人FasL的研究进展

ＦａｓＬ（Ｆａｓ ｌｉｇａｎｄ）为Ⅱ型跨膜糖蛋白,属于肿瘤坏死因子（ｔｕｍｏｒ ｎｅｃｒｏｓｉｓ ｆａｃｔｏｒ,ＴＮＦ）家族成员,与其它成员包括ＴＮＦα、ＴＮＦβ、ＣＤ４０Ｌ、ＣＤ３７Ｌ、ＣＤ３０Ｌ等在结构上有一定的同源笥,但与这些成员（除ＴＮＦ外）诱导细胞分化,增殖的作用不同,ＦａｓＬ的主要生物学活性是诱导Ｆａｓ＾＋细胞的凋亡,因而在淋巴细胞的克隆选择,免疫耐受的形成以及一些疾病的发生过程中都有     

Predicting protein–protein interactions from protein sequences using meta predictor

A novel method is proposed for predicting protein–protein interactions (PPIs) based on the meta approach, which predicts PPIs using support vector machine that combines results by six independent state-of-the-art predictors. Significant improvement in prediction performance is observed, when performed on Saccharomyces cerevisiae and Helicobacter pylori datasets. In addition, we used the final prediction model trained on the PPIs dataset of S. cerevisiae to predict interactions in other species. The results reveal that our meta model is also capable of performing cross-species predictions. The source code and the datasets are available at     

FasL基因表达调控研究进展

FasL基因的表达由其启动子上不同的蛋白 DNA作用模式所控制 ,许多因子诸如NFAT、NFκ B、Egr、IRF、ICER可通过与其顺式元件的结合或与其它蛋白的协同作用来调节FasL的转录。这些因子的表达或活化又受到其它因子的影响 ,就控制FasL基因表达的相关研究作一简要综述。     

Phospho-tyrosine dependent protein–protein interaction network

Post-translational protein modifications, such as tyrosine phosphorylation, regulate protein–protein interactions (PPIs) critical for signal processing and cellular phenotypes. We extended an established yeast two-hybrid system employing human protein kinases for the analyses of phospho-tyrosine (pY)-dependent PPIs in a direct experimental, large-scale approach. We identified 292 mostly novel pY-dependent PPIs which showed high specificity with respect to kinases and interacting proteins and validated a large fraction in co-immunoprecipitation experiments from mammalian cells. About one-sixth of the interactions are mediated by known linear sequence binding motifs while the majority of pY-PPIs are mediated by other linear epitopes or governed by alternative recognition modes. Network analysis revealed that pY-mediated recognition events are tied to a highly connected protein module dedicated to signaling and cell growth pathways related to cancer. Using binding assays, protein complementation and phenotypic readouts to characterize the pY-dependent interactions of TSPAN2 (tetraspanin 2) and GRB2 or PIK3R3 (p55γ), we exemplarily provide evidence that the two pY-dependent PPIs dictate cellular cancer phenotypes.     

Scoring functions for protein–protein interactions

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Assessment and significance of protein–protein interactions during development of protein biopharmaceuticals

Early development of protein biotherapeutics using recombinant DNA technology involved progress in the areas of cloning, screening, expression and recovery/purification. As the biotechnology industry matured, resulting in marketed products, a greater emphasis was placed on development of formulations and delivery systems requiring a better understanding of the chemical and physical properties of newly developed protein drugs. Biophysical techniques such as analytical ultracentrifugation, dynamic and static light scattering, and circular dichroism were used to study protein–protein interactions during various stages of development of protein therapeutics. These studies included investigation of protein self-association in many of the early development projects including analysis of highly glycosylated proteins expressed in mammalian CHO cell cultures. Assessment of protein–protein interactions during development of an IgG1 monoclonal antibody that binds to IgE were important in understanding the pharmacokinetics and dosing for this important biotherapeutic used to treat severe allergic IgE-mediated asthma. These studies were extended to the investigation of monoclonal antibody–antigen interactions in human serum using the fluorescent detection system of the analytical ultracentrifuge. Analysis by sedimentation velocity analytical ultracentrifugation was also used to investigate competitive binding to monoclonal antibody targets. Recent development of high concentration protein formulations for subcutaneous administration of therapeutics posed challenges, which resulted in the use of dynamic and static light scattering, and preparative analytical ultracentrifugation to understand the self-association and rheological properties of concentrated monoclonal antibody solutions.     

PCS-based structure determination of protein–protein complexes

A simple and fast nuclear magnetic resonance method for docking proteins using pseudo-contact shift (PCS) and ¹H^N/¹⁵N chemical shift perturbation is presented. PCS is induced by a paramagnetic lanthanide ion that is attached to a target protein using a lanthanide binding peptide tag anchored at two points. PCS provides long-range (~40 Å) distance and angular restraints between the lanthanide ion and the observed nuclei, while the ¹H^N/¹⁵N chemical shift perturbation data provide loose contact-surface information. The usefulness of this method was demonstrated through the structure determination of the p62 PB1-PB1 complex, which forms a front-to-back 20 kDa homo-oligomer. As p62 PB1 does not intrinsically bind metal ions, the lanthanide binding peptide tag was attached to one subunit of the dimer at two anchoring points. Each monomer was treated as a rigid body and was docked based on the backbone PCS and backbone chemical shift perturbation data. Unlike NOE-based structural determination, this method only requires resonance assignments of the backbone ¹H^N/¹⁵N signals and the PCS data obtained from several sets of two-dimensional ¹⁵N-heteronuclear single quantum coherence spectra, thus facilitating rapid structure determination of the protein–protein complex.     

Structure-based method for analyzing protein–protein interfaces

Hydrogen bond, hydrophobic and vdW interactions are the three major non-covalent interactions at protein–protein interfaces. We have developed a method that uses only these properties to describe interactions between proteins, which can qualitatively estimate the individual contribution of each interfacial residue to the binding and gives the results in a graphic display way. This method has been applied to analyze alanine mutation data at protein–protein interfaces. A dataset containing 13 protein–protein complexes with 250 alanine mutations of interfacial residues has been tested. For the 75 hot-spot residues (G1.5 kcal mol^-1), 66 can be predicted correctly with a success rate of 88%. In order to test the tolerance of this method to conformational changes upon binding, we utilize a set of 26 complexes with one or both of their components available in the unbound form. The difference of key residues exported by the program is 11% between the results using complexed proteins and those from unbound ones. As this method gives the characteristics of the binding partner for a particular protein, in-depth studies on protein–protein recognition can be carried out. Furthermore, this method can be used to compare the difference between protein–protein interactions and look for correlated mutation. Figure Key interaction grids at the interface between barnase and barstar. Key interaction grid for barnase and barstar are presented in one figure according to their coordinates. In order to distinguish the two proteins, different icons were assigned. Crosses represent key grids for barstar and dots represent key grids for barnase. The four residues in ball and stick are Asp40 in barstar and Arg83, Arg87, His102 in barnase.     

Molecular glues to stabilise protein–protein interactions

Targeting protein–protein interactions (PPIs) has become a common approach to tackle various diseases whose pathobiology is driven by their mis-regulation in important signalling pathways. Modulating PPIs has tremendous untapped therapeutic potential and different approaches can be used to modulate PPIs. Initially, therapeutic effects were mostly sought by inhibiting PPIs. However, by gaining insight in the mode of action of certain therapeutic compounds, it became clear that stabilising (i.e. enhancing) PPIs can also be useful. The latter strategy is recently gaining a lot of attention, as stabilising physiologic, or even inducing novel interactions of a target protein with E3 ubiquitin ligases forms the basis of the targeted protein degradation (TPD) approach. An emerging additional example for drug discovery based on PPI stabilisation are the 14-3-3 proteins, a family of regulatory proteins, which engages in many protein–protein interactions, some of which might become therapeutical targets.     

Transient protein–protein complexes in base excision repair

Abstract

Transient protein–protein complexes are of great importance for organizing multiple enzymatic reactions into productive reaction pathways. Base excision repair (BER), a process of critical importance for maintaining genome stability against a plethora of DNA-damaging factors, involves several enzymes, including DNA glycosylases, AP endonucleases, DNA polymerases, DNA ligases and accessory proteins acting sequentially on the same damaged site in DNA. Rather than being assembled into one stable multisubunit complex, these enzymes pass the repair intermediates between them in a highly coordinated manner. In this review, we discuss the nature and the role of transient complexes arising during BER as deduced from structural and kinetic data. Almost all of the transient complexes are DNA-mediated, although some may also exist in solution and strengthen under specific conditions. The best-studied example, the interactions between DNA glycosylases and AP endonucleases, is discussed in more detail to provide a framework for distinguishing between stable and transient complexes based on the kinetic data.

Communicated by Ramaswamy H. Sarma     

Tracking a protein following dissociation from a protein–protein complex using a split SNAP-tag system

Protein–protein interactions (PPIs) are important for various biological processes in living cells. Several methods have been developed for the visualization of PPIs in vivo; however, these methods are unsuitable for visualization of post-PPI events such as dissociation and translocation. In this study, we applied a split SNAP-tag system for the visualization of post-PPI events. This method enabled tracking of the protein following dissociation from the protein–protein complex. Thus, the split SNAP-tag system should prove to be a useful tool for visualization of post-PPI events.     

Fas与FasL的分子生物学研究进展

Fas是细胞表面诱导凋亡的分子,是I型膜蛋白,属TNF受体家族成员。而Fas配体（FasL)是Ⅱ型膜蛋白,属TNF家族成员。Fas与FasL结合,可向细胞传递死亡信号,引发细胞凋亡。Fas在人体中表达及功能正常与否,对人体免疫调控起重要作用。     

FasL在睾丸细胞的定位

FasL/Fas系统介导的胞外信号凋亡途径是哺乳动物睾丸生殖细胞凋亡的一条主要途径,然而,关于FasL在睾丸细胞中的定位却存在争议。本文对近年来国内外关于FasL在睾丸中的细胞定位研究进行了综述,为阐明FasL/Fas系统介导生殖细胞凋亡的机制提供资料,对深入理解睾丸中Sertoli细胞和生殖细胞间的调控关系及临床实践具有一定的指导作用。     

Direct molecular fishing in molecular partners investigation in protein–protein and protein–peptide interactions

An original experimental method of direct molecular fishing has been developed for identification of potential partners of protein–protein and protein–peptide interactions. It is based on combination of surface plasmon resonance technology (SPR), size exclusion and affinity chromatography and mass spectrometric identification of proteins (LC-MS/MS). Previously, we demonstrated applicability of this method for protein interactomics using experimental model system, as well as in the pilot study in the frame of the Human Proteome Project (HPP). In the present paper, this method was successfully applied to identify possible molecular partners of 7 target proteins encoded by genes of 18 chromosome (also in the frame of the HPP). Fishing on the affinity sorbents with immobilized target proteins as ligands was carried out using total lysate of human liver tissue as well as pooled sets of fractions (individual for each bait-protein) obtained by means of a combination of size exclusion chromatography and SPR analysis for the presence of potential prey-proteins in each fraction. As a result we obtained lists of possible molecular partners of all 7 proteins and performed a comparative evaluation of direct fishing specificity for these target proteins. Direct molecular fishing was also successfully used for search of potential protein partners interacting with different isoforms of amyloid-beta peptide, playing a key role in the development of Alzheimer’s disease. The synthetic peptides that are analogues of the metal-binding domain isoforms of beta-amyloid were used as molecular baits and the fishing was performed in various fractions of immortalized human neural cells. As a result, 13 potential partner proteins were identified in the cytosol fraction of the cells by fishing on amyloid-beta peptide (1-16).