Human long intrinsically disordered protein regions are frequent targets of positive selection

Intrinsically disordered regions occur frequently in proteins and are characterized by a lack of a well-defined three-dimensional structure. Although these regions do not show a higher-order of structural organization, they are known to be functionally important. Disordered regions are rapidly evolving, largely attributed to relaxed purifying selection and an increased role of genetic drift. It has also been suggested that positive selection might contribute to their rapid diversification. However, for our own species it is currently unknown whether positive selection has played a role during the evolution of these protein regions. Here we address this question by investigating the evolutionary pattern of more than 6,600 human proteins with intrinsically disordered regions and their ordered counterparts. Our comparative approach with data from more than 90 mammalian genomes uses a-priori knowledge of disordered protein regions and we show that this increases the power to detect positive selection by an order of magnitude. We can confirm that human intrinsically disordered regions evolve more rapidly, not only within humans but also across the entire mammalian phylogeny. They have, however, experienced substantial evolutionary constraint, hinting at their fundamental functional importance. We find compelling evidence that disordered protein regions are frequent targets of positive selection and estimate that the relative rate of adaptive substitutions differs 4-fold between disordered and ordered protein regions in humans. Our results suggest that disordered protein regions are important targets of genetic innovation and that the contribution of positive selection in these regions is more pronounced than in other protein parts

Mechanisms of NF-κB p65 and strategies for therapeutic manipulation

The transcription factor NF-κB is a critical regulator of immune and inflammatory responses. In mammals, the NF-κB/Rel family comprises five members: p50, p52, p65 (Rel-A), c-Rel, and Rel-B proteins, which form homo- or heterodimers and remain as an inactive complex with the inhibitory molecules called IκB proteins in resting cells. Two distinct NF-κB signaling pathways have been described: 1) the canonical pathway primarily activated by pathogens and inflammatory mediators, and 2) the noncanonical pathway mostly activated by developmental cues. The most abundant form of NF-κB activated by pathologic stimuli via the canonical pathway is the p65:p50 heterodimer. Disproportionate increase in activated p65 and subsequent transactivation of effector molecules is integral to the pathogenesis of many chronic diseases such as the rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, and even neurodegenerative pathologies. Hence, the NF-κB p65 signaling pathway has been a pivotal point for intense drug discovery and development. This review begins with an overview of p65-mediated signaling followed by discussion of strategies that directly target NF-κB p65 in the context of chronic inflammation

Intrinsically disordered proteins link alternative splicing and post-translational modifications to complex cell signaling and regulation

Intrinsically disordered proteins and regions (IDPs and IDRs) lack well-defined tertiary structures, yet carry out various important cellular functions, especially those associated with cell signaling and regulation. In eukaryotes, IDPs and IDRs contain the preferred loci for both alternative splicing (AS) and many post-translational modifications (PTMs). Furthermore, AS and/or PTMs at these loci generally alter the signaling outcomes associated with these IDPs or IDRs, where the functional cooperation of these three features is named the IDP-AS-PTM toolkit. However, the prevalence of such functional modulations remains unknown. Also, the signal-altering mechanisms by which AS, and PTMs modulate function and the extent to which AS and PTMs collaborate in their signaling modulations have not been well defined for particular protein examples. Here we focus on three important signaling and regulatory IDR-containing protein families in humans, namely G-protein coupled receptors (GPCRs), which are transmembrane proteins, the nuclear factors of activated T-cells (NFATs), which are transcription factors (TFs), and the Src family kinases (SFKs), which are signaling enzymes. The goal here is to determine how AS and PTMs individually alter the outcomes of the signaling carried out by the various IDRs and to determine whether AS and PTMs work together to bring about differential cellular responses. We also present data indicating that a wide range of other signaling IDPs or IDRs undergo both AS- and PTM-based modifications, suggesting that they, too, likely take advantage of signal outcome modulations that result from collaboration between these two events. Hence, we propose that the widespread cooperation of IDPs, AS and/or PTMs provides a IDP-AS-PTM toolkit and substantially contributes to the vast complexity of eukaryotic cell signaling systems

Rozponávání pomocí neuspořádaných oblastí proteinů

Intrinsic disorder is one of the many traits that can affect the functionality of multiple naturally occurring proteins in biological systems. This thesis reports on the latest findings on mechanisms that intrinsically disordered proteins or intrinsically disordered regions utilize in specific recognition at the molecular level. Here, the general characteristics of intrinsically disordered proteins are summarized, along with the extent of their abundance throughout different lifeforms and the variety of their molecular recognition mechanisms depicted on specific examples. Furthermore, this thesis focuses on protein transitions between ordered and disordered states induced by interaction with its' binding partner. In the last two chapters, characteristic features of intrinsically disordered proteins are described, and attention is paid to the way these features influence cellular signaling pathways such as interactional promiscuity, the role of signaling hubs, alternative splicing, and post- translational modification.Neuspořádanost je jedna z vlastností mnoha přirozeně se vyskytujících proteinů, která ovlivňuje jejich funkčnost v biologických systémech. Tato práce popisuje poznatky o mechanismech, kterými mohou neuspořádané proteiny nebo neuspořádané proteinové domény přispívat ke specifickému rozpoznávání na molekulární úrovni. Jsou zde shrnuty obecné charakteristiky neuspořádaných proteinů, míra jejich zastoupení napříč různými organismy a na konkrétních příkladech jsou zde představeny možné způsoby molekulárního rozpoznávání. Dále se tato práce zaměřuje na přechody mezi uspořádaným a neuspořádaným stavem vyvolané interakcí s vazebným partnerem. V posledních dvou kapitolách se věnuje charakteristickým rysům neuspořádaných proteinů ovlivňujících buněčnou signalizaci, kterými jsou vazebná promiskuita v podobě signaling hubs, alternativní splicing nebo post-translační modifikace.Katedra buněčné biologieDepartment of Cell BiologyFaculty of SciencePřírodovědecká fakult

The crystal structure of the Leishmania infantum Silent Information Regulator 2 related protein 1: implications to protein function and drug design.

The research leading to these results received funding from the European Community’s Seventh Framework Programme under grant agreement No.602773 (Project KINDRED).The de novo crystal structure of the Leishmania infantum Silent Information Regulator 2 related protein 1 (LiSir2rp1) has been solved at 1.99Å in complex with an acetyl-lysine peptide substrate. The structure is broadly commensurate with Hst2/SIRT2 proteins of yeast and human origin, reproducing many of the structural features common to these sirtuin deacetylases, including the characteristic small zinc-binding domain, and the larger Rossmann-fold domain involved in NAD+-binding interactions. The two domains are linked via a cofactor binding loop ordered in open conformation. The peptide substrate binds to the LiSir2rp1 protein via a cleft formed between the small and large domains, with the acetyl-lysine side chain inserting further into the resultant hydrophobic tunnel. Crystals were obtained only with recombinant LiSir2rp1 possessing an extensive internal deletion of a proteolytically-sensitive region unique to the sirtuins of kinetoplastid origin. Deletion of 51 internal amino acids (P253-E303) from LiSir2rp1 did not appear to alter peptide substrate interactions in deacetylation assays, but was indispensable to obtain crystals. Removal of this potentially flexible region, that otherwise extends from the classical structural elements of the Rossmann-fold, specifically the β8-β9 connector, appears to result in lower accumulation of the protein when expressed from episomal vectors in L. infantum SIR2rp1 single knockout promastigotes. The biological function of the large serine-rich insertion in kinetoplastid/trypanosomatid sirtuins, highlighted as a disordered region with strong potential for post-translational modification, remains unknown but may confer additional cellular functions that are distinct from their human counterparts. These unique molecular features, along with the resolution of the first kinetoplastid sirtuin deacetylase structure, present novel opportunities for drug design against a protein target previously established as essential to parasite survival and proliferation.Publisher PDFPeer reviewe

Structural biology of calcium phosphate nanoclusters sequestered by phosphoproteins

Biofluids that contain stable calcium phosphate nanoclusters sequestered by phosphopeptides make it possible for soft and hard tissues to co-exist in the same organism with relative ease. The stability diagram of a solution of nanocluster complexes shows how the minimum concentration of phosphopeptide needed for stability increases with pH. In the stable region, amorphous calcium phosphate cannot precipitate. Nevertheless, if the solution is brought into contact with hydroxyapatite, the crystalline phase will grow at the expense of the nanocluster complexes. The physico-chemical principles governing the formation, composition, size, structure, and stability of the complexes are described. Examples are given of complexes formed by casein, osteopontin, and recombinant phosphopeptides. Application of these principles and properties to blood serum, milk, urine, and resting saliva is described to show that under physiological conditions they are in the stable region of their stability diagram and so cannot cause soft tissue calcification. Stimulated saliva, however, is in the metastable region, consistent with its role in tooth remineralization. Destabilization of biofluids, with consequential ill-effects, can occur when there is a failure of homeostasis, such as an increase in pH without a balancing increase in the concentration of sequestering phosphopeptides

A Systems Biology Analysis of PP2A Functions in Cancer Cells

Cancer is characterized by aberrant activation of phosphorylation signalling cascades. However, despite the critical role of phosphatases in protein phosphorylation, their contribution to cancer cell signalling is only emerging. Notably, Protein phosphatase 2A (PP2A) has a well-established tumor suppressor function but it is poorly understood which of its many targets are relevant for this function. This is partly due to the wide range of activities that PP2A participates in and partly due to the fact that PP2A activity regulation, as well as the deregulation in cancer, occurs via many auxiliary subunits and endogenous inhibitor proteins. In this MD-PhD thesis, we have used various systems biology approaches, including phosphoproteomics, high throughput drug sensitivity screening, and transcriptomics to study the functions of the most frequently mutated PP2A subunit, PPP2R1A, as well as three of its endogenous inhibitor proteins, CIP2A, PME1, and SET in cancer cells. This study demonstrates that PP2A reactivation is poorly tolerated by several types of cancer cells and results in downregulation of multiple oncogenic pathways, as well as induction of senescence. Specifically, CIP2A is a regulator of MYC transactivation in basal type breast cancers and our results indicate multiple cooperative mechanisms by which PP2A regulates MYC. Analysis of PP2A dephosphorylome also provided novel insights into general organization of phosphorylation signalling and emphasized the role of PP2A inhibition in the nucleus. By combining the phosphoprotemics data with cancer cell responses to over 300 drugs, we have identified mechanistically distinct types of interactions between drug sensitivity and PP2A activity. We further validated that inhibition of PP2A in KRAS mutant lung cancers confers resistance to MAPK pathway inhibitors including the combination of Raf and MEK inhibitors. Together, these findings provide new evidence to support PP2A reactivation as cancer therapeutic strategy and to support evaluating PP2A activity as a predicitive marker for cancer therapy responses.Syövälle ominainen piirre on fosforylaation perustuvien signaalipolkujen poikkeava aktivoituminen. Huolimatta fosfataasien keskeisestä tehtävästä proteiinien fosforylaatiossa, niiden merkityksestä syöpäsolujen signlaalinvälityksessä on vasta vähän tietoa. Erityisesti Proteiinifosfataasi 2A:lla (PP2A) on selkeästi osoitettu olevan kasvunestäjproteiini-ominaisuuksia, mutta se mitkä PP2A:n monista kohdeproteiineista ovat tärkeitä syövän kannalta tunnetaan huonosti. Osittain tämä johtuu PP2A:n toimintojen moninaisuudesta ja osittain siitä, että PP2A:n säätely, sekä syövissä esiintyvät säätelyn häiriöt, tapahtuvat ylimääräisten alayksiköiden ja inhibiiittoriproteiinien kautta Tässä väitöskirjatutkimuksessa olemme tutkineet PP2A:n yleisimmin mutatoituneen alayksikön, PPP2R1A:n, sekä kolmen inhibiittoriproteiinin, CIP2A:n, PME-1:n ja SETin, toimintoja syöpäsoluissa käyttäen erilaisia systeemibilogisia lähestymistapoja, mukaan lukien fosfoproteomiikkaa, lääkeherkkyysseulontaa ja transkriptomi-analyysiä. Tämä tutkimus osoittaa, että monet syöpäsolut sietävät huonosti PP2A:n uudelleen aktivoimista, joka johtaa useiden onkogeenisten signaalipolkujen estymiseen ja senesenssin käynnistymiseen. Tuloksemme viittaavat siihen, että PP2A säätelee MYC-onkogeeniä useilla toisiaan tukevilla mekanismeilla, ja CIP2A:lla on merkitystä MYCin transaktivaation säätelyssä basaalityypin rintasyövässä. PP2A:n defosforylomista saatu tieto auttaa myös ymmärtämään yleisellä tasolla fosforylaatiosignaloinnin järjestäytymistä soluissa ja osoittaa, että PP2A:n inhibitiolla on keskeinen merkitys tumassa. Yhdistämällä fosfoproteomiikan ja syöpäsolujen vasteet yli 300 lääkkeelle olemme tunnistaneet useita mekanismeiltaan erilaisia yhteisvaikutuksia PP2A:n aktiivisuuden ja lääkeherkkyyksien välillä. PP2A:n estämisestä aiheutuva resistenssin MAPK-signaalipolun inhbiittoreille KRAS-mutaatioita kantasvissa syöpäsoluissa vahvistettiin lisätutukimuksilla. PP2A:n estäminen teki solut resistenteiksi myös MEK ja RAF inhibiittorien yhdistelmälle. Yhdessä nämä tulokset puoltavat PP2A:n reaktivaatiota syövän hoitostrategiana ja PP2A:n aktiivisuuden määrittämistä syöpähoitojen ennustekijänä.Siirretty Doriast

Mechanisms of binding diversity in protein disorder : molecular recognition features mediating protein interaction networks

Indiana University-Purdue University Indianapolis (IUPUI)Intrinsically disordered proteins are proteins characterized by lack of stable tertiary structures under physiological conditions. Evidence shows that disordered proteins are not only highly involved in protein interactions, but also have the capability to associate with more than one partner. Short disordered protein fragments, called “molecular recognition features” (MoRFs), were hypothesized to facilitate the binding diversity of highly-connected proteins termed “hubs”. MoRFs often couple folding with binding while forming interaction complexes. Two protein disorder mechanisms were proposed to facilitate multiple partner binding and enable hub proteins to bind to multiple partners: 1. One region of disorder could bind to many different partners (one-to-many binding), so the hub protein itself uses disorder for multiple partner binding; and 2. Many different regions of disorder could bind to a single partner (many-to-one binding), so the hub protein is structured but binds to many disordered partners via interaction with disorder. Thousands of MoRF-partner protein complexes were collected from Protein Data Bank in this study, including 321 one-to-many binding examples and 514 many-to-one binding examples. The conformational flexibility of MoRFs was observed at atomic resolution to help the MoRFs to adapt themselves to various binding surfaces of partners or to enable different MoRFs with non-identical sequences to associate with one specific binding pocket. Strikingly, in one-to-many binding, post-translational modification, alternative splicing and partner topology were revealed to play key roles for partner selection of these fuzzy complexes. On the other hand, three distinct binding profiles were identified in the collected many-to-one dataset: similar, intersecting and independent. For the similar binding profile, the distinct MoRFs interact with almost identical binding sites on the same partner. The MoRFs can also interact with a partially the same but partially different binding site, giving the intersecting binding profile. Finally, the MoRFs can interact with completely different binding sites, thus giving the independent binding profile. In conclusion, we suggest that protein disorder with post-translational modifications and alternative splicing are all working together to rewire the protein interaction networks

Intrinsic Disorder and Allostery in Glucocorticoid Receptor

Intrinsically disordered (ID) regions of proteins, lacking stable tertiary structure, are malleable and sensitive regulators of cell functions. Allostery is transmittance of a perturbation at one region to distant sites of the same molecule, allowing for precise control of macromolecular function. Nature preferentially uses both ID regions and allostery to regulate protein function, as observed in transcription factors. This motivated us to investigate whether and how ID regions can facilitate allostery. The classic allosteric models all feature a static structural and single molecule view of allostery and are not applicable to study allostery mediated by ID regions. To address this limitation, our group has developed the Ensemble Allosteric Model (EAM), which views allostery as an effector binding driven shift in ensemble probabilities. It is a thermodynamic and quantitative model that can be applied to structured proteins, ID proteins and mixed proteins. In this thesis project, I investigated the intra- and inter- domain allostery mediated by the ID regions in human glucocorticoid receptor (GR). Through thermodynamic and functional studies on eight GR translational isoforms, I found that the ID GR N terminal domain (NTD) is composed of two functionally distinct regions, unfavorably coupled with each other, and both of them are favorably coupled to the DNA binding domain (DBD). Based on these experimental constraints, an EAM was built for GR and reasonable thermodynamic parameter combinations were found to describe both the transcriptional activity and binding affinity of different translational isoforms. We found that GR uses these competing energetic couplings, which are modulated in different translational isoforms, to provide tunable responses to environmental cues. In the context of the EAM predictions, mutagenesis was carried out in different regions of GR and the influence on transcriptional activity and binding affinity was assayed to pinpoint the molecular basis of allostery, which paves a way for allosteric drug design. This study suggests a unifying strategy to investigate thermodynamics and the molecular basis of allostery in any complicated system