ResearchPad - Biochemistry https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Analysis of the data on titration of native and peroxynitrite modified αA- and αB-crystallins by Cu2+-ions]]> https://www.researchpad.co/product?articleinfo=N53acec24-e321-43b9-bc94-a3812bb8ff83

The interaction of αA- and αB-crystallins with Cu2+ ion modulates their structure and chaperone-like activity which is important for lens transparency. Theoretical analysis of the dependences of fluorescence intensity of native αA- and αB-crystallins and αA- and αB-crystallins modified by peroxynitrite on concentration of Cu2+ ions has been carried out. It has been shown that one subunit of native αA-crystallin contains two equivalent Cu2+-binding sites. The microscopic dissociation constant for Cu2+–αA-crystallin complex (Kdiss) was found to be equal to 9.7 µM. For peroxynitrite modified αA-crystallin the Kdiss value is equal to 17 µM. One subunit of native αB-crystallin contains two non-equivalent Cu2+-binding sites. The corresponding microscopic dissociation constants for Cu2+–αB-crystallin complexes (K1 and K2) were found to be equal to 0.94 and 36 µM. For peroxynitrite modified αB-crystallin the K1 and K2 values are equal to 4.3 and 70 µM, respectively.

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<![CDATA[Whole genome sequencing data of Escherichia coli isolated from bloodstream infection patients in Cipto Mangunkusumo National Hospital, Jakarta, Indonesia]]> https://www.researchpad.co/product?articleinfo=N6fa874a6-425d-4db7-a898-e947a595746a

Bloodstream infections (BSIs) are some of the most devastating preventable complications in critical care units. Of the bacterial causes of BSIs, Escherichia coli is the most common among Enterobacteriaceae. Bacteria resistant to therapeutic antibiotics represent a significant global health challenge. In this study, we present whole genome sequence data of 22 E. coli isolates that were obtained from bloodstream infection patients admitted to Cipto Mangunkusumo National Hospital, Jakarta, Indonesia. These data will be useful for analysing the serotypes, virulence genes, and antimicrobial resistance genes of E. coli. DNA sequences of E. coli were obtained using the Illumina MiSeq platform. The FASTQ raw files of these sequences are available under BioProject accession number PRJNA596854 and Sequence Read Archive accession numbers SRR10761126–SRR10761147.

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<![CDATA[Impact of Ice Slurry Ingestion During Break-Times on Repeated-Sprint Exercise in the Heat]]> https://www.researchpad.co/product?articleinfo=N4803c751-34ee-4b32-b63e-5a1f5e8c6b04

The study aimed to investigate the effects of ice slurry ingestion during break times and half-time (HT) on repeated-sprint performance and core temperature in the heat. Seven males performed two different trials as follows: ice slurry (−1°C) or room temperature water ingestion at each break and HT break at 36.5°C, 50% relative humidity. Participants performed 30 sets of 1-min periods of repeated- sprint exercises protocol using a cycling ergometer. Each period consisted of 5 sec of maximal pedaling, 25 sec of pedaling with no workload, and 30 sec of rest; two sets of exercise periods were separated by 10 min of rest. Each break was implemented for 1 min after every 5 sets. The rectal temperature in ice slurry ingestion was significantly lower than that of the room temperature water at 45 set (p=0.04). Total and mean work done was greater in ice slurry ingestion compared to room temperature water ingestion (p < 0.05). These results suggested that ice slurry ingestion during break times and HT break may be an effective cooling strategy to attenuate the rise of core temperature in the second half of exercise and improve the repeated-sprint exercise capacity in the heat.

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<![CDATA[Extending thermotolerance to tomato seedlings by inoculation with SA1 isolate of Bacillus cereus and comparison with exogenous humic acid application]]> https://www.researchpad.co/product?articleinfo=N5b151d82-6b14-4a7f-beb8-82f649a56498

Heat stress is one of the major abiotic stresses that impair plant growth and crop productivity. Plant growth-promoting endophytic bacteria (PGPEB) and humic acid (HA) are used as bio-stimulants and ecofriendly approaches to improve agriculture crop production and counteract the negative effects of heat stress. Current study aimed to analyze the effect of thermotolerant SA1 an isolate of Bacillus cereus and HA on tomato seedlings. The results showed that combine application of SA1+HA significantly improved the biomass and chlorophyll fluorescence of tomato plants under normal and heat stress conditions. Heat stress increased abscisic acid (ABA) and reduced salicylic acid (SA) content; however, combined application of SA1+HA markedly reduced ABA and increased SA. Antioxidant enzymes activities revealed that SA1 and HA treated plants exhibited increased levels of ascorbate peroxidase (APX), superoxide dismutase (SOD), and reduced glutathione (GSH). In addition, heat stress markedly reduced the amino acid contents; however, the amino acids were increased with co-application of SA1+HA. Similarly, inductively-coupled plasma mass-spectrometry results showed that plants treated with SA1+HA exhibited significantly higher iron (Fe+), phosphorus (P), and potassium (K+) uptake during heat stress. Heat stress increased the relative expression of SlWRKY33b and autophagy-related (SlATG5) genes, whereas co-application of SA1+HA augmented the heat stress response and reduced SlWRKY33b and SlATG5 expression. The heat stress-responsive transcription factor (SlHsfA1a) and high-affinity potassium transporter (SlHKT1) were upregulated in SA1+HA-treated plants. In conclusion, current findings suggest that co-application with SA1+HA can be used for the mitigation of heat stress damage in tomato plants and can be commercialized as a biofertilizer.

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<![CDATA[Enhancing flavonoid production by promiscuous activity of prenyltransferase, BrPT2 from Boesenbergia rotunda]]> https://www.researchpad.co/product?articleinfo=N7adc3fc8-502e-4a64-99a2-eacda43411c6

Flavonoids and prenylated flavonoids are active components in medicinal plant extracts which exhibit beneficial effects on human health. Prenylated flavonoids consist of a flavonoid core with a prenyl group attached to it. This prenylation process is catalyzed by prenyltranferases (PTs). At present, only a few flavonoid-related PT genes have been identified. In this study, we aimed to investigate the roles of PT in flavonoid production. We isolated a putative PT gene (designated as BrPT2) from a medicinal ginger, Boesenbergia rotunda. The deduced protein sequence shared highest gene sequence homology (81%) with the predicted homogentisate phytyltransferase 2 chloroplastic isoform X1 from Musa acuminata subsp. Malaccensis. We then cloned the BrPT2 into pRI vector and expressed in B. rotunda cell suspension cultures via Agrobacterium-mediated transformation. The BrPT2-expressing cells were fed with substrate, pinostrobin chalcone, and their products were analyzed by liquid chromatography mass spectrometry. We found that the amount of flavonoids, namely alpinetin, pinostrobin, naringenin and pinocembrin, in BrPT2-expressing cells was higher than those obtained from the wild type cells. However, we were unable to detect any targeted prenylated flavonoids. Further in-vitro assay revealed that the reaction containing the BrPT2 protein produced the highest accumulation of pinostrobin from the substrate pinostrobin chalcone compared to the reaction without BrPT2 protein, suggesting that BrPT2 was able to accelerate the enzymatic reaction. The finding of this study implied that the isolated BrPT2 may not be involved in the prenylation of pinostrobin chalcone but resulted in high yield and production of other flavonoids, which is likely related to enzyme promiscuous activities.

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<![CDATA[A single power stroke by ATP binding drives substrate translocation in a heterodimeric ABC transporter]]> https://www.researchpad.co/product?articleinfo=N31301349-16ac-43e0-9228-476ce24b03ef

ATP-binding cassette (ABC) transporters constitute the largest family of primary active transporters, responsible for many physiological processes and human maladies. However, the mechanism how chemical energy of ATP facilitates translocation of chemically diverse compounds across membranes is poorly understood. Here, we advance the quantitative mechanistic understanding of the heterodimeric ABC transporter TmrAB, a functional homolog of the transporter associated with antigen processing (TAP) by single-turnover analyses at single-liposome resolution. We reveal that a single conformational switch by ATP binding drives unidirectional substrate translocation. After this power stroke, ATP hydrolysis and phosphate release launch the return to the resting state, which facilitates nucleotide exchange and a new round of substrate binding and translocation. In contrast to hitherto existing steady-state assays, our single-turnover approach uncovers the power stroke in substrate translocation and the tight chemomechanical coupling in these molecular machines.

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<![CDATA[Vip1 is a kinase and pyrophosphatase switch that regulates inositol diphosphate signaling]]> https://www.researchpad.co/product?articleinfo=Nd04d9fc2-a864-4158-9156-bedee43244a3

Significance

Our studies demonstrate that Vip1 represents a rare class of bifunctional enzyme capable of synthesizing and destroying signaling molecules important for nutrient adaptation, cellular architecture, and organelle morphology. We find that Vip1 contains two tethered autonomous catalytic active sites, which modulate levels of 1-IP7 and 1,5-IP8 through 1-kinase and 1-pyrophosphatase domains. Each activity is critical for maintaining the highly dynamic anabolic and catabolic regulation of cellular pools of IP7 and IP8. That this occurs through a single gene product emphasizes that Vip1 is a key metabolic switch critical for cellular adaptation.

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<![CDATA[Rcf2 revealed in cryo-EM structures of hypoxic isoforms of mature mitochondrial III-IV supercomplexes]]> https://www.researchpad.co/product?articleinfo=Na42cde90-4275-4a05-8e54-55745c7be6e3

Significance

As the terminal electron acceptor of our mitochondrial respiratory chains, complex IV drives and regulates oxidative phosphorylation, the process by which most of our ATP is produced. Complex IV forms supercomplexes (SCs) of different stoichiometries with other respiratory proteins, interacting via its subunits with tissue-specific or oxygen level-dependent expression isoforms, suggesting a link between SC assembly and metabolic/disease state. We investigated the effect of complex IV subunit isoform exchange in yeast using cryo-EM and biochemical assays and found no significant differences in overall SC formation, architecture, or catalytic activities. However, our structural work unexpectedly revealed the presence of a Hig1 protein which we propose is a stoichiometric subunit of complex IV, at least when within a SC with complex III.

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<![CDATA[Transient expression and purification of β-caryophyllene synthase in Nicotiana benthamiana to produce β-caryophyllene in vitro]]> https://www.researchpad.co/product?articleinfo=Nc2169696-659d-42ae-9d3d-d108e0c26eb0

The sesquiterpene β-caryophyllene is an ubiquitous component in many plants that has commercially been used as an aroma in cosmetics and perfumes. Recent studies have shown its potential use as a therapeutic agent and biofuel. Currently, β-caryophyllene is isolated from large amounts of plant material. Molecular farming based on the Nicotiana benthamiana transient expression system may be used for a more sustainable production of β-caryophyllene. In this study, a full-length cDNA of a new duplicated β-caryophyllene synthase from Artemisia annua (AaCPS1) was isolated and functionally characterized. In order to produce β-caryophyllene in vitro, the AaCPS1 was cloned into a plant viral-based vector pEAQ-HT. Subsequently, the plasmid was transferred into the Agrobacterium and agroinfiltrated into N. benthamiana leaves. The AaCPS1 expression was analyzed by quantitative PCR at different time points after agroinfiltration. The highest level of transcripts was observed at 9 days post infiltration (dpi). The AaCPS1 protein was extracted from the leaves at 9 dpi and purified by cobalt–nitrilotriacetate (Co-NTA) affinity chromatography using histidine tag with a yield of 89 mg kg−1 fresh weight of leaves. The protein expression of AaCPS1 was also confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analyses. AaCPS1 protein uses farnesyl diphosphate (FPP) as a substrate to produce β-caryophyllene. Product identification and determination of the activity of purified AaCPS1 were done by gas chromatography–mass spectrometry (GC–MS). GC–MS results revealed that the AaCPS1 produced maximum 26.5 ± 1 mg of β-caryophyllene per kilogram fresh weight of leaves after assaying with FPP for 6 h. Using AaCPS1 as a proof of concept, we demonstrate that N. benthamiana can be considered as an expression system for production of plant proteins that catalyze the formation of valuable chemicals for industrial applications.

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<![CDATA[A common coupling mechanism for A-type heme-copper oxidases from bacteria to mitochondria]]> https://www.researchpad.co/product?articleinfo=N3c240945-18cb-4c51-94cc-0f62213cf780

Significance

We present a comprehensive investigation of mitochondrial DNA-encoded variants of cytochrome c oxidase (CcO) that harbor mutations within their core catalytic subunit I, designed to interrogate the presently disputed functions of the three putative proton channels. We assess overall respiratory competence, specific CcO catalytic activity, and, most importantly, proton/electron (H+/e) stoichiometry from adenosine diphosphate to oxygen ratio measurements on preparations of intact mitochondria. We unequivocally show that yeast mitochondrial CcO uses the D-channel to translocate protons across its hydrophilic core, providing direct evidence in support of a common proton pumping mechanism across all members of the A-type heme-copper oxidase superfamily, independent of their bacterial or mitochondrial origin.

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<![CDATA[Data on protein changes of chick vitreous during normal eye growth using data-independent acquisition (SWATH-MS)]]> https://www.researchpad.co/product?articleinfo=Ncf19086a-57b8-4a37-91c3-da4ead271845

Myopia is the most common refractive error which is estimated to affect half the population of the world by 2050. It has been suggested that it could be determined by multiple factors such as environmental and genetic, but the mechanism behind the cause of myopia is still yet to be identified. Vitreous humor (VH) is a transparent gelatin-like substance that takes up to 80% of the volume of the eye, making it the largest component of the eye. Although VH is the main contributor to axial elongation of the eye including normal eye growth (emmetropization) and myopia, the diluted nature of VH (made up of 99% of water) made it difficult for less abundant molecules to be identified and therefore often overlooked. Using the more sensitive label-free mass spectrometry approach with data-independent acquisition (SWATH-MS), we established a comprehensive VH proteome library in chick animal model and quantified possible protein biomarkers that are responsible for the axial elongation during emmetropization (7, 14, 21, 28 days after hatching, n = 48 eyes). Raw data files for both information-dependent acquisition (IDA) and data-independent acquisition (SWATH-MS) were uploaded on PeptideAtlas for public access (http://www.peptideatlas.org/PASS/PASS01258).

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<![CDATA[Correction: Endothelial PKA activity regulates angiogenesis by limiting autophagy through phosphorylation of ATG16L1]]> https://www.researchpad.co/product?articleinfo=N63d19ade-13da-4b37-a1de-55afc96d0697 ]]> <![CDATA[Bambara groundnut soil metagenomics data]]> https://www.researchpad.co/product?articleinfo=N31f244a1-b1c4-4686-bd5e-a9b2199fe692

Metagenomics analysis was carried out on extracted DNA of Rhizospheric soil samples from Bambara groundnut. This dataset presented reports on the bacterial communities at the different growth stages of Bambara groundnut and the bulk soil. Paired-end Illumina-Miseq sequencing of 16S rRNA genes was carried on the soil samples of the bacterial community with the phyla dominated by Actinobacteria (30.1%), Proteobacteria (22%), Acidobacteria (20.9%), Bacteroides (8.4%), Chloroflex (4.5%) and Firmicutes (4.4%) in all the soil samples. Samples from the bulk soil had the least average percent phyla, while samples at seed maturity stage had the highest average percent phyla. The alpha diversity at p = 0.05 was highest at this stage compared to the others and the control. Rubrobacter was the most predominant genera, after which is Acidobacterium and Skermanella. The biodiversity profile generated from the metagenomics analysis is useful in increasing knowledge of the drought-tolerance ability of Bambara groundnut. The data generated can be used to compare bacterial diversity at different growth stages of plants.

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<![CDATA[Data on the cancer risk and mortalities induced by annual background radiations at various ages in Kohgiluyeh and Boyer-Ahmad province, Iran]]> https://www.researchpad.co/product?articleinfo=N9eff0af1-e09d-4fe4-9012-b4dc71a8355a

Measurement of background radiations (BRs) as the sources of cancer risk, is important. The aim of this study was to measure the BR, as well as its cancer risk and mortalities in Kohgiluyeh and Boyer-Ahmad province (KBAp). Indoors and outdoors BRs were measured in eight cities utilizing a Geiger-Muller detector. Five main locations (north, east, west, south, and center) were chosen for measuring outdoor and indoor BRs in each city of KBAp. The BEIR VII-Phase 2 model was used to calculate the BRs induced cancer risks and mortalities of various cancer types at different ages. The average dose rates of outdoor and indoor were 136.9 ± 12.5 and 149.3 ± 19.8 nSv.h−1, respectively. The average annual effective doses (AEDs) for adults, children, and infants were 0.17, 0.19, and 0.22 mSv.y−1 due to the outdoor, and 0.73, 0.84, and 0.94 mSv.y−1 resulting from the indoor exposure, respectively. The average lifetime risk for one year BRs induced cancers was 164.8 ± 15.7 and 307.1 ± 32.3 (in 100,000 people) for new-borns male and female, in that order. This risk decreased with age and reached 11.2 ± 1.6 and 13.8 ± 1.6 (in 100,000 people) for men and women at the age of 80, respectively. The average lifetime risk of mortality due to cancers induced by annual BRs was 70.7 ± 8.3 and 113.8 ± 10.6 (incidence probability in 100,000 people) for new-borns male and female respectively. This risk decreased with age and reached 9.8 ± 1.3 and 12.2 ± 1.3 (in 100,000 people) for men and women at the age of 80 years, respectively.

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<![CDATA[Quantification of chitooligosaccharides by FACE method: Determination of combinatory effects of mouse chitinases]]> https://www.researchpad.co/product?articleinfo=N191e2ad7-0ee5-4784-9b1c-5fb2cc206b07

Highlights

  • FACE is a simple, qualitative and quantitative method.

  • The standard curve warrants quantification of chitooligosaccharides of up to 10 nmol regardless on used buffer system.

  • Our improved FACE method enable us to quantify chitooligosaccharides produced by chitinases at pH 2.0–8.0.

  • Determination of the combinatory effects of the Chit1 and AMCase using the FACE method.

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<![CDATA[Dataset of HOXB7, HOXB8 and HOXB9 expression profiles in cell lines representative of the breast cancer molecular subtypes Luminal a (MCF7), Luminal b (BT474), HER2+ (SKBR3) and triple-negative (MDA231, MDA468), compared to a model of normal cells (MCF10A)]]> https://www.researchpad.co/product?articleinfo=Nb771d94c-b0a4-4cc4-80c4-4bbf9a9cfeac

Alterations in HOXB genes expression in breast cancer have been described and related to therapy response and disease progression. However, due to breast cancer complexity and heterogeneity, added to the use of different technical approaches, the observed expression profiles are sometimes contradictory. Here, we provided the analyses of HOXB7, HOXB8 and HOXB9 expression profiles in cell lines extensively used in the literature addressing the putative role of HOXB genes in breast cancer (MCF7, BT474, SKBR3, MDA231 and MDA468) and representative of the clinical breast cancer molecular subtypes (Luminal A, Luminal B, HER2+ and Triple-negatives Claudin-low/Basal), compared to a normal breast model (MCF10A), using quantitative-PCR (qPCR). This technique allows a very sensitive quantification of gene expression and was performed using the fluorophore SYBR Green in order to obtain the expression levels relative to a reference gene, GAPDH in this case. We showed that HOXB7 is upregulated in all breast cancer cells analyzed, while HOXB8 and HOXB9 are significantly upregulated in MCF7 (Luminal A), BT474 (Luminal B) and MDA231 cells (Triple-negative Claudin-low). In addition, we found that the magnitude of the upregulation is highly subtype-specific, being the HER2+ cells the model with lowest HOXB7 upregulation, presenting very low or even null expression for HOXB8 and HOXB9, respectively. These results are analyzed in more detail in “HOX genes function in Breast Cancer development” [1] and are potentially relevant for a better understanding of the molecular heterogeneity of breast cancer, in addition to be a valuable tool assisting researchers in the choice of the most suitable cell models to perform functional assays concerning HOXB7, HOXB8 and HOXB9 genes.

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<![CDATA[Dataset of foraminiferal sedimentary DNA (sedDNA) sequences from Svalbard]]> https://www.researchpad.co/product?articleinfo=Ne76dccf2-4ee5-4d09-b71a-a8ab57ec17a2

Environmental DNA (eDNA) is usually defined as genetic material obtained directly from environmental samples, such as soil, water, or ice. Coupled to DNA metabarcoding, eDNA is a powerful tool in biodiversity assessments. Results from eDNA approach provided valuable insights to the studies of past and contemporary biodiversity in terrestrial and aquatic environments. However, the state and fate of eDNA are still investigated and the knowledge about the form of eDNA (i.e., extracellular vs. intracellular) or the DNA degradation under different environmental conditions is limited. Here, we tackle this issue by analyzing foraminiferal sedimentary DNA (sedDNA) from different size fractions of marine sediments: >500 µm, 500–100 µm, 100–63 µm, and < 63 µm. Surface sediment samples were collected at 15 sampling stations located in the Svalbard archipelago. Sequences of the foraminifera-specific 37f region were generated using Illumina technology. The presented data may be used as a reference for a wide range of eDNA-based studies, including biomonitoring and biodiversity assessments across time and space.

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<![CDATA[Icariin protects against sodium azide—induced neurotoxicity by activating the PI3K/Akt/GSK-3β signaling pathway]]> https://www.researchpad.co/product?articleinfo=Ncbd124ac-94d4-4eb8-bed6-5403ae50ae27

Background

Icariin (ICA) is one of the major active flavonoids extracted from the traditional Chinese herb Epimedium brevicornum Maxim and has been shown to have neuroprotective effects. This study was designed to investigate the effect of ICA on sodium azide (NaN3)-induced rat adrenal pheochromocytoma (PC12) cell damage and to further examine the underlying mechanisms.

Methods

To explore its possible mechanism, we used NaN3 (50 mM)-induced neuronal PC12 cell damage. Cell viability was evaluated by CCK-8 and lactate dehydrogenase (LDH) assays. Mitochondrial membrane potential (MMP) was detected by JC-1. Glucose concentration was assessed by the glucose oxidase method. The role of ICA in the PI3K/Akt/GSK-3β signaling pathway was explored by Western blotting.

Results

The results indicate that pretreatment with ICA reduced NaN3-induced cell damage and significantly reduced the leakage rate of LDH in PC12 cells. ICA pretreatment increased the MMP and a decrease in glucose concentration indicate increased glucose consumption. Furthermore, the protein levels of p-PI3K (p85), PI3K-110α, p-Ser473-Akt and p-Ser9-GSK-3β were markedly decreased in PC12 cells after NaN3 treatment for 24 h, whereas these effects were reverted after pretreatment with ICA. Tau phosphorylation at the Ser396/404 and Thr217 sites was significantly decreased by pretreatment with ICA.

Conclusions

These results suggest that ICA protects against NaN3-induced neurotoxicity in PC12 cells by activating the PI3K/Akt/GSK-3β signaling pathway.

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<![CDATA[Periprotein lipidomes of Saccharomyces cerevisiae provide a flexible environment for conformational changes of membrane proteins]]> https://www.researchpad.co/product?articleinfo=N7474e69a-1332-42c7-a745-986216ca8672

Yeast tolerates a low pH and high solvent concentrations. The permeability of the plasma membrane (PM) for small molecules is low and lateral diffusion of proteins is slow. These findings suggest a high degree of lipid order, which raises the question of how membrane proteins function in such an environment. The yeast PM is segregated into the Micro-Compartment-of-Can1 (MCC) and Pma1 (MCP), which have different lipid compositions. We extracted proteins from these microdomains via stoichiometric capture of lipids and proteins in styrene-maleic-acid-lipid-particles (SMALPs). We purified SMALP-lipid-protein complexes by chromatography and quantitatively analyzed periprotein lipids located within the diameter defined by one SMALP. Phospholipid and sterol concentrations are similar for MCC and MCP, but sphingolipids are enriched in MCP. Ergosterol is depleted from this periprotein lipidome, whereas phosphatidylserine is enriched relative to the bulk of the plasma membrane. Direct detection of PM lipids in the 'periprotein space' supports the conclusion that proteins function in the presence of a locally disordered lipid state.

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<![CDATA[Analysis of the nucleocytoplasmic shuttling RNA-binding protein HNRNPU using optimized HITS-CLIP method]]> https://www.researchpad.co/product?articleinfo=Nb5a6160c-8969-498c-b6ff-671487ce7810

RNA-binding proteins (RBPs) control many types of post-transcriptional regulation, including mRNA splicing, mRNA stability, and translational efficiency, by directly binding to their target RNAs and their mutation and dysfunction are often associated with several human neurological diseases and tumorigenesis. Crosslinking immunoprecipitation (CLIP), coupled with high-throughput sequencing (HITS-CLIP), is a powerful technique for investigating the molecular mechanisms underlying disease pathogenesis by comprehensive identification of RBP target sequences at the transcriptome level. However, HITS-CLIP protocol is still required for some optimization due to experimental complication, low efficiency and time-consuming, whose library has to be generated from very small amounts of RNAs. Here we improved a more efficient, rapid, and reproducible CLIP method by optimizing BrdU-CLIP. Our protocol produced a 10-fold greater yield of pre-amplified CLIP library, which resulted in a low duplicate rate of CLIP-tag reads because the number of PCR cycles required for library amplification was reduced. Variance of the yields was also reduced, and the experimental period was shortened by 2 days. Using this, we validated IL-6 expression by a nuclear RBP, HNRNPU, which directly binds the 3’-UTR of IL-6 mRNA in HeLa cells. Importantly, this interaction was only observed in the cytoplasmic fraction, suggesting a role of cytoplasmic HNRNPU in mRNA stability control. This optimized method enables us to accurately identify target genes and provides a snapshot of the protein-RNA interactions of nucleocytoplasmic shuttling RBPs.

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