AIFM1 variants associated with auditory neuropathy spectrum disorder cause apoptosis due to impaired apoptosis-inducing factor dimerization / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Auditory neuropathy spectrum disorder (ANSD) represents a variety of sensorineural deafness conditions characterized by abnormal inner hair cells and/or auditory nerve function, but with the preservation of outer hair cell function. ANSD represents up to 15% of individuals with hearing impairments. Through mutation screening, bioinformatic analysis and expression studies, we have previously identified several apoptosis-inducing factor (AIF) mitochondria-associated 1 (AIFM1) variants in ANSD families and in some other sporadic cases. Here, to elucidate the pathogenic mechanisms underlying each AIFM1 variant, we generated AIF-null cells using the clustered regularly interspersed short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system and constructed AIF-wild type (WT) and AIF-mutant (mut) (p.‍T260A, p.‍R422W, and p.‍R451Q) stable transfection cell lines. We then analyzed AIF structure, coenzyme-binding affinity, apoptosis, and other aspects. Results revealed that these variants resulted in impaired dimerization, compromising AIF function. The reduction reaction of AIF variants had proceeded slower than that of AIF-WT. The average levels of AIF dimerization in AIF variant cells were only 34.5%‍‒‍49.7% of that of AIF-WT cells, resulting in caspase-independent apoptosis. The average percentage of apoptotic cells in the variants was 12.3%‍‒‍17.9%, which was significantly higher than that (6.9%‍‒‍7.4%) in controls. However, nicotinamide adenine dinucleotide (NADH) treatment promoted the reduction of apoptosis by rescuing AIF dimerization in AIF variant cells. Our findings show that the impairment of AIF dimerization by AIFM1 variants causes apoptosis contributing to ANSD, and introduce NADH as a potential drug for ANSD treatment. Our results help elucidate the mechanisms of ANSD and may lead to the provision of novel therapies.

Dimerized, Not Monomeric, Translationally Controlled Tumor Protein Induces Basophil Activation and Mast Cell Degranulation in Chronic Urticaria

Translationally controlled tumor protein (TCTP) is also known as histamine releasing factor as it has the ability to activate mast cells. To investigate the role of TCTP in the pathogenesis of chronic spontaneous urticaria (CSU), we evaluated serum level of TCTP and effect of TCTP on basophil and mast cell degranulation. TCTP levels in the sera from 116 CSU patients and 70 normal healthy controls (NCs) were measured by ELISA. CD203c expression on basophils from CSU patients and β-hexosaminidase release from Laboratory of Allergic Disease 2 mast cells were measured upon stimulation monomeric and dimeric TCTP. Non-reducing Western blot analysis was used for detecting dimeric TCTP. No difference was observed in serum TCTP levels between CSU patients and NCs (p=0.676). However, dimeric TCTP intensity on Western blot was stronger in CSU patients than in NCs. TCTP levels were higher in patients with severe CSU (p=0.049) and with IgG positivity to FcɛRIα (p=0.038). A significant positive correlation was observed between TCTP and eosinophil cationic protein levels (Spearman's rho=0.341; p=0.001). Both basophil and mast cell degranulation were significantly increased after stimulation with dimeric TCTP, but not with monomic TCTP. The ability of TCTP to activate basophil and mast cells is dependent on dimerization, suggesting that the inhibition of TCTP dimerization can be a therapeutic option for CSU. Association between TCTP levels and the presence of IgG to high affinity Fc epsilon receptor I alpha subunit in CSU patients indicates that autoimmune mechanisms may be involved in the dimerization of TCTP.

Development of a Molecular Marker Linked to the A4 Locus and the Structure of HD Genes in Pleurotus eryngii

Allelic differences in A and B mating-type loci are a prerequisite for the progression of mating in the genus Pleurotus eryngii; thus, the crossing is hampered by this biological barrier in inbreeding. Molecular markers linked to mating types of P. eryngii KNR2312 were investigated with randomly amplified polymorphic DNA to enhance crossing efficiency. An A4-linked sequence was identified and used to find the adjacent genomic region with the entire motif of the A locus from a contig sequenced by PacBio. The sequence-characterized amplified region marker 7-2299 distinguished A4 mating-type monokaryons from KNR2312 and other strains. A BLAST search of flanked sequences revealed that the A4 locus had a general feature consisting of the putative HD1 and HD2 genes. Both putative HD transcription factors contain a homeodomain sequence and a nuclear localization sequence; however, valid dimerization motifs were found only in the HD1 protein. The ACAAT motif, which was reported to have relevance to sex determination, was found in the intergenic region. The SCAR marker could be applicable in the classification of mating types in the P. eryngii breeding program, and the A4 locus could be the basis for a multi-allele detection marker.

Generation of dimeric single-chain antibodies neutralizing the cytolytic activity of vaginolysin

Background: Gardnerella vaginalis is a bacterial vaginosis (BV)-associated vaginal bacterium that produces the toxin vaginolysin (VLY). VLY is a pore-forming toxin that is suggested to be the main virulence factor of G. vaginalis. The high recurrence rate of BV and the emergence of antibiotic-resistant bacterial species demonstrate the need for the development of recombinant antibodies as novel therapeutic agents for disease treatment. Single-chain variable fragments (scFvs) generated against VLY exhibited reduced efficacy to neutralize VLY activity compared to the respective full-length antibodies. To improve the properties of scFvs, monospecific dimeric scFvs were generated by the genetic fusion of two anti-VLY scFv molecules connected by an alpha-helix-forming peptide linker. Results: N-terminal hexahistidine-tagged dimeric scFvs were constructed and produced in Escherichia coli and purified using metal chelate affinity chromatography. Inhibition of VLY-mediated human erythrocyte lysis by dimeric and monomeric scFvs was detected by in vitro hemolytic assay. The circulating half-life of purified scFvs in the blood plasma of mice was determined by ELISA. Dimeric anti-VLY scFvs showed higher neutralizing potency and extended circulating half-life than parental monomeric scFv. Conclusions: The protein obtained by the genetic fusion of two anti-VLY scFvs into a dimeric molecule exhibited improved properties in comparison with monomeric scFv. This new recombinant antibody might implement new possibilities for the prophylaxis and treatment of the diseases caused by the bacteria G. vaginalis.

Development of Inhibitors Targeting Hypoxia-Inducible Factor 1 and 2 for Cancer Therapy

Hypoxia is frequently observed in solid tumors and also one of the major obstacles for effective cancer therapies. Cancer cells take advantage of their ability to adapt hypoxia to initiate a special transcriptional program that renders them more aggressive biological behaviors. Hypoxia-inducible factors (HIFs) are the key factors that control hypoxia-inducible pathways by regulating the expression of a vast array of genes involved in cancer progression and treatment resistance. HIFs, mainly HIF-1 and -2, have become potential targets for developing novel cancer therapeutics. This article reviews the updated information in tumor HIF pathways, particularly recent advances in the development of HIF inhibitors. These inhibitors interfere with mRNA expression, protein synthesis, protein degradation and dimerization, DNA binding and transcriptional activity of HIF-1 and -2, or both. Despite efforts in the past two decades, no agents directly inhibiting HIFs have been approved for treating cancer patients. By analyzing results of the published reports, we put the perspectives at the end of the article. The therapeutic efficacy of HIF inhibitors may be improved if more efforts are devoted on developing agents that are able to simultaneously target HIF-1 and -2, increasing the penetrating capacity of HIF inhibitors, and selecting suitable patient subpopulations for clinical trials.

Structural insights into the regulatory mechanism of the Pseudomonas aeruginosa YfiBNR system

YfiBNR is a recently identified bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) signaling system in opportunistic pathogens. It is a key regulator of biofilm formation, which is correlated with prolonged persistence of infection and antibiotic drug resistance. In response to cell stress, YfiB in the outer membrane can sequester the periplasmic protein YfiR, releasing its inhibition of YfiN on the inner membrane and thus provoking the diguanylate cyclase activity of YfiN to induce c-di-GMP production. However, the detailed regulatory mechanism remains elusive. Here, we report the crystal structures of YfiB alone and of an active mutant YfiB(L43P) complexed with YfiR with 2:2 stoichiometry. Structural analyses revealed that in contrast to the compact conformation of the dimeric YfiB alone, YfiB(L43P) adopts a stretched conformation allowing activated YfiB to penetrate the peptidoglycan (PG) layer and access YfiR. YfiB(L43P) shows a more compact PG-binding pocket and much higher PG binding affinity than wild-type YfiB, suggesting a tight correlation between PG binding and YfiB activation. In addition, our crystallographic analyses revealed that YfiR binds Vitamin B6 (VB6) or L-Trp at a YfiB-binding site and that both VB6 and L-Trp are able to reduce YfiB(L43P)-induced biofilm formation. Based on the structural and biochemical data, we propose an updated regulatory model of the YfiBNR system.

Effect of glycemic index on obesity control

Objective Evaluate the effect of glycemic index (GI) on biochemical parameters, food intake, energy metabolism, anthropometric measures and body composition in overweight subjects.Materials and methods Simple blind study, in which nineteen subjects were randomly assigned to consume in the laboratory two daily low GI (n = 10) or high GI (n = 9) meals, for forty-five consecutive days. Habitual food intake was assessed at baseline. Food intake, anthropometric measures and body composition were assessed at each 15 days. Energy metabolism and biochemical parameters were evaluated at baseline and the end of the study.Results Low GI meals increased fat oxidation, and reduced waist circumference and HOMA-IR, while high GI meals increased daily dietary fiber and energy intake compared to baseline. There was a higher reduction on waist circumference and body fat, and a higher increase on postprandial fat oxidation in response to the LGI meals than after high GI meals. High GI meals increased fasting respiratory coefficient compared to baseline and low GI meals.Conclusion The results of the present study showed that the consumption of two daily low GI meals for forty-five consecutive days has a positive effect on obesity control, whereas, the consumption of high GI meals result has the opposite effect. Arch Endocrinol Metab. 2015;59(3):245-51.

Predictors of Success in the Treatment of Obstructive Sleep Apnea Syndrome with Mandibular Repositioning Appliance: A Systematic Review

Introduction Obstructive sleep apnea syndrome affects up to 4% of middle-aged men and 2% of adult women. It is associated with obesity. Objective The objective of this article is to review the literature to determine which factors best correlate with treatment success in patients with obstructive sleep apnea syndrome treated with a mandibular repositioning appliance. Data Synthesis A search was performed of the PubMed, Cochrane, Lilacs, Scielo, and Web of Science databases of articles published from January 1988 to January 2012. Two review authors independently collected data and assessed trial quality. Sixty-nine articles were selected from PubMed and 1 from Cochrane library. Of these, 42 were excluded based on the title and abstract, and 27 were retrieved for complete reading. A total of 13 articles and 1 systematic review were considered eligible for further review and inclusion in this study: 6 studies evaluated anthropomorphic and physiologic factors, 3 articles addressed cephalometric and anatomic factors, and 4 studies evaluated variables related to mandibular repositioning appliance design and activation. All the studies evaluated had low to moderate methodologic quality and were not able to support evidence on prediction of treatment success. Conclusion Based on this systematic review on obstructive sleep apnea syndrome treatment, it remains unclear which predictive factors can be used with confidence to select patients suitable for treatment with a mandibular repositioning appliance. .

Comparison of human and Drosophila atlastin GTPases

Formation of the endoplasmic reticulum (ER) network requires homotypic membrane fusion, which involves a class of atlastin (ATL) GTPases. Purified Drosophila ATL is capable of mediating vesicle fusion in vitro, but such activity has not been reported for any other ATLs. Here, we determined the preliminary crystal structure of the cytosolic segment of Drosophila ATL in a GDP-bound state. The structure reveals a GTPase domain dimer with the subsequent three-helix bundles associating with their own GTPase domains and pointing in opposite directions. This conformation is similar to that of human ATL1, to which GDP and high concentrations of inorganic phosphate, but not GDP only, were included. Drosophila ATL restored ER morphology defects in mammalian cells lacking ATLs, and measurements of nucleotide-dependent dimerization and GTPase activity were comparable for Drosophila ATL and human ATL1. However, purified and reconstituted human ATL1 exhibited no in vitro fusion activity. When the cytosolic segment of human ATL1 was connected to the transmembrane (TM) region and C-terminal tail (CT) of Drosophila ATL, the chimera still exhibited no fusion activity, though its GTPase activity was normal. These results suggest that GDP-bound ATLs may adopt multiple conformations and the in vitro fusion activity of ATL cannot be achieved by a simple collection of functional domains.

A new dimeric lignan from Zanthoxylum simulans / 中国中药杂志

To study the chemical constituents from Zanthoxylum simulans and their anti-inflammatory activity. The constituents of Z. simulans were isolated and purified using various column chromatographies. Their chemical structures were elucidated using extensive spectroscopic methods. The compounds were assayed inhibitory activity against NO production in LPS stimulated RAW 264.7 cells. Four compounds were obtained from the ethanol extract of Z. simulans and determined to be isozanthpodocarpin B(1), kobusin (2), (+)-fargesin (3), and epieudesmin (4). Compound 1 exhibited NO production inhibitory effect with IC50 value of 14.49 µmol · L(-1). Compound 1 is a new dimeric lignan and may be serve as potential anti-inflammatory agent in the future.

Selective Activator of the Glucocorticoid Receptor Compound A Dissociates Therapeutic and Atrophogenic Effects of Glucocorticoid Receptor Signaling in Skin

BACKGROUND: Glucocorticoids are effective anti-inflammatory drugs widely used in dermatology and for the treatment of blood cancer patients. Unfortunately, chronic treatment with glucocorticoids results in serious metabolic and atrophogenic adverse effects including skin atrophy. Glucocorticoids act via the glucocorticoid receptor (GR), a transcription factor that causes either gene transactivation (TA) or transrepression (TR). Compound A (CpdA), a novel non-steroidal GR ligand, does not promote GR dimerization and TA, retains anti-inflammatory potential but induces fewer metabolic side effects compared to classical glucocorticoids when used systemically. As topical effects of CpdA have not been well studied, this work goal was to compare the anti-inflammatory and side effects of topical CpdA and glucocorticoids and to assess their effect on GR TA and TR in keratinocytes. METHODS: We used murine immortalized keratinocytes and F1 C57BlxDBA mice. Effect of glucocorticoid fluocinolone acetonide (FA) and CpdA on gene expression in keratinocytes in vitro and in vivo was evaluated by reverse transcription-PCR. The anti-inflammatory effects were assessed in the model of tumor promoter 12-O-tertradecanoyl-acetate (TPA)-induced dermatitis and in croton oil-induced ear edema test. Skin atrophy was assessed by analysis of epidermal thickness, keratinocyte proliferation, subcutaneous adipose hypoplasia, and dermal changes after chronic treatment with FA and CpdA. RESULTS: In mouse keratinocytes in vitro and in vivo, CpdA did not activate GR-dependent genes but mimicked closely the inhibitory effect of glucocorticoid FA on the expression of inflammatory cytokines and matrix metalloproteinases. When applied topically, CpdA inhibited TPA-induced skin inflammation and hyperplasia. Unlike glucocorticoids, CpdA itself did not induce skin atrophy which correlated with lack of induction of atrophogene regulated in development and DNA damage response 1 (REDD1) causatively involved in skin and muscle steroid-induced atrophy. CONCLUSIONS: Overall, our results suggest that CpdA and its derivatives represent novel promising class of anti-inflammatory compounds with reduced topical side effects.

SARS coronavirus papain-like protease inhibits the type I interferon signaling pathway through interaction with the STING-TRAF3-TBK1 complex

SARS coronavirus (SARS-CoV) develops an antagonistic mechanism by which to evade the antiviral activities of interferon (IFN). Previous studies suggested that SARS-CoV papain-like protease (PLpro) inhibits activation of the IRF3 pathway, which would normally elicit a robust IFN response, but the mechanism(s) used by SARS PLpro to inhibit activation of the IRF3 pathway is not fully known. In this study, we uncovered a novel mechanism that may explain how SARS PLpro efficiently inhibits activation of the IRF3 pathway. We found that expression of the membrane-anchored PLpro domain (PLpro-TM) from SARS-CoV inhibits STING/TBK1/IKKε-mediated activation of type I IFNs and disrupts the phosphorylation and dimerization of IRF3, which are activated by STING and TBK1. Meanwhile, we showed that PLpro-TM physically interacts with TRAF3, TBK1, IKKε, STING, and IRF3, the key components that assemble the STING-TRAF3-TBK1 complex for activation of IFN expression. However, the interaction between the components in STING-TRAF3-TBK1 complex is disrupted by PLpro-TM. Furthermore, SARS PLpro-TM reduces the levels of ubiquitinated forms of RIG-I, STING, TRAF3, TBK1, and IRF3 in the STING-TRAF3-TBK1 complex. These results collectively point to a new mechanism used by SARS-CoV through which PLpro negatively regulates IRF3 activation by interaction with STING-TRAF3-TBK1 complex, yielding a SARS-CoV countermeasure against host innate immunity.

Pertuzumab, primer inhibidor de la dimerización HER / Pertuzumab, first HER dimerization inhibitor

Entre un 15 y un 20% de pacientes con cáncer de mama presentan amplificación o sobre expresión de HER2, lo que le confiere un comportamiento más agresivo. Pese a que el advenimiento del trastuzumab, primer agente dirigido contra HER2, mejoró significativamente el pronóstico de este grupo de pacientes, el 50% de ellas aún progresa dentro del año de tratamiento. Pertuzumab, el primer inhibidor de la dimerización de HER2, muestra un mecanismo de acción complementario a trastuzumab, logrando mejores resultados clínicos en pacientes tratadas con ambos agentes anti-HER2 y quimioterapia. El presente trabajo repasa las propiedades de pertuzumab y su desarrollo clínico.

Approximately 15 to 20% of breast cancers present amplificationor overexpression of HER2, and these tumoursshow a more aggressive behavior. Trastuzumab, the firsttargeted agent against HER2, significantly improved prognosisfor these patients, but still around 50% shows diseaseprogression within the first year of treatment. Pertuzumab,the first HER2 dimerization inhibitor, has a mechanismof action that is complementary to that of trastuzumab,achieving enhanced efficacy for patients treated with bothanti-HER2 agents and chemotepapy. This work reviews themain aspects of pertuzumab and its clinical development.

Binding of bacterial secondary messenger molecule c di-GMP is a STING operation

Initial skirmishes between the host and pathogen result in spillage of the contents of the bacterial cell. Amongst the spillage, the secondary messenger molecule, cyclic dimeric guanosine monophosphate (c di-GMP), was recently shown to be bound by stimulator of interferon genes (STING). Binding of c di-GMP by STING activates the Tank Binding Kinase (TBK1) mediated signaling cascades that galvanize the body's defenses for elimination of the pathogen. In addition to c di-GMP, STING has also been shown to function in innate immune responses against pathogen associated molecular patterns (PAMPs) originating from the DNA or RNA of pathogens. The pivotal role of STING in host defense is exemplified by the fact that STING(-/-) mice die upon infection by HSV-1. Thus, STING plays an essential role in innate immune responses against pathogens. This opens up an exciting possibility of targeting STING for development of adjuvant therapies to boost the immune defenses against invading microbes. Similarly, STING could be targeted for mitigating the inflammatory responses augmented by the innate immune system. This review summarizes and updates our current understanding of the role of STING in innate immune responses and discusses the future challenges in delineating the mechanism of STING-mediated responses.

Structural and biochemical studies of RIG-I antiviral signaling

Retinoic acid-inducible gene I (RIG-I) is an important pattern recognition receptor that detects viral RNA and triggers the production of type-I interferons through the downstream adaptor MAVS (also called IPS-1, CARDIF, or VISA). A series of structural studies have elaborated some of the mechanisms of dsRNA recognition and activation of RIG-I. Recent studies have proposed that K63-linked ubiquitination of, or unanchored K63-linked polyubiquitin binding to RIG-I positively regulates MAVS-mediated antiviral signaling. Conversely phosphorylation of RIG-I appears to play an inhibitory role in controlling RIG-I antiviral signal transduction. Here we performed a combined structural and biochemical study to further define the regulatory features of RIG-I signaling. ATP and dsRNA binding triggered dimerization of RIG-I with conformational rearrangements of the tandem CARD domains. Full length RIG-I appeared to form a complex with dsRNA in a 2:2 molar ratio. Compared with the previously reported crystal structures of RIG-I in inactive state, our electron microscopic structure of full length RIG-I in complex with blunt-ended dsRNA, for the first time, revealed an exposed active conformation of the CARD domains. Moreover, we found that purified recombinant RIG-I proteins could bind to the CARD domain of MAVS independently of dsRNA, while S8E and T170E phosphorylation-mimicking mutants of RIG-I were defective in binding E3 ligase TRIM25, unanchored K63-linked polyubiquitin, and MAVS regardless of dsRNA. These findings suggested that phosphorylation of RIG inhibited downstream signaling by impairing RIG-I binding with polyubiquitin and its interaction with MAVS.

Structural insight into enhanced calcium indicator GCaMP3 and GCaMPJ to promote further improvement

Genetically encoded Ca(2+) indicators (GECI) are important for the measurement of Ca(2+) in vivo. GCaMP2, a widely-used GECI, has recently been iteratively improved. Among the improved variants, GCaMP3 exhibits significantly better fluorescent intensity. In this study, we developed a new GECI called GCaMPJ and determined the crystal structures of GCaMP3 and GCaMPJ. GCaMPJ has a 1.5-fold increase in fluorescence and 1.3-fold increase in calcium affinity over GCaMP3. Upon Ca(2+) binding, GCaMP3 exhibits both monomeric and dimeric forms. The structural superposition of these two forms reveals the role of Arg-376 in improving monomer performance. However, GCaMPJ seldom forms dimers under conditions similar to GCaMP3. St ructural and mutagenesis studies on Tyr-380 confirmed its importance in blocking the cpEGFP β-barrel holes. Our study proposes an efficient tool for mapping Ca(2+) signals in intact organs to facilitate the further improvement of GCaMP sensors.

Glutathione regulates the transfer of iron-sulfur cluster from monothiol and dithiol glutaredoxins to apo ferredoxin

Holo glutaredoxin (Grx) is a homo-dimer that bridges a [2Fe-2S] cluster with two glutathione (GSH) ligands. In this study, both monothiol and dithiol holo Grxs are found capable of transferring their iron-sulfur (FeS) cluster to an apo ferredoxin (Fdx) through direct interaction, regardless of FeS cluster stability in holo Grxs. The ligand GSH molecules in holo Grxs are unstable and can be exchanged with free GSH, which inhibits the FeS cluster transfer from holo Grxs to apo Fdx. This phenomenon suggests a novel role of GSH in FeS cluster trafficking.

MsbA ATP-binding cassette (ABC) transporter of E. coli: Structure and possible flippase mechanism.

ATP-binding cassette (ABC) transporters utilize the energy present in cellular ATP to drive the translocation of structurally diverse set of solutes across the membrane barriers of eubacteria, archaebacteria and eukaryotes. In bacteria, these transporters are considered to be important virulence factors because they play role in nutrient uptake and in the secretion of toxins. The advances in structural determination and functional analysis of bacterial transporters have greatly increased our understanding of the mechanism of transport of these ABC transporters. Although progress in the field of structural biology has been made with the prokaryotic family members, it is likely that eukaryotic transporters will utilize the same mechanisms for translocation process. In this review, we summarize the function of the known MsbA ABC transporters in E. coli and mechanistic insights from structural and possible flippase mechanism studies.

Primary investigation on heterodimerization of kappa-opioid receptor and ORL1 receptor / 药学学报

This study investigates whether kappa-opioid receptor and ORL1 receptor may interact to form a heterodimer. In immunofluorescence and co-immunoprecipitation experiments, differentially epitope-tagged receptors, colocalization and heterodimerization of kappa-opioid receptor and ORL1 receptor were used and examined in primary culturing rat neurons, Chinese hamster ovary (CHO) or human embryonic kidney 293 (HEK293) cells. The results show that fluorescence of both kappa-opioid receptor and ORL1 receptor were overlapping in primary culturing hippocampal and cortical neurons. Similarly in co-expressing CHO or HEK293 cells, HA-KOR and Myc-ORL1 were almost exclusively confined to the membranes, revealing extensive colocalization. When Flag-KOR and Myc-ORL1 were co-expressing in CHO cells, heterodimerization was identified to have the ability to co-immunoprecipitate ORL1-receptors with kappa-opioid receptor and vice versa. In the current study, further evidence was provided for the direct interaction of two subtypes of opioid receptors, kappa-opioid receptor and ORL1-receptor, to form the heterodimerization. The finding represents the novel pharmacological mechanism for modulation of opioid receptor function as well as diversity of G protein-coupled receptors.