A creature with a hundred waggly tails: intrinsically disordered proteins in the ribosome

Intrinsic disorder (i.e., lack of a unique 3-D structure) is a common phenomenon, and many biologically active proteins are disordered as a whole, or contain long disordered regions. These intrinsically disordered proteins/regions constitute a significant part of all proteomes, and their functional repertoire is complementary to functions of ordered proteins. In fact, intrinsic disorder represents an important driving force for many specific functions. An illustrative example of such disorder-centric functional class is RNA-binding proteins. In this study, we present the results of comprehensive bioinformatics analyses of the abundance and roles of intrinsic disorder in 3,411 ribosomal proteins from 32 species. We show that many ribosomal proteins are intrinsically disordered or hybrid proteins that contain ordered and disordered domains. Predicted globular domains of many ribosomal proteins contain noticeable regions of intrinsic disorder. We also show that disorder in ribosomal proteins has different characteristics compared to other proteins that interact with RNA and DNA including overall abundance, evolutionary conservation, and involvement in protein–protein interactions. Furthermore, intrinsic disorder is not only abundant in the ribosomal proteins, but we demonstrate that it is absolutely necessary for their various functions.     

To be disordered or not to be disordered: is that still a question for proteins in the cell?

There is ample evidence that many proteins or regions of proteins lack a well-defined folded structure under native-like conditions. These are called intrinsically disordered proteins (IDPs) or intrinsically disordered regions (IDRs). Whether this intrinsic disorder is also their main structural characteristic in living cells has been a matter of intense debate. The structural analysis of IDPs became an important challenge also because of their involvement in a plethora of human diseases, which made IDPs attractive targets for therapeutic development. Therefore, biophysical approaches are increasingly being employed to probe the structural and dynamical state of proteins, not only in isolation in a test tube, but also in a complex biological environment and even within intact cells. Here, we survey direct and indirect evidence that structural disorder is in fact the physiological state of many proteins in the proteome. The paradigmatic case of α-synuclein is used to illustrate the controversial nature of this topic.     

Comprehensive review of methods for prediction of intrinsic disorder and its molecular functions

Computational prediction of intrinsic disorder in protein sequences dates back to late 1970 and has flourished in the last two decades. We provide a brief historical overview, and we review over 30 recent predictors of disorder. We are the first to also cover predictors of molecular functions of disorder, including 13 methods that focus on disordered linkers and disordered protein–protein, protein–RNA, and protein–DNA binding regions. We overview their predictive models, usability, and predictive performance. We highlight newest methods and predictors that offer strong predictive performance measured based on recent comparative assessments. We conclude that the modern predictors are relatively accurate, enjoy widespread use, and many of them are fast. Their predictions are conveniently accessible to the end users, via web servers and databases that store pre-computed predictions for millions of proteins. However, research into methods that predict many not yet addressed functions of intrinsic disorder remains an outstanding challenge.     

Functions of intrinsic disorder in transmembrane proteins

Intrinsic disorder is common in integral membrane proteins, particularly in the intracellular domains. Despite this observation, these domains are not always recognized as being disordered. In this review, we will discuss the biological functions of intrinsically disordered regions of membrane proteins, and address why the flexibility afforded by disorder is mechanistically important. Intrinsically disordered regions are present in many common classes of membrane proteins including ion channels and transporters; G-protein coupled receptors (GPCRs), receptor tyrosine kinases and cytokine receptors. The functions of the disordered regions are many and varied. We will discuss selected examples including: (1) Organization of receptors, kinases, phosphatases and second messenger sources into signaling complexes. (2) Modulation of the membrane-embedded domain function by ball-and-chain like mechanisms. (3) Trafficking of membrane proteins. (4) Transient membrane associations. (5) Post-translational modifications most notably phosphorylation and (6) disorder-linked isoform dependent function. We finish the review by discussing the future challenges facing the membrane protein community regarding protein disorder.     

Evolution of intrinsic disorder in eukaryotic proteins

Conformational flexibility conferred though regions of intrinsic structural disorder allows proteins to behave as dynamic molecules. While it is well-known that intrinsically disordered regions can undergo disorder-to-order transitions in real-time as part of their function, we also are beginning to learn more about the dynamics of disorder-to-order transitions along evolutionary time-scales. Intrinsically disordered regions endow proteins with functional promiscuity, which is further enhanced by the ability of some of these regions to undergo real-time disorder-to-order transitions. Disorder content affects gene retention after whole genome duplication, but it is not necessarily conserved. Altered patterns of disorder resulting from evolutionary disorder-to-order transitions indicate that disorder evolves to modify function through refining stability, regulation, and interactions. Here, we review the evolution of intrinsically disordered regions in eukaryotic proteins. We discuss the interplay between secondary structure and disorder on evolutionary time-scales, the importance of disorder for eukaryotic proteome expansion and functional divergence, and the evolutionary dynamics of disorder.     

Structural disorder in plant proteins: where plasticity meets sessility

Plants are sessile organisms. This intriguing nature provokes the question of how they survive despite the continual perturbations caused by their constantly changing environment. The large amount of knowledge accumulated to date demonstrates the fascinating dynamic and plastic mechanisms, which underpin the diverse strategies selected in plants in response to the fluctuating environment. This phenotypic plasticity requires an efficient integration of external cues to their growth and developmental programs that can only be achieved through the dynamic and interactive coordination of various signaling networks. Given the versatility of intrinsic structural disorder within proteins, this feature appears as one of the leading characters of such complex functional circuits, critical for plant adaptation and survival in their wild habitats. In this review, we present information of those intrinsically disordered proteins (IDPs) from plants for which their high level of predicted structural disorder has been correlated with a particular function, or where there is experimental evidence linking this structural feature with its protein function. Using examples of plant IDPs involved in the control of cell cycle, metabolism, hormonal signaling and regulation of gene expression, development and responses to stress, we demonstrate the critical importance of IDPs throughout the life of the plant.     

Regulation of microRNA biogenesis and turnover by animals and their viruses

MicroRNAs (miRNAs) are a ubiquitous component of gene regulatory networks that modulate the precise amounts of proteins expressed in a cell. Despite their small size, miRNA genes contain various recognition elements that enable specificity in when, where and to what extent they are expressed. The importance of precise control of miRNA expression is underscored by functional studies in model organisms and by the association between miRNA mis-expression and disease. In the last decade, identification of the pathways by which miRNAs are produced, matured and turned-over has revealed many aspects of their biogenesis that are subject to regulation. Studies in viral systems have revealed a range of mechanisms by which viruses target these pathways through viral proteins or non-coding RNAs in order to regulate cellular gene expression. In parallel, a field of study has evolved around the activation and suppression of antiviral RNA interference (RNAi) by viruses. Virus encoded suppressors of RNAi can impact miRNA biogenesis in cases where miRNA and small interfering RNA pathways converge. Here we review the literature on the mechanisms by which miRNA biogenesis and turnover are regulated in animals and the diverse strategies that viruses use to subvert or inhibit these processes.     

Intrinsic disorder here,there, and everywhere,and nowhere to escape from it

The concept of protein intrinsic disorder persistently penetrates into all areas of modern protein science. It cannot be ignored anymore, and cannot be shrugged off, as it represents a vital feature (or, more correctly, a broad spectrum of important features), which, when added to and mixed with features arising from the well established protein structure-function paradigm, complete the picture of a functioning protein. The field of protein intrinsic disorder is very dynamic and fast developing. This Multi-Author Review represents a snapshot of this field by introducing some recent advances. Articles assembled in this Multi-Author Review introduce some of the new aspects of intrinsic disorder, outline some fascinating ideas related to the intrinsically disordered proteins, their structure, and functionality, and show challenges related to the analysis of proteins carrying intrinsic disorder.     

Intrinsic disorder in proteins involved in amyotrophic lateral sclerosis

Five structurally and functionally different proteins, an enzyme superoxide dismutase 1 (SOD1), a TAR-DNA binding protein-43 (TDP-43), an RNA-binding protein FUS, a cofilin-binding protein C9orf72, and polypeptides generated as a result of its intronic hexanucleotide expansions, and to lesser degree actin-binding profilin-1 (PFN1), are considered to be the major drivers of amyotrophic lateral sclerosis. One of the features common to these proteins is the presence of significant levels of intrinsic disorder. The goal of this study is to consider these neurodegeneration-related proteins from the intrinsic disorder perspective. To this end, we employed a broad set of computational tools for intrinsic disorder analysis and conducted intensive literature search to gain information on the structural peculiarities of SOD1, TDP-43, FUS, C9orf72, and PFN1 and their intrinsic disorder predispositions, and the roles of intrinsic disorder in their normal and pathological functions.     

How order and disorder within paramyxoviral nucleoproteins and phosphoproteins orchestrate the molecular interplay of transcription and replication

In this review, we summarize computational and experimental data gathered so far showing that structural disorder is abundant within paramyxoviral nucleoproteins (N) and phosphoproteins (P). In particular, we focus on measles, Nipah, and Hendra viruses and highlight both commonalities and differences with respect to the closely related Sendai virus. The molecular mechanisms that control the disorder-to-order transition undergone by the intrinsically disordered C-terminal domain (N_TAIL) of their N proteins upon binding to the C-terminal X domain (XD) of the homologous P proteins are described in detail. By having a significant residual disorder, N_TAIL–XD complexes are illustrative examples of “fuzziness”, whose possible functional significance is discussed. Finally, the relevance of N–P interactions as promising targets for innovative antiviral approaches is underscored, and the functional advantages of structural disorder for paramyxoviruses are pinpointed.     

Viral journeys on the intracellular highways

Viruses are obligate intracellular pathogens that are dependent on cellular machineries for their replication. Recent technological breakthroughs have facilitated reliable identification of host factors required for viral infections and better characterization of the virus–host interplay. While these studies have revealed cellular machineries that are uniquely required by individual viruses, accumulating data also indicate the presence of broadly required mechanisms. Among these overlapping cellular functions are components of intracellular membrane trafficking pathways. Here, we review recent discoveries focused on how viruses exploit intracellular membrane trafficking pathways to promote various stages of their life cycle, with an emphasis on cellular factors that are usurped by a broad range of viruses. We describe broadly required components of the endocytic and secretory pathways, the Endosomal Sorting Complexes Required for Transport pathway, and the autophagy pathway. Identification of such overlapping host functions offers new opportunities to develop broad-spectrum host-targeted antiviral strategies.     

Conditionally disordered proteins: bringing the environment back into the fold

For many proteins, biological function requires the folding of the polypeptide chain into a unique and persistent tertiary structure. This review concerns proteins that adopt a specific tertiary structure to function, but are otherwise partially or completely disordered. The biological cue for protein folding is environmental perturbation or minor post-translational modification. Hence, we term these proteins conditionally disordered. Many of these proteins recognize and bind other molecules, and conditional disorder has been hypothesized to allow for more nuanced control and regulation of binding processes. However, this remains largely unproven. The sequences of conditionally disordered proteins suggest their propensity to fold; yet, under the standard laboratory conditions, they do not do so, which may appear surprising. We argue that the surprise results from the failure to consider the role of the environment in protein structure formation and that conditional disorder arises as a natural consequence of the marginal stability of the folded state.     

Plasticins: membrane-damaging peptides with ‘chameleon-like’ properties

Plasticins belong to the dermaseptin superfamily of gene-encoded, membrane-active host defense peptides produced by the skin of hylid frogs. The plasticins, which are rich in Gly and Leu residues arranged in regular 5-mer motifs GXXXG (where X is any amino acid residue), have very similar amino acid sequences, hydrophobicities, and amphipathicities but differ markedly in their net charge, conformational plasticity, and activity spectra. The intrinsic flexibility and structural malleability of plasticins modulate their ability to bind to and disrupt the membranes of prokaryotic and eukaryotic cells, and/or to reach intracellular targets, therefore triggering functional versatility. This family of closely related but functionally divergent peptides constitutes a good model to address the relationships between structural polymorphism, membrane-interacting properties, and the biological activity of antimicrobial, cell-penetrating, and viral fusion peptides. Plasticins could thus serve as templates to design potent multifunctional drugs that could act simultaneously against bacterial pathogens and viruses. Received 26 September 2007; received after revision 22 October 2007; accepted 29 October 2007     

Functions of disordered regions in mammalian early base excision repair proteins

Reactive oxygen species, generated endogenously and induced as a toxic response, produce several dozen oxidized or modified bases and/or single-strand breaks in mammalian and other genomes. These lesions are predominantly repaired via the conserved base excision repair (BER) pathway. BER is initiated with excision of oxidized or modified bases by DNA glycosylases leading to formation of abasic (AP) site or strand break at the lesion site. Structural analysis by experimental and modeling approaches shows the presence of a disordered segment commonly localized at the N- or C-terminus as a characteristic signature of mammalian DNA glycosylases which is absent in their bacterial prototypes. Recent studies on unstructured regions in DNA metabolizing proteins have indicated their essential role in interaction with other proteins and target DNA recognition. In this review, we have discussed the unique presence of disordered segments in human DNA glycosylases, and AP endonuclease involved in the processing of glycosylase products, and their critical role in regulating repair functions. These disordered segments also include sites for posttranslational modifications and nuclear localization signal. The teleological basis for their structural flexibility is discussed.     

Mast cell tryptase,a still enigmatic enzyme

Tryptases constitute a subfamily of trypsin-like proteinases, stored in the mast cell secretory granules of all mammalian organisms. These enzymes are released along with other mediators into the extracellular medium upon mast cell activation/degranulation. Among the trypsin-like enzymes, tryptases are unique: they are present as active enzymes in the mast cell granules, but display activity only extracellularly, and have a specificity which is much more restricted than trypsin. Tryptases are mostly tetrameric, and in only few organisms (not in humans) are they inhibited by endogenous inhibitors in vitro. The enzymatic and molecular properties of tryptases are far better characterized that any of their plausible biological functions. On the basis of its structural and functional features it could be predicted that tryptase would not degrade a large number of proteins in vivo due to low accessibility to the tetramer central pore where the active sites face inwards. Although their biological function has not yet been clarified, tryptases seem to be involved in a number of mast cell-mediated allergic and inflammatory diseases. In particular, the involvement of tryptase in asthma, an inflammatory disease of the airways often caused by allergy, has been proposed. Here we review the present knowledge on the structure-function relationship of tryptases from different organisms, with special emphasis on human enzymes, and on their role in a variety of pathophsyiological processes.Received 29 October 2003; received after revision 3 December 2003; accepted 11 December 2003     

Methods of probing the interactions between small molecules and disordered proteins

It is generally recognized that a large fraction of the human proteome is made up of proteins that remain disordered in their native states. Despite the fact that such proteins play key biological roles and are involved in many major human diseases, they still represent challenging targets for drug discovery. A major bottleneck for the identification of compounds capable of interacting with these proteins and modulating their disease-promoting behaviour is the development of effective techniques to probe such interactions. The difficulties in carrying out binding measurements have resulted in a poor understanding of the mechanisms underlying these interactions. In order to facilitate further methodological advances, here we review the most commonly used techniques to probe three types of interactions involving small molecules: (1) those that disrupt functional interactions between disordered proteins; (2) those that inhibit the aberrant aggregation of disordered proteins, and (3) those that lead to binding disordered proteins in their monomeric states. In discussing these techniques, we also point out directions for future developments.     

Nectin family of cell-adhesion molecules: structural and molecular aspects of function and specificity

Cell–cell adhesive processes are central to the physiology of multicellular organisms. A number of cell surface molecules contribute to cell–cell adhesion, and the dysfunction of adhesive processes underlies numerous developmental defects and inherited diseases. The nectins, a family of four immunoglobulin superfamily members (nectin-1 to -4), interact through their extracellular domains to support cell–cell adhesion. While both homophilic and heterophilic interactions among the nectins are implicated in cell–cell adhesion, cell-based and biochemical studies suggest heterophilic interactions are stronger than homophilic interactions and control a range of physiological processes. In addition to interactions within the nectin family, heterophilic associations with nectin-like molecules, immune receptors, and viral glycoproteins support a wide range of biological functions, including immune modulation, cancer progression, host-pathogen interactions and immune evasion. We review current structural and molecular knowledge of nectin recognition processes, with a focus on the biochemical and biophysical determinants of affinity and selectivity that drive distinct nectin associations. These proteins and interactions are discussed as potential targets for immunotherapy.     

Scaffolding proteins and their role in viral assembly

Scaffolding proteins are proteins that are required to catalyse, regulate or modulate some step in the assembly of a macromolecular complex. They associate specifically with the nascent protein complex during assembly, but are subsequently removed, and are absent from the mature structure. Scaffolding proteins have been described primarily from viral systems, in particular from the double-stranded DNA bacteriophages, but most likely play a more general role in macromolecular assembly, a fundamental process in all biological systems. Scaffolding proteins may act in a specific fashion, by actively encouraging the formation of correct protein-protein interactions, or more generally by nucleating and promoting assembly. They may also work to ensure the fidelity of the assembly process by preventing the formation of improper interactions, in many ways similar to the role of molecular chaperones in protein folding. In viruses, scaffolding proteins are found both in the form of internal cores and external bracing, and may form elaborate and complex structures. This review will focus on the viral scaffolding proteins, for which an increasing amount of structural and functional information has recently become available.     

Platelets and viruses: an ambivalent relationship

Thrombocytopenia is a frequent complication of viral infections providing evidence that interaction of platelets with viruses is an important pathophysiological phenomenon. Multiple mechanisms are involved depending on the nature of the viruses involved. These include immunological platelet destruction, inappropriate platelet activation and consumption, and impaired megakaryopoiesis. Viruses bind platelets through specific receptors and identified ligands, which lead to mutual alterations of both the platelet host and the viral aggressor. We have shown that HIV-1 viruses are internalized specifically in platelets and megakaryocytes, where they can be either sheltered, unaltered (with potential transfer of the viruses into target organs), or come in contact with platelet secretory products leading to virus destruction and facilitated platelet clearance. In this issue, we have reviewed the various pathways that platelets use in order to interact with viruses, HIV and others. This review also shows that more work is still needed to precisely identify platelet roles in viral infections, and to answer the challenge of viral safety in platelet transfusion.     

Signalling in viral entry

Viral infections are serious battles between pathogens and hosts. They can result in cell death, elimination of the virus or latent infection keeping both cells and pathogens alive. The outcome of an infection is often determined by cell signalling. Viruses deliver genomes and proteins with signalling potential into target cells and thereby alter the metabolism of the host. Virus interactions with cell surface receptors can elicit two types of signals, conformational changes of viral particles, and intracellular signals triggering specific cellular reactions. Responses by cells include stimulation of innate and adaptive immunity, growth, proliferation, survival and apoptosis. In addition, virus-activated cell signalling boosts viral entry and gene delivery, as recently shown for adenoviruses and adeno-associated viruses. This review illustrates that multiple activation of host cells during viral entry profoundly impacts the elaborate relationship between hosts and viral pathogens. Received 13 September 2001; received after revision 23 October 2001; accepted 16 November 2001