Interplay between Chaperones and Protein Disorder Promotes the Evolution of Protein Networks

Evolution is driven by mutations, which lead to new protein functions but come at a cost to protein stability. Non-conservative substitutions are of interest in this regard because they may most profoundly affect both function and stability. Accordingly, organisms must balance the benefit of accepting advantageous substitutions with the possible cost of deleterious effects on protein folding and stability. We here examine factors that systematically promote non-conservative mutations at the proteome level. Intrinsically disordered regions in proteins play pivotal roles in protein interactions, but many questions regarding their evolution remain unanswered. Similarly, whether and how molecular chaperones, which have been shown to buffer destabilizing mutations in individual proteins, generally provide robustness during proteome evolution remains unclear. To this end, we introduce an evolutionary parameter λ that directly estimates the rate of non-conservative substitutions. Our analysis of λ in Escherichia coli, Saccharomyces cerevisiae, and Homo sapiens sequences reveals how co- and post-translationally acting chaperones differentially promote non-conservative substitutions in their substrates, likely through buffering of their destabilizing effects. We further find that λ serves well to quantify the evolution of intrinsically disordered proteins even though the unstructured, thus generally variable regions in proteins are often flanked by very conserved sequences. Crucially, we show that both intrinsically disordered proteins and highly re-wired proteins in protein interaction networks, which have evolved new interactions and functions, exhibit a higher λ at the expense of enhanced chaperone assistance. Our findings thus highlight an intricate interplay of molecular chaperones and protein disorder in the evolvability of protein networks. Our results illuminate the role of chaperones in enabling protein evolution, and underline the importance of the cellular context and integrated approaches for understanding proteome evolution. We feel that the development of λ may be a valuable addition to the toolbox applied to understand the molecular basis of evolution.     

Building protein-protein interaction networks with proteomics and informatics tools

The systematic characterization of the whole interactomes of different model organisms has revealed that the eukaryotic proteome is highly interconnected. Therefore, biological research is progressively shifting away from classical approaches that focus only on a few proteins toward whole protein interaction networks to describe the relationship of proteins in biological processes. In this minireview, we survey the most common methods for the systematic identification of protein interactions and exemplify different strategies for the generation of protein interaction networks. In particular, we will focus on the recent development of protein interaction networks derived from quantitative proteomics data sets.     

Distinct Types of Disorder in the Human Proteome: Functional Implications for Alternative Splicing

Intrinsically disordered regions have been associated with various cellular processes and are implicated in several human diseases, but their exact roles remain unclear. We previously defined two classes of conserved disordered regions in budding yeast, referred to as “flexible” and “constrained” conserved disorder. In flexible disorder, the property of disorder has been positionally conserved during evolution, whereas in constrained disorder, both the amino acid sequence and the property of disorder have been conserved. Here, we show that flexible and constrained disorder are widespread in the human proteome, and are particularly common in proteins with regulatory functions. Both classes of disordered sequences are highly enriched in regions of proteins that undergo tissue-specific (TS) alternative splicing (AS), but not in regions of proteins that undergo general (i.e., not tissue-regulated) AS. Flexible disorder is more highly enriched in TS alternative exons, whereas constrained disorder is more highly enriched in exons that flank TS alternative exons. These latter regions are also significantly more enriched in potential phosphosites and other short linear motifs associated with cell signaling. We further show that cancer driver mutations are significantly enriched in regions of proteins associated with TS and general AS. Collectively, our results point to distinct roles for TS alternative exons and flanking exons in the dynamic regulation of protein interaction networks in response to signaling activity, and they further suggest that alternatively spliced regions of proteins are often functionally altered by mutations responsible for cancer.     

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.     

Simple Topological Features Reflect Dynamics and Modularity in Protein Interaction Networks

The availability of large-scale protein-protein interaction networks for numerous organisms provides an opportunity to comprehensively analyze whether simple properties of proteins are predictive of the roles they play in the functional organization of the cell. We begin by re-examining an influential but controversial characterization of the dynamic modularity of the S. cerevisiae interactome that incorporated gene expression data into network analysis. We analyse the protein-protein interaction networks of five organisms, S. cerevisiae, H. sapiens, D. melanogaster, A. thaliana, and E. coli, and confirm significant and consistent functional and structural differences between hub proteins that are co-expressed with their interacting partners and those that are not, and support the view that the former tend to be intramodular whereas the latter tend to be intermodular. However, we also demonstrate that in each of these organisms, simple topological measures are significantly correlated with the average co-expression of a hub with its partners, independent of any classification, and therefore also reflect protein intra- and inter- modularity. Further, cross-interactomic analysis demonstrates that these simple topological characteristics of hub proteins tend to be conserved across organisms. Overall, we give evidence that purely topological features of static interaction networks reflect aspects of the dynamics and modularity of interactomes as well as previous measures incorporating expression data, and are a powerful means for understanding the dynamic roles of hubs in interactomes.     

Comparative interactomics analysis of protein family interaction networks using PSIMAP (protein structural interactome map)

MOTIVATION: Many genomes have been completely sequenced. However, detecting and analyzing their protein-protein interactions by experimental methods such as co-immunoprecipitation, tandem affinity purification and Y2H is not as fast as genome sequencing. Therefore, a computational prediction method based on the known protein structural interactions will be useful to analyze large-scale protein-protein interaction rules within and among complete genomes. RESULTS: We confirmed that all the predicted protein family interactomes (the full set of protein family interactions within a proteome) of 146 species are scale-free networks, and they share a small core network comprising 36 protein families related to indispensable cellular functions. We found two fundamental differences among prokaryotic and eukaryotic interactomes: (1) eukarya had significantly more hub families than archaea and bacteria and (2) certain special hub families determined the topology of the eukaryotic interactomes. Our comparative analysis suggests that a very small number of expansive protein families led to the evolution of interactomes and seemed to have played a key role in species diversification. SUPPLEMENTARY INFORMATION: http://interactomics.org.     

Dynein light chain LC8 is a dimerization hub essential in diverse protein networks

The operations within a living cell depend on the collective activity of networks of proteins, sometimes termed "interactomes". Within these networks, most proteins interact with few partners, while a small proportion of proteins, called hubs, participate in a large number of interactions and play a central role in organizing these interactomes. LC8 was first discovered as an essential component of the microtubule-based molecular motor dynein and as such is involved in fundamental processes, including retrograde vesicular trafficking, ciliary/flagellar motility, and cell division. More recently, evidence has accumulated that LC8 also interacts with proteins that are not clearly connected with dynein or microtubule-based transport, including some with roles in apoptosis, viral pathogenesis, enzyme regulation, and kidney development. Here, we introduce the idea that LC8 is a hub protein essential in diverse protein networks, and its function as a dynein light chain is but one of many. We further propose that the crucial regulatory roles of LC8 in various systems are due to its ability to promote dimerization of partially disordered proteins.     

Evolutionarily Conserved Herpesviral Protein Interaction Networks

Herpesviruses constitute a family of large DNA viruses widely spread in vertebrates and causing a variety of different diseases. They possess dsDNA genomes ranging from 120 to 240 kbp encoding between 70 to 170 open reading frames. We previously reported the protein interaction networks of two herpesviruses, varicella-zoster virus (VZV) and Kaposi''s sarcoma-associated herpesvirus (KSHV). In this study, we systematically tested three additional herpesvirus species, herpes simplex virus 1 (HSV-1), murine cytomegalovirus and Epstein-Barr virus, for protein interactions in order to be able to perform a comparative analysis of all three herpesvirus subfamilies. We identified 735 interactions by genome-wide yeast-two-hybrid screens (Y2H), and, together with the interactomes of VZV and KSHV, included a total of 1,007 intraviral protein interactions in the analysis. Whereas a large number of interactions have not been reported previously, we were able to identify a core set of highly conserved protein interactions, like the interaction between HSV-1 UL33 with the nuclear egress proteins UL31/UL34. Interactions were conserved between orthologous proteins despite generally low sequence similarity, suggesting that function may be more conserved than sequence. By combining interactomes of different species we were able to systematically address the low coverage of the Y2H system and to extract biologically relevant interactions which were not evident from single species.     

Protein Disorder and Short Conserved Motifs in Disordered Regions Are Enriched near the Cytoplasmic Side of Single-Pass Transmembrane Proteins

Intracellular juxtamembrane regions of transmembrane proteins play pivotal roles in cell signalling, mediated by protein-protein interactions. Disordered protein regions, and short conserved motifs within them, are emerging as key determinants of many such interactions. Here, we investigated whether disorder and conserved motifs are enriched in the juxtamembrane area of human single-pass transmembrane proteins. Conserved motifs were defined as short disordered regions that were much more conserved than the adjacent disordered residues. Human single-pass proteins had higher mean disorder in their cytoplasmic segments than their extracellular parts. Some, but not all, of this effect reflected the shorter length of the cytoplasmic tail. A peak of cytoplasmic disorder was seen at around 30 residues from the membrane. We noted a significant increase in the incidence of conserved motifs within the disordered regions at the same location, even after correcting for the extent of disorder. We conclude that elevated disorder within the cytoplasmic tail of many transmembrane proteins is likely to be associated with enrichment for signalling interactions mediated by conserved short motifs.     

Methods for mapping of interaction networks involving membrane proteins

Nearly one-third of all genes in various organisms encode membrane-associated proteins that participate in numerous protein-protein interactions important to the processes of life. However, membrane protein interactions pose significant challenges due to the need to solubilize membranes without disrupting protein-protein interactions. Traditionally, analysis of isolated protein complexes by high-resolution 2D gel electrophoresis has been the main method used to obtain an overall picture of proteome constituents and interactions. However, this method is time consuming, labor intensive, detects only abundant proteins and is limited with respect to the coverage required to elucidate large interaction networks. In this review, we discuss the application of various methods to elucidate interactions involving membrane proteins. These techniques include methods for the direct isolation of single complexes or interactors as well as methods for characterization of entire subcellular and cellular interactomes.     

Correlation of disorder between S. cerevisiae interacting proteins

Protein disorder has been frequently associated with protein-protein interaction. However, our knowledge of how protein disorder evolves within a network is limited. It is expected that physically interacting proteins evolve in a coordinated manner. This has so far been shown in their evolutionary rate, and in their gene expression levels. Here we examine the percentage of predicted disorder residues within binary and complex interacting proteins (physical and functional interactions respectively) to investigate how the disorder of a protein relates to that of its interacting partners. We show that the level of disorder of interacting proteins are correlated, with a greater correlation seen among proteins that are co-members of the same complex, and a lesser correlation between proteins that are documented as binary interactors of each other. There is a striking variation among complexes not only in their disorder, but in the extent to which the proteins within the complex differ in their levels of disorder, with RNA processes and protein binding complexes showing more variation in the disorder of their proteins, whilst other complexes show very little variation in the overall disorder of their constituent proteins. There is likely to be a stronger selection for complex subunits to have similar disorder, than is seen for proteins involved in binary interactions. Thus, binary interactions may be more resilient to changes in disorder than are complex interactions. These results add a new dimension to the role of disorder in protein networks, and highlight the potential importance of maintaining similar disorder in the members of a complex.     

The impact of gene expression regulation on evolution of extracellular signaling pathways

Extracellular protein interactions are crucial to the development of multicellular organisms because they initiate signaling pathways and enable cellular recognition cues. Despite their importance, extracellular protein interactions are often under-represented in large scale protein interaction data sets because most high throughput assays are not designed to detect low affinity extracellular interactions. Due to the lack of a comprehensive data set, the evolution of extracellular signaling pathways has remained largely a mystery. We investigated this question using a combined data set of physical pairwise interactions between zebrafish extracellular proteins, mainly from the immunoglobulin superfamily and leucine-rich repeat families, and their spatiotemporal expression profiles. We took advantage of known homology between proteins to estimate the relative rates of changes of four parameters after gene duplication, namely extracellular protein interaction, expression pattern, and the divergence of extracellular and intracellular protein sequences. We showed that change in expression profile is a major contributor to the evolution of signaling pathways followed by divergence in intracellular protein sequence, whereas extracellular sequence and interaction profiles were relatively more conserved. Rapidly evolving expression profiles will eventually drive other parameters to diverge more quickly because differentially expressed proteins get exposed to different environments and potential binding partners. This allows homologous extracellular receptors to attain specialized functions and become specific to tissues and/or developmental stages.     

Disorder and sequence repeats in hub proteins and their implications for network evolution

Protein interaction networks display approximate scale-free topology, in which hub proteins that interact with a large number of other proteins determine the overall organization of the network. In this study, we aim to determine whether hubs are distinguishable from other networked proteins by specific sequence features. Proteins of different connectednesses were compared in the interaction networks of Saccharomyces cerevisiae, Drosophila melanogaster, Caenorhabditis elegans, and Homo sapienswith respect to the distribution of predicted structural disorder, sequence repeats, low complexity regions, and chain length. Highly connected proteins ("hub proteins") contained significantly more of, and greater proportion of, these sequence features and tended to be longer overall as compared to less connected proteins. These sequence features provide two different functional means for realizing multiple interactions: (1) extended interaction surface and (2) flexibility and adaptability, providing a mechanism for the same region to bind distinct partners. Our view contradicts the prevailing view that scaling in protein interactomes arose from gene duplication and preferential attachment of equivalent proteins. We propose an alternative evolutionary network specialization process, in which certain components of the protein interactome improved their fitness for binding by becoming longer or accruing regions of disorder and/or internal repeats and have therefore become specialized in network organization.     

Association Rate Constants of Ras-Effector Interactions Are Evolutionarily Conserved

Evolutionary conservation of protein interaction properties has been shown to be a valuable indication for functional importance. Here we use homology interface modeling of 10 Ras-effector complexes by selecting ortholog proteins from 12 organisms representing the major eukaryotic branches, except plants. We find that with increasing divergence time the sequence similarity decreases with respect to the human protein, but the affinities and association rate constants are conserved as predicted by the protein design algorithm, FoldX. In parallel we have done computer simulations on a minimal network based on Ras-effector interactions, and our results indicate that in the absence of negative feedback, changes in kinetics that result in similar binding constants have strong consequences on network behavior. This, together with the previous results, suggests an important biological role, not only for equilibrium binding constants but also for kinetics in signaling processes involving Ras-effector interactions. Our findings are important to take into consideration in system biology approaches and simulations of biological networks.     

More than just tails: intrinsic disorder in histone proteins

Many biologically active proteins are disordered as a whole, or contain long disordered regions. These intrinsically disordered proteins/regions are very common in nature, abundantly found in all organisms, where they carry out important biological functions. The functions of these proteins complement the functional repertoire of "normal" ordered proteins, and many protein functional classes are heavily dependent on intrinsic disorder. Among these disorder-centric functions are interactions with nucleic acids and protein complex assembly. In this study, we present the results of comprehensive bioinformatics analyses of the abundance and roles of intrinsic disorder in 2007 histones from 746 species. We show that all the members of the histone family are intrinsically disordered proteins. Furthermore, intrinsic disorder is not only abundant in histones, but is absolutely necessary for various histone functions, starting from heterodimerization to formation of higher order oligomers, to interactions with DNA and other proteins, and to posttranslational modifications.     

Two-hybrid technologies in proteomics research

Given that protein-protein interactions (PPIs) regulate nearly every living process; the exploration of global and pathway-specific protein interaction networks is expected to have major implications in the understanding of diseases and for drug discovery. Consequently, the development and application of methodologies that address physical associations among proteins is of major importance in today's proteomics research. The most widely and successfully used methodology to assess PPIs is the yeast two-hybrid system (YTH). Here we present an overview on the current applications of YTH and variant technologies in yeast and mammalian systems. Two-hybrid-based methods will not only continue to have a dominant role in the assessment of protein interactomes but will also become important in the development of novel compounds that target protein interaction interfaces for therapeutic intervention.     

Evidence for a coiled-coil interaction mode of disordered proteins from bacterial type III secretion systems

Gene clusters encoding various type III secretion system (T3SS) injectisomes, frequently code downstream of the conserved atpase gene for small hydrophilic proteins whose amino acid sequences display a propensity for intrinsic disorder and coiled-coil formation. These properties were confirmed experimentally for a member of this class, the HrpO protein from the T3SS of Pseudomonas syringae pv phaseolicola: HrpO exhibits high alpha-helical content with coiled-coil characteristics, strikingly low melting temperature, structural properties that are typical for disordered proteins, and a pronounced self-association propensity, most likely via coiled-coil interactions, resulting in heterogeneous populations of quaternary complexes. HrpO interacts in vivo with HrpE, a T3SS protein for which coiled-coil formation is also strongly predicted. Evidence from HrpO analogues from all T3SS families and the flagellum suggests that the extreme flexibility and propensity for coiled-coil interactions of this diverse class of small, intrinsically disordered proteins, whose structures may alter as they bind to their cognate folded protein targets, might be important elements in the establishment of protein-protein interaction networks required for T3SS function.