Drug binding to human serum albumin: abridged review of results obtained with high-performance liquid chromatography and circular dichroism

The drug binding to plasma and tissue proteins are fundamental factors in determining the overall pharmacological activity of a drug. Human serum albumin (HSA), together with alpha1-acid glycoprotein (AGP), are the most important plasma proteins, which act as drug carriers, with drug pharmacokinetic implications, resulting in important clinical impacts for drugs that have a relatively narrow therapeutic index. This review focuses on the combination of biochromatography and circular dichroism as an effective approach for the characterization of albumin binding sites and their enantioselectivity. Furthermore, their applications to the study of changes in the binding properties of the protein arising by the reversible or covalent binding of drugs are discussed, and examples of physiological relevance reported. Perspectives of these studies reside in supporting the development of new drugs, which require miniaturization to facilitate the screening of classes of compounds for their binding to the target protein, and a deeper characterization of the mechanisms involved in the molecular recognition processes.     

NEDD化修饰系统及其在肿瘤发展中的作用

目前已经鉴定出17种类泛素蛋白（ubiquitin like proteins,UBLs）,这些蛋白与底物的结合方式与泛素相似．根据进化特征,可将UBLs分为9类,分别为：NEDD8、SUMO、ISG15、FUB1、FAT10、Atg8、Atg12、Urm1和UFM1．NEDD8是目前研究最多的UBLs之一,与泛素的氨基酸序列具有高度相似性．NEDD化修饰是一种动态的可逆蛋白质翻译后修饰方式,可以将NEDD8共价结合到靶蛋白之上,也可以将NEDD8从靶蛋白上去除．NEDD化修饰对蛋白功能具有重要的调节作用,如改变蛋白质的空间构象、阻碍底物与其它蛋白质的相互作用和招募与NEDD8相互作用的蛋白等．最新研究表明,NEDD化与肿瘤的发生发展密切相关,但具体的机制还不清楚．本文将就NEDD化修饰在肿瘤发展过程中的作用机制做一综述．     

Distinguishing the optimal binding mechanism of an E3 ubiquitin ligase: Covalent versus noncovalent inhibition

The development of covalent drugs, specifically in cancer therapeutics, has recently sparked interest among the pharmaceutical research community. While representing a significant fraction of the drugs in the market, very few have been deliberately designed to interact covalently with their biological target. One of the enzymes that have been both covalently and noncovalently targeted is the Neural Precursor Cell Expressed Developmentally Downregulated gene 4-1 (Nedd4-1). This enzyme has been found to have multiple physiological implications, including its involvement in cancer invasion. A critical gap still remains in the molecular understanding of the structural mechanism upon the covalent and noncovalent binding to Nedd4-1. In this study, we explore the most optimal binding mechanism in the inhibition of the catalytic site of the Nedd4-1. Our results exhibited a greater stability in the covalent complex compared with the noncovalent complex. This was supported by the secondary structure elements that were more dominant in the covalently inhibited complex. This complex disclosed an optimal free binding energy landscape, induced by the catalytic site energy contributions that showed to be more favorable. The insights demonstrating the above binding mechanism of Nedd4-1 establishes covalent inhibition as the preferred method of inhibition of the enzyme. This investigation aids in the understanding of the structural mechanism of Nedd4-1 inhibition and would assist in the design of more potent covalent inhibitors at the catalytic site of Nedd4-1.     

PICK1：一个功能多样的药靶蛋白

蛋白质是生命功能的执行者.生命体中某些关键蛋白的功能异常往往是导致疾病发生的根本原因.这些疾病相关蛋白极有可能成为药物靶点,为新药研发和疾病治疗提供重要线索. PICK1蛋白（protein interacting with Cα kinase 1）结合能力广泛、功能多样以及在多种重要疾病（如：癌症、精神分裂症、疼痛、帕金森综合症等）的发生发展过程中发挥潜在的作用,使其成为一个可能的药靶蛋白. PICK1与绝大多数配体蛋白的相互作用是通过其PDZ结构域与配体C末端区域的结合介导的,使PICK1的PDZ结构域成为一个潜在的药物靶点.因此,可以利用生物小分子物质特异性地结合PICK1的PDZ结构域,干扰或阻断PICK1与配体蛋白的天然相互作用,最终达到治疗相关疾病的目的.     

Development of nanoparticle libraries for biosensing

Magnetic and magnetofluorescent nanoparticles have become important materials for biological applications especially for sensing, separation, and imaging. To achieve target specificity, these nanomaterials are often covalently modified with binding proteins such as antibodies or proteins. Here we report on the creation of nanoparticle libraries that achieve specificity through multivalent modification with small molecules. We explore different synthetic routes to attach small molecules with anhydride, amine, hydroxyl carboxyl, thiol, and epoxy handles. We show that the derived nanomaterials have unique biological functions, possess different behaviors in cell screens, and can be used as substrates for biological screens.     

Interaction of S100 proteins with the antiallergic drugs, olopatadine, amlexanox, and cromolyn: identification of putative drug binding sites on S100A1 protein

S100 proteins are a multigenic family of low-molecular-weight Ca(2+)-binding proteins comprising 19 members. These proteins undergo a conformational change by Ca(2+)-binding and consequently interact with their target proteins. Recently, we reported that two antiallergic drugs, Amlexanox and Cromolyn, bind to S100A12 and S100A13 of the S100 protein family. In the present study, we used a newly developed antiallergic drug, Olopatadine, as a ligand for affinity chromatography and examined binding specificity of the drug to S100 protein family. Olopatadine binds specifically to S100 proteins, such as S100A1, S100B, S100L, S100A12, and S100A13, in a Ca(2+)-dependent manner but not to calmodulin. Mutagenesis study showed that amino acid residues 76-85 in S100A1 are necessary for its binding to Olopatadine. In contrast, residues 89-94 were identified as an Amlexanox-binding site in S100A1. Moreover, Olopatadine did not competitively inhibit S100A1-binding site of Amlexanox. Furthermore, we showed that Olopatadine inhibited the binding of S100A1 target protein's binding site peptides to S100A1. These results indicate that C-terminal region of S100A1 is important for antiallergic drug binding, although the drug binding sites are different according to each antiallergic drug. Differences in the binding sites of S100A1 to antiallergic drugs suggest that the regulatory functions of S100 proteins may exist in several regions. Therefore, these drugs may serve as useful tools for evaluating the physiological significance of S100 protein family.     

Modelling topoisomerase I inhibition by minor groove binders

Topoisomerase inhibition is an extremely useful target for anticancer and antimicrobial drugs, and an undesirable side effect of some drugs targeting other proteins. Published modelling studies are sparse, and have used small data sets with relatively low molecular diversity. Given the important role of minor groove binding in the mechanism of topoisomerase I inhibition, we have conducted the first 3D QSAR study of topoisomerase I inhibition of a large, diverse set of minor groove binders using the minor groove binding conformation as the alignment template. The highly significant QSAR models resulting from this alignment identify the roles played by molecular features, most importantly the hydrogen bond donor properties.     

Autoinhibition of X11/Mint scaffold proteins revealed by the closed conformation of the PDZ tandem

Members of the X11/Mint family of multidomain adaptor proteins are composed of a divergent N terminus, a conserved PTB domain and a pair of C-terminal PDZ domains. Many proteins can interact with the PDZ tandem of X11 proteins, although the mechanism of such interactions is unclear. Here we show that the highly conserved C-terminal tail of X11alpha folds back and inserts into the target-binding groove of the first PDZ domain. The binding of this tail occludes the binding of other target peptides. This autoinhibited conformation of X11 requires that the two PDZ domains and the entire C-terminal tail be covalently connected to form an integral structural unit. The autoinhibited conformation of the X11 PDZ tandem provides a mechanistic explanation for the unique target-binding properties of the protein and hints at potential regulatory mechanisms for the X11-target interactions.     

Protein-protein interactions regulate Ubl conjugation

The ubiquitin-like proteins (Ubls) can be covalently linked to target proteins to provide a critical signal in diverse cellular processes. Members of the Ubl family include ubiquitin itself and a growing number of homologs such as SUMO, Nedd8, ISG15 and Atg8. The enzymatic mechanism of Ubl conjugation involves an E1, E2, E3 cascade of enzymes that is well conserved between the Ubls. In the past two years, novel structural details of Ubl conjugation were uncovered through analysis of protein-protein complexes. This has given insight in activation of E1, the role of the target lysine in E2-dependent catalysis, the role of noncovalent Ubl binding in Ubl chain formation and the importance of dimerization of Ring-type E3 ligases.     

Aptamer directly evolved from live cells recognizes membrane bound immunoglobin heavy mu chain in Burkitt's lymphoma cells

The identification of tumor related cell membrane protein targets is important in understanding tumor progression, the development of new diagnostic tools, and potentially for identifying new therapeutic targets. Here we present a novel strategy for identifying proteins that are altered in their expression levels in a diseased cell using cell specific aptamers. Using an intact viable B-cell Burkitt's lymphoma cell line (Ramos cells) as the target, we have selected aptamers that recognize cell membrane proteins with high affinity. Among the selected aptamers that showed different recognition patterns with different cell lines of leukemia, the aptamer TD05 showed binding with Ramos cells. By chemically modifying TD05 to covalently cross-link with its target on Ramos cells to capture and to enrich the target receptors using streptavidin coated magnetic beads followed by mass spectrometry, we were able to identify membrane bound immunoglobin heavy mu chain as the target for TD05 aptamer. Immunoglobin heavy mu chain is a major component of the B-cell antigen receptor, which is expressed in Burkitt's lymphoma cells. This study demonstrates that this two step strategy, the development of high quality aptamer probes and then the identification of their target proteins, can be used to discover new disease related potential markers and thus enhance tumor diagnosis and therapy. The aptamer based strategy will enable effective molecular elucidation of disease related biomarkers and other interesting molecules.     

InTRIMsic immunity: Positive and negative regulation of immune signaling by tripartite motif proteins

During the immune response, striking the right balance between positive and negative regulation is critical to effectively mount an anti-microbial defense while preventing detrimental effects from exacerbated immune activation. Intra-cellular immune signaling is tightly regulated by various post-translational modifications, which allow for this dynamic response. One of the post-translational modifiers critical for immune control is ubiquitin, which can be covalently conjugated to lysines in target molecules, thereby altering their functional properties. This is achieved in a process involving E3 ligases which determine ubiquitination target specificity.One of the most prominent E3 ligase families is that of the tripartite motif (TRIM) proteins, which counts over 70 members in humans. Over the last years, various studies have contributed to the notion that many members of this protein family are important immune regulators. Recent studies into the mechanisms by which some of the TRIMs regulate the innate immune system have uncovered important immune regulatory roles of both covalently attached, as well as unanchored poly-ubiquitin chains. This review highlights TRIM evolution, recent findings in TRIM-mediated immune regulation, and provides an outlook to current research hurdles and future directions.     

Metal chelate affinity precipitation: a new approach to protein purification

Metal chelate affinity precipitation of proteins, a method combining metal–protein interaction and affinity precipitation is being discussed as a selective separation process for proteins. The technique utilizes a flexible soluble–insoluble thermo-responsive polymer with a covalently linked ligand loaded with metal ions. The affinity binding of the target protein varies with different metal ions. Copolymers of N-isopropylacrylamide with 1-vinylimidazole loaded with Cu(II) ions are designed as a potential carriers for affinity purification and proved to be successful for purification of protein inhibitors from a variety of cereals. This revised version was published online in July 2006 with corrections to the Cover Date.     

Covalent binding to tubulin by isothiocyanates. A mechanism of cell growth arrest and apoptosis

Isothiocyanates (ITCs) found in cruciferous vegetables, including benzyl-ITC (BITC), phenethyl-ITC (PEITC), and sulforaphane (SFN), inhibit carcinogenesis in animal models and induce apoptosis and cell cycle arrest in various cell types. The biochemical mechanisms of cell growth inhibition by ITCs are not fully understood. Our recent study showed that ITC binding to intracellular proteins may be an important initiating event for the induction of apoptosis. However, the specific protein target(s) and molecular mechanisms were not identified. In this study, two-dimensional gel electrophoresis of human lung cancer A549 cells treated with radiolabeled PEITC and SFN revealed that tubulin may be a major in vivo binding target for ITC. We examined whether binding to tubulin by ITCs could lead to cell growth arrest. The proliferation of A549 cells was significantly reduced by ITCs, with relative activities of BITC > PEITC > SFN. All three ITCs also induced mitotic arrest and apoptosis with the same order of activity. We found that ITCs disrupted microtubule polymerization in vitro and in vivo with the same order of potency. Mass spectrometry demonstrated that cysteines in tubulin were covalently modified by ITCs. Ellman assay results indicated that the modification levels follow the same order, BITC > PEITC > SFN. Together, these results support the notion that tubulin is a target of ITCs and that ITC-tubulin interaction can lead to downstream growth inhibition. This is the first study directly linking tubulin-ITC adduct formation to cell growth inhibition.     

Determinants of RNA Binding and Translational Repression by the Bicaudal-C Regulatory Protein

Bicaudal-C (Bic-C) RNA binding proteins function as important translational repressors in multiple biological contexts within metazoans. However, their RNA binding sites are unknown. We recently demonstrated that Bic-C functions in spatially regulated translational repression of the xCR1 mRNA during Xenopus development. This repression contributes to normal development by confining the xCR1 protein, a regulator of key signaling pathways, to specific cells of the embryo. In this report, we combined biochemical approaches with in vivo mRNA reporter assays to define the minimal Bic-C target site within the xCR1 mRNA. This 32-nucleotide Bic-C target site is predicted to fold into a stem-loop secondary structure. Mutational analyses provided evidence that this stem-loop structure is important for Bic-C binding. The Bic-C target site was sufficient for Bic-C mediated repression in vivo. Thus, we describe the first RNA binding site for a Bic-C protein. This identification provides an important step toward understanding the mechanisms by which evolutionarily conserved Bic-C proteins control cellular function in metazoans.     

Soluble expression of aggregating proteins by covalent coupling to the ribosome

Ribosomes are extremely soluble ribonucleoprotein complexes. Heterologous target proteins were fused to ribosomal protein L23 (rpL23) and expressed in an rpL23 deficient Escherichia coli strain. This enabled the isolation of 70S ribosomes with covalently bound target protein. Isolation of recombinant proteins from 70S ribosomes was achieved by specific proteolytic cleavage followed by efficient removal of ribosomes by centrifugation. By this procedure we isolated active green fluorescent protein, streptavidin (SA), and murine interleukin-6 (mIL-6). Approximately 500microg of each protein was isolated per gram cellular wet weight. By pull-down assays we demonstrate that SA covalently bound to the ribosome binds d-biotin. Ribosomal coupling is therefore suggested as a method for the investigation of protein interactions. The presented strategy is in particular efficient for the expression, purification, and investigation of proteins forming inclusion bodies in the E. coli cytoplasm.     

Structural basis of 5-nitroimidazole antibiotic resistance: the crystal structure of NimA from Deinococcus radiodurans

5-Nitroimidazole-based antibiotics are compounds extensively used for treating infections in humans and animals caused by several important pathogens. They are administered as prodrugs, and their activation depends upon an anaerobic 1-electron reduction of the nitro group by a reduction pathway in the cells. Bacterial resistance toward these drugs is thought to be caused by decreased drug uptake and/or an altered reduction efficiency. One class of resistant strains, identified in Bacteroides, has been shown to carry Nim genes (NimA, -B, -C, -D, and -E), which encode for reductases that convert the nitro group on the antibiotic into a non-bactericidal amine. In this paper, we have described the crystal structure of NimA from Deinococcus radiodurans (drNimA) at 1.6 A resolution. We have shown that drNimA is a homodimer in which each monomer adopts a beta-barrel fold. We have identified the catalytically important His-71 along with the cofactor pyruvate and antibiotic binding sites, all of which are found at the monomer-monomer interface. We have reported three additional crystal structures of drNimA, one in which the antibiotic metronidazole is bound to the protein, one with pyruvate covalently bound to His-71, and one with lactate covalently bound to His-71. Based on these structures, a reaction mechanism has been proposed in which the 2-electron reduction of the antibiotic prevents accumulation of the toxic nitro radical. This mechanism suggests that Nim proteins form a new class of reductases, conferring resistance against 5-nitroimidazole-based antibiotics.     

Crystal structure of Ufc1, the Ufm1-conjugating enzyme

Ubiquitin and ubiquitin-like protein-conjugating enzymes play central roles in posttranslational modification processes. The ubiquitin-fold modifier 1 (Ufm1), one of a variety of ubiquitin-like modifiers, is covalently attached to target proteins via Uba5 and Ufm1-conjugating enzyme 1 (Ufc1), which are analogous to the E1 and E2 ubiquitylation enzymes. As Ufm1-related proteins are conserved in metazoa and plants, the Ufm1 system likely plays important roles in various multicellular organisms. Herein, we report the X-ray structure of human Ufc1 determined at 1.6 A resolution. The Ufc1 structure comprises a canonical E2 domain and an additional N-terminal domain. The Uba5 binding site on Ufc1 was assigned by structural comparison of Ufc1 and Ubc12 and related mutational analyses. In addition, we show that the N-terminal unique domain of Ufc1 contributes to thermal stability.     

Molecular recognition of 15-deoxy-delta(12,14)-prostaglandin J2 by nuclear factor-kappa B and other cellular proteins

15-Deoxy-delta(12,14)-prostaglandin J2 (15d-PGJ2), a dehydration product of prostaglandin D2, is an important pharmacological molecule, which with the virtue of its electrophilicity, has been reported to covalently modify some cellular proteins (such as nuclear factor-kappa B (NF-kappaB), AP-1, p53, and thioredoxin) and elicit its physiological effects. The aim of the present computational study is to understand the role molecular recognition plays in the association of 15d-PGJ2 with NF-kappaB and other proteins. Another aim is to characterize whether p53 is a direct target for covalent modification by 15d-PGJ2. A docking strategy is applied along with calculation of ab initio electrostatic potential maps to analyze the mode of binding of prostaglandin molecule with critical cysteine-containing sites in each protein. The results provide identification of important sites in the target proteins, which provide recognition and stability to the prostaglandin molecule. Fit of shape and complementarity of electrostatic interactions are derived as significant determinants of molecular recognition of 15d-PGJ2. Further, comparative results indicate that p53 protein may also be a target for direct modification by 15d-PGJ2. The molecular models obtained should allow the rational design of more specific analogs of 15d-PGJ2.     

植物蛋白质翻译后修饰组学研究进展

蛋白质翻译后修饰(Protein post-translational modification,PTMs)是一种重要的细胞调控机制,通过在蛋白质的氨基酸侧链上共价结合一些化学小分子基团来调节蛋白质的活性、结构、定位和蛋白质间的互作关系,从而精细调控蛋白质生物学功能的动态变化。PTMs是植物对环境变化最快、最早的反应之一,是植物蛋白质组多样性的关键机制,在植物生长发育和对环境适应中起重要作用。主要介绍了近年来植物磷酸化、乙酰化、琥珀酰化、糖基化、泛素化、巴豆酰化、S-亚硝基化及2-羟基异丁酰化等PTMs研究进展,旨为认识植物PTMs的关键生物学功能和研究前景提供参考。     

Sequence specificity of drug-DNA interactions

Methods for determining sequence specificities of anticancer drugs, carcinogens, and mutagens which interact with natural DNA's are presented. For drugs which nick or covalently bind to DNA and thus leave a permanent record of their residence position on the helix, the sequences important in drug action can be readily determined. For agents which interact with DNA in an equilibrium fashion, "footprinting" analysis, a technique used to investigate protein-DNA binding, has proved to be useful in studying drug-DNA interactions. The sequence specificities of a number of small ligands which interact with natural DNA's are also presented.