protein. This truncation leads to the loss of its interaction site with the MHF1-MHF2 complex, preventing its entry into the nucleus and the subsequent recognition of DNA damage. Consequently, the localization of the FA core complex on chromatin is disrupted, impeding the normal activation of the FA pathway and reducing the cell's ability to repair damaged ICLs. By disrupting the rapid proliferation and meiotic division processes of primordial germ cells, the reserve of oocytes is depleted, thereby triggering premature ovarian insufficiency in females.
Topics: Female; Primary Ovarian Insufficiency; Humans; Mutation; Fanconi Anemia; Adult; Infertility, Female; DNA Helicases
PubMed: 38948269
DOI: 10.12182/20240560207 -
ACS Central Science Jun 2024Using directed evolution, aminoacyl-tRNA synthetases (aaRSs) have been engineered to incorporate numerous noncanonical amino acids (ncAAs). Until now, the selection of...
Using directed evolution, aminoacyl-tRNA synthetases (aaRSs) have been engineered to incorporate numerous noncanonical amino acids (ncAAs). Until now, the selection of such novel aaRS mutants has relied on the expression of a selectable reporter protein. However, such translation-dependent selections are incompatible with exotic monomers that are suboptimal substrates for the ribosome. A two-step solution is needed to overcome this limitation: (A) engineering an aaRS to charge the exotic monomer, without ribosomal translation; (B) subsequent engineering of the ribosome to accept the resulting acyl-tRNA for translation. Here, we report a platform for aaRS engineering that directly selects tRNA-acylation without ribosomal translation (START). In START, each distinct aaRS mutant is correlated to a cognate tRNA containing a unique sequence barcode. Acylation by an active aaRS mutant protects the corresponding barcode-containing tRNAs from oxidative treatment designed to damage the 3'-terminus of the uncharged tRNAs. Sequencing of these surviving barcode-containing tRNAs is then used to reveal the identity of the aaRS mutants that acylated the correlated tRNA sequences. The efficacy of START was demonstrated by identifying novel mutants of the pyrrolysyl-tRNA synthetase from a naïve library that enables incorporation of ncAAs into proteins in living cells.
PubMed: 38947215
DOI: 10.1021/acscentsci.3c01557 -
Zhonghua Yi Xue Yi Chuan Xue Za Zhi =... Jul 2024To investigate the clinical and genetic features of a child with Dyschromatosis symmetrica hereditaria (DSH) and variant of the ADAR1 gene.
OBJECTIVE
To investigate the clinical and genetic features of a child with Dyschromatosis symmetrica hereditaria (DSH) and variant of the ADAR1 gene.
METHODS
A child who was admitted to the Department of Dermatology of the First Affiliated Hospital of Zhengzhou University in June 2020 due to irregular pigmented maculopapular rash on the dorsum of hands was selected as the study subject. Whole exome sequencing (WES) was carried out for the child and his similarly affected father, and Sanger sequencing was used to verify the candidate variant. SWISS-MODEL was used to predict the secondary and tertiary structures of the wild-type and mutant ADAR1 proteins.
RESULTS
The child, a 13-year-old boy, had symmetrical hyperpigmented and depigmented spots on the back of his hands and was clinically diagnosed with DSH. WES and Sanger sequencing results showed that he and his father had both harbored a heterozygous c.2858dup (p.T954Dfs*20) truncating variant in exon 10 of the ADAR1 gene. Based on the guidelines from the American College of Medical Genetics and Genomics, the variant was predicted as pathogenic (PVS1+PM2_Supporting+PM1+PP3).
CONCLUSION
The c.2858dup (p.T954Dfs*20) variant of the ADAR1 gene probably underlay the DSH in this pedigree.
Topics: Humans; Male; Adenosine Deaminase; Pigmentation Disorders; RNA-Binding Proteins; Adolescent; Mutation; Exome Sequencing; Exons; Genetic Testing; Pedigree
PubMed: 38946371
DOI: 10.3760/cma.j.cn511374-20230612-00352 -
Zhonghua Yi Xue Yi Chuan Xue Za Zhi =... Jul 2024To explore the genetic basis for a child featuring global developmental delay and epilepsy.
OBJECTIVE
To explore the genetic basis for a child featuring global developmental delay and epilepsy.
METHODS
A child who had presented at Guangzhou Women and Children's Medical Center Liuzhou Hospital on February 19, 2023 was selected as the study subject. Clinical data of the child was collected. The child was subjected to whole exome sequencing, and candidate variant was validated by Sanger sequencing and bioinformatic analysis.
RESULTS
The child, an 8-month-old girl, had manifested with global developmental delay, epilepsy, and hyperlactacidemia. Cranial MRI revealed diverse hypomyelinating leukodystrophies. Electroencephalogram showed slow background activities. Genetic testing revealed that she has harbored a homozygous variant of the SLC25A12 gene, namely c.115T>G (p.Phe39Val), for which both of her parents were heterozygous carriers. Based on the guidelines from the American College of Medical Genetics and Genomics, the variant was predicted to be of uncertain significance (PM2_Supporting+PM3_Supporting+PP3_Moderate+PP4_Moderate). I-Mutant v3.0 software predicted that the variant may affect the stability of protein product.
CONCLUSION
The homozygous c.115T>G (p.Phe39Val) variant of the SLC25A12 gene probably underlay the pathogenesis of the disease in this child.
Topics: Humans; Female; Infant; Epilepsy; Homozygote; Developmental Disabilities; Mutation; Mitochondrial Membrane Transport Proteins; Exome Sequencing
PubMed: 38946367
DOI: 10.3760/cma.j.cn511374-20230428-00252 -
American Journal of Physiology. Cell... Jul 2024Euryhaline fish experience variable osmotic environments requiring physiological adjustments to tolerate elevated salinity. Mozambique tilapia () possess one of the...
Euryhaline fish experience variable osmotic environments requiring physiological adjustments to tolerate elevated salinity. Mozambique tilapia () possess one of the highest salinity tolerance limits of any fish. In tilapia and other euryhaline fish species the -inositol biosynthesis (MIB) pathway enzymes, -inositol phosphate synthase (MIPS) and inositol monophosphatase 1 (IMPA1.1), are among the most upregulated mRNAs and proteins indicating the high importance of this pathway for hyper-osmotic (HO) stress tolerance. These abundance changes must be precluded by HO perception and signaling mechanism activation to regulate the expression of and genes. In previous work using a cell line (OmB), a reoccurring osmosensitive enhancer element (OSRE1) in both and was shown to transcriptionally upregulate these enzymes in response to HO stress. The OSRE1 core consensus (5'-GGAAA-3') matches the core binding sequence of the predominant mammalian HO response transcription factor, nuclear factor of activated T-cells (NFAT5). HO challenged OmB cells showed an increase in mRNA suggesting NFAT5 may contribute to MIB pathway regulation in euryhaline fish. Ectopic expression of wild-type NFAT5 induced an promoter-driven reporter by 5.1-fold (p < 0.01). Moreover, expression of dominant negative NFAT5 in HO media resulted in a 47% suppression of the reporter signal (p<0.005). Furthermore, reductions of IMPA1.1 (37-49%) and MIPS (6-37%) mRNA abundance were observed in HO challenged NFAT5 knockout cells relative to control cells. Collectively, these multiple lines of experimental evidence establish NFAT5 as a tilapia transcription factor contributing to HO induced activation of the MIB pathway.
PubMed: 38946247
DOI: 10.1152/ajpcell.00187.2024