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Activating signal co-integrator complex 1 (ASCC1) acts with ASCC-ALKBH3 complex in alkylation damage responses. ASCC1 uniquely combines two evolutionarily ancient domains: nucleotide-binding K-Homology (KH) (associated with regulating splicing, transcriptional, and translation) and two-histidine phosphodiesterase (PDE; associated with hydrolysis of cyclic nucleotide phosphate bonds). Germline mutations link loss of ASCC1 function to spinal muscular atrophy with congenital bone fractures 2 (SMABF2). Herein analysis of The Cancer Genome Atlas (TCGA) suggests ASCC1 RNA overexpression in certain tumors correlates with poor survival, Signatures 29 and 3 mutations, and genetic instability markers. We determined crystal structures of Alvinella pompejana (Ap) ASCC1 and Human (Hs) PDE domain revealing high-resolution details and features conserved over 500 million years of evolution. Extending our understanding of the KH domain Gly-X-X-Gly sequence motif, we define a novel structural Helix-Clasp-Helix (HCH) nucleotide binding motif and show ASCC1 sequence-specific binding to CGCG-containing RNA. The V-shaped PDE nucleotide binding channel has two His-Φ-Ser/Thr-Φ (HXT) motifs (Φ being hydrophobic) positioned to initiate cyclic phosphate bond hydrolysis. A conserved atypical active-site histidine torsion angle implies a novel PDE substrate. Flexible active site loop and arginine-rich domain linker appear regulatory. Small-angle X-ray scattering (SAXS) revealed aligned KH-PDE RNA binding sites with limited flexibility in solution. Quantitative evolutionary bioinformatic analyses of disease and cancer-associated mutations support implied functional roles for RNA binding, phosphodiesterase activity, and regulation. Collective results inform ASCC1's roles in transactivation and alkylation damage responses, its targeting by structure-based inhibitors, and how ASCC1 mutations may impact inherited disease and cancer.

Background Insufficient awareness of foot self-care among diabetic individuals results in diabetic foot ulcers especially in the Indian population considering its varied ethnicity and lifestyle practices. The management of diabetes and diabetic foot ulcers therefore demands a well-coordinated approach that involves multiple healthcare providers (HCPs). The present study aims to develop and validate an interprofessional collaborative (IPC) educational program involving HCPs to efficiently oversee and instruct the public on appropriate strategies for self-managing diabetic foot health in the Indian population. Methods The research group worked on creating an educational module titled ‘An Interprofessional Collaborative Educational Module on Self-Management of Foot for Individuals with Type II Diabetes Mellitus.’ The objective of this module was to promote the adoption of proper practices in self-managing foot health for individuals with type 2 diabetes mellitus. A panel of 13 experts participated in a two-stage validation process using the Delphi method to assess the module and its educational resources. Subsequently, the module was tested on a group of 30 participants, i.e., individuals with diabetes, with its efficacy evaluated through conversation analysis and in-depth interviews. Results The three-month-long module included three sessions • 1: Diabetes and its health implications • 2: Diabetic foot and self-management • 3: Interprofessional education in diabetic foot care The mode of content delivery was via Whatsapp, and the educational resources, in the form of pamphlets, flowcharts, handouts, case-based cartoons, and videos on diabetes, including diabetic foot, its risks, and self-management, were shared regularly. All participating experts consensually validated the module and educational resources. Analysis of in-depth interviews revealed that the module immensely benefitted the participants and helped them improve their knowledge and practices of foot care in diabetes. Conclusions The study highlights the importance of developing and validating IPC educational modules tailored to diabetic foot care. It can aid in enhancing adherence to proper diabetic foot care practices.

Background: Long-term exposure of pancreatic islets to fatty acids (FAs), common in obesity, metabolic syndrome, and type 2 diabetes, leads to a compensatory hyperactivity followed by inflammation, apoptosis, dysfunctional beta cells, and results in insulin dependence of the patient. Restriction of fatty uptake by islet beta cells may protect them from lipotoxicity. Purpose: Pancreatic islet beta cells express the fatty acid binding protein 3 (FABP3) to bind FAs and to orchestrate lipid signals. Based on this, we investigated whether downregulation of FABP3, by Fabp3 silencing, might slow lipid metabolism and protect against lipotoxicity in insulin-secreting cells. Results: Neither Fabp3 silencing, nor overexpression affected the glucose-stimulated insulin secretion in absence of FAs. Fabp3 silencing decreased FA-uptake, lipid droplets formation, and the expression of the lipid accumulation-regulating gene Dgat1 in Ins1E cells. It reduced FA-induced inflammation by deactivation of NF-κB, which was associated with upregulation of IκBα and deactivation of the NF-κB p65 nuclear translocation, and the downregulation of the cytokines ILl-6, IL-1β, and TNFα. Ins1E cells were protected from the FA-induced apoptosis as assessed by different parameters including DNA degradation and cleaved caspase-3 immunoblotting. Furthermore, FABP3 silencing improved the viability, Pdx1 gene expression, and the insulin-secreting function in cells long-term cultured with palmitic acid. All results were confirmed by the opposite action rendered by FABP3 overexpression. Conclusion: The present data reveals that pancreatic beta cells can be protected from lipotoxicity by inhibition of FA-uptake, intracellular utilization and accumulation. FABP3 inhibition, hence, may be a useful pharmaceutical approach in obesity, metabolic syndrome, and type 2 diabetes. [ABSTRACT FROM AUTHOR]

Noroviruses are a prevalent cause of human viral gastroenteritis, yet the precise mechanisms underlying their infection cycle, particularly their interactions with and entry into cells, remain poorly understood. Human norovirus (HuNoV) primarily targets human small intestinal epithelial cells, within which 3- O -sulfogalactosylceramide (sulfatide) ranks among the most abundant glycosphingolipids (GSLs). While sulfatide involvement in the binding and infection mechanism of several viruses has been documented, its interaction with noroviruses remains underexplored. This study investigated whether noroviruses interact with sulfatide. We found that the recombinant viral capsid protein VP1 of HuNoV (genogroups I and II) and murine norovirus (genogroup V) exhibited robust binding to sulfatide compared with other tested GSLs using enzyme-linked immunosorbent assay, thin-layer chromatography binding assay and real-time quantitative reverse transcription polymerase chain reaction binding assay. VP1 also bound 3- O -sulfated lactosylceramide, which shares the 3- O -sulfated galactose moiety with sulfatide. However, both VP1 and its P domain, identified as the sulfatide-binding domain, exhibited limited binding to structural analogues of sulfatide and other sulfated compounds. These findings suggest a specific recognition of the 3- O -sulfated galactose moiety. Notably, we found that sulfatide is a novel binding target for norovirus particles. Overall, our findings reveal a previously unknown norovirus–sulfatide interaction, proposing sulfatide as a potential candidate for norovirus infection receptors. [ABSTRACT FROM AUTHOR]

The Qinghai-Xizang Plateau is a unique geographical unit with the highest elevation in the world and a natural laboratory for studying organism adaptation to extreme environment. Among the fundamental and abundant cell types in vertebrate life, erythrocytes play a vital role in sustaining vertebrates. Spatially and temporally coordinated regulatory networks and feedback mechanisms are essential for fish erythropoiesis and typical development. Fish species belonging to the subfamily Sohizothoraoinae, after a long period of adaptation to high temperature and low oxygen, have become one of the significant components of the present fish fauna of the Qinghai-Tibetan Plateau. In order to study the regulatory mechanism of erythrocyte development, we selected Gymnocypris eckloni, a representative species of the Sohizothoraoinae subfamily, due to its unique geographical distribution and life history responses. Based on previous research in the laboratory, the whole genome data of G. eckloni was used to identify the gene involved in erythroid development, namely KLF17, and to obtain the promoter sequence of KLF17. The KLF17 promoter DNA was used as bait to screen the total proteins from the kidney tissues of G. eckloni and identify the candidate binding proteins by LC-MS/MS. The identified candidate binding proteins of the KLF17 promoter were subjected to GO function enrichment, and KEGG pathway analysis by bioinformatics methods. The results showed that a total of 576 proteins were screened for specific binding to the KLF17 promoter region in G. eckloni kidney tissue. After removing proteins that could not be identified, there were 306 candidate binding proteins. These include eukaryotic translation initiation factor 2, cytochrome P450 enzymes, transferrin, protein-like metalloproteinases with type I platelet binding protein motifs, pyruvate dehydrogenase, erythrocyte membrane protein band 4.1 proteins, and other hematopoiesis-related proteins. Furthermore, GO functional enrichment analysis indicated that these proteins were involved in various biological functions, including cell growth, cell cycle, immune response, signaling and nucleic acid binding to transcription factors. KEGG pathway analysis showed that these proteins participated in several signaling pathways, such as amino acid metabolism pathway, apoptosis, PPAR signaling, Cytochrome P450 metabolism, HIF-1signaling, Hippo signaling and the neurotrophic factor pathway. The candidate binding proteins of the KLF17 promoter screened in this study mainly include eukaryotic translation initiation factor 2, cytochrome P450 enzymes and transferrin. Functional analyses revealed that they are involved in the HIF-1signaling pathway, which stimulates erythropoiesis and erythrocyte development through the regulation of hemoglobin and cadherin gene expression, iron metabolism, angiogenesis, glycolysis and other pathways. KLF17 is involved in regulating the interactions between haem, iron and globin proteins in erythrocytes at the transcriptional level, providing important information for studying the mechanism of erythrocyte differentiation. Meanwhile, transferrin, as a cross-regulator involved in the regulation of iron and oxygen, can promote the transport of iron in the blood and enhance the binding capacity of blood oxygen, which enables the organism to survive for a long time in a low-oxygen environment, which also shows that the mechanism of hypoxic adaptation in G. eckloni is related to transferrin downstream of HIF-1. In summary, the present study provide a solid foundation for future investigations into the interaction mechanism between this protein and KLF17. It also provide scientific data for dissecting the molecular mechanism underlying hematopoietic system development and adaptation to low-oxygen environment in native plateau fishes. [ABSTRACT FROM AUTHOR]

Background and aim: Gut microbiota influences energy homeostasis in part through circulating hormones. Insulin-like growth factor-binding protein (IGFBP)-2 is a biomarker whose increase in systemic circulation is associated with positive effects on body weight and metabolism. In a recent clinical trial, probiotic Lacticaseibacillus rhamnosus HA-114 supplementation showed positive effects on eating behaviors and insulin resistance in overweight participants undergoing a weight-loss intervention. In this context, this ancillary study aimed at assessing the impact of L. rhamnosus HA-114 supplementation on plasma IGFBP-2 levels in these individuals, and whether this modulation correlated with changes in fat mass, energy metabolism, and eating behaviors. Methods: Fasting plasma IGFBP-2 concentrations were quantified in 100 overweight or obese men and women enrolled in a 12-week diet-based weight reduction program (−500 kcal/day), in combination with probiotic L. rhamnosus HA-114 or placebo supplementation. Baseline and changes in circulating IGFBP-2 concentrations were correlated with anthropometric parameter, glucose and lipid metabolism, cardiorespiratory function and eating behaviors. Results: On average, the intervention reduced BMI by 4.6 % and increased IGFBP-2 by 13 %, regardless of supplementation group. Individuals who presented an increase in IGFBP-2 levels had significantly greater reductions in BMI. Changes in IGFBP-2 levels were correlated with loss in fat mass (r = 0.2, p

To date, more than 150 chemical modifications have been disclosed in different RNA species, which are employed to diversify the structure and function of RNA in living organisms. The N 6-methyladenosine (m6A) modification, which is found in the adenosine N 6 site of RNA, has been demonstrated to be the most heavy modification in the mRNA in cells. Moreover, the m6A modification in mRNAs of mammalian and other eukaryotic cells is highly conserved and mandatorily encoded. Increasing evidence indicates that the m6A modification plays a pivotal role in gene-expression regulation and cell-fate decisions. Here, we summarize the most recent m6A-sequencing technology, as well as the molecular mechanism underlying its occurrence, development, and potential use as a target for the treatment of human diseases. Furthermore, our review highlights other newly discovered chemical modifications of RNA that are associated with human disease, as well as their underlying molecular mechanisms. Thus, significant advancements have been made in qualitative/quantitative m6A detection and high-throughput sequencing, and research linking this RNA modification to disease. Efforts toward simplified and more accessible chemical/biological technologies that contribute to precision medicine are ongoing, to benefit society and patients alike.

Destruxin A (DA) is a cyclo-hexadepsipeptidic insecticidal mycotoxin isolated from the entomopathogenic fungi, Metarhizium spp. However, its mode of action is unknown. In this study, we isolated 149 candidate DA-binding proteins by drug affinity response target stability, and determined the interactions of 80 canditates with DA in vitro by surface plasmon resonance. The affinity coefficients (KD) ranged from 24 to 469 μM. Binding proteins were functionally diverse and included cytoskeletal components and cell motility, protein transcription and translation pathways, ubiquitin dependent protein metabolic processes, nucleus pore entry and exit, and endoplasmic reticulum vesicle transport and etc. Electron microscopy revealed that DA damaged the cytoskeleton and multiple organelles, disrupted cell adhesion and motility, and led to cell death. DA appeared to have a multi-targeted approach to cellular structures and multiple life processes, leading to cell death. The results of this study could provide molecular evidence for the analysis of the insecticidal toxicology of DA and further improve the study of the pathogenic insect mechanism of Metarhizium. [ABSTRACT FROM AUTHOR]

Simple Summary: Animal growth depends on various factors, such as breed, nutrition, growth hormones, physiological stage, and environment. Therefore, the decrease in piglets' growth after weaning is not only related to a decrease in feed intake but may also be related to the breed and growth hormones. Thus, the present study investigated the insulin-like growth factor (IGF) system components in Chinese indigenous pig breeds (Taoyuan black and Xiangcun black pigs) and commercial pig breeds (Duroc pigs) during weaning. Our findings indicated that weaning can affect the expressions of IGF system components in piglets, and the expressions of IGF system components in response to weaning stress differed in the three pig breeds. The differences in IGF system components in the three pig breeds might be related to their anti-stress tolerance characteristics. The present study investigated the insulin-like growth factors (IGFs) and their receptors and binding proteins among three pig breeds during weaning. Sixty Duroc (DR), Taoyuan black (TYB), and Xiangcun black (XCB) piglets (20 piglets per breed) were selected at 21 and 24 (3 days of post-weaning) days of age to analyze organ indices, plasma concentrations of IGF and IGF-binding proteins (IGFBPs) using ELISA kits, and gene expression of IGF-system-related components in different tissues. The plasma IGFBP-3 concentration in TYB piglets was higher (p > 0.05) than in the XCB and DR piglets at 21 days of age. At 21 days of age, compared with the DR piglets, the IGF-1 expression was lower (p < 0.05) in the kidney, but it was higher (p < 0.05) in the spleen of XCB and TYB piglets. At 24 days of age, the IGF-1 expression was higher (p < 0.05) in the kidney of TYB piglets than in the XCB and DR piglets, while IGFBP-3 in the stomach and IGFBP-4 in the liver of XCB and TYB piglets were lower (p < 0.05) compared with the DR piglets. Weaning down-regulated (p < 0.05) IGF-1 expression in the jejunum, spleen, and liver of piglets, while it up-regulated (p < 0.05) IGFBP-3 expression in the stomach, IGFBP-4 in the liver, IGFBP-5 in the ileum, and IGFBP-6 in the jejunum of DR piglets. Spearman's correlation analysis showed a negative correlation (p < 0.05) between plasma IGFBP-2 and IGFBP-5 concentration and the organ indices of piglets. Collectively, there were significant differences in the IGF system components among the three pig breeds. The IGF system components were altered during weaning, which might be involved in weaning stress to decrease the growth of piglets. [ABSTRACT FROM AUTHOR]

Phosphatidylinositol (3,4,5)-trisphosphate (PIP3)-dependent Rac exchanger 1 (P-Rex1) catalyzes the exchange of GDP for GTP on Rac GTPases, thereby triggering changes in the actin cytoskeleton and in transcription. Its overexpression is highly correlated with the metastasis of certain cancers. P-Rex1 recruitment to the plasma membrane and its activity are regulated via interactions with heterotrimeric Gβγ subunits, PIP3, and protein kinase A (PKA). Deletion analysis has further shown that domains C-terminal to its catalytic Dbl homology (DH) domain confer autoinhibition. Among these, the first dishevelled, Egl-10, and pleckstrin domain (DEP1) remains to be structurally characterized. DEP1 also harbors the primary PKA phosphorylation site, suggesting that an improved understanding of this region could substantially increase our knowledge of P-Rex1 signaling and open the door to new selective chemotherapeutics. Here we show that the DEP1 domain alone can autoinhibit activity in context of the DH/PH-DEP1 fragment of P-Rex1 and interacts with the DH/PH domains in solution. The 3.1 Å crystal structure of DEP1 features a domain swap, similar to that observed previously in the Dvl2 DEP domain, involving an exposed basic loop that contains the PKA site. Using purified proteins, we show that although DEP1 phosphorylation has no effect on the activity or solution conformation of the DH/PH-DEP1 fragment, it inhibits binding of the DEP1 domain to liposomes containing phosphatidic acid. Thus, we propose that PKA phosphorylation of the DEP1 domain hampers P-Rex1 binding to negatively charged membranes in cells, freeing the DEP1 domain to associate with and inhibit the DH/PH module.

Pseudomonas plecoglossicida can infect Pseudosciaena crocea and cause visceral white spot disease, resulting in substantial economic losses to the P. crocea industry. Pseudomonas plecoglossicida is a temperate-sensitive bacterium and only infects P. crocea below 22 ℃. Aptamers are oligonucleotide molecules selected by the systematic evolution of ligands by exponential enrichment (SELEX) and can identify all kinds of targets. In the present study, five aptamers (M17, M19, M48, M56 and M81) for P. plecoglossicida cultured at 18 ℃ were selected by SELEX and based on comparative analysis of the high frequency sequences. The five aptamers had significantly higher affinities for the pathogenic P. plecoglossicida at 18 ℃ than for the nonpathogenic P. plecoglossicida at 28 ℃ and the other six bacteria (Escherichia coli, Edwardsiella tarda, Aeromonas hydrophila, Vibiro harveryi, Vibiro alginolyticus, Pseudomonas aeruginosa) (P

Long noncoding RNAs (lncRNAs) participate in plant biological processes under biotic and abiotic stresses. However, little is known about the function and regulation mechanism of lncRNAs related to the pathogen at a molecular level. A banana lncRNA, Malnc2310, is a Fusarium oxysporum f. sp. cubense inducible lncRNA in roots. In this study, we demonstrate the nuclear localization of Malnc2310 by fluorescence in situ hybridization and it can bind to several proteins that are related to flavonoid pathway, pathogen response and programmed cell death. Overexpression of Malnc2310 increases susceptibility to Fusarium crude extract (Fu), salinity, and cold in transgenic Arabidopsis. In addition, Malnc2310 transgenic Arabidopsis accumulated more anthocyanins under Fusarium crude extract and cold treatments that are related to upregulation of these genes involved in anthocyanin biosynthesis. Based on our findings, we propose that Malnc2310 may participate in flavonoid metabolism in plants under stress. Furthermore, phenylalanine ammonia lyase (PAL) protein expression was enhanced in Malnc2310 overexpressed transgenic Arabidopsis, and Malnc2310 may participate in PAL regulation by binding to it. This study provides new insights into the role of Malnc2310 in mediating plant stress adaptation. Key message: The lncRNA Malnc2310 is induced in banana roots by Fusarium. The 35S::Malnc2310 transgenic Arabidopsis accumulate more anthocyanin under stress, verifying that Malnc2310 involved in flavonoid pathway through regulating PAL proteins. [ABSTRACT FROM AUTHOR]

Midgut receptors play a critical role in the specificity of Cry toxins for individual insect species. Cadherin proteins are essential putative receptors of Cry1A toxins in lepidopteran larvae. Cry2A family members share common binding sites in Helicoverpa armigera, and one of them, Cry2Aa, has been widely reported to interact with midgut cadherin. Here, we studied the binding interaction and functional role of H. armigera cadherin in the mechanism of Cry2Ab toxicity. A region spanning from cadherin repeat 6 (CR6) to the membrane-proximal region (MPR) of cadherin protein was produced as six overlapping peptides to identify the specific binding regions of Cry2Ab. Binding assays showed that Cry2Ab binds nonspecifically to peptides containing CR7 and CR11 regions in a denatured state but binds specifically only to CR7-containing peptides in the native state. The peptides CR6-11 and CR6-8 were transiently expressed in Sf9 cells to assess the functional role of cadherin. Cytotoxicity assays showed that Cry2Ab is not toxic to the cells expressing any of the cadherin peptides. However, ABCA2-expressing cells showed high sensitivity to Cry2Ab toxin. Neither increased nor decreased sensitivity to Cry2Ab was observed when the peptide CR6-11 was coexpressed with the ABCA2 gene in Sf9 cells. Instead, treating ABCA2-expressing cells with a mixture of Cry2Ab and CR6-8 peptides resulted in significantly reduced cell death compared with treatment with Cry2Ab alone. Moreover, silencing of the cadherin gene in H. armigera larvae showed no significant effect on Cry2Ab toxicity, in contrast to the reduced mortality in ABCA2-silenced larvae. [ABSTRACT FROM AUTHOR]

Ezrin is the cytoskeletal organizer and functions in the modulation of membrane–cytoskeleton interaction, maintenance of cell shape and structure, and regulation of cell–cell adhesion and movement, as well as cell survival. Ezrin plays a critical role in regulating tumor metastasis through interaction with other binding proteins. Notably, Ezrin has been reported to interact with immune cells, allowing tumor cells to escape immune attack in metastasis. Here, we review the main functions of Ezrin, the mechanisms through which it acts, its role in tumor metastasis, and its potential as a therapeutic target. [ABSTRACT FROM AUTHOR]

Calcium/calmodulin-stimulated protein kinase II (CaMKII) is a family of broad substrate specificity serine (Ser)/threonine (Thr) protein kinases widely expressed in many tissues that is capable of mediating diverse functional responses depending on its cellular and molecular microenvironment. This review briefly summarises current knowledge on the structure and regulation of CaMKII and focuses on how the molecular environment, and interaction with binding partner proteins, can produce different populations of CaMKII in different cells, or in different subcellular locations within the same cell, and how these different populations of CaMKII can produce diverse functional responses to activation following an increase in intracellular calcium concentration. This review also explores the possibility that identifying and characterising the molecular interactions responsible for the molecular targeting of CaMKII in different cells in vivo, and identifying the sites on CaMKII and/or the binding proteins through which these interactions occur, could lead to the development of highly selective inhibitors of specific CaMKII-mediated functional responses in specific cells that would not affect CaMKII-mediated responses in other cells. This may result in the development of new pharmacological agents with therapeutic potential for many clinical conditions. [ABSTRACT FROM AUTHOR]

Destruxin A (DA), a hexa-cyclodepsipeptidic mycotoxin produced by the entomopathogenic fungus Metarhizium anisopliae, has insecticidal activity, but its molecular mechanism of action is still not clear. Three proteins with modification-related functions, calreticulin (BmCRT), dipeptidyl peptidase Ⅲ (BmDPP3), and protein disulfide isomerase A5 (BmPDIA5), were selected to verify the interactions with DA in this study. The kinetic data of the interactions were measured by surface plasmon resonance (SPR) and bio-layer interferometry (BLI) in vitro. The KD values of DA with BmCRT, BmDPP3, and BmPDIA5 ranged from 10−4 to 10−5 mol/L, which suggested that the three proteins all had fairly strong interactions with DA. Then, it was found that DA in a dose-dependent manner affected the interactions of the three proteins with their partners in insect two-hybrid tests in SF-9 cells. Furthermore, the results of enzyme activities by ELISA indicated that DA could inhibit the activity of BmDPP3 but had no significant effect on BmPDIA5. In addition, DA induced the upregulation of BmDPP3 and the downregulation of BmCRT. The results prove that BmCRT, BmDPP3, and BmPDIA5 are all binding proteins of DA. This study might provide new insights to elucidate the molecular mechanism of DA. [ABSTRACT FROM AUTHOR]

Thyroid stimulating antibodies (TSAB) cause Graves’ disease and contribute to Graves’ Orbitopathy (GO) pathogenesis. We hypothesise that the presence of TSH binding proteins (truncated TSHR variants (TSHRv)) and/or nonclassical ligands such as thyrostimulin (α2β5) might provide a mechanism to protect against or exacerbate GO. We analysed primary human orbital preadipocyte-fibroblasts (OF) from GO patients and people free of GO (non-GO). Transcript (QPCR) and protein (western blot) expression levels of TSHRv were measured through an adipogenesis differentiation process. Cyclic-AMP production by TSHR activation was studied using luciferase-reporter and RIA assays. After differentiation, TSHRv levels in OF from GO were significantly higher than non-GO (p = 0.039), and confirmed in ex vivo analysis of orbital adipose samples. TSHRv western blot revealed a positive signal at 46 kDa in cell lysates and culture media (CM) from non-GO and GO-OF. Cyclic-AMP decreased from basal levels when OF were stimulated with TSH or Monoclonal TSAB (M22) before differentiation protocol, but increased in differentiated cells, and was inversely correlated with the TSHRv:TSHR ratio (Spearman correlation: TSH r = −0.55, p = 0.23, M22 r = 0.87, p = 0.03). In the bioassay, TSH/M22 induced luciferase-light was lower in CM from differentiated GO-OF than non-GO, suggesting that secreted TSHRv had neutralised their effects. α2 transcripts were present but reduced during adipogenesis (p < 0.005) with no difference observed between non-GO and GO. β5 transcripts were at the limit of detection. Our work demonstrated that TSHRv transcripts are expressed as protein, are more abundant in GO than non-GO OF and have the capacity to regulate signalling via the TSHR.

Carotenoids play important roles in living organisms. However, animals cannot synthesize carotenoids by themselves, and they must absorb and accumulate carotenoids from their diets in which some key genes are involved. In present study, a gene named StAR-like-3 was characterized in the noble scallop Chlamys nobilis, and its function was identified using golden scallops with higher carotenoids content and brown scallops with less carotenoids content by immunohistochemistry, carotenoid binding assay and RNAi. Results showed that the StAR-like-3 encodes a 54.7 kDa transmembrane protein (named as StAR3) of 481 amino acids containing a MENTAL domain and a START (Steroidogenic acute regulatory protein-related lipid transfer) domain, and its expression level in hemocytes and intestine of golden scallops were significantly higher than those of brown ones. Subsequently, the StAR3 protein was detected in the intestinal epithelial cells of golden scallops, and recombinant StAR3 could bind lutein conjugated to protein G and antibody to form a yellow complex, suggesting it is a carotenoid binding protein involving in carotenoids accumulation in golden scallops. Furthermore, total carotenoids content of hemolymph in golden scallops was significantly decreased when the expression of StAR-like-3 suppressed, suggesting this gene plays an important role in transport of carotenoids. Conclusion, the present results indicated that the StAR-like-3 is a key gene responsible for the carotenoids accumulation in the scallop.

Objectives: Establishment of reference interval for M2BPGI glyco-biomarker in the serum of relatively healthy Mongolian people. Methods: Serum analysis for M2BPGI glyco-biomarker, hepatitis virus markers, and AFP analysis were performed using a fully automated chemiluminescence immunoassay analyzer. Non-parametric methods were used for establishment of reference interval according to the guideline of the Clinical Laboratory Standards Institute. Statistical analysis was done by using SPSS ver. 20.0; SPSS Inc., Chicago, IL software. Results: Total of 3384 people were involved in this study. Of the total participants, 40% were overweight and 31.3% were obese. Also, of the population 10.1 percent were positive for HBsAg, 17 percent were positive for Anti-HCV, and 1.1 percent had co infection of both HBV and HCV. A total of the 316 individuals met the inclusion criteria for reference interval establishment and 77 (24.4%) of them were men and 239 (75.6%) were women. The reference interval of M2BPGI glyco-biomarker for men was 0.883 ± 0.311C.O.I and for women was 0.951 ± 0.411C.O.I. Conclusion: It is determined that the reference interval for M2BPGI glyco-biomarker in the serum of relatively healthy Mongolian people was 0.936 ± 0.391C.O.I.

The covalent attachment of a 14-carbon aliphatic tail on a glycine residue of nascent translated peptide chains is catalyzed in human cells by two N-myristoyltransferase (NMT) enzymes using the rare myristoyl-CoA (C(14)-CoA) molecule as fatty acid donor. Although, NMT enzymes can only transfer a myristate group, they lack specificity for C(14)-CoA and can also bind the far more abundant palmitoyl-CoA (C(16)-CoA) molecule. We determined that the acyl-CoA binding protein, acyl-CoA binding domain (ACBD)6, stimulated the NMT reaction of NMT2. This stimulatory effect required interaction between ACBD6 and NMT2, and was enhanced by binding of ACBD6 to its ligand, C(18:2)-CoA. ACBD6 also interacted with the second human NMT enzyme, NMT1. The presence of ACBD6 prevented competition of the NMT reaction by C(16)-CoA. Mutants of ACBD6 that were either deficient in ligand binding to the N-terminal ACBD or unable to interact with NMT2 did not stimulate activity of NMT2, nor could they protect the enzyme from utilizing the competitor C(16)-CoA. These results indicate that ACBD6 can locally sequester C(16)-CoA and prevent its access to the enzyme binding site via interaction with NMT2. Thus, the ligand binding properties of the NMT/ACBD6 complex can explain how the NMT reaction can proceed in the presence of the very abundant competitive substrate, C(16)-CoA.

Abstract Epitranscriptomics, also known as “RNA epigenetics”, is a chemical modification for RNA regulation. Ribonucleic acid (RNA) methylation is considered to be a major discovery following the deoxyribonucleic acid (DNA) and histone methylation. Messenger RNA (mRNA) methylation modification accounts for more than 60% of all RNA modifications and N6-methyladenosine (m6A) is known as one of the most common type of eukaryotic mRNA methylation modifications in current. The m6A modification is a dynamic reversible modification, which can directly or indirectly affect biological processes, such as RNA degradation, translation and splicing, and can play important biological roles in vivo. This article introduces the mRNA m6A methylation modification enzymes and binding proteins, and reviews the research progress and related mechanisms of the role of mRNA m6A methylation in the nervous system from the aspects of neural stem cells, learning and memory, brain development, axon growth and glioblastoma.

Eukaryotic messenger mRNAs contain many RNA methyl chemical modifications, in which N6-methyladenosine (m6A) plays a very important role. The modification process of RNA methylation is a dynamic reversible regulatory process that is mainly catalyzed by “Writer” m6A methyltransferase, removed by “Eraser” m6A demethylase, and recognized by the m6A binding protein, thereby, linking m6A modification with other mRNA pathways. At various stages of the life cycle, m6A modification plays an extremely important role in regulating mRNA splicing, processing, translation, as well as degradation, and is associated with gametogenesis and fertility for both sexes. Normal gametogenesis is a basic guarantee of fertility. Infertility leads to trauma, affects harmony in the family and seriously affects the quality of life. We review the roles and mechanisms of RNA m6A methylation modification in infertility and provide a potential target for infertility treatment, which can be used for drug development.

Substrate‐binding proteins (SBPs) mediate ligand translocation and have been classified into seven clusters (A‐G). Although the substrate specificities of these clusters are known to some extent, their ligand‐binding mechanism(s) remain(s) incompletely understood. In this study, the list of SBPs belonging to different clusters was updated (764 SBPs) compared to the previously reported study (504 SBPs). Furthermore, a new cluster referred to as cluster H was identified. Results reveal that SBPs follow different ligand‐binding mechanisms. Intriguingly, the majority of the SBPs follow the 'one domain movement' rather than the well‐known 'Venus Fly‐trap' mechanism. Moreover, SBPs of a few clusters display subdomain conformational movement rather than the complete movement of the N‐ and C‐terminal domains. [ABSTRACT FROM AUTHOR]

It has been found that the non‐B form DNA structures, like G‐quadruplex (G4) and i‐motif, are involved in many important biological processes. Our previous study showed that the silkworm transcription factor BmLARK binds to the G4 structure in the promoter of the transcription factor BmPOUM2 and regulates its promoter activity. However, the binding mechanism between BmLARK and BmPOUM2 G4 structure remains unclear. In this study, binding domains and key amino acid residues involved in the interaction between BmLARK and BmPOUM2 G4 were studied. The electrophoretic mobility shift assay results indicated that the two RNA‐recognition motifs (RRM) of BmLARK are simultaneously required for the binding with the G4 structure. Either RRM1 or RRM2 alone could not bind with the G4 structure. The zinc‐finger motif was not involved in the binding. A series of mutant proteins with specific amino acid mutations were expressed and used to identify the key amino acid residues involving the interaction. The results indicated that β sheets, especially the β1 and β3 sheets, in the RRM domains of BmLARK played critical roles in the binding with the G4 structure. Several amino acid mutations of RRM1/2 in ribonucleoprotein domain 1 (RNP1) (motif in β3 strand) and RNP2 (motif in β1 strand) caused loss of binding ability, indicating that these amino acids are the key sites for the binding. All the results suggest that RRM domains, particularly their the RNP1 and RNP2 motifs, play important roles not only in RNA recognition, but also in the G4 structure binding. [ABSTRACT FROM AUTHOR]

DestruxinA(DA), a hexa-cyclodepsipeptidic mycotoxin produced by the entomopathogenic fungus Metarhizium anisopliae, exhibits insecticidal activities in a wide range of pests and is known as an innate immunity inhibitor. However, its mechanism of action requires further investigation. In this research, the interactions of DA with the six aminoacyl tRNA synthetases (ARSs) of Bombyx mori, BmAlaRS, BmCysRS, BmMetRS, BmValRS, BmIleRS, and BmGluProRS, were analyzed. The six ARSs were expressed and purified. The BLI (biolayer interferometry) results indicated that DA binds these ARSs with the affinity indices (KD) of 10-4 to 10-5 M. The molecular docking suggested a similar interaction mode of DA with ARSs, whereby DA settled into a pocket through hydrogen bonds with Asn, Arg, His, Lys, and Tyr of ARSs. Furthermore, DA treatments decreased the contents of soluble protein and free amino acids in Bm12 cells, which suggested that DA impedes protein synthesis. Lastly, the ARSs in Bm12 cells were all downregulated by DA stress. This study sheds light on exploring and answering the molecular target of DA against target insects. [ABSTRACT FROM AUTHOR]

Tobacco can induce reactive oxygen species (ROS) production extensively in cells, which is a major risk factor for oral leukoplakia (OLK) development. Peroxiredoxin 1 (Prx1) is a key antioxidant protein, upregulated in a variety of malignant tumors. We previously found that nicotine, the main ingredient of tobacco, promotes oral carcinogenesis via regulating Prx1. The aim of the present study was to screen and identify the Prx1 interacting proteins and investigate the mechanisms of nicotine on the development of OLK. Through liquid chromatography-tandem mass spectrometry combined with bioinformatics analysis, the candidate Prx1 interacting proteins of cofilin-1 (CFL1), tropomyosin alpha-3 chain (TPM3), and serine/threonine-protein phosphatase 2A 65 kDa regulatory subunit A alpha isoform (PPP2R1A) were screened in human dysplastic oral keratinocyte cells treated with nicotine. CFL1, TPM3, and PPP2R1A were highly expressed in human OLK tissues. The expression of CFL1 increased and the expression of PPP2R1A decreased in OLK of smokers compared to that in OLK of non-smokers. Nicotine upregulated CFL1 and downregulated PPP2R1A in 4-nitro-quinoline-1-oxide (4NQO)-induced OLK tissues in mice in part dependent on Prx1. Furthermore, the in-situ interaction of CFL1, TPM3, and PPP2R1A with Prx1 were validated in human OLK tissues. Our results suggested that tobacco might promote the development of OLK via regulating Prx1 and its interacting proteins CFL1 and PPP2R1A.

Summary: Insulin-like growth factor-binding protein (IGFBP)-2 is a circulating biomarker of cardiometabolic health. Here, we report that circulating IGFBP-2 concentrations robustly increase after different bariatric procedures in humans, reaching higher levels after biliopancreatic diversion with duodenal switch (BPD-DS) than after Roux-en-Y gastric bypass (RYGB) and sleeve gastrectomy (SG). This increase is closely associated with insulin sensitization. In mice and rats, BPD-DS and RYGB operations also increase circulating IGFBP-2 levels, which are not affected by SG or caloric restriction. In mice, Igfbp2 deficiency significantly impairs surgery-induced loss in adiposity and early improvement in insulin sensitivity but does not affect long-term enhancement in glucose homeostasis. This study demonstrates that the modulation of circulating IGFBP-2 may play a role in the early improvement of insulin sensitivity and loss of adiposity brought about by bariatric surgery.

The essential role of the Crem gene in normal sperm development is widely accepted and is confirmed by azoospermia in male mice lacking the Crem gene. The exact number of genes affected by Crem absence is not known, however a large difference has been observed recently between the estimated number of differentially expressed genes found in Crem knock-out (KO) mice compared to the number of gene loci bound by CREM. We therefore re-examined global gene expression in male mice lacking the Crem gene using whole genome transcriptome analysis with Affymetrix microarrays and compared the lists of differentially expressed genes from Crem−/− mice to a dataset of genes where binding of CREM was determined by Chip-seq. We determined the global effect of CREM on spermatogenesis as well as distinguished between primary and secondary effects of the CREM absence. We demonstrated that the absence of Crem deregulates over 4700 genes in KO testis. Among them are 101 genes associated with spermatogenesis 41 of which are bound by CREM and are deregulated in Crem KO testis. Absence of several of these genes in mouse models has proven their importance for normal spermatogenesis and male fertility. Our study showed that the absence of Crem plays a more important role on different aspects of spermatogenesis as estimated previously, with its impact ranging from apoptosis induction to deregulation of major circadian clock genes, steroidogenesis and the cell-cell junction dynamics. Several new genes important for normal spermatogenesis and fertility are down-regulated in KO testis and are therefore possible novel targets of CREM.

The carbohydrate response element binding protein (ChREBP), a basic helix-loop-helix/leucine zipper transcription factor, plays a critical role in the control of lipogenesis in the liver. To identify the direct targets of ChREBP on a genome-wide scale and provide more insight into the mechanism by which ChREBP regulates glucose-responsive gene expression, we performed chromatin immunoprecipitation-sequencing and gene expression analysis. We identified 1153 ChREBP binding sites and 783 target genes using the chromatin from HepG2, a human hepatocellular carcinoma cell line. A motif search revealed a refined consensus sequence (CABGTG-nnCnG-nGnSTG) to better represent critical elements of a functional ChREBP binding sequence. Gene ontology analysis shows that ChREBP target genes are particularly associated with lipid, fatty acid and steroid metabolism. In addition, other functional gene clusters related to transport, development and cell motility are significantly enriched. Gene set enrichment analysis reveals that ChREBP target genes are highly correlated with genes regulated by high glucose, providing a functional relevance to the genome-wide binding study. Furthermore, we have demonstrated that ChREBP may function as a transcriptional repressor as well as an activator.

Members of the human acyl-CoA binding domain-containing (ACBD) family regulate processes as diverse as viral replication, stem-cell self-renewal, organelle organization, and protein acylation. These functions are defined by nonconserved motifs present downstream of the ACBD. The human ankyrin-repeat-containing ACBD6 protein supports the reaction catalyzed by the human and Plasmodium N-myristoyltransferase (NMT) enzymes. Likewise, the newly identified Plasmodium ACBD6 homologue regulates the activity of the NMT enzymes. The relatively low abundance of myristoyl-CoA in the cell limits myristoylation. Binding of myristoyl-CoA to NMT is competed by more abundant acyl-CoA species such as palmitoyl-CoA. ACBD6 also protects the Plasmodium NMT enzyme from lauryl-CoA and forces the utilization of the myristoyl-CoA substrate. The phosphorylation of two serine residues of the acyl-CoA binding domain of human ACBD6 improves ligand binding capacity, prevents competition by unbound acyl-CoAs, and further enhances the activity of NMT. Thus, ACBD6 proteins promote N-myristoylation in mammalian cells and in one of their intracellular parasites under unfavorable substrate-limiting conditions.

Caused by a polyglutamine expansion in the huntingtin protein, Huntington's disease leads to stnatal degeneration via the transcriptional dysregulation of a number of genes, including those involved in mitochondnal biogenesis. Here we show that transglutaminase 2, which is upregulated in HD, exacerbates transcriptional dysregulation by acting as a selective corepressor of nuclear genes; transglutaminase 2 interacts directly with histone H3 in the nucleus. In a cellular model of HD, transglutaminase inhibition de-repressed two established regulators of mitochondrial function, PGC-l alpha and cytochrome c and reversed susceptibility of human HD cells to the mitochondrial toxin, 3-nitroproprionic acid; however, protection mediated by transglutaminase inhibition was not associated with improved mitochondnal bioenergetics. A gene microarray analysis indicated that transglutaminase inhibition normalized expression of not only mitochondrial genes but also 40% of genes that are dysregulated in HD striatal neurons, including chaperone and histone genes. Moreover, transglutaminase inhibition attenuated degeneration in a Drosophila model of HD and protected mouse HD stnatal neurons from excitotoxicity. Altogether these findings demonstrate that selective TG inhibition broadly corrects transcriptional dysregulation in HD and defines a novel HDAC-independent epigenetic strategy for treating neurodegeneration.

Introduction: Hematoporphyrin is a photosensitizer used in photodynamic therapy of various malignant diseases. It is carried to the cancer tissue by serum albumins. Spectrofluorimetric spectra of hematoporphyrin--serum albumin complexes were examined in vitro. Material and methods: The chemicals were: hematoporphyrin, human serum albumin and bovine serum albumin. The spectra were recorded on a Kontron SFM-25 Instrument AG at two excitation wavelengths: λex = 280 nm and λex = 295 nm. The spectra of hematoporphyrin 1.5 x 10-5 M as well as spectra of complexes of hematoporphyrin--human serum albumin (1.5 x 10-5 M Hp -- 1.25 x 10-6 M HSA) and hematoporphyrin--bovine serum albumin (1.5 x 10-5 M Hp -- 3.5 x 10-7 M BSA) were recorded repetitively for 8 days and compared to the initial spectrum. Results: Formation of a complex with human serum albumin extends the stability of the hematoporphyrin spectrum. This extension is greater at excitation λex = 295 nm. Different stability of complexes with bovine and human serum albumins most likely does not result from an actual lower stability of bovine serum albumin complexes, but from the fact that dissimilarity in the structure of both albumins enables additional spectroscopic observations within subdomain IB in the bovine serum albumin molecule. Conclusions: Spectrofluorimetric spectra are stable longer when hematoporphyrin forms a complex with human serum albumin. The present data may be important for understanding the mechanism of hematoporphyrin transportation to the target cancer tissue and effectiveness of photodynamic therapy. [ABSTRACT FROM AUTHOR]

Babesia microti, an intraerythrocytic protozoa, can cause an emerging tick-borne disease—Human babesiosis. The parasite can successfully invade host red blood cells owing to the assistance of molecules expressed by babesia. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the housekeeping intracellular glycolytic enzyme, can also be expressed in the external of cells, where contributes to binding to several molecules such as plasminogen and actin. In the present study, we identified B. microti GAPDH (BmGAPDH) and generated the recombinant BmGAPDH (rBmGAPDH) via an E. coli expression system. Furthermore, we confirmed its catalytic dehydration activity in vitro. Moreover, we also demonstrated that rBmGAPDH could bind to human plasminogen and mouse α-actin. In addition, we demonstrated that rBmGAPDH could recognize anti-B. microti mouse serum. In conclusion, BmGAPDH is a multifunctional glycolytic enzyme, which can bind to host plasminogen and α-actin.

The cDNA encoding p43, a DNA binding protein from pea chloroplasts (ct) that binds to cognate DNA polymerase and stimulates the polymerase activity, has been cloned and characterised. The characteristic sequence motifs of hydroxyproline-rich glyco-proteins (HRGP) are present in the cDNA corres-ponding to the N-terminal domain of the mature p43. The protein was found to be highly O-arabinosylated. Chemically deglycosylated p43 (i.e. p29) retains its binding to both DNA and pea ct-DNA polymerase but fails to stimulate the DNA polymerase activity. The mature p43 is synthesised as a pre-p43 protein containing a 59 amino acid long transit peptide which undergoes stromal cleavage as evidenced from the post-translational in vitro import of the precursor protein into the isolated intact pea chloroplasts. Surprisingly, p43 is found only in pea chloroplasts. The unique features present in the cloned cDNA indicate that p43 is a novel member of the HRGP family of proteins. Besides p43, no other DNA-polymerase accessory protein with O-glycosylation has been reported yet.

Ticks are hematophagous arachnids that parasitize mammals and other hosts, feeding on their blood. Ticks secrete numerous salivary factors that enhance host blood flow or suppress the host inflammatory response. The recruitment of leukocytes, a hallmark of inflammation, is regulated by chemokines, which activate chemokine receptors on the leukocytes. Ticks target this process by secreting glycoproteins called Evasins, which bind to chemokines and prevent leukocyte recruitment. This review describes the recent discovery of numerous Evasins produced by ticks, their classification into two structural and functional classes, and the efficacy of Evasins in animal models of inflammatory diseases. The review also proposes a standard nomenclature system for Evasins and discusses the potential of repurposing or engineering Evasins as therapeutic anti-inflammatory agents. Chemokines are mammalian proteins that are secreted at the sites of tissue insult and stimulate trafficking of leukocytes to the affected tissues, a key component of the inflammatory response. Evasins are tick salivary glycoproteins that bind to chemokines, thereby suppressing the host inflammatory response, an apparent mechanism to prolong the residence times of ticks on their hosts. Bioinformatics searches of salivary transcriptomic and genomic sequence databases and yeast surface display screening methods have enabled discovery of Evasins produced by numerous tick species spread across at least three genera. Two families of Evasins (Classes A and B) have been identified. Evasins from the two families have different 3D structures, different conserved sequence features (including patterns of disulfide bonds), and selectivity for different families of chemokines (CC and CXC, respectively). Evasins each bind to several (or many) chemokines but different Evasins have distinct selectivities for target chemokines. Structures, mutational experiments, and sequence comparisons are beginning to reveal the critical elements of Evasins for chemokine recognition. Evasins show efficacy in animal models of inflammatory diseases, suggesting that either natural Evasins or engineered variants have potential as therapeutic anti-inflammatory agents. [ABSTRACT FROM AUTHOR]

Per- and poly-fluorinated substances (PFASs) are known to have the potential of binding to certain proteins. Protein binding is important to the understanding of cellular toxicities, biotransformation pathways, and the fate of selected PFASs. In this work, we provide a systematic review of major approaches to characterizing PFAS-protein binding, including the techniques based on separation, calorimetry, spectroscopy, mass spectrometry, surface plasmon resonance, molecular docking, and surfactant nature of PFASs. These approaches enable qualitative and/or quantitative characterization of binding parameters, such as the binding affinity constant and binding stoichiometry. For each approach, we review its principles and evaluate inherent strengths and limitations. In addition, the main methodologies for the identification of binding sites and target proteins are reviewed. Through the integrated review and identification of knowledge gaps and challenges, our work will guide the selection of methodologies for better characterizations of the mechanisms, kinetics, and adverse effects of PFAS-protein binding. • A systematic review of major approaches to characterizing PFAS-protein binding. • Approaches for the identification of binding sites and target proteins are reviewed. • The strengths and weaknesses of each method are summarized. • Knowledge gaps and challenges are identified. [ABSTRACT FROM AUTHOR]

The insulin-like growth factor (IGF) axis may be involved in the development of type 2 diabetes. We examined the associations of IGF-I and IGF binding proteins (IGFBP)-1 and -3 with diabetes risk and evaluated macronutrient intakes related to the observed associations. In a nested case–control study of the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study of Finnish male smokers aged 50–69 years, the IGF variables were measured from baseline serum samples for a random sample of 310 men with diabetes diagnosed during a 12-year follow-up and for 310 controls matched by age, recruitment day and intervention group. Diet at baseline was assessed using a validated FFQ. The associations of IGF proteins with diabetes risk were estimated using conditional logistic regression and the associations with macronutrient intakes using linear regression. IGF-I and IGFBP-3 were not associated with the incidence of diabetes. Higher IGFBP-1 was associated with lower diabetes risk in an unadjusted crude model (OR 0·25; 95 % CI 0·15, 0·42 in the highest quartile compared with the lowest), but not after adjustment for BMI (corresponding OR 0·76; 95 % CI 0·41, 1·40). Intakes of carbohydrates, plant protein and milk protein associated positively and intake of meat protein and fat negatively with IGFBP-1 (P <0·005). IGFBP-1 was inversely associated with diabetes risk, but the association was substantially dependent on BMI. The associations between macronutrient intakes and IGFBP-1 may reflect influences of nutrients or foods on insulin concentrations. [ABSTRACT FROM AUTHOR]

Insulin-like growth factors (IGFs) play a critical role in fetal growth, and components of the IGF system have been associated with fetal growth restriction in women. In human pregnancy, the proteolytic cleavage of insulin-like growth factor binding proteins (IGFBPs), particularly IGFBP-4, releases free IGF for respective action at the tissue level. The aim of the present study was to determine IGFBP-2, IGFBP-3, and IGFBP-4 concentrations by Western ligand blotting during pregnancy until day 100 in cows and to compare these concentrations with those of non-pregnant cows and cows undergoing embryonic/fetal mortality. Therefore, two study trials (I and II) and an in vitro study were conducted. In study I, 43 cows were not pregnant, 34 cows were pregnant, and 4 cows were undergoing fm. In study II, 500 cows were examined, and 7 cases of pregnancy loss between days 24–27 and 34–37 after artificial insemination (AI, late embryonic mortality; em) and 8 cases of pregnancy loss between days 34–37 and 54–57 after AI (late embryonic mortality and early fetal mortality; em/fm) were defined from the analyses of 30 pregnant and 20 non-pregnant cows randomly selected for insulin-like growth factor 1 and IGFBP analyses. In vitro serum from pregnant (n = 3) and non-pregnant (n = 3) cows spiked after incubation with recombinant human (rh) IGFBP-4 for 24 h, and IGFBP-4 levels were analyzed before and after incubation to detect proteolytic degradation. The IGFBP-2, -3, and -4 concentrations did not decline during early pregnancy in cows, while IGFBP-4 concentrations were comparable between pregnant and non-pregnant cows, irrespective of low proteolytic activity, which was also demonstrated in cows. Interestingly, cows with em or fm showed distinct IGFBP patterns. The IGFBP-2 and -3 concentrations were higher (P

The covalent attachment of a 14-carbon aliphatic tail on a glycine residue of nascent translated peptide chains is catalyzed in human cells by two N-myristoyltransferase (NMT) enzymes using the rare myristoyl-CoA (C14-CoA) molecule as fatty acid donor. Although, NMT enzymes can only transfer a myristate group, they lack specificity for C14-CoA and can also bind the far more abundant palmitoyl-CoA (C16-CoA) molecule. We determined that the acyl-CoA binding protein, acyl-CoA binding domain (ACBD)6, stimulated the NMT reaction of NMT2. This stimulatory effect required interaction between ACBD6 and NMT2, and was enhanced by binding of ACBD6 to its ligand, C18:2-CoA. ACBD6 also interacted with the second human NMT enzyme, NMT1. The presence of ACBD6 prevented competition of the NMT reaction by C16-CoA. Mutants of ACBD6 that were either deficient in ligand binding to the N-terminal ACBD or unable to interact with NMT2 did not stimulate activity of NMT2, nor could they protect the enzyme from utilizing the competitor C16-CoA. These results indicate that ACBD6 can locally sequester C16-CoA and prevent its access to the enzyme binding site via interaction with NMT2. Thus, the ligand binding properties of the NMT/ACBD6 complex can explain how the NMT reaction can proceed in the presence of the very abundant competitive substrate, C16-CoA.

(-)-Epigallocatechin-3-gallate (EGCg), a major constituent of green tea extract, is well-known to exhibit many beneficial actions for human health by interacting with numerous proteins. In this study we identified synaptic vesicle membrane protein VAT-1 homolog (VAT1) as a novel EGCg-binding protein in human neuroglioma cell extracts using a magnetic pull-down assay and LC-tandem mass spectrometry. We prepared recombinant human VAT1 and analyzed its direct binding to EGCg and its alkylated derivatives using surface plasmon resonance. For EGCg and the derivative NUP-15, we measured an association constant of 0.02-0.85 ×10 3 M -1 s -1 and a dissociation constant of nearly 8 × 10 -4 s -1 . The affinity K m(affinity) of their binding to VAT1 was in the 10-20 µM range and comparable with that of other EGCg-binding proteins reported previously. Based on the common structure of the compounds, VAT1 appeared to recognize a catechol or pyrogallol moiety around the B-, C- and G-rings of EGCg. Next, we examined whether VAT1 mediates the effects of EGCg and NUP-15 on expression of neprilysin (NEP). Treatments of mock cells with these compounds upregulated NEP, as observed previously, whereas no effect was observed in the VAT1-overexpressing cells, indicating that VAT1 prevented the effects of EGCg or NUP-15 by binding to and inactivating them in the cells overexpressing VAT1. Further investigation is required to determine the biological significance of the VAT1-EGCg interaction.

The binding determinants of the human acyl-CoA binding domain-containing protein (ACBD) 6 and its function in lipid renewal of membranes were investigated. ACBD6 binds acyl-CoAs of a chain length of 6 to 20 carbons. The stoichiometry of the association could not be fitted to a 1-to-1 model. Saturation of ACBD6 by C16:0-CoA required higher concentration than less abundant acyl-CoAs. In contrast to ACBD1 and ACBD3, ligand binding did not result in the dimerization of ACBD6. The presence of fatty acids affected the binding of C18:1-CoA to ACBD6, dependent on the length, the degree of unsaturation, and the stereoisomeric conformation of their aliphatic chain. ACBD1 and ACBD6 negatively affected the formation of phosphatidylcholine (PC) and phosphatidylethanolamine in the red blood cell membrane. The acylation rate of lysophosphatidylcholine into PC catalyzed by the red cell lysophosphatidylcholine-acyltransferase 1 protein was limited by the transfer of the acyl-CoA substrate from ACBD6 to the acyltransferase enzyme. These findings provide evidence that the binding properties of ACBD6 are adapted to prevent its constant saturation by the very abundant C16:0-CoA and protect membrane systems from the detergent nature of free acyl-CoAs by controlling their release to acyl-CoA-utilizing enzymes.

Celangulin V, an insecticidal compound isolated from the root bark of Chinese bittersweet, can affect the digestive system of insects. However, the mechanism of how Celangulin V induces a series of symptoms is still unknown. In this study, affinity chromatography was conducted through coupling of Celangulin V-6-aminoacetic acid ester to the CNBr-activated Sepharose 4B. SDS-PAGE was used to analyze the collected fraction eluted by Celangulin V. Eight binding proteins (Zinc finger protein, Thioredoxin peroxidase (TPx), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), SUMO E3 ligase RanBP2, Transmembrane protein 1, Actin, APN and V-ATPase) were obtained and identified by LC/Q-TOF-MS from the midgut of Mythimna separata larvae. The potential of these proteins to serve as target proteins involved in the insecticidal activity of Celangulin V is discussed.

Pleiotropic protein promoting DNA repair A (PprA) is a key protein facilitating the extreme radiation resistance of Deinococcus radiodurans. PprA is a unique protein to the genus Deinococcus and exists as an oligomer ranging from a tetramer to an ~100-mer depending on protein concentrations. Here, the X-ray crystal structure of PprA was determined to clarify how PprA confers radiation resistance. The tertiary structure of dimeric PprA was elucidated by using mutants obtained with random and site-directed mutagenesis methods (W183R and A139R); these mutants have disabled DNA binding and polymerization functions. Because the mutant A139R and W183R proteins have dimeric assemblies with 2 different interfaces (Interfaces 1 and 2), the linear and oligomerized PprA model was constructed as a left-handed face-to-face periodic screw structure. In addition, the linear structure in solution was confirmed by small-angle scattering experiments. The site-directed mutational analysis identified essential basic amino acids for DNA binding. These analytical data support the hypothesis that a complex assembly of PprA molecules, which are extended and have a screw structure, surrounds and stretches the DNA strand, acting as a novel guide to colocalize the DNA strands for efficient DNA repairs. [ABSTRACT FROM AUTHOR]