蛋白质相互作用网络进化分析研究进展

近年来,随着高通量实验技术的发展和广泛应用,越来越多可利用的蛋白质相互作用网络数据开始出现．这些数据为进化研究提供了新的视角．从蛋白质、蛋白质相互作用、模体、模块直到整个网络五个层次,综述了近年来蛋白质相互作用网络进化研究领域的主要进展,侧重于探讨蛋白质相互作用、模体、模块直到整个网络对蛋白质进化的约束作用,以及蛋白质相互作用网络不同于随机网络特性的起源和进化等问题．总结了前人工作给学术界的启示,探讨了该领域未来可能的发展方向．     

用于蛋白质分子设计的三维模体搜索

三维结构模体,常是蛋白质中离散残基片断组成的功能性子结构,传统的序列依赖结构比较方法无法地其搜索,本文采用序列无关的结构比较算法,实现了搜索程序,经多例测试,证明本程序是快速和良好的识别三维结构模式的工具,在小分子设计和多肽模拟中有重要的应用。     

蛋白质相互作用的生物信息学研究进展

生命过程的分子基础在于生物分子之间的相互作用,其中蛋白质分子之间的相互作用占有极其重要的地位。研究蛋白质相互作用对于理解生命的真谛、探讨致病微生物的致病机理,以及研究新药提高人们的健康水平具有重要的作用。用生物信息学的方法研究蛋白质的相互作用已经取得了许多重要的成果,但也有很多问题还需解决。本文从蛋白质相互作用的数据库、预测方法、可预测蛋白质相互作用的网上服务、蛋白质相互作用网络等几方面,对蛋白质相互作用的生物信息学研究成果及其存在的问题做了概述。     

锚蛋白重复序列介导的蛋白质-蛋白质相互作用

锚蛋白重复序列(ANK)是生物体中广泛利用的一种序列模体.ANK模体在ANK结构域中折叠成β₂α₂结构,在空间上则形成L型结构.数目不等的ANK串联起来, 依靠氢键和疏水相互作用,组成紧密、稳定的结构域,并且形成了种类众多但功能各异的ANK蛋白质分子.ANK结构域介导蛋白质与蛋白质的相互作用,它能够和多种配体结合,实现纷繁复杂的生物功能.着重介绍几类结构已知的ANK家族蛋白质分子及复合物的结构特征、生理功能及与疾病的关系.     

TPR基序——一种介导蛋白质相互作用的结构域

ＴＰＲ蛋白家庭成员功能复杂,均含有ＴＰＲ基序。该基序是一个含有３４个氨基酸的重复序列,通常串行排列。通过形成特殊的空间结构,介导蛋白质的相互作用,并在一些重要的蛋白复合物形成中非常重要。本文拟就ＴＰＲ基序的结构及功能作一简要综述。     

用于蛋白质分子设计的三维模体搜索

三维结构模体,常是蛋白质中离散残基片断组成的功能性子结构,传统的序列依赖结构比较方法无法对其搜索。本文采用序列无关的结构比较算法,实现了搜索程序。经多例测试,证明本程序是快速和良好的识别三维结构模式的工具,在小分子设计和多肽模拟中有重要的应用     

用于蛋白质间相互作用研究的新型双杂交系统

近年来,一些不依赖于转录因子活性的新型双杂交系统相继建立,如分离的泛素系统、蛋白质片段互补分析、阻遏物重构分析和SOS恢复系统等。同利用转录因子活性的酵母双杂交系统相似,这些方法也利用了一些活性蛋白的结构与功能特点来研究蛋白质间相互作用,这些活性蛋白不是转录因子,但也可在结构上进行分离可通过重构使其生物活性得以恢复。由于这些新型双杂交系统的各自特点,使得它们成为酵母双杂交系统的有益补充和研究蛋白质间相互作用的有力工具。     

蛋白质相互作用的研究方法

蛋白质相互作用的研究是现代分子生物学研究领域一个很重要的方面。该介绍了经典的和最近新建立的研究蛋白质相互作用的方法,并比较了各种方法的优缺点和所适用的领域。     

基于结构比较的蛋白质模建系统及其评估：Ⅲ.灵敏的蛋…

本文按二级结构、疏水性和侧链氢链把蛋白质的局部结构环境划分为６４类,按环境依赖的残基替代频数表构成残基与环境的兼容性分数表,可用来评估一个蛋白质的整体折叠正确与否和检测蛋白质折叠的局部错误。     

福鼎大白茶蛋白质相互作用网络研究

蛋白质相互作用网络的构建可以为探究茶树生长过程中的关键蛋白并预测其功能提供理论参考。以贵州都匀地区福鼎大白茶为研究对象,利用三代Nanopore测序技术和同源比对方法构建福鼎大白茶根、茎、叶差异基因蛋白质相互作用网络,通过网络进一步预测关键蛋白及其功能。结果表明,叶与根、叶与茎、茎与根和根茎叶差异基因蛋白质相互作用网络中的关键蛋白分别为53、39、42和53个,并且预测出了关键蛋白的功能,如TEA003744的功能可能为腺苷酸激酶活性、蛋白质丝氨酸/苏氨酸激酶活性和ATP结合,参与光合作用和蛋白质磷酸化过程;TEA026776可能为发育蛋白,参与细胞分化过程和蛋白质磷酸化过程,还具有ATP结合活性和蛋白激酶活性;TEA019056的功能可能为ATP结合、GTP结合和GTP酶活性,参与过氧化物酶体组织的组成和蛋白质磷酸化等。随后预测出4个网络中打分最高功能模块的功能,并进行拓扑属性分析、功能模块分析、集群注释和聚类分析。研究结果可为鉴定蛋白质的功能、寻找关键蛋白及选育优良品种等提供理论依据。     

Hidden Multivalency in Phosphatase Recruitment by a Disordered AKAP Scaffold

Disordered scaffold proteins provide multivalent landing pads that, via a series of embedded Short Linear Motifs (SLiMs), bring together the components of a complex to orchestrate precise spatial and temporal regulation of cellular processes. One such protein is AKAP5 (previously AKAP79), which contains SLiMs that anchor PKA and Calcineurin, and recruit substrate (the TRPV1 receptor). Calcineurin is anchored to AKAP5 by a well-characterised PxIxIT SLiM. Here we show, using a combination of biochemical and biophysical approaches, that the Calcineurin PxIxIT-binding groove also recognises several hitherto unknown lower-affinity SLiMs in addition to the PxIxIT motif. We demonstrate that the assembly is in reality a complex system with conserved SLiMs spanning a wide affinity range. The capture is analogous to that seen for many DNA-binding proteins that have a weak non-specific affinity for DNA outside the canonical binding site, but different in that it involves (i) two proteins, and (ii) hydrophobic rather than electrostatic interactions. It is also compatible with the requirement for both stable anchoring of the enzyme and responsive downstream signalling. We conclude that the AKAP5 C-terminus is enriched in lower-affinity/mini-SLiMs that, together with the canonical SLiM, maintain a structurally disordered but tightly regulated signalosome.     

Biochemical and Structural Characterization of the Interaction between the Siderocalin NGAL/LCN2 (Neutrophil Gelatinase-associated Lipocalin/Lipocalin 2) and the N-terminal Domain of Its Endocytic Receptor SLC22A17

The neutrophil gelatinase-associated lipocalin (NGAL, also known as LCN2) and its cellular receptor (LCN2-R, SLC22A17) are involved in many physiological and pathological processes such as cell differentiation, apoptosis, and inflammation. These pleiotropic functions mainly rely on NGAL''s siderophore-mediated iron transport properties. However, the molecular determinants underlying the interaction between NGAL and its cellular receptor remain largely unknown. Here, using solution-state biomolecular NMR in conjunction with other biophysical methods, we show that the N-terminal domain of LCN2-R is a soluble extracellular domain that is intrinsically disordered and interacts with NGAL preferentially in its apo state to form a fuzzy complex. The relatively weak affinity (≈10 μm) between human LCN2-R-NTD and apoNGAL suggests that the N terminus on its own cannot account for the internalization of NGAL by LCN2-R. However, human LCN2-R-NTD could be involved in the fine-tuning of the interaction between NGAL and its cellular receptor or in a biochemical mechanism allowing the receptor to discriminate between apo- and holo-NGAL.     

Interaction between Intrinsically Disordered Proteins Frequently Occurs in a Human Protein-Protein Interaction Network

Intrinsic protein disorder is a widespread phenomenon characterised by a lack of stable three-dimensional structures and is considered to play an important role in protein-protein interactions (PPIs). This study examined the genome-wide preference of disorder in PPIs by using exhaustive disorder prediction in human PPIs. We categorised the PPIs into three types (interaction between disordered proteins, interaction between structured proteins, and interaction between a disordered protein and a structured protein) with regard to the flexibility of molecular recognition and compared these three interaction types in an existing human PPI network with those in a randomised network. Although the structured regions were expected to become the identifiers for binding recognition, this comparative analysis revealed unexpected results. The occurrence of interactions between disordered proteins was significantly frequent, and that between a disordered protein and a structured protein was significantly infrequent. We found that this propensity was much stronger in interactions between nonhub proteins. We also analysed the interaction types from a functional standpoint by using GO, which revealed that the interaction between disordered proteins frequently occurred in cellular processes, regulation, and metabolic processes. The number of interactions, especially in metabolic processes between disordered proteins, was 1.8 times as large as that in the randomised network. Another analysis conducted by using KEGG pathways provided results where several signaling pathways and disease-related pathways included many interactions between disordered proteins. All of these analyses suggest that human PPIs preferably occur between disordered proteins and that the flexibility of the interacting protein pairs may play an important role in human PPI networks.     

A decade and a half of protein intrinsic disorder: Biology still waits for physics

The abundant existence of proteins and regions that possess specific functions without being uniquely folded into unique 3D structures has become accepted by a significant number of protein scientists. Sequences of these intrinsically disordered proteins (IDPs) and IDP regions (IDPRs) are characterized by a number of specific features, such as low overall hydrophobicity and high net charge which makes these proteins predictable. IDPs/IDPRs possess large hydrodynamic volumes, low contents of ordered secondary structure, and are characterized by high structural heterogeneity. They are very flexible, but some may undergo disorder to order transitions in the presence of natural ligands. The degree of these structural rearrangements varies over a very wide range. IDPs/IDPRs are tightly controlled under the normal conditions and have numerous specific functions that complement functions of ordered proteins and domains. When lacking proper control, they have multiple roles in pathogenesis of various human diseases. Gaining structural and functional information about these proteins is a challenge, since they do not typically “freeze” while their “pictures are taken.” However, despite or perhaps because of the experimental challenges, these fuzzy objects with fuzzy structures and fuzzy functions are among the most interesting targets for modern protein research. This review briefly summarizes some of the recent advances in this exciting field and considers some of the basic lessons learned from the analysis of physics, chemistry, and biology of IDPs.     

Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

Membrane proteins are essential for cell viability and are therefore important therapeutic targets^1-3. Since they function in complexes⁴, methods to identify and characterize their interactions are necessary⁵. To this end, we developed the Membrane Strep-protein interaction experiment, called Membrane-SPINE⁶. This technique combines in vivo cross-linking using the reversible cross-linker formaldehyde with affinity purification of a Strep-tagged membrane bait protein. During the procedure, cross-linked prey proteins are co-purified with the membrane bait protein and subsequently separated by boiling. Hence, two major tasks can be executed when analyzing protein-protein interactions (PPIs) of membrane proteins using Membrane-SPINE: first, the confirmation of a proposed interaction partner by immunoblotting, and second, the identification of new interaction partners by mass spectrometry analysis. Moreover, even low affinity, transient PPIs are detectable by this technique. Finally, Membrane-SPINE is adaptable to almost any cell type, making it applicable as a powerful screening tool to identify PPIs of membrane proteins.     

Tubulin Tail Sequences and Post-translational Modifications Regulate Closure of Mitochondrial Voltage-dependent Anion Channel (VDAC)

It was previously shown that tubulin dimer interaction with the mitochondrial outer membrane protein voltage-dependent anion channel (VDAC) blocks traffic through the channel and reduces oxidative metabolism and that this requires the unstructured anionic C-terminal tail peptides found on both α- and β-tubulin subunits. It was unclear whether the α- and β-tubulin tails contribute equally to VDAC blockade and what effects might be due to sequence variations in these tail peptides or to tubulin post-translational modifications, which mostly occur on the tails. The nature of the contribution of the tubulin body beyond acting as an anchor for the tails had not been clarified either. Here we present peptide-protein chimeras to address these questions. These constructs allow us to easily combine a tail peptide with different proteins or combine different tail peptides with a particular protein. The results show that a single tail grafted to an inert protein is sufficient to produce channel closure similar to that observed with tubulin. We show that the β-tail is more than an order of magnitude more potent than the α-tail and that the lower α-tail activity is largely due to the presence of a terminal tyrosine. Detyrosination activates the α-tail, and activation is reversed by the removal of the glutamic acid penultimate to the tyrosine. Nitration of tyrosine reverses the tyrosine inhibition of binding and even induces prolonged VDAC closures. Our results demonstrate that small changes in sequence or post-translational modification of the unstructured tails of tubulin result in substantial changes in VDAC closure.     

A weakened interface in the P182L variant of HSP27 associated with severe Charcot‐Marie‐Tooth neuropathy causes aberrant binding to interacting proteins

HSP27 is a human molecular chaperone that forms large, dynamic oligomers and functions in many aspects of cellular homeostasis. Mutations in HSP27 cause Charcot‐Marie‐Tooth (CMT) disease, the most common inherited disorder of the peripheral nervous system. A particularly severe form of CMT disease is triggered by the P182L mutation in the highly conserved IxI/V motif of the disordered C‐terminal region, which interacts weakly with the structured core domain of HSP27. Here, we observed that the P182L mutation disrupts the chaperone activity and significantly increases the size of HSP27 oligomers formed in vivo, including in motor neurons differentiated from CMT patient‐derived stem cells. Using NMR spectroscopy, we determined that the P182L mutation decreases the affinity of the HSP27 IxI/V motif for its own core domain, leaving this binding site more accessible for other IxI/V‐containing proteins. We identified multiple IxI/V‐bearing proteins that bind with higher affinity to the P182L variant due to the increased availability of the IxI/V‐binding site. Our results provide a mechanistic basis for the impact of the P182L mutation on HSP27 and suggest that the IxI/V motif plays an important, regulatory role in modulating protein–protein interactions.     

Exploring the binding diversity of intrinsically disordered proteins involved in one‐to‐many binding

Molecular recognition features (MoRFs) are intrinsically disordered protein regions that bind to partners via disorder‐to‐order transitions. In one‐to‐many binding, a single MoRF binds to two or more different partners individually. MoRF‐based one‐to‐many protein–protein interaction (PPI) examples were collected from the Protein Data Bank, yielding 23 MoRFs bound to 2–9 partners, with all pairs of same‐MoRF partners having less than 25% sequence identity. Of these, 8 MoRFs were bound to 2–9 partners having completely different folds, whereas 15 MoRFs were bound to 2–5 partners having the same folds but with low sequence identities. For both types of partner variation, backbone and side chain torsion angle rotations were used to bring about the conformational changes needed to enable close fits between a single MoRF and distinct partners. Alternative splicing events (ASEs) and posttranslational modifications (PTMs) were also found to contribute to distinct partner binding. Because ASEs and PTMs both commonly occur in disordered regions, and because both ASEs and PTMs are often tissue‐specific, these data suggest that MoRFs, ASEs, and PTMs may collaborate to alter PPI networks in different cell types. These data enlarge the set of carefully studied MoRFs that use inherent flexibility and that also use ASE‐based and/or PTM‐based surface modifications to enable the same disordered segment to selectively associate with two or more partners. The small number of residues involved in MoRFs and in their modifications by ASEs or PTMs may simplify the evolvability of signaling network diversity.     

Resilience of death: intrinsic disorder in proteins involved in the programmed cell death

It is recognized now that intrinsically disordered proteins (IDPs), which do not have unique 3D structures as a whole or in noticeable parts, constitute a significant fraction of any given proteome. IDPs are characterized by an astonishing structural and functional diversity that defines their ability to be universal regulators of various cellular pathways. Programmed cell death (PCD) is one of the most intricate cellular processes where the cell uses specialized cellular machinery and intracellular programs to kill itself. This cell-suicide mechanism enables metazoans to control cell numbers and to eliminate cells that threaten the animal''s survival. PCD includes several specific modules, such as apoptosis, autophagy, and programmed necrosis (necroptosis). These modules are not only tightly regulated but also intimately interconnected and are jointly controlled via a complex set of protein–protein interactions. To understand the role of the intrinsic disorder in controlling and regulating the PCD, several large sets of PCD-related proteins across 28 species were analyzed using a wide array of modern bioinformatics tools. This study indicates that the intrinsic disorder phenomenon has to be taken into consideration to generate a complete picture of the interconnected processes, pathways, and modules that determine the essence of the PCD. We demonstrate that proteins involved in regulation and execution of PCD possess substantial amount of intrinsic disorder. We annotate functional roles of disorder across and within apoptosis, autophagy, and necroptosis processes. Disordered regions are shown to be implemented in a number of crucial functions, such as protein–protein interactions, interactions with other partners including nucleic acids and other ligands, are enriched in post-translational modification sites, and are characterized by specific evolutionary patterns. We mapped the disorder into an integrated network of PCD pathways and into the interactomes of selected proteins that are involved in the p53-mediated apoptotic signaling pathway.     

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification (TAP)

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. However, the use of biochemical screens aimed at extending the knowledge gained from genetic analysis was explored only recently. Here we describe a method to purify the protein complex that associates with any protein of interest from adult fly heads. This method takes advantage of the Drosophila GAL4/UAS system to express a bait protein fused with a Tandem Affinity Purification (TAP) tag in fly neurons in vivo, and then implements two rounds of purification using a TAP procedure similar to the one originally established in yeast¹ to purify the interacting protein complex. At the end of this procedure, a mixture of multiple protein complexes is obtained whose molecular identities can be determined by mass spectrometry. Validation of the candidate proteins will benefit from the resource and ease of performing loss-of-function studies in flies. Similar approaches can be applied to other fly tissues. We believe that the combination of genetic manipulations and this proteomic approach in the fly model system holds tremendous potential for tackling fundamental problems in the field of neurobiology and beyond.