Biology subjects

Inoue, H.

Publications and source records attributed to Inoue, H..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Rapid screening and identification of genes involved in bacterial extracellular membrane vesicle production using a curvature-sensing peptide

Bacteria secrete extracellular membrane vesicles (EMVs). Physiology and biotechnological applications of these lipid nanoparticles have been attracting significant attention. However, the molecular basis of EMV biogenesis has not yet been fully elucidated. In the previous research, an N-terminus-substituted FAAV peptide labeled with NBD (nFAAV5-NBD) was developed, which can sense a curvature of a lipid bilayer and selectively bind to EMVs in the presence of the cells. Herein, we applied nFAAV5-NBD to a screening of hyper- and hypo-vesiculation transposon mutants of a Gram-negative bacterium, Shewanella vesiculosa HM13, to identify the genes involved in EMV production. As a result, for 16 and six genes, we found that transposon insertion within or near the gene caused hyper- and hypo-vesiculation, respectively. Targeted gene-disrupted mutants of the identified genes demonstrated that the lack of putative dipeptidyl carboxypeptidase, glutamate synthase {beta}-subunit, LapG protease, metallohydrolase, RNA polymerase sigma-54 factor, inactive transglutaminase, PepSY domain-containing protein, and Rhs-family protein caused EMV overproduction. On the other hand, disruption of the genes encoding putative phosphoenolpyruvate synthase, D-hexose-6-phosphate epimerase, NAD-specific glutamate dehydrogenase, and sensory box histidine kinase/response regulator decreased EMV production. This study shows the utility of a novel screening method using a curvature-sensing peptide and fundamental information on the genes related to EMV production. IMPORTANCEConventional methods for isolation and quantification of EMVs are generally time-consuming. nFAAV5-NBD can detect EMVs in the culture without separating EMVs from cells. In situ detection of EMVs using this peptide facilitated screening of the genes related to EMV production. We succeeded in identifying a number of genes involved in the EMV production of S. vesiculosa HM13, and the strains obtained in this study would contribute to the elucidation of bacterial EMV formation mechanisms. Additionally, the hyper-vesiculating mutants obtained in this study would be useful in the application of this bacterium, such as in the production of useful substances as cargoes of EMVs and in the production of surface-engineered vesicles.

microbiology↗

bioRxiv2024

Highly reduced complementary genomes of dual bacterial symbionts in the mulberry psyllid Anomoneura mori

The genomes of obligately host-restricted bacteria suffer from accumulating mildly deleterious mutations, resulting in a drastic size reduction. Psyllids (Hemiptera) are phloem sap-sucking insects with a specialized organ called the bacteriome, which typically harbors two vertically transmitted bacterial symbionts: the primary symbiont "Candidatus Carsonella ruddii" (Gammaproteobacteria) and a secondary symbiont phylogenetically diverse among psyllid lineages. Genomes of several Carsonella lineages were revealed to be drastically reduced (158-174 kb), AT-rich (14.0-17.9% GC), and structurally conserved with similar gene inventories devoted for synthesizing essential amino acids scarce in the phloem sap. However, genomic information for secondary symbionts was limited. Hence, this study analyzed genomes of the bacteriome-associated dual symbionts, Secondary_AM (Gammaproteobacteria) and Carsonella_AM, in the mulberry psyllid Anomoneura mori (Psyllidae). This revealed that the Secondary_AM genome is as small and AT-rich (229,822 bp, 17.3% GC) as those of Carsonella, including Carsonella_AM (169,120 bp, 16.2% GC), implying that Secondary_AM is an evolutionarily ancient obligate mutualist like Carsonella. Phylogenomic analyses demonstrated that Secondary_AM is sister to "Candidatus Psyllophila symbiotica" of Cacopsylla spp. (Psyllidae), whose genomes (221-237 kb, 17.3-18.6% GC) were recently reported. The Secondary_AM and Psyllophila genomes showed highly conserved synteny, sharing all genes for complementing the incomplete tryptophan biosynthetic pathway of Carsonella and genes for synthesizing B vitamins. However, sulfur assimilation and carotenoid synthesizing genes were retained only in Secondary_AM and Psyllophila, respectively, indicating ongoing gene silencing. Average nucleotide identity, gene ortholog similarity, genome-wide synteny, and substitution rates suggested that the Secondary_AM/Psyllophila genomes are more labile than the Carsonella genomes.

genomics↗

bioRxiv2024

Legionella uses host Rab GTPases and BAP31 to create a unique ER niche

Upon entry into host cells, the facultative intracellular bacterium Legionella pneumophila (L.p.) uses its type IV secretion system, Dot/Icm, to secrete ~330 bacterial effector proteins into the host cell. Some of these effectors hijack endoplasmic reticulum (ER)-derived vesicles to form the Legionella-containing vacuole (LCV). Despite extensive investigation over decades, the fundamental question persists: Is the LCV membrane distinct from or contiguous with the host ER network? Here, we employ advanced photobleaching techniques, revealing a temporal acquisition of both smooth and rough ER (sER and rER) markers on the LCV. In the early stages of infection, the sER intimately associates with the LCV. Remarkably, as the infection progresses, the LCV evolves into a distinct niche comprising an rER membrane that is independent of the host ER network. We discover that the L.p. effector LidA binds to and recruits two host proteins of the Rab superfamily, Rab10, and Rab4, that play significant roles in acquiring sER and rER membranes, respectively. Additionally, we identify the pivotal role of a host ER-resident protein, BAP31, in orchestrating the transition from sER to rER. While previously recognized for shuttling between sER and rER, we demonstrate BAP31s role as a Rab effector, mediating communication between these ER sub-compartments. Furthermore, using genomic deletion strains, we uncover a novel L.p. effector, Lpg1152, essential for recruiting BAP31 to the LCV and facilitating its transition from sER to rER. Depletion of BAP31 or infection with an isogenic L.p. strain lacking Lpg1152 results in a growth defect. Collectively, our findings illuminate the intricate interplay between molecular players from both host and pathogen, elucidating how L.p. orchestrates the transformation of its residing vacuole membrane from a host-associated sER to a distinct rER membrane that is not contiguous with the host ER network.

cell biology↗

bioRxiv2024

Microbiome of psyllids of the family Aphalaridae, including Aphalara itadori, a potential biocontrol agent against Reynoutria spp.

Several European and North American countries have started releasing the Japanese knotweed psyllid Aphalara itadori (Hemiptera: Aphalaridae) to control the Japanese knotweed Reynoutria japonica (Caryophyllales: Polygonaceae) and its relatives, which are among the worst invasive exotic plants. However, establishing populations of the current Kyushu and Hokkaido strains in the field has not been successful, desiring new lineages. Moreover, little is known about the microbiome of the current strains, which potentially impacts properties as biocontrol agents. Hence, this study analyzed the microbiota of an A. itadori strain newly collected on Honshu Island, along with related species of the family Aphalaridae, using amplicon sequencing of 16S rRNA genes. The A. itadori symbionts were further located using fluorescence in situ hybridization. The results demonstrated that the analyzed A. itadori strain has a dual symbiotic system with "Candidatus Carsonella ruddii" (Gammaproteobacteria: Oceanospirillales) and Sodalis sp. (Gammaproteobacteria: Enterobacterales) harbored in the bacteriome, suggesting their evolutionarily stable mutualistic relationships with A. itadori. The central area of the bacteriome harboring Sodalis appeared to comprise uninucleate bacteriocytes with nuclei larger than those of bacteriocytes for Carsonella, rather than a syncytium with smaller nuclei as previously reported for various psyllid lineages. No known plant pathogens or manipulators of insect reproduction were identified in the analyzed strain, indicating its suitability as a biocontrol agent, posing a minimum risk to the ecosystem. Besides distinct Carsonella lineages, the analysis identified Sodalis independently acquired by Craspedolepta miyatakeai, and an ambiguous Enterobacterales symbiont in Epheloscyta kalopanacis. Only Carsonella was identified in Togepsylla matsumurana.

molecular biology↗

bioRxiv2024

Enrichment of allelic editing outcomes by Prime Editing in iPS cells

Gene editing in human induced pluripotent stem (iPS) cells with programmable nucleases facilitates reliable disease models, but methods using double-strand break repair often produce random on-target by-products. Prime editing (PE) combines Cas9 nickase with reverse transcriptase (RT) and a prime editing guide RNA (pegRNA) encoding a repair template to reduce by-products. We implemented a GMP-compatible protocol for transfecting Cas9- or PE-2A-mCherry plasmids to track and fractionate human iPS cells based on PE expression level. We compared the editing outcomes of Cas9- and PE-based methods in a GFP-to-BFP conversion assay, at the HEK3 benchmark locus, and at the APOE Alzheimers risk locus, revealing superior precision of PE at high expression levels. Moreover, sorting cells for PE expression level influenced allelic editing outcomes at the target loci. We expect that our findings will aid in the creation of gene-edited human iPS cells with intentional heterozygous and homozygous genotypes. HighlightsO_LIDelivered large plasmids to human iPS cells under GMP-compliant conditions C_LIO_LIDeveloped a flow cytometry-based approach to enrich for PE in human iPS cells C_LIO_LIDemonstrated few on-target indels in cells regardless of PE expression C_LIO_LISorted iPS cells based on PE expression level to influence mono- or bi-allelic editing C_LI

bioengineering↗

bioRxiv2023

Trans-omic analysis reveals opposite metabolic dysregulation between feeding and fasting in liver associated with obesity

Dysregulation of liver metabolism associated with obesity during feeding and fasting leads to the breakdown of metabolic homeostasis. However, the underlying mechanism remains unknown. Here, we measured multi-omics data in the liver of wild-type and leptin-deficient obese (ob/ob) mice at ad libitum feeding, and constructed a differential regulatory trans-omic network of metabolic reactions. We compared the trans-omic network at feeding with that at 16 h-fasting constructed in our previous study. Intermediate metabolites in glycolytic and nucleotide metabolism decreased in ob/ob mice at feeding but increased at fasting. Allosteric regulation reversely shifted between feeding and fasting, generally showing activation at feeding while inhibition at fasting in ob/ob mice. Transcriptional regulation was similar between feeding and fasting, generally showing inhibiting transcription factor regulations, activating enzyme protein regulations in ob/ob mice. The opposite metabolic dysregulation between feeding and fasting characterizes breakdown of metabolic homeostasis associated with obesity.

systems biology↗

bioRxiv2023

Atf3 controls transitioning in female mitochondrial cardiomyopathy as identified by single-cell transcriptomics

Oxidative phosphorylation defects results in mitochondrial diseases, with cardiac involvement markedly impacting prognosis. However, the mechanisms underlying the transition from compensation to dysfunction in response to metabolic deficiency remain unclear, impeding the development of effective treatments. Here, we employed single-nucleus RNA sequencing (snRNA-seq) on hearts from mitochondrial cardiomyopathy (MCM) mice with cardiac-specific Ndufs6 knockdown (FS6KD). Pseudotime trajectory analysis of cardiomyocytes from early stage of female FS6KD hearts revealed dynamic cellular state transitioning from compensation to severe compromise, coincided with transient upregulation of a critical transcription factor, activating transcription factor 3 (Atf3). Genetic ablation or adeno-associated virus-mediated Atf3 knockdown in FS6KD mice effectively delayed cardiomyopathy progression in a female-specific manner. Notably, human MCM snRNA-seq revealed a similar transition, including the dynamic expression of ATF3. In conclusion, our findings highlight a fate-determining role of Atf3 in female MCM progression, providing a promising therapeutic candidate for the currently intractable disease.

cell biology↗

bioRxiv2023

Prolonged exposure to hypergravity increases number and size of cells and enhances lignin deposition in the stem of Arabidopsis thaliana (L.) Heynh.

We have performed a lab-based hypergravity cultivation experiment using a centrifuge equipped with a lighting system and examined long-term effects of hypergravity on the development of the main axis (stem) of the Arabidopsis (Arabidopsis thaliana (L.) Heynh.) primary inflorescence. Plants grown under 1 x g (gravitational acceleration on Earth) conditions for 20-23 days and having the first visible flower bud were exposed to hypergravity at 8 x g for 10 days. We analyzed the effect of prolonged hypergravity conditions on growth, lignin deposition, and tissue anatomy of the main axis. As a result, the length of the main axis decreased and cross-sectional area, dry mass per unit length, cell number, lignin content of the main axis significantly increased under hypergravity. Lignin content in the rosette leaves also increased when they were exposed to hypergravity during their development. Except for interfascicular fibers, cross-sectional areas of the tissues composing the internode significantly increased under hypergravity in most type of the tissues in the basal part than the apical part of the main axis, indicating that the effect of hypergravity is more pronounced in the basal part than the apical part. The number of cells in fascicular cambium and xylem significantly increased under hypergravity both in the apical and basal internodes of the main axis, indicating a possibility that hypergravity stimulates procambium activity to produce xylem element more than phloem element. The main axis was suggested to be strengthened through changes in its morphological characteristics as well as lignin deposition under prolonged hypergravity conditions.

plant biology↗

bioRxiv2023

Pseudoirreversible inhibition elicits persistent efficacy of a sphigosine-1-phosphate receptor-1 antagonist

Sphingosine 1-phosphate receptor 1 (S1PR1), a G protein-coupled receptor, is required for lymphocyte trafficking, and is a promising therapeutic target in inflammatory diseases. We synthesized a competitive antagonist, KSI-6666, that persistently inhibits S1PR1 activity and effectively suppresses pathogenic inflammation. Metadynamics simulation proposed that the interaction of KSI-6666 with a methionine residue in the ligand-binding pocket of S1PR1 may inhibit the dissociation of KSI-6666 from S1PR1. Consistently, in vitro functional and mutational analyses revealed that KSI-6666 causes pseudoirreversible inhibition of S1PR1, dependent on the methionine residue of the protein and substituents on the distal benzene ring of KSI-6666. Moreover, in vivo study suggested that this pseudoirreversible inhibition is responsible for the persistent activity of KSI-6666. In this study, we discover that KSI-6666 is a potent S1PR1 antagonist, achieving pseudoirreversible inhibition of S1PR1 activity through interacting with a specific methionine residue.

pharmacology and toxicology↗

bioRxiv2022

Progressive spreading of DNA methylation in the GSTP1 promoter CpG island across transitions from precursors to invasive prostate cancer

Glutathione S-transferase pi 1 (GSTP1) is lowly expressed in normal prostate luminal cells and becomes induced in most proliferative inflammatory atrophy lesions (PIA). GSTP1 becomes silenced in prostatic intraepithelial neoplasia (PIN) and prostate adenocarcinoma (CaP) via cytosine-phospho-guanine (CpG) island promoter hypermethylation. However, GSTP1 methylation patterns in PIA and PIN, and their relationship to patterns in CaP are poorly understood. We used bisulfite genomic sequencing to examine patterns of GSTP1 promoter CpG island methylation in laser capture microdissected benign, PIA, PIN, and CaP regions from 32 subjects that underwent radical prostatectomy. We analyzed 908 sequence clones across 24 normal epithelium, 37 PIA, 18 PIN, and 23 CaP regions, allowing assessment of 34,863 CpG sites with allelic phasing. Normal and PIA lesions were mostly unmethylated with 0.52 and 1.3% of total CpG sites methylated, respectively. PIN and CaP lesions had greater methylation with 24% and 51% of total CpG sites methylated, respectively. The degree of GSTP1 methylation showed progression from PIA << PIN < CaP. PIN lesions showed more partial methylation compared to CaP lesions. Partially methylated lesions were enriched for methylation changes at AP1 and SP1 transcription factor binding sites. These results demonstrate that methylation density in the GSTP1 CpG island in PIN was intermediate relative to that in normal prostate epithelium/PIA and CaP lesions. These results are consistent with gradual spreading of DNA methylation centered at the SP1/AP1 transcription factor binding sites in precursor lesions, with subsequent spreading of methylation across the entire CpG island in transition to CaP.

cancer biology↗

bioRxiv2022

In vivo transomic analyses of glucose-responsive metabolism in skeletal muscle reveal core differences between the healthy and obese states

Metabolic regulation in skeletal muscle is essential for blood glucose homeostasis. Obesity causes insulin resistance in skeletal muscle, leading to hyperglycemia and type 2 diabetes. In this study, we performed multiomic analysis of the skeletal muscle of wild-type (WT) and genetically obese (ob/ob) mice, and constructed regulatory transomic networks for metabolism after oral glucose administration. Our network revealed that metabolic regulation by glucose-responsive metabolites had a major effect on WT mice, especially carbohydrate metabolic pathways. By contrast, in ob/ob mice, much of the metabolic regulation by glucose-responsive metabolites was lost and metabolic regulation by glucose-responsive genes was largely increased, especially in carbohydrate and lipid metabolic pathways. We present some characteristic metabolic regulatory pathways found in central carbon, branched amino acids, and ketone body metabolism. Our transomic analysis will provide insights into how skeletal muscle responds to changes in blood glucose and how it fails to respond in obesity.

systems biology↗

bioRxiv2021

C9ORF72 dipeptide repeat proteins disrupt formation of GEM bodies and induce aberrant accumulation of survival of motor neuron protein

A GGGGCC repeat expansion in the C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS), a devastating motor neuron disease. In the neurons of ALS patients, dipeptide repeat proteins (DPRs) are produced from repeat-containing RNAs by an unconventional form of translation, and some of these proteins, especially those containing poly(glycine-arginine) and poly(proline-arginine), are toxic to neurons. Gemini of coiled bodies (GEMs) are nuclear structures that harbor survival of motor neuron (SMN) protein, and SMN is essential for the assembly of U-rich small nuclear ribonucleoproteins (snRNPs) that are central for splicing. We previously reported that GEMs are lost and that snRNP biogenesis is misregulated in the motor neurons of ALS patients. Here we show that DPRs interfere with GEM formation and proper SMN localization in HeLa cells and iPSC-derived motor neurons from an ALS patient with the C9ORF72 mutation. The accumulation of poly(glycine-arginine) markedly reduced the number of GEMs and caused the formation of aberrant cytoplasmic RNA granules that sequestered SMN. These findings indicate the functional impairment of SMN in motor neurons expressing DPRs and may provide a mechanism to explain the vulnerability of motor neurons of C9ORF72-ALS patients.

neuroscience↗

bioRxiv2020

β-catenin signaling via astrocyte-encoded TCF7L2 regulates neuronal excitability and social behavior

Astrocytes play essential roles in supporting neuronal activity and synapse formation; however, mechanisms by which these functions are regulated are unclear. The Wnt/{beta}-catenin signaling pathway plays a crucial role in brain development and is implicated in neurodevelopmental disorders including autism spectrum disorder (ASD). We sought to investigate if some impacts of Wnt signaling are mediated via astrocytes. Here we show that the canonical Wnt/{beta}-catenin pathway is active in postnatal cortical astrocytes and that its effector, the transcription factor TCF7L2 -is expressed in astrocyte lineage cells during embryonic and postnatal development in both mouse and human. Astrocyte-specific deletion of Tcf7l2 in the early postnatal period led to alterations in astrocyte morphology, membrane depolarization and decreased cortical neuron excitability. Mice with the conditional knockout exhibited increased sociability and social preference in a naturalistic setting. Taken together, these data reveal a key role of astrocytic Wnt signaling in shaping postnatal neuronal development and adult social behavior.

neuroscience↗

bioRxiv2020

Zinc-binding motif acts as an oxidative stress sensor to regulate TRPM7 channel activity

TRPM7 channel activity is negatively regulated by intracellular Mg2+. We previously reported that TRPM7 was inhibited by oxidative stress due to an enhancement of the inhibition by intracellular Mg2+. In the present study, we aimed to clarify the precise mechanism underlying the TRPM7 inhibition by oxidative stress induced by hydrogen peroxide (H2O2). Site-directed mutagenesis on full-length TRPM7 revealed that none of the cysteines other than C1809 and C1813 within the zinc-binding motif of the TRPM7 kinase domain were involved in the H2O2-induced TRPM7 inhibition. When C1809 or C1813 was mutated, full-length TRPM7 was not expressed on the plasma membrane. We, therefore, developed a novel approach in which the full-length TRPM7 is functionally reconstituted by co-expressing the TRPM7 channel domain (M7cd) and the TRPM7 kinase domain (M7kd) as separate individual proteins in HEK293 cells. When M7cd was expressed alone, the current was inhibited by intracellular Mg2+ more strongly than in full-length TRPM7. Co-expression of M7cd and M7kd attenuated the current inhibition by intracellular Mg2+, and the current was sensitive to oxidative stress, indicating successful reconstitution of a full-length TRPM7-like current. A similar current reconstitution was observed when M7cd was co-expressed with the kinase inactive mutant M7kd-K1645R. Thus, it is suggested that the kinase activity is not essential for the reconstitution. Co-expression of M7cd and M7kd carrying a mutation at C1809 or C1813 failed to restore the full-length TRPM7-like current. No reconstitution was also observed with M7kd carrying a mutation at H1750 and H1807, which are involved in the zinc-binding motif formation together with C1809 and C1813. These data suggest that the zinc-binding motif is essential for the intracellular Mg2+-dependent regulation of the TRPM7 channel activity by M7kd, and the cysteines in the zinc-binding motif might play a role in the oxidative stress response of TRPM7.

physiology↗

bioRxiv2020

Erythropoietin production?by the kidney and the liver in response to severe hypoxia evaluated by Western blotting with deglycosylation

The detection of erythropoietin (Epo) protein by Western blotting has required pre-purification of the sample. We developed a new Western blot method to detect plasma and urinary Epo using deglycosylation. Epo in urine and tissue and erythropoiesis-stimulating agents (ESAs) in urine were directly detected by our Western blotting. Plasma Epo and ESAs were detected by our Western blotting after deglycosylation. The broad bands of Epo and ESAs were shifted to 22 kDa by deglycosylation except PEG-bound epoetin {beta} pegol. The 22 kDa band from anemic patient urine was confirmed by Liquid Chromatography/Mass Spectrometry (LC/MS) to contain human Epo. Sever hypoxia (7% O2, 4 hr) caused a 400-fold increase in deglycosylated Epo expression in rat kidneys, which is consistent with the increases in both Epo gene expression and plasma Epo concentration. Immunohistochemistry showed Epo expression in nephrons but not in interstitial cells under control conditions, and hypoxia increased Epo expression in interstitial cells but not in tubules. These data show that intrinsic Epo and all ESAs can be detected by Western blot either directly in urine or after deglycosylation in blood, and that the kidney is the main and sole site of Epo production in control and severe hypoxia. Our method will completely change Epo doping and detection.

physiology↗

bioRxiv2019

An integrated approach unravels a crucial structural property for the function of the insect steroidogenic Halloween protein Noppera-bo

Ecdysteroids are the principal insect steroid hormones essential for insect development and physiology. In the last 18 years, several enzymes responsible for ecdysteroid biosynthesis, encoded by Halloween genes, have been identified and well characterized, both genetically and biochemically. However, none of these proteins have yet been characterized at the tertiary structure level. Here, we report an integrated in silico, in vitro, and in vivo analyses of the Halloween glutathione S-transferase (GST) protein, Noppera-bo (Nobo). We determine crystal structures of Drosophila melanogaster Nobo (DmNobo) complexed with glutathione and 17{beta}-estradiol, a DmNobo inhibitor. 17{beta}-estradiol almost fully occupied the putative ligand-binding pocket, and a prominent hydrogen bond formed between Asp113 of DmNobo and 17{beta}-estradiol. Asp113 is essential for inhibiting DmNobo enzymatic activity by 17{beta}-estradiol, as 17{beta}-estradiol does not inhibit and physically interacts less with the Asp113Ala DmNobo point mutant. Asp113 is highly conserved among Nobo proteins, but not among other GSTs, implying that Asp113 is important for endogenous Nobo function. Indeed, a homozygous nobo allele possessing the Asp113Ala point mutation exhibits embryonic lethality with undifferentiated cuticle structure, a phenocopy of complete loss-of-function nobo homozygotes. These results suggest that the nobo family of GST proteins has acquired a unique amino acid residue, which seems to be essential for binding an endogenous sterol substrate to regulate ecdysteroid biosynthesis. This is the first study to reveal the structural characteristics of insect steroidogenic Halloween proteins. This study also provides basic insight into applied entomology for developing a new type of insecticides that specifically inhibit ecdysteroid biosynthesis.\n\nSignificance StatementInsect molting and metamorphosis are drastic and dynamic biological processes and, therefore, have fascinated many scientists. Ecdysteroids represent one class of insect hormones that are indispensable for inducing molting and metamorphosis. It is well known that proteins responsible for catalyzing ecdysteroid biosynthesis reactions are encoded by \"Halloween\" genes, most of which have names of ghosts and phantoms. However, no studies have focused on the structural properties of these biosynthetic proteins. In this study, we addressed this unsolved issue and successfully unraveled a structural property that is crucial for the function of the fruit fly Halloween protein, Noppera-bo (a Japanese faceless ghost). This is the first study to reveal the structural characteristics of an insect steroidogenic Halloween protein.

biochemistry↗

bioRxiv2019

SCAP promotes CARTS biogenesis at endoplasmic reticulum-Golgi contact sites

In response to cholesterol deprivation, SCAP escorts SREBP transcription factors from the endoplasmic reticulum (ER) to the Golgi complex for their proteolytic activation, leading to gene expression for cholesterol synthesis and uptake. Here we show that in cholesterol-fed cells ER-localized SCAP interacts through Sac1 phosphoinositide 4-phosphate (PI4P) phosphatase with a VAP/OSBP complex, which mediates counter-transport of ER cholesterol and Golgi PI4P at ER-Golgi contact sites. SCAP knockdown inhibited the turnover of PI4P perhaps due to a cholesterol transport defect and altered the subcellular distribution of the VAP/OSBP complex. As in the case of perturbation of lipid transfer complexes at ER-Golgi contact sites, SCAP knockdown inhibited the biogenesis of the trans-Golgi network-derived transport carriers CARTS, which was reversed by expression of wild-type SCAP but not cholesterol sensing-defective mutants. Altogether, our findings reveal a new role of SCAP under cholesterol-fed conditions in the facilitation of CARTS biogenesis at ER-Golgi contact sites, depending on the ER cholesterol. SummarySCAP is the key regulatory protein in cholesterol metabolism. Wakana et al. describe a new role of SCAP in controlling Golgi PI4P turnover and the biogenesis of the Golgi-derived transport carries CARTS via cholesterol/PI4P exchange machinery at ER-Golgi contact sites.

cell biology↗

bioRxiv2019

Trans-omic analysis reveals allosteric and gene regulation-axes for altered glucose-responsive liver metabolism associated with obesity

Impaired glucose tolerance associated with obesity causes postprandial hyperglycemia and can lead to type 2 diabetes. To study the differences in liver metabolism in the healthy and obese states, we constructed and analyzed trans-omic glucose-responsive metabolic networks with layers for metabolites, expression data for metabolic enzyme genes, transcription factors, and insulin signaling proteins from the livers of healthy and obese mice. We integrated multi-omic time-course data from wild-type (WT) and leptin-deficient obese (ob/ob) mice after orally administered glucose. In WT mice, metabolic reactions were rapidly regulated (within 10 minutes of oral glucose administration) primarily by glucose-responsive metabolites, especially ATP and NADP+, which functioned as allosteric regulators and substrates of metabolic enzymes, and by Akt-dependent glucose-responsive genes encoding metabolic enzymes. In ob/ob mice, most rapid regulation by glucose-responsive metabolites was absent; instead, glucose administration produced slow changes in the expression of metabolic enzyme-encoding genes to alter metabolic reactions in a time scale of hours. Few common regulatory events occurred in both the healthy and obese mice. Thus, our trans-omic network analysis revealed regulation of liver metabolism in response to glucose is mediated through different mechanisms in the healthy and obese states: Rapid changes in allosteric regulators and substrates and in gene expression dominate the healthy state, and slow transcriptional regulation dominates the obese state.\n\nOne Sentence SummaryRapid changes in regulatory metabolites and gene expression dominate the healthy state, and slow transcriptional regulation dominates the obese state.

systems biology↗