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Neuropsychiatric disorders seriously affect the health of patients, and early diagnosis and treatment are crucial to improve the quality of patients’ life. Machine learning and other related methods can be used for disease diagnosis and prediction, among which multi-classifier fusion method has been widely studied due to its significant performance over single classifiers. In this paper, we propose a multi-classifier fusion classification framework based on belief-valuefor the neuropsychiatric disorders diagnosis. Specifically, the belief-value measures the belief level of different samples by considering information from two perspectives, which are distance information (the output distance of the classifier) and local density information (the weight of the nearest neighbor samples on the test samples). The proposed belief-value is more representative compared to the belief-value which only uses a single type of information. Further, based on the concept of multi-view learning, we performed the calculation of the belief-values under the sample space with different features, and the complementary relationship between different belief-values was captured by a multilayer perceptual (MLP) network. Compared with majority voting and linear fusion methods, the MLP network can better capture the nonlinear relationship between belief-values, which produces better diagnostic results. Experimental results show that the proposed method outperforms single classifier and multi-classifier linear fusion methods for the diagnosis of neuropsychiatric disorders.

Background: Host defense peptides (HDPs) have emerged as a novel therapeutic paradigm for wound management; however, their clinical applications remain a challenge owing to their poor pharmacological properties and lack of suitable pharmaceutical formulations. Nanodefensin (ND), a nanoengineered human α-defensin 5 (HD5), has shown improved pharmacological properties relative to the parent compound. In this study, we engineered a nanodefensin-encased hydrogel (NDEFgel), investigated the effects of NDEFgel on wound healing, and elucidated underlying mechanisms. Method: ND was chemically synthesized and tested functions by in vitro antimicrobial and scratch assays and western blotting. Different NDEFgels were evaluated by in vitro characterizations including degradation, drug release and antimicrobial activity. In full-thickness excisional murine models, the optimal NDEFgel was directly applied onto wound sites, and the efficacy was assessed. Moreover, the underlying mechanisms of pro-regenerative effect developed by NDEFgel were also explored. Results: Apart from bactericidal effects, ND modulated fibroblast behaviors by promoting migration and differentiation. Among the tested hydrogels, the Pluronic F127 (Plu) hydrogel represented the most desirable carrier for ND delivery owing to its favorable controlled release and compatibility with ND. Local treatment of NDEFgel on the wound bed resulted in accelerated wound regeneration and attenuated bacterial burden. We further demonstrated that NDEFgel therapy significantly upregulated genes related to collagen deposition and fibroblasts, and increased the expression of myofibroblasts and Rac1. We therefore found that Rac1 is a critical factor in the ND-induced modulation of fibroblast behaviors in vitro through a Rac1-dependent cytoskeletal rearrangement. Conclusion: Our results indicate that NDEFgel may be a promising dual-action therapeutic option for advanced wound management in the future.

The degeneracy of the genetic code confers a wide array of properties to coding sequences. Yet, its origin is still unclear. A structural analysis has shown that the stability of the Watson–Crick base pair at the second position of the anticodon–codon interaction is a critical parameter controlling the extent of non-specific pairings accepted at the third position by the ribosome, a flexibility at the root of degeneracy. Based on recent cryo-EM analyses, the present work shows that residue A1493 of the decoding center provides a significant contribution to the stability of this base pair, revealing that the ribosome is directly involved in the establishment of degeneracy. Building on existing evolutionary models, we show the evidence that the early appearance of A1493 and A1492 established the basis of degeneracy when an elementary kinetic scheme of translation was prevailing. Logical considerations on the expansion of this kinetic scheme indicate that the acquisition of the peptidyl transferase center was the next major evolutionary step, while the induced-fit mechanism, that enables a sharp selection of the tRNAs, necessarily arose later when G530 was acquired by the decoding center.

Polyinosinic-polycytidylic acid (poly(I:C)) is the agonist of Toll-like receptor 3 (TLR3), which participates in innate immune responses under the condition of myocardial ischemia/reperfusion injury (MIRI). It has been shown that poly(I:C) exhibited cardioprotective activities through the PI3K/Akt pathway, which is the main signal transduction pathway during autophagy. However, the precise mechanism by whether poly(I:C) regulates autophagy remains poorly understood. Thus, this study was designed to investigate the therapeutic effect of poly(I:C) against MIRI and the underlying pathway connection with autophagy. We demonstrated that 1.25 and 5 mg/kg poly(I:C) preconditioning significantly reduced myocardial infarct size and cardiac dysfunction. Moreover, poly(I:C) significantly promoted cell survival by restoring autophagy flux and then regulating it to an adequate level Increased autophagy protein Beclin1 and LC3II together with p62 degradation after additional chloroquine. In addition, mRFP-GFP-LC3 adenoviruses exhibited autophagy activity in neonatal rat cardiac myocytes (NRCMs). Mechanistically, poly(I:C) activated the PI3K/AKT/mTOR pathway to induce autophagy, which was abolished by LY294002 (PI3K antagonist), rapamycin (autophagy activator and mTOR inhibitor), or 3-methyladenine (autophagy inhibitor), suggesting either inhibition of the PI3K/Akt/mTOR pathway or autophagy activity interrupt the beneficial effect of poly(I:C) preconditioning. In conclusion, poly(I:C) promotes cardiomyocyte survival from ischemia/reperfusion injury by regulating autophagy via the PI3K/Akt/mTOR pathway.

Optogenetics combined with protein engineering based on natural light‑sensitive dimerizing proteins has evolved as a powerful strategy to study cellular functions. The present study focused on tropomyosin kinase receptors (Trks) that have been engineered to be light‑sensitive. Trk belongs to the superfamily of receptor tyrosine kinases (RTKs), which are single‑pass transmembrane receptors that are activated by natural ligands and serve crucial roles in cellular growth, differentiation, metabolism and motility. However, functional variations exist among receptors fused with light‑sensitive proteins. The present study proposed a signal transduction model for light‑induced receptor activation. This model is based on analysis of previous light‑induced Trk receptors reported to date and comparisons to the activation mechanism of natural receptors. In this model, quantitative differences on the dimerization induced from either top‑to‑bottom or bottom‑to‑up may lead to the varying amplitude of intracellular signals. We hypothesize that the top‑to‑bottom propagation is more favourable for activation and yields better results compared with the bottom‑to‑top direction. The careful delineation of the dimerization mechanisms fine‑tuning activation will guide future design for an optimum cellular output with the precision of light.

Esophageal squamous cell carcinoma (ESCC) is a predominant subtype of esophageal cancer (EC) and has a poor prognosis due to its aggressive nature. Accordingly, it is necessary to find novel prognostic biomarkers and therapeutic targets for ESCC. Lysine-specific histone demethylase 1 (LSD1) plays a core role in the regulation of ESCC oncogenesis. However, the detailed mechanism of LSD1-regulated ESCC growth has not been elucidated. This study aims to explore molecular mechanism underlying the LSD1-regulated ESCC's oncogenesis. After LSD1 silencing, we detected differentially expressed genes (DEGs) in human ESCC cell line, TE-1, by transcriptome sequencing. Subsequently, we investigated expression pattern of the selected molecules in the ESCC tissues and cell lines by qRT-PCR and Western blotting. Furthermore, we explored the roles of selected molecules in ESCC using gene silencing and overexpression assays. Transcriptome sequencing showed that the expression of dual specificity phosphatase 4 (DUSP4) in TE-1 was significantly attenuated after the LSD1 silencing. In addition, the DUSP4 mRNA expression level was significantly higher in the ESCC tissues, especially in those derived from patients with invasion or metastasis. Moreover, the DUSP4 expression was positively associated with the LSD1 expression in the ESCC tissues. DUSP4 overexpression promoted proliferation, invasion, and migration of the ESCC cells, while DUSP4 silencing had an opposite effect. DUSP4 overexpression also enhanced tumorigenicity of the ESCC cells in vivo, while DUSP4 silencing inhibited tumor growth. Importantly, inhibition of cell proliferation, invasion, and migration by the LSD1 inhibitor (ZY0511) was reversed by DUSP4 overexpression. Conclusively, we found that LSD1 promotes ESCC's oncogenesis by upregulating DUSP4, the potential therapeutic and diagnostic target in ESCC.

Background: Mutations in the mitochondrial translation optimization 1 (MTO1) gene can cause hypertrophic cardiomyopathy. Although the functional role of MTO1 deficiency in certain cells is gradually confirmed, the phenotype of MTO1 deficiency in a lymphoblastoid cybrid line is not yet reported. In this study, we characterized changes of mitochondrial function in MTO1 silenced cybrid cells derived from human lymphoblastoids, immature white blood cells that give rise to lymphocytes. Results: We showed that MTO1 silence decreased the levels of 2-thiourylation of mitochondrial tRNALys, tRNAGlu, and tRNAGln, nevertheless, the aminoacylation efficiency of tRNALys and the steady state of mitochondrial tRNAs were elevated. These aberrant tRNA changes caused a significant decrease in protein levels of oxidative phosphorylation complex subunits including complex I, III, IV and V. Furthermore, dysfunctional mitochondria promoted apoptosis in stress, evidenced by elevated ratios of apoptotic cells and increased levels of apoptosis-activated proteins in the MTO1 knockdown cell lines, as compared to the controls. Conclusions: Our data provide new insights into the important functional role of MTO1 in lymphoblastoid mitochondria. We envision the cybrid cell line approach we have established provides an alternative model for the cardiac tissue of high-energy demands characteristics, and they hold promises for the diagnosis and drug screening for the therapeutic agents of hypertrophic cardiomyopathy caused by MTO1 dysfunction.

Mutations in mitochondrial DNA (mtDNA) have been associated with deafness and their pathophysiology remains poorly understood. In this study, we investigated the pathogenic mechanism of deafness-associated 7505A > G variant in the mitochondrial tRNASer(UCN). The m.7505A > G variant affected the highly conserved adenine at position 11 (A11), disrupted the highly conserved A11-U24 base-pairing of DHU stem of tRNASer(UCN) and introduced a tertiary base pairing (G11-C56) with the C56 in the TΨC loop. We therefore hypothesized that the m.7505A > G variant altered both structure and function of tRNASer(UCN). We demonstrated that the m.7505A > G variant perturbed the conformation and stability of tRNASer(UCN), as indicated by an increased melting temperature and electrophoretic mobility of the mutated tRNA compared with the wild type molecule. Using the cybrids constructed by transferring mitochondria from the Chinese family into mitochondrial DNA (mtDNA)-less cells, we demonstrated the m.7505A > G variant led to significantly decreased steady-state levels of tRNASer(UCN) in the mutant cybrids, as compared with those of control cybrids. The aberrant tRNASer(UCN) metabolism resulted in the variable decreases in mtDNA-encoded polypeptides in the mutant cybrids. Furthermore, we demonstrated that the m.7505A > G variant decreased the activities of mitochondrial respiratory complexes I, III and IV, markedly diminished mitochondrial ATP levels and membrane potential, and increased the production of reactive oxygen species in the mutant cybrids. These results demonstrated that the m.7505A > G variant affected both structure and function of tRNASer(UCN) and consequently altered mitochondrial function. Our findings highlighted critical insights into the pathophysiology of maternally inherited deafness, which is manifested by the aberrant tRNA metabolism.

Membrane protein interactions are crucial for diverse biological processes. We report the application of genetic code expansion in combination with photo-crosslinking chemistry, as we termed "optoproteomics", to identify proteins interacting with the human L-type membrane amino acid transporter 3 (LAT3, also known as SLC43A1). The site-specifically incorporated photo-cross-linker p-azido-L-phenylalanine (AzF), which reacts with proteins in their proximity, enabled the capture of weak and transient partners of LAT3 in living cells. We identify 11 unique interacting proteins which are light-sensitive and 19 unique proteins that are site-specific, validating the approach and providing insights into the LAT3 protein-protein interaction network currently unavailable.

Mutations in the mitochondrial translation optimization 1 (MTO1) gene can cause hypertrophic cardiomyopathy. Although the functional role of MTO1 deficiency in certain cells is gradually confirmed, the phenotype of MTO1 deficiency in a lymphoblastoid cybrid line is not yet reported. In this study, we characterized changes of mitochondrial function in MTO1 silenced cybrid cells derived from human lymphoblastoids, immature white blood cells that give rise to lymphocytes. We showed that MTO1 silence decreased the levels of 2-thiourylation of mitochondrial tRNALys, tRNAGlu, and tRNAGln, nevertheless, the aminoacylation efficiency of tRNALys and the steady state of mitochondrial tRNAs were elevated. These aberrant tRNA changes caused a significant decrease in protein levels of oxidative phosphorylation complex subunits including complex I, III, IV and V. Furthermore, dysfunctional mitochondria promoted apoptosis in stress, evidenced by elevated ratios of apoptotic cells and increased levels of apoptosis-activated proteins in the MTO1 knockdown cell lines, as compared to the controls. Our data provide new insights into the important functional role of MTO1 in lymphoblastoid mitochondria. We envision the cybrid cell line approach we have established provides an alternative model for the cardiac tissue of high-energy demands characteristics, and they hold promises for the diagnosis and drug screening for the therapeutic agents of hypertrophic cardiomyopathy caused by MTO1 dysfunction.

Light-sensitive protein with the light-induced allostery has great applications in bio-medical researches, which can be obtained by introducing the mutation at certain site in the protein. In the present work, we investigate the experimental data on NMDA receptors with light-sensitive mutations, and propose empirical formulas to connect the structural factors to the observable electric current change, which is the signature of the light-induced allosteric effect. We found our formulas present reasonable agreement with experimental data using the full-length structure of NMDA receptor 4tll. It can be used to predict the light-sensitive phenomena of this protein with other types of mutation. Present method can be directly extended to other proteins beyond the current 4tll.

Human-induced pluripotent stem cells (hiPSCs) can be self-renewed for many generations on nanofibrous substrates. Herein, a casting method is developed to replicate the nanofibrous morphology into a thin layer of polymethylsiloxane (PDMS). The template is obtained by electrospinning and chemical crosslinking of gelatin nanofibers on a glass slide. The replicas of the template are surface-functionalized by gelatin and used for propagation of hiPSCs over tenth generations. The performance of the propagated hiPSCs is checked by immunofluorescence imaging, flowcytometry, and RT-PCR, confirming the practicability of this method. The results are also compared to those obtained using electrospun nanofiber substrates. Inherently, the PDMS replica is of low stiffness and can be reproduced easily. Compared to other patterning techniques, casting is more flexible and cost-effective, suggesting that this method might find applications in cell-based assays that rely on stringent consideration of both substrate stiffness and surface morphology.

The use of light to control the expression of genes and the activity of proteins is a rapidly expanding field. Whereas many of these approaches use fusion between a light-activable protein and the protein of interest to control the activity of the latter, it is also possible to control the activity of a protein by uncaging a specific ligand. In that context, controlling the activation of a protein fused to the modified estrogen receptor (ERT) by uncaging its ligand cyclofen-OH has emerged as a generic and versatile method to control the activation of proteins quantitatively, quickly, and locally in a live organism. We present that approach and its uses in a variety of physiological contexts.

The possible mechanism(s) by which ribosomes make peptide bonds during protein synthesis have been explored for decades. Yet, there is no agreement on how the catalytic site, the peptidyl transferase center (PTC), promotes this reaction. Here, we discuss the results of recent investigations of translation with D amino acids that provide fresh insights into that longstanding question.

Although posterior mediastinal (PM) route and retrosternal (RS) route have been used for reconstruction after minimally invasive esophagectomy (MIE), the optimal route remains controversial. This study reviewed our experiences with McKeown MIEs for esophageal cancer and aimed to investigate which route was better for esophageal reconstruction.From December 2011 to December 2013, 103 patients who underwent McKeown MIE and esophageal reconstruction by PM or RS routes were reviewed. The decision regarding which approach was appropriated mainly depended on the first surgeon's preference and experience. Baseline demographics, operative, and postoperative data of the patients were analyzed.Fifty-six and forty-seven patients receiving PM and RS route reconstruction were reviewed, respectively. Shorter operation time (P = 0.001), less blood loss (P = 0.029), and longer route length (P0.001) were observed in PM route compared with RS route. No difference was observed in the resection type, harvested lymph node, intensive care unit and hospital stay, postoperative complications, and in-hospital mortality between the two routes (all P0.05).Both RS route and PM route were safe and effective. PM route was associated with shorter operation time, less blood loss, but longer route length compared with RS route.

D-Amino acids are largely excluded from protein synthesis, yet they are of great interest in biotechnology. Unnatural amino acids have been introduced into proteins using engineered aminoacyl-tRNA synthetases (aaRSs), and this strategy might be applicable to D-amino acids. Several aaRSs can aminoacylate their tRNA with a D-amino acid; of these, tyrosyl-tRNA synthetase (TyrRS) has the weakest stereospecificity. We use computational protein design to suggest active site mutations in Escherichia coli TyrRS that could increase its D-Tyr binding further, relative to L-Tyr. The mutations selected all modify one or more sidechain charges in the Tyr binding pocket. We test their effect by probing the aminoacyl-adenylation reaction through pyrophosphate exchange experiments. We also perform extensive alchemical free energy simulations to obtain L-Tyr/D-Tyr binding free energy differences. Agreement with experiment is good, validating the structural models and detailed thermodynamic predictions the simulations provide. The TyrRS stereospecificity proves hard to engineer through charge-altering mutations in the first and second coordination shells of the Tyr ammonium group. Of six mutants tested, two are active towards D-Tyr; one of these has an inverted stereospecificity, with a large preference for D-Tyr. However, its activity is low. Evidently, the TyrRS stereospecificity is robust towards charge rearrangements near the ligand. Future design may have to consider more distant and/or electrically neutral target mutations, and possibly design for binding of the transition state, whose structure however can only be modeled.

Mutations in mitochondrial DNA (mtDNA) have been associated with deafness and their pathophysiology remains poorly understood. In this study, we investigated the pathogenic mechanism of deafness-associated 7505A G variant in the mitochondrial tRNA

Genetic code expansion exploiting unnatural amino acids (Uaas) is a powerful technique to create novel protein function in vivo. Here, we provide a protocol for the incorporation of two UV-sensitive crosslinking Uaas into NMDA receptors (NMDARs), a type of glutamate-gated ion channels mediating fast synaptic transmission. Through heterologous expression in Xenopus laevis oocytes, we have identified light-sensitive NMDARs of GluN2B subtype by using the two-electrode voltage electrophysiology measurement in combination with online-UV application. Immunoblotting analysis has been used to confirm inter-subunit crosslinking.

In the past decade, genetic code expansion technology has emerged as discovery tools in studies of GPCRs for monitoring of dynamic protein conformational changes, for the screening of ligand–protein and protein–protein interactions, and as alternatives to conventional labeling approaches for the site-specific labeling of GPCRs with spectroscopy probes. Interactome mapping among GPCRs, their ligands, and interactive proteins discovered using genetically encoded photo-cross-linking unnatural amino acids (Uaas) display methods that link substrate specificity to binding pockets revealed by static X-ray crystal structures are inaccessible by other methodologies. Fluorescent-based analysis to directly monitor the GPCR conformational changes are beginning to move forward into cell-based assays for high-throughput drug screening platforms. This review details the significant progress in Uaa containing GPCRs discovery platforms, as well as advances in understanding the structure activity relationship of GPCRs in the “post structural biology” era.

Genetic code expansion exploiting unnatural amino acids (Uaas) is a powerful technique to create novel protein function in vivo. Here, we provide a protocol for the incorporation of two UV-sensitive crosslinking Uaas into NMDA receptors (NMDARs), a type of glutamate-gated ion channels mediating fast synaptic transmission. Through heterologous expression in Xenopus laevis oocytes, we have identified light-sensitive NMDARs of GluN2B subtype by using the two-electrode voltage electrophysiology measurement in combination with online-UV application. Immunoblotting analysis has been used to confirm inter-subunit crosslinking.

// Shuiliang Wang 1 , Ling Zhu 1 , Weimin Zuo 1 , Zhiyong Zeng 2 , Lianghu Huang 1 , Fengjin Lin 1 , Rong Lin 1 , Jin Wang 1 , Jun Lu 1 , Qinghua Wang 1 , Lingjing Lin 1 , Huiyue Dong 1 , Weizhen Wu 1 , Kai Zheng 1 , Jinquan Cai 1 , Shunliang Yang 1 , Yujie Ma 1 , Shixin Ye 2 , Wei Liu 3 , Yinghao Yu 3 , Jianming Tan 1 , Bolin Liu 4 1 Fujian Key Laboratory of Transplant Biology, Fuzhou General Hospital, Xiamen University, Fuzhou, Fujian, China 2 Department of Thoracic Surgery, Fuzhou General Hospital, Xiamen University, Fuzhou, Fujian, China 3 Department of Pathology, Fuzhou General Hospital, Xiamen University, Fuzhou, Fujian, China 4 Department of Pathology, School of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO, USA Correspondence to: Shuiliang Wang, email: shuiliang.wang@xmu.edu.cn Jianming Tan, email: tanjm156@xmu.edu.cn Bolin Liu, email: bolin.liu@ucdenver.edu Keywords: entinostat, Survivin, miRNA, DNMT1, paclitaxel Received: January 26, 2016 Accepted: April 26, 2016 Published: May 10, 2016 ABSTRACT Elevated expression of Survivin correlates with poor prognosis, tumor recurrence, and drug resistance in various human cancers, including non-small cell lung cancer (NSCLC). The underlying mechanism of Survivin upregulation in cancer cells remains elusive. To date, no Survivin-targeted therapy has been approved for cancer treatment. Here, we explored the molecular basis resulting in Survivin overexpression in NSCLC and investigated the antitumor activity of the class I HDAC inhibitor entinostat in combination with paclitaxel. Our data showed that entinostat significantly enhanced paclitaxel-mediated anti-proliferative/anti-survival effects on NSCLC cells in vitro and in vivo . Mechanistically, entinostat selectively decreased expression of Survivin via induction of miR-203 ( in vitro and in vivo ) and miR-542-3p ( in vitro ). Moreover, analysis of NSCLC patient samples revealed that the expression levels of miR-203 were downregulated due to promoter hypermethylation in 45% of NSCLC tumors. In contrast, increased expression of both DNA methytransferase I (DNMT1) and Survivin was observed and significantly correlated with the reduced miR-203 in NSCLC. Collectively, these data shed new lights on the molecular mechanism of Survivin upregulation in NSCLC. Our findings also support that the combinatorial treatments of entinostat and paclitaxel will likely exhibit survival benefit in the NSCLC patients with overexpression of DNMT1 and/or Survivin. The DNMT1-miR-203-Survivin signaling axis may provide a new avenue for the development of novel epigenetic approaches to enhance the chemotherapeutic efficacy against NSCLC.

Allostery is essential to neuronal receptor function, but its transient nature poses a challenge for characterization. The N-terminal domains (NTDs) distinct from ligand binding domains are a major locus for allosteric regulation of NMDA receptors (NMDARs), where different modulatory binding sites have been observed. The inhibitor ifenprodil, and related phenylethanoamine compounds specifically targeting GluN1/GluN2B NMDARs have neuroprotective activity. However, whether they use differential structural pathways than the endogenous inhibitor Zn2+ for regulation is unknown. We applied genetically encoded unnatural amino acids (Uaas) and monitored the functional changes in living cells with photo-cross-linkers specifically incorporated at the ifenprodil binding interface between GluN1 and GluN2B subunits. We report constraining the NTD domain movement, by a light induced crosslinking bond that introduces minimal perturbation to the ligand binding, specifically impedes the transduction of ifenprodil but not Zn2+ inhibition. Subtle distance changes reveal interfacial flexibility and NTD rearrangements in the presence of modulators. Our results present a much richer dynamic picture of allostery than conventional approaches targeting the same interface, and highlight key residues that determine functional and subtype specificity of NMDARs. The light-sensitive mutant neuronal receptors provide complementary tools to the photo-switchable ligands for opto-neuropharmacology.

Rhodopsin is a prototypical heptahelical family A G-protein-coupled receptor (GPCR) responsible for dim-light vision. Light isomerizes rhodopsin's retinal chromophore and triggers concerted movements of transmembrane helices, including an outward tilting of helix 6 (H6) and a smaller movement of H5, to create a site for G-protein binding and activation. However, the precise temporal sequence and mechanism underlying these helix rearrangements is unclear. We used site-directed non-natural amino acid mutagenesis to engineer rhodopsin with p-azido-l-phenylalanine residues incorporated at selected sites, and monitored the azido vibrational signatures using infrared spectroscopy as rhodopsin proceeded along its activation pathway. Here we report significant changes in electrostatic environments of the azido probes even in the inactive photoproduct Meta I, well before the active receptor state was formed. These early changes suggest a significant rotation of H6 and movement of the cytoplasmic part of H5 away from H3. Subsequently, a large outward tilt of H6 leads to opening of the cytoplasmic surface to form the active receptor photoproduct Meta II. Thus, our results reveal early conformational changes that precede larger rigid-body helix movements, and provide a basis to interpret recent GPCR crystal structures and to understand conformational sub-states observed during the activation of other GPCRs.

D-Amino acids are largely excluded from protein synthesis, yet they are of great interest in biotechnology. Unnatural amino acids have been introduced into proteins using engineered aminoacyl-tRNA synthetases (aaRSs), and this strategy might be applicable to D-amino acids. Several aaRSs can aminoacylate their tRNA with a D-amino acid; of these, tyrosyl-tRNA synthetase (TyrRS) has the weakest stereospecificity. We use computational protein design to suggest active site mutations in Escherichia coli TyrRS that could increase its D-Tyr binding further, relative to L-Tyr. The mutations selected all modify one or more sidechain charges in the Tyr binding pocket. We test their effect by probing the aminoacyl-adenylation reaction through pyrophosphate exchange experiments. We also perform extensive alchemical free energy simulations to obtain L-Tyr/D-Tyr binding free energy differences. Agreement with experiment is good, validating the structural models and detailed thermodynamic predictions the simulations provide. The TyrRS stereospecificity proves hard to engineer through charge-altering mutations in the first and second coordination shells of the Tyr ammonium group. Of six mutants tested, two are active towards D-Tyr; one of these has an inverted stereospecificity, with a large preference for D-Tyr. However, its activity is low. Evidently, the TyrRS stereospecificity is robust towards charge rearrangements near the ligand. Future design may have to consider more distant and/or electrically neutral target mutations, and possibly design for binding of the transition state, whose structure however can only be modeled.

Background Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer mortality. MicroRNAs (miRNAs), small noncoding RNAs, regulate the expression of genes that play roles in human cancer via posttranscriptional inhibition. Methods To identify the potential miRNA biomarkers in NSCLC, we downloaded the miRNA expression profile (ID: GSE29248) of NSCLC from the Gene Expression Omnibus (GEO) database and analyzed the differentially expressed miRNAs in NSCLC tissue compared with normal control tissue. Then the targets of these differentially expressed miRNAs were screened and used in network construction and functional enrichment analysis. Results We identified a total of 17 miRNAs that showed a significantly differential expression in NSCLC tissue. We found that miR-34b and miR-520h might play important roles in the regulation of NSCLC, miR-22 might be a novel biomarker as an oncogene, and miR-448 might promote, while miR-654-3p prevents, NSCLC progression. Conclusions Our study may provide the groundwork for further clinical molecular target therapy experiments in NSCLC.

We have designed polypeptides combining selected lipophilic (LP) and hydrophilic (HP) sequences that assemble into amphiphilic (AP) alpha-helical bundles to reproduce key structure characteristics and functional elements of natural membrane proteins. The principal AP maquette (AP1) developed here joins 14 residues of a heme binding sequence from a structured diheme-four-alpha-helical bundle (HP1), with 24 residues of a membrane-spanning LP domain from the natural four-alpha-helical M2 channel of the influenza virus, through a flexible linking sequence (GGNG) to make a 42 amino acid peptide. The individual AP1 helices (without connecting loops) assemble in detergent into four-alpha-helical bundles as observed by analytical ultracentrifugation. The helices are oriented parallel as indicated by interactions typical of adjacent hemes. AP1 orients vectorially at nonpolar-polar interfaces and readily incorporates into phospholipid vesicles with97% efficiency, although most probably without vectorial bias. Mono- and diheme-AP1 in membranes enhance functional elements well established in related HP analogues. These include strong redox charge coupling of heme with interior glutamates and internal electric field effects eliciting a remarkable 160 mV splitting of the redox potentials of adjacent hemes that leads to differential heme binding affinities. The AP maquette variants, AP2 and AP3, removed heme-ligating histidines from the HP domain and included heme-ligating histidines in LP domains by selecting the b(H) heme binding sequence from the membrane-spanning d-helix of respiratory cytochrome bc(1). These represent the first examples of AP maquettes with heme and bacteriochlorophyll binding sites located within the LP domains.

We sought to construct a monolayer composed of four-α-helical bundles formed by association of di-α-helical peptides. Specular X-ray reflectivity showed that dihelices that have been made amphiphilic by attachment of C16 hydrocarbon chains to their N-termini can be vectorially oriented in Langmuir monolayers at an air−water interface with their helical axes normal to the interface. But off-specular X-ray reflectivity indicated that these dihelices did not associate to form four-helix bundles possibly because they were constrained to be of parallel topology. To achieve four-helix bundles vectorially oriented at the interface, we relaxed this constraint to allow for a 1:1 association of the amphiphilic dihelices with their water-soluble counterparts. Electron density profiles for the monolayers derived from specular X-ray reflectivity demonstrated four-helix bundle formation only when the association between dihelices is directed via designed attractive electrostatic interactions between the polar faces of ...

Reprogramming receptors to artificially respond to light has strong potential for molecular studies and interrogation of biological functions. Here, we design a light-controlled ionotropic glutamate receptor by genetically encoding a photoreactive unnatural amino acid (UAA). The photo–cross-linker p-azido-l-phenylalanine (AzF) was encoded in NMDA receptors (NMDARs), a class of glutamate-gated ion channels that play key roles in neuronal development and plasticity. AzF incorporation in the obligatory GluN1 subunit at the GluN1/GluN2B N-terminal domain (NTD) upper lobe dimer interface leads to an irreversible allosteric inhibition of channel activity upon UV illumination. In contrast, when pairing the UAA-containing GluN1 subunit with the GluN2A subunit, light-dependent inactivation is completely absent. By combining electrophysiological and biochemical analyses, we identify subunit-specific structural determinants at the GluN1/GluN2 NTD dimer interfaces that critically dictate UV-controlled inactivation. Our work reveals that the two major NMDAR subtypes differ in their ectodomain-subunit interactions, in particular their electrostatic contacts, resulting in GluN1 NTD coupling more tightly to the GluN2B NTD than to the GluN2A NTD. It also paves the way for engineering light-sensitive ligand-gated ion channels with subtype specificity through the genetic code expansion.

A RT-PCR method is developed to isolate RNA aminoacylated on their 3’ end from large pools of RNA. The method is being applied in two separate projects. We are interested in isolating a new class of ribozymes that could successively catalyze the two chemical reactions leading to their own 3’ aminoacylation (ATP activation of an amino acid followed by 3' esterification of the RNA). The catalysis of each of the two reactions has independently been demonstrated for some RNA isolated with the SELEX methodology [1-2]. However, the coupling of both reactions on a same molecule has not been achieved yet. The identification of these still hypothetical ribozymes may help understand how the former translation system started in the absence of the aminoacyltRNA Synthetase, which catalyzes the above two reactions on tRNA in modern cells. In another project, we would like to identify the whole repertoire of aminoacylated RNA (the “aminoacylome”) in cells. There are strong indications that other RNA besides tRNA and tmRNA may be aminoacylated for biological purposes [3-4].

We demonstrate the site-directed incorporation of an IR-active amino acid, p-azido-L-phenylalanine (azidoF, 1), into the G protein-coupled receptor rhodopsin using amber codon suppression technology. The antisymmetric stretch vibration of the azido group absorbs at approximately 2,100 cm(-1) in a clear spectral window and is sensitive to its electrostatic environment. We used FTIR difference spectroscopy to monitor the azido probe and show that the electrostatic environments of specific interhelical networks change during receptor activation.

To advance dynamic, temporal, and kinetic studies of the G protein-coupled receptor (GPCR) signalosome, new approaches are required to introduce non- or minimally perturbing labels or probes into expressed receptors. We report here a series of methods that are based on unnatural amino acid mutagenesis of GPCRs using amber codon suppression technology. We show that labeling reactions at genetically introduced keto moieties (p-acetyl-L-Phe/AcF and p-benzoyl-L-Phe/BzF) are not completely bioorthogonal due to protein oxidation, which causes high background. However, labeling reactions that target p-azido-L-Phe (azF) using the Staudinger-Bertozzi ligation and the strain-promoted alkyne-azide cycloaddition are bioorthogonal and are satisfactory for introducing labels or probes at near quantitative efficiency under mild labeling conditions. To our knowledge, this is the first report of a site-specific modification of an azF residue with a dibenzocyclooctyne-derivatized fluorophore. The methodologies we discuss are general, in that they can be applied in principle to any amino acid position in any expressed GPCR.

To advance dynamic, temporal, and kinetic studies of the G protein-coupled receptor (GPCR) signalosome, new approaches are required to introduce non- or minimally perturbing labels or probes into expressed receptors. We report here a series of methods that are based on unnatural amino acid mutagenesis of GPCRs using amber codon suppression technology. We show that labeling reactions at genetically introduced keto moieties (p-acetyl-L-Phe/AcF and p-benzoyl-L-Phe/BzF) are not completely bioorthogonal due to protein oxidation, which causes high background. However, labeling reactions that target p-azido-L-Phe (azF) using the Staudinger-Bertozzi ligation and the strain-promoted alkyne-azide cycloaddition are bioorthogonal and are satisfactory for introducing labels or probes at near quantitative efficiency under mild labeling conditions. To our knowledge, this is the first report of a site-specific modification of an azF residue with a dibenzocyclooctyne-derivatized fluorophore. The methodologies we discuss are general, in that they can be applied in principle to any amino acid position in any expressed GPCR.

Heterologous expression of ligand-gated ion channels (LGICs) in Xenopus laevis oocytes combined with site-directed mutagenesis has been demonstrated to be a powerful approach to study structure-function relationships. In particular, introducing unnatural amino acids (UAAs) has enabled modifications that are not found in natural proteins. However, the current strategy relies on the technically demanding in vitro synthesis of aminoacylated suppressor tRNA. We report here a general method that circumvents this limitation by utilizing orthogonal aminoacyl-tRNA synthetase (aaRS)/suppressor tRNA(CUA) pairs to genetically encode UAAs in Xenopus oocytes. We show that UAAs inserted in the N-terminal domain of N-methyl-D-aspartate receptors (NMDARs) serve as photo-crosslinkers that lock the receptor in a discrete conformational state in response to UV photo treatment. Our method should be generally applicable to studies of other LGICs in Xenopus oocytes.

G protein-coupled receptors (GPCRs) are ubiquitous heptahelical transmembrane proteins involved in a wide variety of signaling pathways. The work described here on application of unnatural amino acid mutagenesis to two GPCRs, the chemokine receptor CCR5 (a major co-receptor for the human immunodeficiency virus) and rhodopsin (the visual photoreceptor), adds a new dimension to studies of GPCRs. We incorporated the unnatural amino acids p-acetyl-L-phenylalanine (Acp) and p-benzoyl-L-phenylalanine (Bzp) into CCR5 at high efficiency in mammalian cells to produce functional receptors harboring reactive keto groups at three specific positions. We obtained functional mutant CCR5, at levels up to approximately 50% of wild type as judged by immunoblotting, cell surface expression, and ligand-dependent calcium flux. Rhodopsin containing Acp at three different sites was also purified in high yield (0.5-2 microg/10(7) cells) and reacted with fluorescein hydrazide in vitro to produce fluorescently labeled rhodopsin. The incorporation of reactive keto groups such as Acp or Bzp into GPCRs allows their reaction with different reagents to introduce a variety of spectroscopic and other probes. Bzp also provides the possibility of photo-cross-linking to identify precise sites of protein-protein interactions, including GPCR binding to G proteins and arrestins, and for understanding the molecular basis of ligand recognition by chemokine receptors.

Lung cancer is the leading cause of cancer-related deaths worldwide and has a low 5-year survival rate. The current treatment for lung cancer patients is surgical resection followed by chemotherapy. However, the majority of patients will eventually experience disease progression and require further treatment. Increased evidence indicates that overexpression of survivin, the smallest member of IAP (inhibitor of apoptosis) family, results in poor prognosis, tumor recurrence, and drug resistance in various cancers, including non-small-cell lung cancer (NSCLC), which accounts for approximately 85% of all lung cancer cases. Although the precise mechanism leading to survivin overexpression in cancer cells is not fully understood, approach to effectively downregulate survivin is thought to be required to enhance chemotherapy, reduce relapse, and improve the survival of NSCLC patients. In the present study, we have explored the therapeutic potential of the class I HDAC inhibitor (HDACi) entinostat (or MS-275, SNDX-275, LC Laboratories, Woburn, MA) in combination with paclitaxel in the treatment of NSCLC. We show that entinostat significantly enhances paclitaxel-induced anti-proliferative/anti-survival effects and apoptosis in NSCLC cells. It appears that this HDACi selectively reduces the expression levels of survivin, but not the functionally-related molecules Mcl-1 and Bcl-xL. Further studies reveal that entinostat induces expression of two survivin-targeting miRNAs, miR-203 and miR-542-3p; and that both survivin-specific shRNAs and the mimics of miR-203 and/or miR-542-3p effectively downregulate survivin and dramatically promote NSCLC cells undergoing apoptosis upon paclitaxel treatment. Moreover, in a tumor xenografts model-established from NSCLC cells, entinostat maintains its capability to upregulate miR-203 and downregulate survivin in vivo. Importantly, the combinations of entinostat and paclitaxel exhibit a striking activity, as compared to either agent alone, to inhibit tumor growth. These data indicate that entinostat is efficacious to enhance paclitaxel-mediated anti-NSCLC activity. Our findings suggest that epigenetic targeting of survivin with entinostat or miRNA-replacement therapy may be a novel strategy to re-sensitize NSCLC to chemotherapy. Numerous studies demonstrate that entinostat exerts potent antitumor activity in various human cancers. Its clinic activity in NSCLC is currently being tested in combination with the DNA methyltransferase inhibitor Azacitidine or EGFR inhibitor erlotinib, but not conventional chemotherapy (http://www.clinicaltrial.gov/ct2/results?term = entinostat&Search = Search). Our studies provide a strong rationale to initiate clinical evaluation of the combinatorial effects of entinostat and paclitaxel on NSCLC patients. Citation Format: Shuiliang Wang, Ling Zhu, Zhiyong Zeng, Lianghu Huang, Fengjin Lin, Rong Lin, Jin Wang, Jun Lu, Qinghua Wang, Lingjing Lin, Huiyue Dong, Weizhen Wu, Kai Zheng, Jinquan Cai, Shunliang Yang, Yujie Ma, Shixin Ye, Wei Liu, Yinghao Yu, Bolin Liu, Jianming Tan. Epigenetic targeting of survivin enhances paclitaxel-mediated antitumor activity against non-small-cell lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5382. doi:10.1158/1538-7445.AM2015-5382