121 research outputs found

    Molecular Dynamics Simulation of Ligand Dissociation from Liver Fatty Acid Binding Protein

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    The mechanisms of how ligands enter and leave the binding cavity of fatty acid binding proteins (FABPs) have been a puzzling question over decades. Liver fatty acid binding protein (LFABP) is a unique family member which accommodates two molecules of fatty acids in its cavity and exhibits the capability of interacting with a variety of ligands with different chemical structures and properties. Investigating the ligand dissociation processes of LFABP is thus a quite interesting topic, which however is rather difficult for both experimental approaches and ordinary simulation strategies. In the current study, random expulsion molecular dynamics simulation, which accelerates ligand motions for rapid dissociation, was used to explore the potential egress routes of ligands from LFABP. The results showed that the previously hypothesized “portal region” could be readily used for the dissociation of ligands at both the low affinity site and the high affinity site. Besides, one alternative portal was shown to be highly favorable for ligand egress from the high affinity site and be related to the unique structural feature of LFABP. This result lends strong support to the hypothesis from the previous NMR exchange studies, which in turn indicates an important role for this alternative portal. Another less favored potential portal located near the N-terminal end was also identified. Identification of the dissociation pathways will allow further mechanistic understanding of fatty acid uptake and release by computational and/or experimental techniques

    (1)H, (15)N and (13)C backbone resonance assignments of the Kelch domain of mouse Keap1.

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    Kelch-like ECH-associated Protein 1 (Keap1) is a multi-domain protein that functions as an inhibitor of the transcription factor nuclear factor E2-related factor 2 (Nrf2) in the cellular response to oxidative stress. Under normal conditions, Keap1 binds to Nrf2 via its C-terminal Kelch domain and the interaction ultimately leads to the ubiquitin-dependent degradation of Nrf2. It has been proposed that designing molecules to selectively disrupt the Keap1-Nrf2 interaction can be a potential therapeutic approach for enhancing the expression of cytoprotective genes. Here, we reported the (1)H, (13)C, and (15)N backbone chemical shift assignments of the Kelch domain of mouse Keap1. Further, unlabeled Nrf2 peptide containing the Kelch-binding motif was added to the (15)N-labeled Kelch sample. (1)H-(15)N HSQC spectra of the protein in the absence and presence of an equimolar concentration of the Nrf2 peptide were presented. A significant number of resonance signals were shifted upon addition of the peptide, confirming the protein-peptide interaction. The results here will not just facilitate the further studies of the binding between Keap1 and Nrf2, it will also be valuable for probing interactions between the Kelch domain and small molecules, as well as a growing list of protein targets that have been identified recently

    Fuzzy complex formation between the intrinsically disordered prothymosin α and the Kelch domain of Keap1 involved in the oxidative stress response.

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    Kelch-like ECH-associated protein 1 (Keap1) is an inhibitor of nuclear factor erythroid 2-related factor 2 (Nrf2), a key transcription factor for cytoprotective gene activation in the oxidative stress response. Under unstressed conditions, Keap1 interacts with Nrf2 in the cytoplasm via its Kelch domain and suppresses the transcriptional activity of Nrf2. During oxidative stress, Nrf2 is released from Keap1 and is translocated into the nucleus, where it interacts with the small Maf protein to initiate gene transcription. Prothymosin α (ProTα), an intrinsically disordered protein, also interacts with the Kelch domain of Keap1 and mediates the import of Keap1 into the nucleus to inhibit Nrf2 activity. To gain a molecular basis understanding of the oxidative stress response mechanism, we have characterized the interaction between ProTα and the Kelch domain of Keap1 by using nuclear magnetic resonance spectroscopy, isothermal titration calorimetry, peptide array analysis, site-directed mutagenesis, and molecular dynamic simulations. The results of nuclear magnetic resonance chemical shift mapping, amide hydrogen exchange, and spin relaxation measurements revealed that ProTα retains a high level of flexibility, even in the bound state with Kelch. This finding is in agreement with the observations from the molecular dynamic simulations of the ProTα-Kelch complex. Mutational analysis of ProTα, guided by peptide array data and isothermal titration calorimetry, further pinpointed that the region (38)NANEENGE(45) of ProTα is crucial for the interaction with the Kelch domain, while the flanking residues play relatively minor roles in the affinity of binding

    Molecular effects of cancer-associated somatic mutations on the structural and target recognition properties of Keap1.

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    Kelch-like ECH-associated protein 1 (Keap1) plays an important regulatory role in the nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent oxidative stress response pathway. It functions as a repressor of Nrf2, a key transcription factor that initiates the expression of cytoprotective enzymes during oxidative stress to protect cells from damage caused by reactive oxygen species. Recent studies show that mutations of Keap1 can lead to aberrant activation of the antioxidant pathway, which is associated with different types of cancers. To gain a mechanistic understanding of the links between Keap1 mutations and cancer pathogenesis, we have investigated the molecular effects of a series of mutations (G333C, G350S, G364C, G379D, R413L, R415G, A427V, G430C and G476R) on the structural and target recognition properties of Keap1 by using nuclear magnetic resonance (NMR) spectroscopy, circular dichroism (CD) and isothermal titration calorimetry (ITC). Depending on their locations in the protein, these mutations are found to exert differential effects on the protein stability and target binding. Together with the proposed hinge-and-latch mechanism of Nrf2-Keap1 binding in the literature, our results provide important insight into the molecular affect of different somatic mutations on Keap1\u27s function as an Nrf2 repressor

    Transcriptome analysis of stem development in the tumourous stem mustard Brassica juncea var. tumida Tsen et Lee by RNA sequencing

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    <p>Abstract</p> <p>Background</p> <p>Tumourous stem mustard (<it>Brassica juncea </it>var. <it>tumida </it>Tsen et Lee) is an economically and nutritionally important vegetable crop of the <it>Cruciferae </it>family that also provides the raw material for <it>Fuling </it>mustard. The genetics breeding, physiology, biochemistry and classification of mustards have been extensively studied, but little information is available on tumourous stem mustard at the molecular level. To gain greater insight into the molecular mechanisms underlying stem swelling in this vegetable and to provide additional information for molecular research and breeding, we sequenced the transcriptome of tumourous stem mustard at various stem developmental stages and compared it with that of a mutant variety lacking swollen stems.</p> <p>Results</p> <p>Using Illumina short-read technology with a tag-based digital gene expression (DGE) system, we performed <it>de novo </it>transcriptome assembly and gene expression analysis. In our analysis, we assembled genetic information for tumourous stem mustard at various stem developmental stages. In addition, we constructed five DGE libraries, which covered the strains <it>Yong'an </it>and <it>Dayejie </it>at various development stages. Illumina sequencing identified 146,265 unigenes, including 11,245 clusters and 135,020 singletons. The unigenes were subjected to a BLAST search and annotated using the GO and KO databases. We also compared the gene expression profiles of three swollen stem samples with those of two non-swollen stem samples. A total of 1,042 genes with significantly different expression levels occurring simultaneously in the six comparison groups were screened out. Finally, the altered expression levels of a number of randomly selected genes were confirmed by quantitative real-time PCR.</p> <p>Conclusions</p> <p>Our data provide comprehensive gene expression information at the transcriptional level and the first insight into the understanding of the molecular mechanisms and regulatory pathways of stem swelling and development in this plant, and will help define new mechanisms of stem development in non-model plant organisms.</p

    Hadis-hadis Antropomorfisme: Analisis Terhadap Takwil Ibn Hajar Al-‘asqalânî Dalam Fath Al-bârî

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    : Anthropomorphism in Hadith: An Analysis of Ibn Hajar al- ‘Asqalânî\u27s Ta\u27wîl in Fath al-Bârî. In the history of Islamic theology, discussion on Quranic verses and the Prophetic traditions that deal with anthropomorphism has undergone long history starting from heated debate between literal hadith centrists with those of rationalists theologians and the Muktazilah. This essay attempts to elaborate Ibn Hajar\u27s view, as an advocate of tradition, in understanding the hadiths that describe the attributes similar to that of His creatures. In order to avoid potential error and confusion in understanding the attributes of God, Ibn Hajar utilized ta\u27wîl method and departed from his root due to socio-political condition and the prevailing theological teachings that led him to support the tenets of Asy‘ariyah. Conversely, he was very keen on safeguarding the Muslim\u27s creed from equating God\u27s attributes with His creatures

    l-Isoleucine Administration Alleviates Rotavirus Infection and Immune Response in the Weaned Piglet Model

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    Rotavirus (RV) infection is one of the main pathogenic causes of severe gastroenteritis and diarrhea in infants and young animals. This study aimed to determine how dietary l-isoleucine supplementation improves the growth performance and immune response in weaned piglets with RV infection. In cell culture experiment, after IPEC-J2 and 3D4/31 cells were treated by 8 mM l-isoleucine for 24 h, the gene expressions of β-defensins and pattern recognition receptors (PRR) signaling pathway were significantly increased. Then, in the in vivo experiment, 28 crossbred weaned pigs were randomly divided into two groups fed with basal diet with or without l-isoleucine for 18 days. On the 15th day, the oral RV gavage was executed in the half of piglets. Average daily feed intake and gain of piglets were impaired by RV infection (P &lt; 0.05). RV infection also induced severe diarrhea and the increasing serum urea nitrogen concentration (P &lt; 0.05), and decreased CD4+ lymphocyte and CD4+/CD8+ ratio of peripheral blood (P &lt; 0.05). However, dietary l-isoleucine supplementation attenuated diarrhea and decreasing growth performance (P &lt; 0.05), decreased the NSP4 concentration in ileal mucosa, and enhanced the productions and/or expressions of immunoglobulins, RV antibody, cytokines, and β-defensins in serum, ileum, and/or mesenteric lymph nodes of weaned piglets (P &lt; 0.05), which could be relative with activation of PRR signaling pathway and the related signaling pathway (P &lt; 0.05) in the weaned pigs orally infused by RV. These results indicate that dietary l-isoleucine could improve the growth performance and immune function, which could be derived from l-isoleucine treatment improving the innate and adaptive immune responses via activation of PRR signaling pathway in RV-infected piglets. It is possible that l-isoleucine can be used in the therapy of RV infection in infants and young animals

    Solution Structures of the Acyl Carrier Protein Domain from the Highly Reducing Type I Iterative Polyketide Synthase CalE8

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    Biosynthesis of the enediyne natural product calicheamicins γ1I in Micromonospora echinospora ssp. calichensis is initiated by the iterative polyketide synthase (PKS) CalE8. Recent studies showed that CalE8 produces highly conjugated polyenes as potential biosynthetic intermediates and thus belongs to a family of highly-reducing (HR) type I iterative PKSs. We have determined the NMR structure of the ACP domain (meACP) of CalE8, which represents the first structure of a HR type I iterative PKS ACP domain. Featured by a distinct hydrophobic patch and a glutamate-residue rich acidic patch, meACP adopts a twisted three-helix bundle structure rather than the canonical four-helix bundle structure. The so-called ‘recognition helix’ (α2) of meACP is less negatively charged than the typical type II ACPs. Although loop-2 exhibits greater conformational mobility than other regions of the protein with a missing short helix that can be observed in most ACPs, two bulky non-polar residues (Met992, Phe996) from loop-2 packed against the hydrophobic protein core seem to restrict large movement of the loop and impede the opening of the hydrophobic pocket for sequestering the acyl chains. NMR studies of the hydroxybutyryl- and octanoyl-meACP confirm that meACP is unable to sequester the hydrophobic chains in a well-defined central cavity. Instead, meACP seems to interact with the octanoyl tail through a distinct hydrophobic patch without involving large conformational change of loop-2. NMR titration study of the interaction between meACP and the cognate thioesterase partner CalE7 further suggests that their interaction is likely through the binding of CalE7 to the meACP-tethered polyene moiety rather than direct specific protein-protein interaction