Biology subjects

Miyamoto, J.

Publications and source records attributed to Miyamoto, J..

7 recordsLinked to original sources

bioRxiv2025

Arabidopsis TITAN LIKE is required for U12-type intron splicing, especially for AT-AC subtypes

Many eukaryotes possess two types of spliceosomes: the U2-dependent and U12-dependent spliceosomes. The U2-dependent spliceosome processes more than 99% of all introns, whereas the U12-dependent spliceosome acts on only about 0.3% of introns, one-third of which start with AT and end with AC, with the remainder having GT-AG termini. How the U12-dependent spliceosome splices two types of introns with different terminal sequences remains poorly understood. Human CENATAC is a subunit of the U12-dependent spliceosome that is particularly required for the splicing of the AT-AC subtype. The Arabidopsis genome contains a single homolog, TITAN LIKE (TTL), but its function in splicing remains unknown. Here, we generated TTL mutants and isolated two viable alleles, of which we analyzed one, designated ttl-142, to investigate TTL function in splicing. ttl-142 carries a 42-nucleotide deletion that removes 14 amino acid residues from the predicted protein, and homozygous mutants exhibit morphological abnormalities. Most U12-dependent introns were less efficiently spliced in ttl-142 than in the wild-type, with the splicing of AT-AC introns particularly suppressed. Splicing suppression in ttl-142 was more extensive than that in a DROL1 mutant, which carries a mutation in a gene specifically required for AT-AC intron splicing. Conversely, fewer genes showed altered expression levels in ttl-142 than in drol1, and most differentially expressed genes differed between the two mutants. These results suggest that the phenotypes of ttl-142 and drol1 mutants may reflect the impairment of distinct spliceosomal functions.

plant biology↗

bioRxiv2024

PEX1G843D remains functional in peroxisome biogenesis but is rapidly degraded by the proteasome

The PEX1/PEX6 AAA-ATPase is required for the biogenesis and maintenance of peroxisomes. Mutations in HsPEX1 and HsPEX6 disrupt peroxisomal matrix protein import and are the leading cause of Peroxisome Biogenesis Disorders (PBDs). The most common disease-causing mutation in PEX1 is the HsPEX1G843D allele, which results in a reduction of peroxisomal protein import. Here we demonstrate that in vitro the homologous yeast mutant, ScPex1G700D, reduces the stability of Pex1s active D2 ATPase domain and impairs assembly with Pex6, but can still form an active AAA-ATPase motor. In vivo, ScPex1G700D exhibits only a slight defect in peroxisome import. We generated model human HsPEX1G843D cell lines and show that PEX1G843D is rapidly degraded by the proteasome, but that induced overexpression of PEX1G843D can restore peroxisome import. Additionally, we found that the G843D mutation reduces PEX1s affinity for PEX6, and that impaired assembly is sufficient to induce degradation of PEX1WT. Lastly, we found that fusing a deubiquitinase to PEX1G843D significantly hinders its degradation in mammalian cells. Altogether, our findings suggest a novel regulatory mechanism for PEX1/PEX6 hexamer assembly and highlight the potential of protein stabilization as a therapeutic strategy for PBDs arising from the G843D mutation and other PEX1 hypomorphs.

biochemistry↗

bioRxiv2024

Data-independent acquisition coupled with electron-activated dissociation for in-depth structure elucidation of fatty acid ester of hydroxy fatty acids

Fatty acid esters of hydroxy fatty acid (FAHFAs) are a biologically important class of lipids known for their anti-inflammatory and anti-diabetic effects in animals. The physiological activity of FAHFAs varies depending on the length of the carbon chain, number and position of double bonds (DBs), and the position of the hydroxyl (OH) group. Moreover, gut bacteria produce FAHFAs with more diverse structures than those produced by the host, which necessitates a FAHFA-lipidomics approach grasping their diverse structures to fully understand the physiological and metabolic significance of FAHFAs. In this study, we developed a methodology for in-depth structural elucidation of FAHFAs. First, FAHFAs were enriched using a solid-phase extraction (SPE) system coated with titanium and zirconium dioxide, which separated these analytes from neutral lipids and phospholipids. The fractionated metabolites were then derivatized using N,N-dimethylethylenediamine (DMED) to facilitate FAHFA detection in the positive ion mode of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) system. A data-independent acquisition technique known as sequential window acquisition of all theoretical mass spectra (SWATH-DIA) was used to collect sequential MS/MS spectra of the DMED-derivatized fatty acid metabolites. Structural elucidation was based on the fragment ions generated by electron-activated dissociation (EAD). DMED-FAHFAs were annotated using the newly updated MS-DIAL program, and FAHFA isomers were quantified using the MRMPROBS program, which quantifies lipids based on SWATH-MS/MS chromatograms. This procedure was applied to profile the FAHFAs present in mouse fecal samples, characterizing seven structures at the molecular species level, 63 structures at the OH position-resolved level, and 15 structures at both the DB and OH position-resolved levels using the MS-DIAL program. In the MRMPROBS analysis, 2OH and 3OH hydroxy fatty acids with more than 20 carbon atoms were predominantly expressed, while 5OH-13OH hydroxy fatty acids with 16 or 18 carbon atoms were the major components, abundant at positions 5, 7, 9, and 10. Furthermore, age-related changes in FAHFA isomers were also observed, where FAHFA 4:0/2O(FA 26:0) and FAHFA 16:0/10O(FA 16:0) significantly increased with age. In conclusion, our study offers a novel LC-SWATH-EAD-MS/MS technique with the updates of computational MS to facilitate in-depth structural lipidomics of FAHFAs. TOC graphics O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=101 SRC="FIGDIR/small/627939v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1d33c09org.highwire.dtl.DTLVardef@10a97b5org.highwire.dtl.DTLVardef@6e2c35org.highwire.dtl.DTLVardef@1186da5_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Maternal progesterone and adipose mPRε in pregnancy regulate the embryonic nutritional state

Sex steroid hormones such as progesterone play a pivotal role in reproductive functions and maintaining pregnancy; however, the impact of progesterone on the interaction between mother and embryo is unclear. Here, we demonstrate that the relationship between maternal progesterone and membrane progesterone receptor epsilon (mPR{varepsilon}) in adipose tissue regulates embryonic nutritional environment and growth after birth in mice. The activation of adipose mPR{varepsilon} by increased progesterone during pregnancy enhanced maternal insulin resistance through the production of prostaglandins, thereby efficiently providing glucose to embryos. The offspring of mPR{varepsilon}-deficient mothers exhibited metabolic dysfunction, whereas mPR{varepsilon}-deficient mothers with high-fat-diet-induced obesity exhibited improved insulin sensitivity. These findings establish the importance of progesterone as a nutritional regulator between mother and embryo, and suggest that mPR{varepsilon} modulators could be developed to treat pregnant glycemic control disorders such as gestational diabetes mellitus, as well as metabolic syndrome in offspring. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/609823v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1ecd968org.highwire.dtl.DTLVardef@b5492aorg.highwire.dtl.DTLVardef@1d692ecorg.highwire.dtl.DTLVardef@47de8a_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2024

MS-DIAL 5 multimodal mass spectrometry data mining unveils lipidome complexities

Lipidomics and metabolomics communities comprise various informatics tools; however, software programs that can handle multimodal mass spectrometry (MS) data with structural annotations guided by the Lipidomics Standards Initiative are limited. Here, we provide MS-DIAL 5 to facilitate the in-depth structural elucidation of lipids through electron-activated dissociation (EAD)-based tandem MS, as well as determine their molecular localization through MS imaging (MSI) data using a species/tissue-specific lipidome database containing the predicted collision-cross section (CCS) values. With the optimized EAD settings using 14 eV kinetic energy conditions, the program correctly delineated the lipid structures based on EAD-MS/MS data from 96.4% of authentic standards. Our workflow was showcased by annotating the sn- and double-bond positions of eye-specific phosphatidylcholine molecules containing very-long-chain polyunsaturated fatty acids (VLC-PUFAs), characterized as PC n-3-VLC-PUFA/FA. Using MSI data from the eye and HeLa cells supplemented with n-3-VLC-PUFA, we identified glycerol 3-phosphate (G3P) acyltransferase (GPAT) as an enzyme candidate responsible for incorporating n-3 VLC-PUFAs into the sn-1 position of phospholipids in mammalian cells, which was confirmed using recombinant proteins in a cell-free system. Therefore, the MS-DIAL 5 environment, combined with optimized MS data acquisition methods, facilitates a better understanding of lipid structures and their localization, offering novel insights into lipid biology.

bioinformatics↗

bioRxiv2023

A procedure for solid phase extractions using metal oxide coated silica column in lipidomics

Lipid enrichment is indispensable for enhancing the coverage of targeted molecules in mass spectrometry (MS)-based lipidomics studies. In this study, we developed a simple stepwise fractionation method using a titanium- and zirconium-dioxide-coated solid-phase extraction (SPE) silica column that separates neutral lipids, phospholipids, and other lipids, including fatty acids (FAs) and glycolipids. Chloroform was used to dissolve the lipids, and neutral lipids, including steryl esters and di- and triacylglycerols, were collected in the loading fraction. Second, methanol with formic acid (99:1, v/v) was used to retrieve FAs, ceramides, and glycolipids, including glycosylated ceramides and glycosylated diacylglycerols, by competing for affinity with the Lewis acid sites on the metal oxide surface. Finally, phospholipids strongly retained via chemoaffinity interactions were eluted using a solution containing 5% ammonia and high water content (45:50 v/v, 2-propanol:water), which canceled the electrostatic and chelating interactions with the SPE column. High average reproducibility of <10% and coverage of [~]100% compared to those of the non-SPE samples were demonstrated by untargeted lipidomics of human plasma and mouse brain, testis, and feces. The advantage of our procedure was showcased by characterizing minor lipid subclasses, including dihexosylceramides containing very long-chain polyunsaturated FA in the testis, mono- and digalactosyl monoacylglycerols in feces, and acetylated and glycolylated derivatives of gangliosides in the brain that were not detected using conventional solvent extraction methods. Likewise, the value of our method in biology is maximized during glycolipidome profiling in the absence of neutral lipids and phospholipids that cover more than 80% of the chromatographic peaks. Table of Contents artwork O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/562428v2_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@170b451org.highwire.dtl.DTLVardef@4e4d3forg.highwire.dtl.DTLVardef@b1a1d4org.highwire.dtl.DTLVardef@1f77b31_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2023

Using data-dependent and independent hybrid acquisitions for fast liquid chromatography-based untargeted lipidomics

Untargeted lipidomics using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) has become an essential technique for large cohort studies. When a fast LC gradient of less than 10 min is used for the rapid screening of lipids, the annotation rate decreases because of the lower coverage of the MS/MS spectra caused by the narrow peak width. We propose a systematic procedure to achieve a high annotation rate in fast LC-based untargeted lipidomics by integrating data-dependent acquisition (DDA), and sequential window acquisition of all theoretical mass spectra data-independent acquisition (SWATH-DIA) techniques with the updated MS-DIAL program. Our strategy uses variable SWATH-DIA methods for quality control (QC) samples, which are a mixture of biological samples analyzed multiple times to correct MS signal drifts. In contrast, biological samples are analyzed using DDA to facilitate the structural elucidation of lipids using the pure spectrum to the maximum extent. We demonstrate our workflow using an 8.6 min LC gradient, where QCs are analyzed using five different SWATH-DIA methods. The results indicated that using both DDA and SWATH-DIA achieves 2.0-fold annotation coverage from publicly available benchmark data obtained by a fast LC-DDA-MS technique and offers 94.5% lipid coverage compared with the benchmark dataset from a 25 min LC gradient. Our study demonstrated that harmonized improvements in the analytical conditions and informatics tools provide a comprehensive lipidome in fast LC-based untargeted lipidomics, not only for large-scale studies but also for small-scale experiments, contributing to both clinical applications and basic biology.

bioinformatics↗