Structure-based design of ester compounds to inhibit MLL complex catalytic activity by targeting mixed lineage leukemia 1 (MLL1)–WDR5 interaction

WDR5 is an essential protein for enzymatic activity of MLL1. Targeting the protein–protein interaction (PPI) between MLL1 and WDR5 represents a new potential therapeutic strategy for MLL leukemia. Based on the structure of reported inhibitor WDR5-0103, a class of ester compounds were designed and synthetized to disturb MLL1–WDR5 PPI. These inhibitors efficiently inhibited the histone methyltransferase activity in vitro. Especially, WL-15 was one of the most potent inhibitors, blocking the interaction of MLL1–WDR5 with IC₅₀ value of 26.4 nM in competitive binding assay and inhibiting the catalytic activity of MLL1 complex with IC₅₀ value of 5.4 μM. Docking model indicated that ester compounds suitably occupied the central cavity of WDR5 protein and recapitulated the interactions of WDR5-0103 and the hydrophobic groups and key amino greatly increased the activity in blocking MLL1–WDR5 PPI.     

Kinetic analysis of a high-affinity antibody/antigen interaction performed by multiple Biacore users

To explore the reliability of Biacore-based assays, 22 study participants measured the binding of prostate-specific antigen (PSA) to a monoclonal antibody (mAb). Each participant was provided with the same reagents and a detailed experimental protocol. The mAb was immobilized on the sensor chip at three different densities and a two-step assay was used to determine the kinetic and affinity parameters of the PSA/mAb complex. First, PSA was tested over a concentration range of 2.5-600 nM to obtain k(a) information. Second, to define the k(d) of this stable antigen/antibody complex accurately, the highest PSA concentration was retested with the dissociation phase of each binding cycle monitored for 1h. All participants collected data that could be analyzed to obtain kinetic parameters for the interaction. The association and the extended-dissociation data derived from the three antibody surfaces were globally fit using a simple 1:1 interaction model. The average k(a) and k(d) for the PSA/mAb interaction as calculated from the 22 analyses were (4.1+/-0.6) x 10(4) M(-1) s(-1) and (4.5+/-0.6) x 10(-5) s(-1), respectively. Overall, the experimental standard errors in the rate constants were only approximately 14%. Based on the kinetic rate constants, the affinity (K(D)) of the PSA/mAb interaction was 1.1+/-0.2 nM.     

基于雷帕霉素与FRB结合的蛋白质间相互作用的相关技术

FKBP12-rapamycin复合物的结合位点(FKBP1 2-rapamycin binding,FRB)为雷帕霉素(rapamycin,RAP)与哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)结合的结构城,基于RAP介导FK506结合蛋白12(12 kD FK506-binging protein,FKBP12)与FRB蛋白质相互作用相关研究技术的发展与应用,使人们对小分子介导的蛋白质相互作用有了更多的认识.就研究RAP作用于FRB域及研究FKBP12-RAP-FRB三元复合物形成的相关技术作一综述,为确认新的mTOR抑制剂的作用机制及认识其他蛋白质间相互作用提供参考.     

Fluorescent protein pair emit intracellular FRET signal suitable for FACS screening

The fluorescent proteins ECFP and HcRed were shown to give an easily resolved FRET-signal when expressed as a fusion inside mammalian cells. HeLa-tat cells expressing ECFP, pHcRed, or the fusion protein pHcRed-ECFP were analyzed by flow cytometry after excitation of ECFP. Cells expressing HcRed-ECFP, or ECFP and HcRed, were mixed and FACS-sorted for FRET positive cells: HcRed-ECFP cells were greatly enriched (72 times). Next, cloned human antibodies were fused with ECFP and expressed anchored to the ER membrane. Their cognate antigens (HIV-1 gp120 or gp41) were fused to HcRed and co-expressed in the ER. An increase of 13.5+/-1.5% (mean+/-SEM) and 8.0+/-0.7% in ECFP fluorescence for the specific antibodies reacting with gp120 or gp41, respectively, was noted after photobleaching. A positive control (HcRed-ECFP) gave a 14.8+/-2.6% increase. Surprisingly, the unspecific antibody (anti-TT) showed 12.1+/-1.1% increase, possibly because overexpression in the limited ER compartment gave false FRET signals.     

Polyamines: Naturally occurring small molecule modulators of electrostatic protein-protein interactions

Modulations of protein-protein interactions are a key step in regulating protein function, especially in networks. Modulators of these interactions are supposed to be candidates for the development of novel drugs. Here, we describe the role of the small, polycationic and highly abundant natural polyamines that could efficiently bind to charged spots at protein interfaces as modulators of such protein-protein interactions. Using the mitochondrial cytochrome P45011A1 (CYP11A1) electron transfer system as a model, we have analyzed the capability of putrescine, spermidine, and spermine at physiologically relevant concentrations to affect the protein-protein interactions between adrenodoxin reductase (AdR), adrenodoxin (Adx), and CYP11A1. The actions of polyamines on the individual components, on their association/dissociation, on electron transfer, and on substrate conversion were examined. These studies revealed modulating effects of polyamines on distinct interactions and on the entire system in a complex way. Modulation via changed protein-protein interactions appeared plausible from docking experiments that suggested favourable high-affinity binding sites of polyamines (spermine > spermidine > putrescine) at the AdR-Adx interface. Our findings imply for the first time that small endogenous compounds are capable of interfering with distinct components of transient protein complexes and might control protein functions by modulating electrostatic protein-protein interactions.     

Cross-linking of human cytochrome P450 2B6 to NADPH-cytochrome P450 reductase: Identification of a potential site of interaction

The site(s) of interaction between human cytochrome P450 2B6 and NADPH-cytochrome P450 reductase (P450 reductase) have yet to be identified. To investigate this, the cross-linking agent 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) was used to covalently link P450 2B6-P450 reductase. Following digestion with trypsin, the cross-linked peptides were identified by reconstituting the peptides in ¹⁸O-water based on the principle that the cross-linked peptides would be expected to incorporate twice as many ¹⁸O atoms as the non-cross-linked peptides. Subsequent mass spectrometric analyses of the resulting peptides led to the identification of one cross-linked peptide candidate. De novo sequencing of the peptide indicated that it is a complex between residues in the C-helix of the P450 (based upon solved X-ray crystal structures of P450 2B4) and the connecting domain of the P450 reductase. To confirm this experimentally, the P450 2B6 peptide identified through the cross-linking studies was synthesized and peptide competition studies were performed. In the presence of the synthetic peptide, P450 catalytic activity was decreased by up to 60% when compared to competition studies performed using a nonsense peptide. Taken together, these studies indicate that residues in the C-helix of P450 2B6 play a major role in the interaction with the P450 reductase.     

蛋白质相互作用的研究方法

随着基因组测序工程数量的增加,未知功能蛋白质测序工作也呈现指数式增长。生物学进程主要是由蛋白质执行和控制的,因此,阐明未知或已知蛋白质的生物学功能和从细胞水平上确定细胞机制,已成为蛋白质组学研究的主要目标。目前,随着酵母[1]、果蝇[2]、线虫[3]相互作用图谱的相继完成,蛋白质相互作用研究方法也不断发展和完善。综述了当前研究蛋白质相互作用的主要技术方法,包括酵母双杂交技术,GSTpull-down技术,免疫共沉淀技术和串联亲和纯化技术等多种研究方法,分析了各种技术方法的优缺点以及各种方法的改进,在试验中可根据不同的要求和目的选择适宜的方法。     

酵母双杂交衍生系统

酵母双杂交系统是在酵母体内分析蛋白质蛋白质相互作用的基因系统,由Fields等人于1989年首次建立并得到广泛地应用。十余年来,随着酵母双杂交迅速推广,不断涌现出一些衍生系统,其中包括酵母双杂交的二元诱饵系统,逆向双杂交系统,非转录读出特点的双杂交系统(如Sos蛋白招募系统、PI3K介导的靶蛋白识别系统和断裂泛素为基础的双杂交系统)以及转录激活因子与其相关蛋白之间的相互作用的双杂交系统(如以polⅢ为基础的杂交系统和RTA系统)等。它们的建立在很大程度上克服了传统酵母双杂交系统的局限性,扩大了被研究的蛋白质的范围,提高了系统的灵敏度。     

Synthesis of cell-permeable stapled peptide dual inhibitors of the p53-Mdm2/Mdmx interactions via photoinduced cycloaddition

We report the first application of a photoinduced 1,3-dipolar cycloaddition reaction to ‘staple’ a peptide dual inhibitor of the p53-Mdm2/Mdmx interactions. A series of stapled peptide inhibitors were efficiently synthesized and showed excellent dual inhibitory activity in ELISA assay. Furthermore, the positively charged, stapled peptides showed enhanced cellular uptake along with modest in vivo activity.     

Extracellular matrix protein 1 interacts with the domain III of fibulin-1C and 1D variants through its central tandem repeat 2

Extracellular matrix protein 1 (ECM1), a widely expressed glycoprotein, has been shown to harbor mutations in lipoid proteinosis (LP), an autosomal recessive disorder characterized by profound alterations in the extracellular matrix of connective tissue. The biological function of ECM1 and its role in the pathomechanisms of LP are unknown. Fibulins comprise a family of extracellular matrix components, and the prototype of this family, fibulin-1, is expressed in various connective tissues and plays a role in developmental and pathologic processes. In this study, we demonstrate that ECM1, and specifically the second tandem repeat domain which is alternatively spliced, interacts with the C-terminal segments of fibulins 1C and 1D splice variants which differ in their C-terminal domain III. The interactions were detected by yeast two-hybrid genetic system and confirmed by co-immunoprecipitations. Kinetics of the binding between ECM1 and fibulin-1D, measured by biosensor assay, revealed a K(d) of 5.71 x 10(-8) M, indicating a strong protein-protein interaction. Since distinct splice variants of ECM1 and fibulin-1 have been shown to be co-expressed in tissues affected in LP, we propose that altered ECM1/fibulin-1 interactions may play a role in the pathogenesis of this disease as well as in a number of processes involving the extracellular matrix of connective tissues.     

Functional form of Caveolin-1 is necessary for the assembly of alpha-hemolysin

The assembly of alpha-HL was shown to rapidly progress upon its interaction with Caveolin-1. Treatment of A431 cells with alpha-HL has resulted in clustering of Caveolin-1 at cell-cell contacts. Consistent with this observation, alpha-HL mutants devoid of assembly property have not induced the clustering of Caveolin-1. While cholesterol depletion of A431 cells completely arrests the assembly of alpha-HL, chelation of membrane cholesterol results in its retarded assembly. Interestingly, HT29 cells, with low Caveolin-1 levels, are resistant to alpha-HL attack. Clustering of Caveolin-1, as seen in case of A431 cells, was readily observed in case of HT29 cells transfected with Caveolin-1 construct, thus overexpressing the full length Caveolin-1, upon alpha-HL treatment. A model was constructed to visualize the interactions between alpha-HL and Caveolin-1 which suggests that facile penetration of alpha-HL's beta-barrel might occur through protein-protein interactions with the surrounding 7 alpha-helices of Caveolin-1.     

Overrepresentation of interactions between homologous proteins in interactomes

It is well proved that the probability that a protein interacts with itself is higher than that it interacts with another protein. It has been recently shown that the probability of interaction is also higher for proteins with significant sequence similarity. In this paper we show that proteins sharing identical PFAM domains interact more often than expected by chance in Saccharomyces cerevisiae and Escherichia coli. We also analyze the variety of domain interfaces used by homologous proteins to interact and show that the overrepresentation of interactions between homological proteins is not caused by small number of pairs of identical "sticky domains" shared between interacting proteins.     

Identification of novel PDEδ interacting proteins

Prenylation is a post-translational modification that increases the affinity of proteins for membranes and mediates protein-protein interactions. The retinal rod rhodopsin-sensitive cGMP 3′,5′-cyclic phosphodiesterase subunit delta (PDEδ) is a prenyl binding protein that is essential for the shuttling of small GTPases between different membrane compartments and, thus, for their proper functioning. Although the prenylome comprises up to 2% of the mammalian proteome, only few prenylated proteins are known to interact with PDEδ. A proteome-wide approach was employed to map the PDEδ interactome among the prenylome and revealed RAB23, CDC42 and CNP as novel PDEδ interacting proteins. Moreover, PDEδ associates with the lamin A mutant progerin in a prenyl-dependent manner. These findings shed new light on the role of PDEδ in binding (and regulating) prenylated proteins in cells.