Inositol Phosphate Research Articles

A naturally occurring membrane-anchored Gαs variant, XLαs, activates phospholipase Cβ4

Extra-large stimulatory Gα (XLαs) is a large variant of G protein αs subunit (Gαs) that uses an alternative promoter and thus differs from Gαs at the first exon. XLαs activation by G protein–coupled receptors mediates cAMP generation, similarly to Gαs; however, Gαs and XLαs have been shown to have distinct cellular and physiological functions. For example, previous work suggests that XLαs can stimulate inositol phosphate production in renal proximal tubules and thereby regulate serum phosphate levels. In this study, we show that XLαs directly and specifically stimulates a specific isoform of phospholipase Cβ (PLCβ), PLCβ4, both in transfected cells and with purified protein components. We demonstrate that neither the ability of XLαs to activate cAMP generation nor the canonical G protein switch II regions are required for PLCβ stimulation. Furthermore, this activation is nucleotide independent but is inhibited by Gβγ, suggesting a mechanism of activation that relies on Gβγ subunit dissociation. Surprisingly, our results indicate that enhanced membrane targeting of XLαs relative to Gαs confers the ability to activate PLCβ4. We also show that PLCβ4 is required for isoproterenol-induced inositol phosphate accumulation in osteocyte-like Ocy454 cells. Taken together, we demonstrate a novel mechanism for activation of phosphoinositide turnover downstream of Gs-coupled receptors that may have a critical role in endocrine physiology.

Discovery of the cyclotide caripe 11 as a ligand of the cholecystokinin-2 receptor

The cholecystokinin-2 receptor (CCK2R) is a G protein-coupled receptor (GPCR) that is expressed in peripheral tissues and the central nervous system and constitutes a promising target for drug development in several diseases, such as gastrointestinal cancer. The search for ligands of this receptor over the past years mainly resulted in the discovery of a set of distinct synthetic small molecule chemicals. Here, we carried out a pharmacological screening of cyclotide-containing plant extracts using HEK293 cells transiently-expressing mouse CCK2R, and inositol phosphate (IP1) production as a readout. Our data demonstrated that cyclotide-enriched plant extracts from Oldenlandia affinis, Viola tricolor and Carapichea ipecacuanha activate the CCK2R as measured by the production of IP1. These findings prompted the isolation of a representative cyclotide, namely caripe 11 from C. ipecacuanha for detailed pharmacological analysis. Caripe 11 is a partial agonist of the CCK2R (Emax = 71%) with a moderate potency of 8.5 µM, in comparison to the endogenous full agonist cholecystokinin-8 (CCK-8; EC50 = 11.5 nM). The partial agonism of caripe 11 is further characterized by an increase on basal activity (at low concentrations) and a dextral-shift of the potency of CCK-8 (at higher concentrations) following its co-incubation with the cyclotide. Therefore, cyclotides such as caripe 11 may be explored in the future for the design and development of cyclotide-based ligands or imaging probes targeting the CCK2R and related peptide GPCRs.

Potential Phosphorus Uptake Mechanisms in the Deep Sedimentary Biosphere

Our understanding of phosphorus (P) dynamics in the deep subseafloor environment remains limited. Here we investigate potential microbial P uptake mechanisms in oligotrophic marine sediments beneath the North Atlantic Gyre and their effects on the relative distribution of organic P compounds as a function of burial depth and changing redox conditions. We use metagenomic analyses to determine the presence of microbial functional genes pertaining to P uptake and metabolism, and solution 31P nuclear magnetic resonance spectroscopy (31P NMR) to characterize and quantify P substrates. Phosphorus compounds or compound classes identified with 31P NMR include inorganic P compounds (orthophosphate, pyrophosphate, polyphosphate), phosphonates, orthophosphate monoesters (including inositol hexakisphosphate stereoisomers) and orthophosphate diesters (including DNA and phospholipid degradation products). Some of the genes identified include genes related to phosphate transport, phosphonate and polyphosphate metabolism, as well as phosphite uptake. Our findings suggest that the deep sedimentary biosphere may have adapted to take advantage of a wide array of P substrates and could play a role in the gradual breakdown of inositol and sugar phosphates, as well as reduced P compounds and polyphosphates.

Genomic characterization of primary cervical cancer and lung metastasis.

e17527 Background: Cervical cancer ranks fourth in incidence and mortality among female tumors in the world. Lung metastases are the most common metastatic foci of cervical cancer, which were associated with poor prognosis. Exploring the different genomic landscape between primary tumor and lung metastasis would help to understand the mechanism of cervical cancer lung metastasis. Methods: A total of 52 Chinese cervical cancer patients with lung metastasis were enrolled between 2016 and 2020. Among whom 37 patients had paired samples (primary tumor and lung metastasis), 6 had only primary tumor samples, and 9 had only lung metastasis samples. Lung metastasis samples were confirmed as cervical cancer metastasis via immunohistochemistry. All the formalin-fixed paraffin-embedded (FFPE) tumor samples and matched peripheral blood were collected and underwent whole-exome sequencing (WES). The Illumina NovaSeq 6000 (Illumina, San Diego, CA, USA) sequencing platform was used to perform 150-nt paired-end multiplex sequencing of DNA samples. The average coverage depth was 340× in FFPE samples and 170× in white blood cell controls. Normally distributed data were compared using the t-test and data with abnormal distribution were compared using the Mann–Whitney U test. Results: There were 4,212 and 6,341 somatic mutations in primary tumor and lung metastasis, respectively. The consistencies for SNVs/indels and CNVs were 41.22% (2.86%–76.11%) and 13.65% (3.69%–76.11%). The most frequently altered genes in primary tumor and lung metastasis were PIK3CA (23.26% vs. 36.96%; p = 0.26), TTN (23.26% vs. 39.96%; p = 0.18), MUC4 (16.28% vs. 39.96%; p = 0.03), ARMCX4 (25.58% vs. 26.09%; p = 1), and MUC16 (18.60% vs. 26.09%; p = 0.45). MUC4 had a significantly higher mutation frequency in lung metastasis than in primary tumor. To explore the mutation characteristics, co-mutations in primary tumor and lung metastasis were analyzed. EP300/MUC16 co-mutation (p < 0.01) was identified in primary tumor. KMT2D/MUC16 co-mutation (p < 0.01) was identified in lung metastasis. Among the paired samples, the Cosmic Signature analysis had shown that Signature 2, which was attributed to activity of the AID/APOBEC family of cytidine deaminases, was higher in lung metastasis than primary tumor (p = 0.042). Meanwhile, the amplification of 17q12 was more frequently observed in lung metastasis in contrast to primary tumor (p = 0.035). Furthermore, the GO/KEGG analysis had revealed that the mutant genes which only observed in lung metastasis were significantly enriched in ATPase activity and inositol phosphate metabolism. Conclusions: In summary, comprehensive genomic profiling of primary cervical cancer and lung metastasis can be of help to identify metastasis-specific features and understand the mechanism of cervical cancer lung metastasis.

Trait Stacking Simultaneously Enhances Mineral and Provitamin a Carotenoid Bioaccessibility in Biofortified Sorghum bicolor

ObjectivesVitamin A, iron, and zinc deficiencies represent major dietary inadequacies in Sub-Saharan Africa and disproportionately affect women and children. Biotechnology strategies have been tested to individually improve carotenoid or mineral content and/or bioaccessibility in relevant cereal crops such as sorghum (Sorghum bicolor). However, approaches combining carotenoid and mineral enhancement in the same event have not been thoroughly evaluated. This work evaluated genetically engineered sorghum events that simultaneously enhanced provitamin A carotenoid accumulation and reduced the mineral limiting antinutrient phytate.MethodsTwo sorghum transformation constructs containing HGGT, to increase vitamin E accumulation and stabilize provitamin A carotenoids during grain storage, CRTI, to increase provitamin A biosynthesis, PSY1 or CRTB, to increase flux through the carotenoid pathway, and PhyA, to decrease phytate, were engineered to produce transgenic events. These sorghum events were processed into model porridges and evaluated for carotenoid and mineral content as well as bioaccessibility.ResultsAll transgenic events produced markedly higher amounts of carotenoids compared to corresponding null segregants and wild-type control (Tx430). A steeping step prior to porridge production to pre-activate phytase drastically reduced phytate content, altered the profile of inositol phosphate conversion products, and reduced molar ratios of phytate to iron and zinc; preventing the chelation of minerals by phytate and enhancing their bioaccessibility. The subsequent release of minerals did not affect micellarization efficiency and the bioaccessible fraction of provitamin A carotenoids were over 2300% greater in transgenic events compared to corresponding null segregants and wild-type controls; providing 53.7% of a 4–8-year-old child's vitamin A estimated average requirement in a standard 200 g serving of porridge.ConclusionsThese data suggest that a combination of strategies to enhance micronutrient content and bioaccessibility are feasible and warrant further assessment in human studies.Funding SourcesThis work was supported in part by the U.S. Department of Agriculture, Agricultural Research Service as well as the Bill and Melinda Gates Foundation.

Structural and catalytic analyses of the InsP6 kinase activities of higher plant ITPKs.

Inositol phosphate signaling in plants is of substantial agricultural interest, with a considerable focus on the inositol tris/tetrakisphosphate kinase (ITPK) family of inositol phosphate kinases. Historically, the 4-6 isoforms of ITPKs that higher plants each express have been studied for their multiplexing a metabolic pathway to synthesize inositol hexakisphosphate (ie InsP6 or phytate), through the phosphorylation and dephosphorylation of multiple inositol phosphates, including Ins(1,3,4,5,6)P5 (inositol-1,3,4,5,6-pentakisphosphate). A more recent discovery is ITPK-catalyzed phosphorylation of InsP6 to inositol pyrophosphates, which regulate plant immunity and phosphate homeostasis. However, a molecular-based explanation for these alternate catalytic activities has been missing, because no plant ITPK structure has previously been solved. Herein, we provide biochemical and structural analyses of ITPKs from Zea mays and Glycine max. For this work we introduce a simple, enzyme-coupled microplate-based assay of InsP6 kinase activity that should promote more general access to this important field. Furthermore, a ZmITPK1/InsP6 crystal complex is described at a resolution of 2.6Å, which identifies a number of catalytically important residues; their functionality is confirmed by mutagenesis. We further demonstrate that ZmITPK1 adds a β-phosphate to the 3-position of Ins(1,2,3,4,5)P5 , yielding a candidate signal for regulating phosphate homeostasis. An impactful discovery is our description of a 29-residue catalytic specificity element; by interchanging this element between GmITPK1 and GmITPK2, we demonstrate how its isoform-specific sequence specifically determines whether the host protein phosphorylates InsP6 , without substantially affecting Ins(1,3,4,5,6)P5 metabolism. Our structural rationalization of key catalytic differences between alternate ITPK isoforms will complement future research into their functional diversity.

Antioxidant Effect of Chrysanthemum morifolium (Chuju) Extract on H2O2-Treated L-O2 Cells as Revealed by LC/MS-Based Metabolic Profiling.

Chrysanthemum has a long history of being used to attenuate various oxidative stress-related discomforts and diseases; however, its mechanisms remain unclear. In this study, the antioxidant effect of chrysanthemum aqueous extract was investigated, and the potential mechanisms were explored via a metabolomics study. Chrysanthemum extract could significantly inhibit hydrogen peroxide (H2O2)-mediated cell death in L-O2 hepatocytes. Propidium iodide staining and annexin V-PI dual staining revealed that the antioxidant effect of chrysanthemum extract was related to the relief of cell cycle arrest and inhibition of non-apoptotic cell damage. The activities of antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) were also upregulated by chrysanthemum extract. Through metabolomics studies, it was found that chrysanthemum extract mainly targeted the arginine synthesis pathway and purine metabolism pathway, in which antioxidation-related endogenous substrates including L-arginosuccinate, citrulline and inositol monophosphate were significantly upregulated by chrysanthemum extract. These results indicated that chrysanthemum extract can antagonize oxidative stress through multiple pathways and have potential therapeutic applications.

The Response and Survival Mechanisms of Staphylococcus aureus under High Salinity Stress in Salted Foods.

Staphylococcus aureus (S. aureus) has a strong tolerance to high salt stress. It is a major reason as to why the contamination of S. aureus in salted food cannot be eradicated. To elucidate its response and survival mechanisms, changes in the morphology, biofilm formation, virulence, transcriptome, and metabolome of S. aureus were investigated. IsaA positively regulates and participates in the formation of biofilm. Virulence was downregulated to reduce the depletion of nonessential cellular functions. Inositol phosphate metabolism was downregulated to reduce the conversion of functional molecules. The MtsABC transport system was downregulated to reduce ion transport and signaling. Aminoacyl-tRNA biosynthesis was upregulated to improve cellular homeostasis. The betaine biosynthesis pathway was upregulated to protect the active structure of proteins and nucleic acids. Within a 10% NaCl concentration, the L-proline content was upregulated to increase osmotic stability. In addition, 20 hub genes were identified through an interaction analysis. The findings provide theoretical support for the prevention and control of salt-tolerant bacteria in salted foods.

Cereal grain fiber composition modifies phosphorus digestibility in grower pigs.

Increased fermentable carbohydrates (e.g., β-glucan, amylose) may increase endogenous losses including for P, and thereby reduce apparent total tract digestibility (ATTD) of P. The present study assessed effects of barley cultivars varying in fermentable starch and fiber on apparent ileal digestibility (AID) and ATTD of P, myo-inositol 1,2,3,4,5,6-hexakis (dihydrogen phosphate; InsP6) and Ca, and standardized total tract digestibility (STTD) of P and the presence of lower inositol phosphates (InsP) compared to wheat. In a 6 (period) × 5 (diet) Youden square, seven ileal-cannulated barrows (initial BW, 27.7 kg) were fed diets containing 80% of one of five cereal grains differing in amylose, β-glucan, and fiber content: 1) high-fermentable, high-β-glucan, hull-less barley (HFB); 2) high-fermentable, high-amylose, hull-less barley (HFA); 3) moderate-fermentable, hull-less barley (MFB); 4) low-fermentable, hulled barley (LFB); and 5) low-fermentable, Canadian Western Red Spring wheat (LFW). On dry matter (DM) basis, cereal grains contained between 0.32% to 0.53% total P and 0.24% to 0.50% InsP6-P. The InsP6-2-P was calculated as the sum of all detected InsP-P (InsP6-P to InsP2-P) in the sample. The P release of degraded InsP-P was calculated by using the following equation: sum InsP6-2-Pdiet (g/kg DM) × (AID or ATTD sum InsP6-2-P (%)/ 100). Data were analyzed using a mixed model with diet as fixed effect, and pig and period as random effects. On DM basis, diets contained 41.4% to 50.6% starch, 0.88% to 8.54% β-glucan, 0.81% to 0.89% total P, and 0.19% to 0.35% InsP6-P. The MFB, LFB, and LFW had greater (P < 0.05) diet AID of P than HFB and HFA, and MFB had greater (P < 0.05) diet ATTD and STTD of P than HFB. The ATTD of InsP6-P was greater (P < 0.05) for HFB than LFB and the ATTD of the sum InsP6-2-P was greater (P < 0.05) for HFB and HFA than LFB. Total tract P release was greater (P < 0.001) for HFB, HFA, and LFW than MFB and LFB. The LFW had greater (P < 0.05) ATTD of Ca than LFB. Diet β-glucan content was not correlated with STTD of P (R2 = 0.03) or ATTD of InsP6 (R2 = 0.05). In conclusion, cereal grains high in fermentable fiber, e.g., amylose and β-glucans included in specific hull-less barley cultivars, had lower diet AID, ATTD, and STTD of P, but greater ATTD of InsP6-P and sum InsP6-2-P. Carbohydrate fermentation, thus, results in greater total tract P release from InsP-P hydrolysis.

2-O-Acetyl-3,4,5,6-tetra-O-benzyl-d-myo-inosityl diphenylphosphate: A new useful intermediate to inositol phosphate and phospholipids.

Inositol phosphates and inositol phospholipids are ubiquitous in biochemistry and play a central role in cell signaling and regulation events. For this reason, their synthesis has attracted widespread interest. This paper describes the preparation of a new optically active inositol phosphate derivative, 2‐O‐acetyl‐3,4,5,6‐tetra‐O‐benzyl‐d‐myo‐inosityl diphenylphosphate (6), and its characterization by spectroscopic methods. Compound (6) represents a useful intermediate for the preparation of inositol phosphate and phospholipids, in particular of glycerophosphoinositol (GPI), a natural anti‐inflammatory agent.

Transcriptome profiling of mRNAs in muscle tissue of Pinan cattle and Nanyang cattle.

Mammalian muscle development is regulated by complex gene networks at the molecular level. The revelation of gene regulatory mechanisms is an important basis for the study of muscle development and molecular breeding. To analyze the excellent meat performance of Pinan cattle at the molecular level, we performed high-throughput RNA sequencing to analyze the key regulatory genes that determine the muscle quality traits in Pinan cattle (n=3) and Nanyang cattle (n=3). We identified 57 differentially expressed genes in muscle tissue of Pinan cattle compared to that of Nanyang cattle, including 32 upregulated and 25 downregulated genes. GO enrichment analysis showed that these genes were significantly enriched in 'molecular function', including voltage-gated ion channel activity, calcium channel activity and calcium ion binding, and KEGG pathway analysis results revealed that adrenergic signaling in cardio myocytes, cell adhesion molecules and inositol phosphate metabolism pathway were significantly enriched. We identified the reliability of RNA-Seq data through RT-qPCR. Meanwhile, we found that GSTA3, PLCB1 and ISYNA1 genes are highly expressed in muscle tissue of Pinan cattle, and these genes play important roles in PI3K/Akt, MEK1/2-ERK and p53-ISYNA1 signaling pathway. In summary, our results suggested that these differentially expressed genes may play important roles in muscle development in Pinan cattle. However, the functions and mechanism of these significantly differential expressed genes should be investigated in future studies.

Structural Insights into Phospholipase Cε

PLC (phospholipase C) enzymes hydrolyze phosphatidylinositol lipids to generate inositol phosphates (IPx) and diacylglycerol (DAG). These second messengers stimulate intracellular calcium release and protein kinase C activation. PLCε is the most recently identified member of the PLC family, and is activated downstream of G protein‐coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs) through direct interactions with G proteins. In the cardiovascular system, PLCε is necessary for normal cardiac contractility, and its dysregulation contributes to cardiac hypertrophy and heart failure. PLCε shares a highly conserved core that is observed in other PLC subfamilies, and contains a unique N‐terminal CDC25 domain and two C‐terminal Ras association (RA) domains. However, structural insights into the full‐length protein, or catalytically active fragments that contain two or more regulatory domains, have been lacking. Using a combination of structural and functional studies, we previously showed that the PH domain and first two EF hands are dynamic in solution, providing an explanation as to why high‐resolution structures have been elusive. We are now pursuing high‐resolution structural studies of larger PLCε variants and the full‐length enzyme using single particle cryo‐electron microscopy (cryo‐EM). To facilitate this process, we are utilizing domain‐specific fab antibodies as fiduciary markers and to stabilize this conformationally heterogeneous protein. Understanding the structure of PLCε will be critical in identifying sites that can be targeted by small molecule chemical probes, ultimately validating this enzyme as a therapeutic target in cardiovascular disease.

Extraction and Prioritization of a Gene-Cancer-By-Survival Network Involved in Homeostasis of Intracellular Calcium Concentrations Using TCGA PANCAN Data.

Regulation of intracellular calcium concentrations, [Ca++]i is important in maintaining the viability of normal as well as cancer cells and can be mediated by tumor microenvironment. Calcium release-activated calcium channel protein (ORAI) calcium channels on the plasma membrane (PM) become physically connected by stromal interaction molecules (STIMs) to the endoplasmic reticulum (ER), on which paralogous receptors of inositol phosphate and of ryanodine are also present along with ATP2A/SERCA (sarco/endoplasmic reticulum calcium ATPases) subunits (also known as PM-ER geneset). Proper expression of this functionally and physically interconnected geneset is essential for the maintenance of [Ca++]i , yet has not been interrogated as a whole for its role in cancer prognosis using multivariable Cox regression. In the present study, we examined whether the expression profile of the PM-ER geneset exhibited prognostic significance across different cancers found in The Cancer Genome Atlas (TCGA) by generating gene-cancer-by-survival networks, in which the nodes represented either genes or cancers and the edges, the logarithmically transformed hazard ratios for overall survival (OS). We then applied network clustering to identify the gene-cancer subnetworks with high connectivity, among which uveal melanoma (UVM) emerged exhibiting the highest degree of genes (k = 10). BAP1, a well-known [Ca++]i regulator and a tumor suppressor, was not found to be significant in predicting OS by PM-ER geneset for UVM, yet it was for several others, including mesothelioma (MESO). Moreover, the best subset of the PM-ER geneset obtained by lasso predicted OS in the TCGA UVM cohort with an area under the receiver operating characteristics (AUC) of 91.4%, comparable to or better than previous prognostic signatures in the literature. Our findings indicate that homeostasis of [Ca++]i is an essential determinant of prognosis in multiple cancers and particularly in UVM. The proposed gene-cancer-by-survival network approach can be extended with other gene sets as well as different survival types.

Understanding the Molecular Mechanism of PLCε Regulation and G Protein‐Mediated Activation

Cardiovascular disease is the leading cause of death in the world. The phospholipase C (PLC) family of enzymes, in particular the PLCε subfamily, are essential for normal cardiovascular function. PLCε hydrolyzes phosphatidylinositol phosphates at cellular membranes, producing inositol phosphates (IPx) and diacylglycerol (DAG). These crucial secondary messengers activate multiple downstream pathways, including cardiac contractility and the expression of hypertrophic genes. In the cardiovascular system, PLCε is regulated through direct interactions with the RhoA and Rap1A small GTPases, which in turn are activated downstream of G protein‐coupled receptors (GPCRs). RhoA is reported to activate PLCε at the plasma membrane, whereas Rap1A translocates and activates PLCε at the perinuclear membrane. However, the elements within PLCε that regulate basal activity and membrane association have not been fully identified. Similarly, the domains involved in Rap1A versus RhoA binding, activation and translocation to different subcellular membranes have not been mapped. In this work, we use a structure‐guided approach, together with cell‐based activity assays, epifluorescence, and confocal microscopy to identify the roles of PLCε regulatory elements and domains in basal activity, subcellular localization, and regulation by RhoA and Rap1A GTPases. Functional studies show N‐ and C‐terminal regulatory domains of PLCε dictate its location within the cell, and contribute differently to basal and G protein‐dependent activity. We also show that regulatory insertions within the catalytic TIM barrel, including the X–Y linker and Y‐box, aid in interfacial activation and membrane association. These studies provide much needed insights into the molecular determinants of PLCε that regulate its localization and activity in cells, which is critical for elucidating its roles in cardiovascular function.

Integrating Single Cell RNAseq and Computational Modeling to Infer Hepatocyte Metabolic Functionalities during Liver Regeneration

Hepatocytes make up over 80% of liver mass and carry out all essential liver functions such as protein synthesis and detoxification, among others. Recent studies have shown distinct metabolic functions of proliferating and non‐proliferating hepatocytes as a compensatory mechanism during liver regeneration. However, this mechanism has not been studied in the case of alcoholic liver disease (ALD), which is known to disrupt the regenerative capacity and alter the metabolic processes of the liver. We seek to determine if alcohol consumption alters the balance in metabolic functionality between proliferating and non‐proliferating hepatocyte populations during liver regeneration. In the present study, rats were pair‐fed either a chronic ethanol diet or a carbohydrate control diet per Lieber DeCarli calorie‐matched liquid diet protocol. We performed 70% partial hepatectomy (PHx) and liver samples were collected at the baseline and at 24 hours post PHx. Single hepatocytes were isolated by laser capture microdissection (LCM) where the subset of proliferating hepatocytes were identified by Ki67 staining. Single‐cell RNA‐sequencing (scRNA‐seq) was employed for the purpose of identifying transcriptional differences across hepatocyte subtypes (i.e. PHx, diet, proliferation). Principal component analysis (PCA) of all samples showed distinct separation of cells into clusters based on liver regeneration (baseline and PHx samples), but not on diet (control and ethanol) and cellular proliferation (Ki67‐positive vs. negative staining). PCA of baseline samples showed separation of proliferating and non‐proliferating hepatocytes in the ethanol group. We characterized metabolic differences between hepatocyte subtypes using a computational model of genome‐scale metabolic reaction activities. We found that the metabolic compartmentalization separating proliferating from non‐proliferating hepatocytes in the carbohydrate group is absent in the ethanol group. Several functional differentiating factors arose favoring reactive oxygen species detoxification and steroid metabolism in proliferating hepatocytes from ethanol‐adapted animals at baseline, which were absent post PHx. Furthermore, proliferating cells from the carbohydrate group showed higher reaction activity in CoA synthesis, NAD metabolism and inositol phosphate metabolic pathways as compared to their non‐proliferating counterparts, both at baseline and following PHx. Our results also indicate that these pathways are also active in the non‐proliferating hepatocytes following PHx in the ethanol group. Taken together, our combinatorial transcriptomic analysis and computational modeling showed distinct metabolic pathways differentiating proliferating from non‐proliferating hepatocytes at baseline, which is absent in hepatocytes following PHx in the ethanol group. These results point towards an alternative mechanism of metabolic compensation in ethanol‐fed animals such that similar metabolic pathways are active in both proliferating and non‐proliferating hepatocytes. This lack of metabolic compartmentalization may hinder the regenerative response to PHx in the ethanol‐fed animals. Targeting the metabolic division of labor may be a potential intervention to promote liver regenerative capacity in alcoholic liver disease.

New insights into regulation of P‐Rex1 autoinhibition

Phosphatidylinositol 3,4,5‐trisphosphate (PIP3)–dependent Rac exchanger 1 (P‐Rex1) is a guanine nucleotide exchange factor for Rho GTPases downstream of G protein coupled receptors (GPCRs) and tyrosine kinases. It is primarily expressed in hematopoietic cells and brain. Its ectopic expression is reported in different cancers including breast, prostrate, skin, and ovarian cancers, making it a potential therapeutic target. P‐Rex1 is a multi‐domain protein consisting of a catalytic Dbl‐homology (DH) domain, followed by a PH, two DEP, two PDZ, and an inositolpolyphosphate‐4 phosphatase‐like (IP4P) domain. P‐Rex1 is regulated by PIP3, Gbg binding and phosphorylation via PKC and PKA. In unstimulated cells, P‐Rex1 is autoinhibited by intradomain interactions with its catalytic DH domain. Previously, our lab had shown that first DEP (DEP1) domain plays a key role in inhibition of DH/PH‐DEP1 exchange activity. However, there is no information available as how these domains are engaged in regulation of full length P‐Rex1. In our current study, we addressed this gap by employing single particle cryo‐electron microscopy (cryo‐EM) complemented by SAXS, HDX‐MS, and functional enzymatic assays to understand P‐Rex1 regulation. A structure with the head group of PIP3 (IP4) bound to the PH domain revealed interactions between DH and DEP1 domain that would block GTPase access to DH domain by coercing a novel configuration of the DH‐PH module. In addition, we observed that the IP4P domain was anchored to PH domain via a previously unrecognized FAT domain. Mutagenesis of residues involved in the DH–DEP1 interface led to activation, suggestive of inhibitory role of this interface. IP4 is a soluble second messenger signaling molecule which is reported to compete with PIP3 binding site in other proteins. Therefore, we tested whether it could regulate P‐Rex1 in a similar manner and found that IP4 competes with PIP3 and negatively regulates P‐Rex1 activity. Based on these observations, we propose that in unstimulated cell, P‐Rex1 is autoinhibited by intradomain interactions between the DEP1 and DH and between the FAT and PH domains, and that inositol phosphate levels may add an additional layer of desensitization by opposing P‐Rex1 membrane translocation and favoring a closed autoinhibited state.

Insights into the hippocampus proteome and phosphorylation modification alterations in C57BL/6 revealed the memory improvement mechanisms of a walnut-derived peptide

This work aimed to explore the underlying mechanisms of memory improvement effects of a walnut derived peptide WNP-10. The morris water maze test, combined with ultrastructural observation, hematoxylin and eosin and Nissl staining showed that WNP–10 significantly improved the learning and memory capability of the scopolamine–injured mice. The four–dimensional label-free quantification proteomics analysis identified 88 differentially expressed proteins in the WNP–10-treated group compared with scopolamine-induced impairment group. Pathway enrichment analysis and western blotting demonstrated that the WNP-10 can regulate the phosphatidylinositol-3-phosphate 5-kinase, cathepsin L, N-acetylgalactosamine 6-sulfate sulfatase and AP-3 complex subunit mu-1 expression to affect inositol phosphate metabolism, thereby maintaining lysosome homeostasis in scopolamine–injured mice. Notably, the results of phosphoproteomics demonstrated that WNP–10 administration resulted in the increased phosphorylation of phosphatidylinositol-3-phosphate 5-kinase. These findings provide novel insights into the underlying mechanism of memory improvement of walnut peptides.

Transcriptome analysis reveals the mechanism of zinc ion-mediated plant resistance to TMV in Nicotiana benthamiana

Zinc ions (Zn2+) are used to promote plant growth and treat multiple diseases. However, it is still unclear which pathways in plants respond to Zn2+. In this study, we found that supplying (CH3COO)2Zn can effectively delay tobacco mosaic virus (TMV) replication and movement in Nicotiana benthamiana. To further understand the regulatory mechanism of antiviral activity mediated by Zn2+, we examined the transcriptomic changes of leaves treated with Zn2+. Three days after treatment, 7575 differential expression genes (DEGs) were enriched in the Zn2+ treatment group compared with the control group. Through GO and KEGG analysis, the pathway of phosphatidylinositol signaling system and inositol phosphate metabolism were significantly enriched after treated with Zn2+, and a large number of ethylene-responsive transcription factors (ERFs) involved in inositol phosphate metabolism were found to be enriched. We identified ERF5 performed a positive effect on plant immunity. Our findings demonstrated that Zn2+-mediated resistance in N. benthamiana activated signal transduction and regulated the expression of resistance-related genes. The results of the study uncover a global view of mRNA changes in Zn2+-mediated cellular processes involved in the competition between plants and viruses.

The effect of dehulling of yellow peas and faba beans on the distribution of carbohydrates upon dry fractionation

The impact of dehulling as a pre-treatment for dry fractionation on the distribution of inositol phosphate and the composition and quantity of different carbohydrates in the fine and coarse fractions from yellow peas and faba beans was elucidated. For peas, low molecular weight carbohydrates markedly increased during air classification in the fine fraction whereas for faba beans this tendency was less pronounced. Sucrose and raffinose series oligosaccharides were higher in the coarse and fine fractions, respectively, independently on dehulling pre-treatment. A large part of the insoluble non-starch polysaccharides (NSP) was removed by dehulling and mostly assigned to cellulose. Dehulled faba beans and peas contained similar amount of total and soluble NSP, of which the latter were assigned to pectin and arabinogalactan. In general, dehulling increased the amounts of soluble NSP. Dehulling enriched phytic acid in remaining seeds and it was further enriched with protein in fine fractions. Regarding the aimed product e.g., fine fraction, dehulling gives a slight reduction of non-digestible oligosaccharides and phytic acid in peas but not in faba beans.

Identifying key genes in milk fat metabolism by weighted gene co-expression network analysis

Milk fat is the most important and energy-rich substance in milk, and its content and composition are important reference elements in the evaluation of milk quality. However, the current identification of valuable candidate genes affecting milk fat is limited. IlluminaPE150 was used to sequence bovine mammary epithelial cells (BMECs) with high and low milk fat rates (MFP), the weighted gene co-expression network (WGCNA) was used to analyze mRNA expression profile data in this study. As a result, a total of 10,310 genes were used to construct WGCNA, and the genes were classified into 18 modules. Among them, violet (r = 0.74), yellow (r = 0.75) and darkolivegreen (r = − 0.79) modules were significantly associated with MFP, and 39, 181, 75 hub genes were identified, respectively. Combining enrichment analysis and differential genes (DEs), we screened five key candidate DEs related to lipid metabolism, namely PI4K2A, SLC16A1, ATP8A2, VEGFD and ID1, respectively. Relative to the small intestine, liver, kidney, heart, ovary and uterus, the gene expression of PI4K2A is the highest in mammary gland, and is significantly enriched in GO terms and pathways related to milk fat metabolism, such as monocarboxylic acid transport, phospholipid transport, phosphatidylinositol signaling system, inositol phosphate metabolism and MAPK signaling pathway. This study uses WGCNA to form an overall view of MFP, providing a theoretical basis for identifying potential pathways and hub genes that may be involved in milk fat synthesis.