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Nature Communications May 2024The mammalian SWI/SNF-like BAF complexes play critical roles during animal development and pathological conditions. Previous gene deletion studies and characterization...
The mammalian SWI/SNF-like BAF complexes play critical roles during animal development and pathological conditions. Previous gene deletion studies and characterization of human gene mutations implicate that the complexes both repress and activate a large number of genes. However, the direct function of the complexes in cells remains largely unclear due to the relatively long-term nature of gene deletion or natural mutation. Here we generate a mouse line by knocking in the auxin-inducible degron tag (AID) to the Smarca4 gene, which encodes BRG1, the essential ATPase subunit of the BAF complexes. We show that the tagged BRG1 can be efficiently depleted by osTIR1 expression and auxin treatment for 6 to 10 h in CD4 + T cells, hepatocytes, and fibroblasts isolated from the knock-in mice. The acute depletion of BRG1 leads to decreases in nascent RNAs and RNA polymerase II binding at a large number of genes, which are positively correlated with the loss of BRG1. Further, these changes are correlated with diminished accessibility at DNase I Hypersensitive Sites (DHSs) and p300 binding. The acute BRG1 depletion results in three major patterns of nucleosome shifts leading to narrower nucleosome spacing surrounding transcription factor motifs and at enhancers and transcription start sites (TSSs), which are correlated with loss of BRG1, decreased chromatin accessibility and decreased nascent RNAs. Acute depletion of BRG1 severely compromises the Trichostatin A (TSA) -induced histone acetylation, suggesting a substantial interplay between the chromatin remodeling activity of BRG1 and histone acetylation. Our data suggest BRG1 mainly plays a direct positive role in chromatin accessibility, RNAPII binding, and nascent RNA production by regulating nucleosome positioning and facilitating transcription factor binding to their target sites.
Topics: Animals; Transcription Factors; DNA Helicases; Nuclear Proteins; Mice; Nucleosomes; Indoleacetic Acids; RNA Polymerase II; Fibroblasts; Gene Knock-In Techniques; Hepatocytes; E1A-Associated p300 Protein; Transcriptional Activation; Transcription, Genetic; Histones; Deoxyribonuclease I; Chromatin; Humans
PubMed: 38811575
DOI: 10.1038/s41467-024-48911-z -
Science Advances May 2024Lipid droplets (LDs) comprise a triglyceride core surrounded by a lipid monolayer enriched with proteins, many of which function in LD homeostasis. How proteins are...
Lipid droplets (LDs) comprise a triglyceride core surrounded by a lipid monolayer enriched with proteins, many of which function in LD homeostasis. How proteins are targeted to the growing LD is still unclear. Rab1b, a GTPase regulating secretory transport, was recently associated with targeting proteins to LDs in a Drosophila RNAi screen. LD formation was prevented in human hepatoma cells overexpressing dominant-negative Rab1b. We thus hypothesized that Rab1b recruits lipid-synthesizing enzymes, facilitating LD growth. Here, FRET between diacylglycerol acyltransferase 2 (DGAT2) and Rab1b and activity mutants of the latter demonstrated that Rab1b promotes DGAT2 ER to the LD surface redistribution. Last, alterations in LD metabolism and DGAT2 redistribution, consistent with Rab1b activity, were caused by mutations in the Rab1b-GTPase activating protein TBC1D20 in Warburg Micro syndrome (WARBM) model mice fibroblasts. These data contribute to our understanding of the mechanism of Rab1b in LD homeostasis and WARBM, a devastating autosomal-recessive disorder caused by mutations in TBC1D20.
Topics: Lipid Droplets; Animals; Humans; rab1 GTP-Binding Proteins; Diacylglycerol O-Acyltransferase; Mice; Endoplasmic Reticulum; Mutation; Lipid Metabolism; GTPase-Activating Proteins
PubMed: 38809969
DOI: 10.1126/sciadv.ade7753 -
Scientific Reports May 2024Post-ERCP pancreatitis (PEP) is an acute pancreatitis caused by endoscopic-retrograde-cholangiopancreatography (ERCP). About 10% of patients develop PEP after ERCP. Here...
Post-ERCP pancreatitis (PEP) is an acute pancreatitis caused by endoscopic-retrograde-cholangiopancreatography (ERCP). About 10% of patients develop PEP after ERCP. Here we show that gamma-glutamyltransferase 1 (GGT1)-SNP rs5751901 is an eQTL in pancreatic cells associated with PEP and a positive regulator of the IL-6 amplifier. More PEP patients had the GGT1 SNP rs5751901 risk allele (C) than that of non-PEP patients at Hokkaido University Hospital. Additionally, GGT1 expression and IL-6 amplifier activation were increased in PEP pancreas samples with the risk allele. A mechanistic analysis showed that IL-6-mediated STAT3 nuclear translocation and STAT3 phosphorylation were suppressed in GGT1-deficient cells. Furthermore, GGT1 directly associated with gp130, the signal-transducer of IL-6. Importantly, GGT1-deficiency suppressed inflammation development in a STAT3/NF-κB-dependent disease model. Thus, the risk allele of GGT1-SNP rs5751901 is involved in the pathogenesis of PEP via IL-6 amplifier activation. Therefore, the GGT1-STAT3 axis in pancreas may be a prognosis marker and therapeutic target for PEP.
Topics: STAT3 Transcription Factor; Pancreatitis; Polymorphism, Single Nucleotide; Humans; Interleukin-6; Cholangiopancreatography, Endoscopic Retrograde; Animals; Quantitative Trait Loci; gamma-Glutamyltransferase; Mice; Male; Female; Middle Aged; Alleles; Cytokine Receptor gp130; Genetic Predisposition to Disease; NF-kappa B; Signal Transduction
PubMed: 38806529
DOI: 10.1038/s41598-024-60312-2 -
PLoS Computational Biology May 2024Lecithin:cholesterol acyltransferase (LCAT) exhibits α-activity on high-density and β-activity on low-density lipoproteins. However, the molecular determinants...
Systematic evaluation of lecithin:Cholesterol acyltransferase binding sites in apolipoproteins via peptide based nanodiscs: Regulatory role of charged residues at positions 4 and 7.
Lecithin:cholesterol acyltransferase (LCAT) exhibits α-activity on high-density and β-activity on low-density lipoproteins. However, the molecular determinants governing LCAT activation by different apolipoproteins remain elusive. Uncovering these determinants would offer the opportunity to design and explore advanced therapies against dyslipidemias. Here, we have conducted coarse-grained and all-atom molecular dynamics simulations of LCAT with nanodiscs made with α-helical amphiphilic peptides either derived from apolipoproteins A1 and E (apoA1 and apoE) or apoA1 mimetic peptide 22A that was optimized to activate LCAT. This study aims to explore what drives the binding of peptides to our previously identified interaction site in LCAT. We hypothesized that this approach could be used to screen for binding sites of LCAT in different apolipoproteins and would provide insights to differently localized LCAT activities. Our screening approach was able to discriminate apoA1 helixes 4, 6, and 7 as key contributors to the interaction with LCAT supporting the previous research data. The simulations provided detailed molecular determinants driving the interaction with LCAT: the formation of hydrogen bonds or salt bridges between peptides E4 or D4 and LCAT S236 or K238 residues. Additionally, salt bridging between R7 and D73 was observed, depending on the availability of R7. Expanding our investigation to diverse plasma proteins, we detected novel LCAT binding helixes in apoL1, apoB100, and serum amyloid A. Our findings suggest that the same binding determinants, involving E4 or D4 -S236 and R7-D73 interactions, influence LCAT β-activity on low-density lipoproteins, where apoE and or apoB100 are hypothesized to interact with LCAT.
PubMed: 38805510
DOI: 10.1371/journal.pcbi.1012137 -
Proceedings of the National Academy of... Jun 2024In mammals, CLOCK and BMAL1 proteins form a heterodimer that binds to E-box sequences and activates transcription of target genes, including (. Translated PER proteins...
In mammals, CLOCK and BMAL1 proteins form a heterodimer that binds to E-box sequences and activates transcription of target genes, including (. Translated PER proteins then bind to the CLOCK-BMAL1 complex to inhibit its transcriptional activity. However, the molecular mechanism and the impact of this PER-dependent inhibition on the circadian clock oscillation remain elusive. We previously identified Ser38 and Ser42 in a DNA-binding domain of CLOCK as phosphorylation sites at the PER-dependent inhibition phase. In this study, knockout rescue experiments showed that nonphosphorylatable (Ala) mutations at these sites shortened circadian period, whereas their constitutive-phospho-mimetic (Asp) mutations completely abolished the circadian rhythms. Similarly, we found that nonphosphorylatable (Ala) and constitutive-phospho-mimetic (Glu) mutations at Ser78 in a DNA-binding domain of BMAL1 also shortened the circadian period and abolished the rhythms, respectively. The mathematical modeling predicted that these constitutive-phospho-mimetic mutations weaken the DNA binding of the CLOCK-BMAL1 complex and that the nonphosphorylatable mutations inhibit the PER-dependent displacement (reduction of DNA-binding ability) of the CLOCK-BMAL1 complex from DNA. Biochemical experiments supported the importance of these phosphorylation sites for displacement of the complex in the PER2-dependent inhibition. Our results provide direct evidence that phosphorylation of CLOCK-Ser38/Ser42 and BMAL1-Ser78 plays a crucial role in the PER-dependent inhibition and the determination of the circadian period.
Topics: ARNTL Transcription Factors; Phosphorylation; CLOCK Proteins; Animals; Circadian Clocks; Period Circadian Proteins; Mice; Humans; DNA; Circadian Rhythm; Mutation; Protein Domains; Protein Binding
PubMed: 38805270
DOI: 10.1073/pnas.2316858121 -
Acta Pharmaceutica Sinica. B May 2024Enzymatic malonylation of natural glycosides provides a promising alternative method for drug-like malonylated glycosides supply. However, the catalytic potential and...
Enzymatic malonylation of natural glycosides provides a promising alternative method for drug-like malonylated glycosides supply. However, the catalytic potential and structural basis of plant malonyltransferase are far from being fully elucidated. This work identified a new malonyltransferase CtMaT1 from It displayed unprecedented mono- and/or di-malonylation activity toward diverse glucosides with different aglycons. A "one-pot" system by CtMaT1 and a malonyl-CoA synthetase was established to biosynthesize nine new malonylated glucosides. Structural investigations revealed that CtMaT1 possesses an adequately spacious acyl-acceptor pocket capable of accommodating diverse glucosides. Additionally, it recognizes malonyl-CoA through strong electrotactic and hydrogen interactions. QM/MM calculation revealed the H167-mediated 2 reaction mechanism of CtMaT1, while dynamic simulations detected the formation of stable hydrogen bonds between the glucose-6-OH group and H167, resulting in its high malonylation regiospecificity. Calculated energy profiles of two isomeric glycosides highlighted lower reaction energy barriers towards glucoside substrates, emphasizing CtMaT1's preference for glucosides. Furthermore, a mutant CtMaT1 with notably increased di-malonylation activity was obtained. The underlying molecular mechanism was illuminated through MM/GBSA binding free energy calculation. This study significantly advances the understanding of plant acyltransferases from both functional and protein structural perspectives, while also providing a versatile tool for enzymatic malonylation applications in pharmacology.
PubMed: 38799633
DOI: 10.1016/j.apsb.2024.02.007 -
Nature Communications May 2024To facilitate inter-tissue communication and the exchange of proteins, lipoproteins, and metabolites with the circulation, hepatocytes have an intricate and efficient...
To facilitate inter-tissue communication and the exchange of proteins, lipoproteins, and metabolites with the circulation, hepatocytes have an intricate and efficient intracellular trafficking system regulated by small Rab GTPases. Here, we show that Rab30 is induced in the mouse liver by fasting, which is amplified in liver-specific carnitine palmitoyltransferase 2 knockout mice (Cpt2) lacking the ability to oxidize fatty acids, in a Pparα-dependent manner. Live-cell super-resolution imaging and in vivo proximity labeling demonstrates that Rab30-marked vesicles are highly dynamic and interact with proteins throughout the secretory pathway. Rab30 whole-body, liver-specific, and Rab30; Cpt2 liver-specific double knockout (DKO) mice are viable with intact Golgi ultrastructure, although Rab30 deficiency in DKO mice suppresses the serum dyslipidemia observed in Cpt2 mice. Corresponding with decreased serum triglyceride and cholesterol levels, DKO mice exhibit decreased circulating but not hepatic ApoA4 protein, indicative of a trafficking defect. Together, these data suggest a role for Rab30 in the selective sorting of lipoproteins to influence hepatocyte and circulating triglyceride levels, particularly during times of excessive lipid burden.
Topics: Animals; rab GTP-Binding Proteins; Mice, Knockout; Homeostasis; Fasting; Carnitine O-Palmitoyltransferase; Mice; Hepatocytes; Lipid Metabolism; Liver; Male; Triglycerides; Mice, Inbred C57BL; Cholesterol; Golgi Apparatus
PubMed: 38796472
DOI: 10.1038/s41467-024-48959-x -
Nutrients May 2024To maintain a beneficial concentration of eicosapentaenoic acid (EPA), the efficient conversion of its precursor, α-linolenic acid (α-LA), is important. Here, we...
To maintain a beneficial concentration of eicosapentaenoic acid (EPA), the efficient conversion of its precursor, α-linolenic acid (α-LA), is important. Here, we studied the conversion of α-LA to EPA using ICR and C57BL/6 mice. A single dose of perilla oil rich-in α-LA or free α-LA had not been converted to EPA 18 h following administration. The α-LA was absorbed into the circulation, and its concentration peaked 6 h after administration, after which it rapidly decreased. In contrast, EPA administration was followed by an increase in circulating EPA concentration, but this did not decrease between 6 and 18 h, indicating that the clearance of EPA is slower than that of α-LA. After ≥1 week perilla oil intake, the circulating EPA concentration was >20 times higher than that of the control group which consumed olive oil, indicating that daily consumption, but not a single dose, of α-LA-rich oil might help preserve the physiologic EPA concentration. The consumption of high concentrations of perilla oil for 4 weeks also increased the hepatic expression of , which is involved in fatty acid elongation; however, further studies are needed to characterize the relationship between the expression of this gene and the conversion of α-LA to EPA.
Topics: Animals; alpha-Linolenic Acid; Eicosapentaenoic Acid; Mice, Inbred C57BL; Mice, Inbred ICR; Male; Plant Oils; Mice; Liver; Fatty Acid Elongases; Olive Oil; Acetyltransferases
PubMed: 38794645
DOI: 10.3390/nu16101407 -
International Journal of Molecular... May 2024Disordered eating behavior differs between the restricting subtype (AN-R) and the binging and purging subtype (AN-BP) of anorexia nervosa (AN). Yet, little is known...
Disordered eating behavior differs between the restricting subtype (AN-R) and the binging and purging subtype (AN-BP) of anorexia nervosa (AN). Yet, little is known about how these differences impact fatty acid (FA) dysregulation in AN. To address this question, we analyzed 26 FAs and 7 FA lipogenic enzymes (4 desaturases and 3 elongases) in 96 women: 25 AN-R, 25 AN-BP, and 46 healthy control women. Our goal was to assess subtype-specific patterns. Lauric acid was significantly higher in AN-BP than in AN-R at the fasting timepoint ( = 0.038) and displayed significantly different postprandial changes 2 h after eating. AN-R displayed significantly higher levels of n-3 alpha-linolenic acid, stearidonic acid, eicosapentaenoic acid (EPA), docosapentaenoic acid, and n-6 linoleic acid and gamma-linolenic acid compared to controls. AN-BP showed elevated EPA and saturated lauric acid compared to controls. Higher EPA was associated with elevated anxiety in AN-R ( = 0.035) but was linked to lower anxiety in AN-BP ( = 0.043). These findings suggest distinct disordered eating behaviors in AN subtypes contribute to lipid dysregulation and eating disorder comorbidities. A personalized dietary intervention may improve lipid dysregulation and enhance treatment effectiveness for AN.
Topics: Humans; Female; Anorexia Nervosa; Adult; Fatty Acids; Young Adult; Lipogenesis; Eicosapentaenoic Acid; Lauric Acids; Fatty Acid Elongases; Adolescent; Fatty Acid Desaturases; Case-Control Studies; Fatty Acids, Unsaturated
PubMed: 38791555
DOI: 10.3390/ijms25105516 -
International Journal of Molecular... May 2024Ribosomal RNAs (rRNAs) are extensively modified during the transcription and subsequent maturation. Three types of modifications, 2'-O-methylation of ribose moiety,...
Ribosomal RNAs (rRNAs) are extensively modified during the transcription and subsequent maturation. Three types of modifications, 2'-O-methylation of ribose moiety, pseudouridylation, and base modifications, are introduced either by a snoRNA-driven mechanism or by stand-alone enzymes. Modified nucleotides are clustered at the functionally important sites, including peptidyl transferase center (PTC). Therefore, it has been hypothesised that the modified nucleotides play an important role in ensuring the functionality of the ribosome. In this study, we demonstrate that seven 25S rRNA modifications, including four evolutionarily conserved modifications, in the proximity of PTC can be simultaneously depleted without loss of cell viability. Yeast mutants lacking three snoRNA genes (, and ) and/or expressing enzymatically inactive variants of and were constructed. The results show that rRNA modifications in PTC contribute collectively to efficient translation in eukaryotic cells. The deficiency of seven modified nucleotides in 25S rRNA resulted in reduced cell growth, cold sensitivity, decreased translation levels, and hyperaccurate translation, as indicated by the reduced missense and nonsense suppression. The modification mC2870 is crucial in the absence of the other six modified nucleotides. Thus, the pattern of rRNA-modified nucleotides around the PTC is essential for optimal ribosomal translational activity and translational fidelity.
Topics: RNA, Ribosomal; Protein Biosynthesis; Saccharomyces cerevisiae; Peptidyl Transferases; Saccharomyces cerevisiae Proteins; Saccharomycetales; Ribosomes; RNA, Small Nucleolar; RNA Processing, Post-Transcriptional; RNA, Fungal; Mutation
PubMed: 38791231
DOI: 10.3390/ijms25105194