APEX/APEX2邻近标记方法在膜蛋白研究中的应用

膜蛋白(membrane proteins,MPs)参与人体内各种复杂的生命活动,了解MPs的结构、功能以及它们的相互作用网络是揭示相关蛋白如何参与复杂的生物学过程的前提。随着蛋白结构预测、结构解析等方面的一系列技术的不断发展,结构获得破解的MPs的数量一直在稳步增加,但是,关于MPs结构和功能的信息仍然非常稀缺。近年来,基于工程抗坏血酸过氧化物酶(engineered ascorbate peroxidase,APEX/APEX2)的邻近蛋白标记技术被广泛应用于探索MPs的亚细胞定位、解析MPs的拓扑结构,追踪MPs的动态迁移过程,以及寻找MPs的相互作用蛋白,该方法为空间受限的MPs的系统分析提供了一种高通量的方法。该综述归纳总结了APEX/APEX2依赖的邻近标记方法在MPs研究中的应用,为MPs的深入研究提供了方法和技术参考。

通过分子对接筛选14-3-3σ天然产物抑制剂

14-3-3s通过蛋白–蛋白相互作用来调控多种生物功能,如信号传导、新陈代谢、细胞生长及细胞增殖等。本研究选取14-3-3 σ 作为靶标来筛选蛋白–蛋白相互作用的天然产物抑制剂。通过分子对接方法从台湾天然产物数据库中筛选出可能的抑制剂,并通过分子动力学方法模拟抑制剂与14-3-3 σ 的复合物的结合特性。由MMGBSA方法计算结合自由能定量探讨抑制剂与4-3-3 σ 的结合强度。研究表明存在两种潜在的14-3-3 σ 抑制剂。

Citrate-stabilized CdSe/CdS quantum dots as fluorescence probe for protein determination

A rapid, ultrasensitive and convenient fluorescence measurement technology based on the enhancement of the fluorescence intensity resulting from the interaction of functionalized CdSe/CdS quantum dots （QDs） with bov/ne serum albumin （BSA） was proposed. The citrate-stabilized CdSe/CdS （QDs） were synthesized by using Se powder and Na2S as precursors instead of any pyrophoric organometallic precursors. The modified CdSe/CdS QDs are brighter and more stable against photobleaching in comparison with organic fluorophores. At pH 7.0, the fluorescence signal of CdSe/CdS is enhanced by increasing the concentration of BSA in the range of 0.1-10 μg/mL, and the low detection limit is 0.06 μg/mL. A linear relationship between the enhanced fluorescence peak intensity （△F） and BSA concentration （c） is established using equation △F=50.7c＋16.4 （R=0.996 36）. Results of determination for BSA in three synthetic samples are identical with the true values, and the recovery （98.9%-102.4%） and relative standard deviation （RSD, 1.8%-2.5%） are satisfactory.

Application of heat shock protein expression for detecting natural adaptation and exposure to stress in natural populations

Heat-shock proteins （HSPs） play an undisputed role for maintaining cellular functioning under environmental challenges and protein denaturing conditions. Compelling evidence points to an evolutionary important role of HSPs and a strict evolutionary control of these proteins as a balance between benefits and costs. While there is a great potential for using HSP expression for detecting natural adaptation and exposure to stress in natural populations, some obstacles and key issues await investigation. From an ecological perspective these key issues needs to be resolved in order to fully appreciate the complex responses and adaptations to stress and to increase our understanding of HSPs and other molecular chaperones for stress adaptation and potential use as biomarkers. Here, the current knowledge and understanding of HSPs is reviewed and a number of key issues including the interpretation of elevated HSP levels, the complications of extrapolating between laboratory and field conditions, the effects of choice of traits and methodology and the larger intra-and extracellular networks of interactions that HSPs participate in are discussed [Current Zoology 56 （6）： 703-713, 2010].

Multifunctionality of the linker histones： an emerging role for protein-protein interactions

Linker histones, e.g., H1, are best known for their ability to bind to nucleosomes and stabilize both nucleosome structure and condensed higher-order chromatin structures. However, over the years many investigators have reported specific interactions between linker histones and proteins involved in important cellular processes. The purpose of this review is to highlight evidence indicating an important alternative mode of action for H1, namely protein-protein interactions. We first review key aspects of the traditional view of linker histone action, including the importance of the H1 C-terminal domain. We then discuss the current state of knowledge of linker histone interactions with other proteins, and, where possible, highlight the mechanism of linker histone-mediated protein-protein interactions. Taken together, the data suggest a combinatorial role for the linker histones, functioning both as primary chromatin architectural proteins and simultaneously as recruitment hubs for proteins involved in accessing and modifying the chromatin fiber.

A Cdc2-related protein kinase hPFTAIRE1 from human brain interacting with 14-3-3 proteins

hPFTAIRE1 （PFTK1）, a Cdc2-related protein kinase, is highly expressed in human brain. It exhibits cytoplasmic distribution in Hela cells, although it contains two nuclear localization signals （NLSs） in its N-terminus. To search for its substrates and regulatory components, we screened a two-hybrid library by using the full-length hPFTAIRE1 as a bait. Four 14-3-3 isoforms （β,ε,η,τ） were identified interacting with the hPFTAIRE1. We found a putative 14-3-3 binding consensus motif（RHSSPSS） in the hPFTAIRE 1, which overlapped with its second NLS. Deletion of the RHSSPSS motif or substitution of Ser^119 gwithAla in the conserved binding motif abolished the specific interaction between the hPFTAIRE 1 and the 14-3 -3 proteins. The mutant S 120A hPFTAIRE1 also showed a weak interaction to the 14-3-3 proteins. The results suggested that the Ser^119 is crucial for the interaction between hPFTAIREI and the 14-3-3 proteins. All the hPFTAIRE1 mutants distributed in cytoplasm of Hela cells and human neuroblastoma cells （SH-SY5Y） when fused to the C-terminus of a green fluorescent protein （GFP）, indicating that binding with the 14-3-3 proteins does not contribute to the subcellular localization of the hPFTAIRE1, although the binding may be involved in its signaling regulation.

Thioredoxin and thioredoxin-interacting protein as prognostic markers for gastric cancer recurrence

AIM:To evaluate the potential of thioredoxin (TXN) and thioredoxin-interacting protein (TXNIP) expression as biomarkers for predicting gastric cancer recurrence. METHODS:TXN and TXNIP expression levels were acquired from gene expression microarray data for 65 human gastric cancer tissues. We determined whether each gene expression level was associated with cancer recurrence and investigated the relationship between the two genes. For validation, the expression levels of TXN and TXNIP were measured by quantitative real- time reverse transcription polymerase chain reaction in 68 independent stage Ⅲ gastric cancer patients. The correlation between gene expression and cancer prognosis was evaluated. Immunohistochemical staining was performed to investigate the protein expression levels of TXN and TXNIP and to characterize the expression patterns of each protein. RESULTS:TXN was a prognosis-related gene (P = 0.009), whereas TXNIP, a TXN inhibitor, demonstrated a negative correlation with TXN in the gene expression microarray data. In the 68 stage Ⅲ patients, the expression levels of both TXN and TXNIP had a statistically significant effect on recurrence-free survival (RFS, P = 0.008 and P = 0.036, respectively). The low TXN and high TXNIP expression group exhibited a better prognosis than the other groups, and the high TXN and low TXNIP expression group exhibited a poorer prognosis (P < 0.001 for RFS and P = 0.001 for overall survival). More than half of the patients in the simulta-neously high TXN and low TXNIP expression group ex- perienced a recurrence within 1 year after curative surgery, and the 5-year survival rate of the patients in this group was 29%, compared with 89% in the low TXN and high TXNIP expression group. The TXN protein was overexpressed in 65% of the gastric cancer tissues, whereas the TXNIP protein was underexpressed in 85% of the cancer cells. In a correlation analysis, TXN and TXNIP were highly correlated with many oncogenes and tumor suppressors as well as with genes related to energy, protein synthesis and autophagy. CONCLUSION:TXN and TXNIP are promising prognostic markers for gastric cancer, and performing personalized adjuvant treatment based on TXN and TXNIP expression levels would be an effective practice in the treatment of gastric cancer.

A Co-expression System Based on Phage and Phagemid to Select Cognate Antibody-antigen Pairs in vivo

A modified selectively infective phage (SIP) is developed to facilitate the selection of interacting antibody antigen pairs from a large single chain antibody (scFv) library in vivo. The system is constructed with a modified helper phage M13KO7 and phagemid pCANTAB 5 E. The antigen fused to the C terminal of N1 N2 domain and the scFv to the N terminal of CT domain of the gIIIp of filamentous phage are encoded on the phage and phagemid vectors respectively. The phages produced by co transformants restore infectivity via interaction between antigen and antibody fusions in the cell periplasm. In a model system, the scFv fragment of the anti hemagglutinin 17/9 antibody and its corresponding antigen are detected in the presence of a 10 5 fold excess of a non interacting control pairs, which demonstrates this system to be very sensitive and facile to screen a large single chain antibody library.

Chemical Physics in Living Cells-using Light to Visualize and Control Intracellular Signal Transduction

Cells are crowded microenvironments filled with macromolecules undergoing constant phys- ical and chemical interactions. The physicochemical makeup of the cells aff）cts various cellular responses, determines cell-cell interactions and influences cell decisions. Chemical and physical properties diff）r between cells and within cells. Moreover, these properties are subject to dynamic changes in response to environmental signals, which often demand adjustments in the chemical or physical states of intracellular molecules. Indeed, cellular responses such as gene expression rely on the faithful relay of information from the outside to the inside of the cell, a process terrned signal transduction. The signal often traverses a complex path across subcellular spaces with variable physical chemistry, sometimes even influencing it. Understanding the molecular states of such signaling molecules and their intracellular environments is vital to our understanding of the cell. Exploring such intricate spaces is possible today largely because of experimental and theoretical tools. Here, we focus on one tool that is commonly used in chemical physics studies light. We summarize recent work which uses light to both visualize the cellular environment and also control intracel- lular processes along the axis of signal transduction. We highlight recent accomplishments in optical microscopy and optogenetics, an emerging experimental strategy which utilizes light to control the molecular processes in live cells. We believe that optogenetics lends un- precedented spatiotemporal precision to the manipulation of physicochemical properties in biological contexts. We hope to use this work to demonstrate new opportunities for chemical physicists who are interested in pursuing biological and biomedical questions.

A Stochastic Model for Protein Synthesis and Activation through RNA-Protein Interaction in BioAmbients Calculus

Cells use various RNA （Ribonucleic Acid） regulatory mechanisms in order to temporally and coordinately influence the rate of protein synthesis. A deeper understanding of the dynamics of RNA regulation can ultimately bridge the gap between transcriptional control and protein expression. The nonlinear process of RNA-Protein Interaction （RIP）, which can be viewed as the RNA analog of the better-known chromatin immunoprecipitation application （CHIP） plays a crucial role in post-transcriptional regulation of gene expression. While ChIP identifies DNA （Deoxyribonucleic Acid） targets of DNA-binding proteins in their cellular context, RIP can be used to identify specific RNA molecules associated with specific nuclear or cytoplasmic RNA-binding proteins. In this paper, a stochastic model in BioAmbients calculus for the protein synthesis and activation through RIP process is presemed.

A Multiple Functional Protein:the Herpes Simplex Virus Type 1 Tegument Protein VP22

The herpes simplex virus type 1 （HSV-1） VP22, is one of the most abundant HSV-I tegument proteins with an average stoichiometry of 2 400 copies per virion and conserved among alphaherpesvirinae. Many functions are attributed to VP22, including nuclear localization, chromatin binding, microtubule binding, induction ofmicrotubule reorganization, intercellular transport, interaction with cellular proteins, such as template activating VP16, pU factor I （TAF-I） and nonmuscle myosin II A （NMIIA）, and viral proteins including pUS9 and pUL46, glycoprotein E （gE） and gD. Recently, many novel functions perform tegument protein ed by the HSV-1 VP22 protein have been shown, including promotion of protein synthesis at late times in infection, accumulation of a subset of viral mRNAs at early times in infection and possible transcriptional regulation function .

Function of lncRNAs and approaches to lncRNA-protein interactions

Long non-coding RNAs(lncRNAs),which represent a new frontier in molecular biology,play important roles in regulating gene expression at epigenetic,transcriptional and post-transcriptional levels.More and more lncRNAs have been found to play important roles in normal cell physiological activities,and participate in the development of varieties of tumors and other diseases.Previously,we have only been able to determine the function of lncRNAs through multiple mechanisms,including genetic imprinting,chromatin remodeling,splicing regulation,mRNA decay,and translational regulation.Application of technological advances to research into the function of lncRNAs is extremely important.The major tools for exploring lncRNAs include microarrays,RNA sequencing(RNA-seq),Northern blotting,real-time quantitative reverse transcription-polymerase chain reaction(qRT-PCR),fluorescence in situ hybridization(FISH),RNA interference(RNAi),RNA-binding protein immunoprecipitation(RIP),chromatin isolation by RNA purification(ChIRP),crosslinking-immunopurification(CLIP),and bioinformatic prediction.In this review,we highlight the functions of lncRNAs,and advanced methods to research lncRNA-protein interactions.

Interaction of δ-opioid Receptor with Membrane Transporters: Possible Mechanisms in Pain Suppression by Acupuncture

Objective： To investigate the possible mechanisms in acupuncture analgesia by interaction of δ-opioid receptor with neurotransmitter transport proteins or the Na^＋-K^＋ pump. Methods： Microinjection of respective heterologous cRNA into the Xenopus oocytes as a model system, and measurement of steady-state currents under two-electrode voltage clamp. Results： The co-expression of the 8-opioid receptor with GAT1, EAAC 1 or the sodium pump resulted in reducing activity of the respective transporter. Opioid receptor activation affected transporter activity in different ways： 1） GAT1 was further inhibited; 2） EAAC1 was stimulated; 3） Na^＋-K^＋ pump activity interfered with agonist sensitivity of DOR. Pump inhibition led to higher sensitivity for DPDPE. Conclusion： GABA transporter inhibition and glutamate transporter stimulation may counteract pain sensation by affecting the neurotransmitter concentration in the synaptic cleft and, therefore, may contribute synergistically to pain suppression by acupuncture. Sodium pump inhibition by endogenous ouabain may amplify these effects. These synergistic effects may be the molecular mechanism of inhibiting pain sense and/or acupuncture analgesia.

Insights into the function of tegument proteins from the varicella zoster virus

Chickenpox(varicella) is caused by primary infection with varicella zoster virus(VZV), which can establish long-term latency in the host ganglion. Once reactivated, the virus can cause shingles(zoster) in the host. VZV has a typical herpesvirus virion structure consisting of an inner DNA core, a capsid, a tegument, and an outer envelope. The tegument is an amorphous layer enclosed between the nucleocapsid and the envelope, which contains a variety of proteins. However, the types and functions of VZV tegument proteins have not yet been completely determined. In this review, we describe the current knowledge on the multiple roles played by VZV tegument proteins during viral infection. Moreover, we discuss the VZV tegument protein-protein interactions and their impact on viral tissue tropism in SCID-hu mice. This will help us develop a better understanding of how the tegument proteins aid viral DNA replication, evasion of host immune response, and pathogenesis.

Transcriptome analysis of blood stasis syndrome in subjects with hypertension

OBJECTIVE:To screen for m RNAs associated with blood stasis syndrome and to explore the genetic mechanisms of blood stasis syndrome in hypertension.METHODS:This study involved groups of patients with hypertension and blood stasis,including those with Qi deficiency,Qi stagnation,cold retention and heat retention;as well as hypertensive patients without blood stasis and healthy individuals.Human umbilical vein endothelial cells were co-cultured with the sera of these healthy individuals and patients with blood stasis syndrome.Total RNA was extracted from these cells and assessed by a high-throughput sequencing method(Solexa)and digital gene expression.Differentially expressed genes among these six groups were compared using whole genome sequences,and m RNAs associated with blood stasis syndrome identified.Differences in gene use and gene ontology function were an-alyzed.Genes enriched significantly and their pathways were determined,as were network interactions,and encoded proteins.Gene identities were confirmed by real-time polymerase chain reactions.RESULTS:Compared with cells cultured in sera of the blood stasis groups,those culture in sera of healthy individuals and of the non-blood stasis group showed 11 and 301 differences,respectively in stasis-related genes.Genes identified as differing between the blood stasis and healthy groups included activating transcription factor 4,activating transcription factor 3,DNA-damage inducible transcription factor 3,Tribbles homolog 3,CCAAT/enhancer binding protein-β,and Jun proto-oncogene(JUN).Pathway and protein interaction network analyses showed that these genes were associated with endoplasmic reticulum stress.Cells cultured in sera of patients with blood stasis and Qi deficiency,Qi stagnation,heat retention,and cold retention were compared with cells cultured in sera of patients with the other types blood stasis syndrome.The comparison showed differences in expression of 28,28,34,and 32 specific genes,respectively.CONCLUSION:The pathogenesis of blood stasis syndrome in hypertension is related to endoplasmic reticulum stress and involves the differential expression of the activating transcription factor 4,activating transcription factor 3,DNA-damage inducible transcription factor 3,Tribbles homolog 3,CCAAT/enhancer binding protein-β,and JUN genes.

IgG and fibrinogen driven nanoparticle aggregation

A thorough understanding of how proteins induce nanoparticle （NP） aggregation is crucial when designing in vitro and in vivo assays and interpreting experimental results. This knowledge is also crucial when developing nano-applications and formulation for drug delivery systems. In this study, we found that extraction of immunoglobulin G （IgG） from cow serum results in lower polystyrene NPs aggregation. Moreover, addition of isolated IgG or fibrinogen to fetal cow serum enhanced this aggregation, thus demonstrating that these factors are major drivers of NP aggregation in serum. Counter-intuitively, NP aggregation was inversely dependent on protein concentration; i.e., low protein concentrations induced large aggregates, whereas high protein concentrations induced small aggregates. Protein-induced NP aggregation and aggregate size were monitored by absorbance at 400 nm and dynamic light scattering, respectively. Here, we propose a mechanism behind the protein concentration dependent aggregation; this mechanism involves the effects of multiple protein interactions on the NP surface, surface area limitations, aggregation kinetics, and the influence of other serum proteins.

Disease gene identification by using graph kernels and Markov random fields

Genes associated with similar diseases are often functionally related.This principle is largely supported by many biological data sources,such as disease phenotype similarities,protein complexes,protein-protein interactions,pathways and gene expression profiles.Integrating multiple types of biological data is an effective method to identify disease genes for many genetic diseases.To capture the gene-disease associations based on biological networks,a kernel-based Markov random field(MRF)method is proposed by combining graph kernels and the MRF method.In the proposed method,three kinds of kernels are employed to describe the overall relationships of vertices in five biological networks,respectively,and a novel weighted MRF method is developed to integrate those data.In addition,an improved Gibbs sampling procedure and a novel parameter estimation method are proposed to generate predictions from the kernel-based MRF method.Numerical experiments are carried out by integrating known gene-disease associations,protein complexes,protein-protein interactions,pathways and gene expression profiles.The proposed kernel-based MRF method is evaluated by the leave-one-out cross validation paradigm,achieving an AUC score of 0.771 when integrating all those biological data in our experiments,which indicates that our proposed method is very promising compared with many existing methods.

Structure and function of ALG-2,a penta-EF-hand calcium-dependent adaptor protein

ALG-2(a gene product of PDCD6) is a 22-kD protein containing five serially repetitive EF-hand structures and belongs to the penta-EF-hand(PEF) family,including the subunits of typical calpains.ALG-2 is the most conserved protein among the PEF family members and its homologs are widely found in eukaryotes.X-ray crystal structures of various PEF proteins including ALG-2 have common features:presence of eightα-helices and dimer formation via paired EF5s that are positioned in anti-parallel orientation.ALG-2 forms a homodimer and a heterodimer with its closest paralog peflin.Like calmodulin,a well-known four-EF-hand protein,ALG-2 interacts with various proteins in a Ca2+-dependent fashion,but the binding motifs are completely different.With some exceptions,ALG-2-interacting proteins commonly contain Pro-rich regions,and ALG-2 recognizes at least two distinct Pro-containing motifs:PPYP(X) nYP(X,variable;n=4 in ALIX and PLSCR3) and PXPGF(represented by Sec31A) .A shorter alternatively spliced isoform,lacking two residues and designated ALG-2 GF122,does not bind ALIX but maintains binding capacity to Sec31A.X-ray crystal structural analyses have revealed that binding of calcium ions induces the configuration of the side chain of R125 so that it opens Pocket 1,which accepts PPYP,but Pocket 1 remains closed in the case of ALG-2 GF122.ALG-2 dimer has two ligand-binding sites,each in a monomer molecule,and appears to function as a Ca2+-dependent adaptor protein to either stabilize a preformed complex or to bridge two proteins on scaffolds in systems of the endosomal sorting complex required for transport(ESCRT) and ER-to-Golgi transport.

HSV-1 stimulation-related protein HSRG1 inhibits viral gene transcriptional elongation by interacting with Cyclin T2

The protein encoded by HSRG1(HSV-1 stimulation-related gene 1) is a virally induced protein expressed in HSV-1-infected cells.We have already reported that HSRG1 is capable of interacting with transcriptional regulator proteins.To further analyze the effects of HSRG1 on the regulation of viral gene transcription,we expressed the HSRG1 protein in transfected cells and found that it postpones the proliferation of HSV-1.CAT(chloramphenicol acetyltransferase) assays also revealed that HSRG1 reduces transcription from HSV-1 promoters.Yeast two-hybrid and immunoprecipitation assays indicated that HSRG1 interacts with Cyclin T2,the regulatory subunit of P-TEFb,which is required for transcription elongation by RNA Pol II(RNAP II) ,and that amino acid residues 1-420 in Cyclin T2 are important for binding with HSRG1.Fluorescence assays suggested that the cellular localizations of those two proteins are influenced by their interaction.Further analyses with CAT assays revealed that HSRG1 inhibits the transcriptional activation by Cyclin T2 of viral promoters.Our results suggested that the inhibitory effects of HSRG1 on viral replication and proliferation are probably induced by its binding to Cyclin T2.Therefore,it is likely that HSRG1 inhibits viral gene transcriptional elongation by interacting with Cyclin T2.

Complex roles of necroptosis in cancer

Necroptosis is a tightly regulated form of necrosis that requires the activation of receptor-interacting protein(RIP)kinases RIPK1 and RIPK3,as well as the RIPK3 substrate mixed lineage kinase domain-like protein(MLKL).Because of membrane rupture,necroptotic cells release damage-associated molecular patterns(DAMPs)that evoke immune responses.Necroptosis is emerging as an important cellular response in the modulation of cancer initiation,progression,and metastasis.Necroptosis of cancer cells is considered to be an immunogenic cell death capable of activating anti-tumor immunity.Necroptosis also participates in the promotion of myeloid cell-induced adaptive immune suppression and thus contributes to oncogenesis.In addition,necroptosis of endothelial cells and tumor cells is conducive to tumor metastasis.In this review,we summarize the current knowledge of the complex role of necroptosis in cancer and discuss the potential of targeting necroptosis components for cancer therapies.