The earliest whole protein order/disorder predictor (Uversky et al., Proteins, 41: 415-427 (2000)), herein called the charge-hydropathy (C-H) plot, was originally developed using the Kyte-Doolittle (1982) hydropathy scale (Kyte & Doolittle., J. Mol. Biol, 157: 105-132(1982)). Here the goal is to determine whether the performance of the C-H plot in separating structured and disordered proteins can be improved by using an alternative hydropathy scale.
Using the performance of the CH-plot as the metric, we compared 19 alternative hydropathy scales, with the finding that the Guy (1985) hydropathy scale (Guy, Biophys. J, 47:61-70(1985)) was the best of the tested hydropathy scales for separating large collections structured proteins and intrinsically disordered proteins (IDPs) on the C-H plot. Next, we developed a new scale, named IDP-Hydropathy, which further improves the discrimination between structured proteins and IDPs. Applying the C-H plot to a dataset containing 109 IDPs and 563 non-homologous fully structured proteins, the Kyte-Doolittle (1982) hydropathy scale, the Guy (1985) hydropathy scale, and the IDP-Hydropathy scale gave balanced two-state classification accuracies of 79%, 84%, and 90%, respectively, indicating a very substantial overall improvement is obtained by using different hydropathy scales. A correlation study shows that IDP-Hydropathy is strongly correlated with other hydropathy scales, thus suggesting that IDP-Hydropathy probably has only minor contributions from amino acid properties other than hydropathy.
We suggest that IDP-Hydropathy would likely be the best scale to use for any type of algorithm developed to predict protein disorder.
Intrinsically disordered proteins; natively unstructured or unfolded proteins; structure and disorder prediction; support vector machines
The intracellular environment represents an extremely crowded milieu, with a limited amount of free water and an almost complete lack of unoccupied space. Obviously, slightly salted aqueous solutions containing low concentrations of a biomolecule of interest are too simplistic to mimic the “real life” situation, where the biomolecule of interest scrambles and wades through the tightly packed crowd. In laboratory practice, such macromolecular crowding is typically mimicked by concentrated solutions of various polymers that serve as model “crowding agents”. Studies under these conditions revealed that macromolecular crowding might affect protein structure, folding, shape, conformational stability, binding of small molecules, enzymatic activity, protein-protein interactions, protein-nucleic acid interactions, and pathological aggregation. The goal of this review is to systematically analyze currently available experimental data on the variety of effects of macromolecular crowding on a protein molecule. The review covers more than 320 papers and therefore represents one of the most comprehensive compendia of the current knowledge in this exciting area.
macromolecular crowding; excluded volume; protein structure; protein folding; protein function; protein-protein interaction; intrinsically disordered protein; protein aggregation
Intrinsically disordered, highly charged protein sequences act as entropic bristles (EBs), which, when translationally fused to partner proteins, serve as effective solubilizers by creating both large favorable surface area for water interactions and large excluded volumes around the partner. By extending away from the partner and sweeping out large molecules, EBs can enable the target protein to fold free from interference. Using both naturally-occurring and artificial polypeptides we demonstrate the successful implementation of intrinsically disordered fusions as protein solubilizers. The artificial fusions discussed herein have low sequence complexity and high net charge, but are diversified by means of distinctive amino acid compositions and lengths. Using 6xHis fusions as controls, soluble protein expression enhancements from 65% (EB60A) to 100% (EB250) were observed for a 20-protein portfolio. Additionally, these EBs were able to more effectively solubilize targets compared to frequently-used fusions such as maltose-binding-protein, glutathione S-transferase, thioredoxin, and N utilization substance A. Finally, although these EBs possess very distinct physio-chemical properties they did not perturb the structure, conformational stability nor function of the green fluorescent protein or the glutathione S-transferase protein. This work thus illustrates the successful de novo design of intrinsically-disordered fusions, and presents a promising technology and complementary resource for researchers attempting to solubilize recalcitrant proteins.
intrinsic disorder; protein; solubility; aggregation; translational fusion
The axis inhibition (Axin) scaffold protein colocalizes β-catenin, casein kinase Iα, and glycogen synthetase kinase 3β by their binding to Axin's long intrinsically disordered region, thereby yielding structured domains with flexible linkers. This complex leads to the phosphorylation of β-catenin, marking it for destruction. Fusing proteins with flexible linkers vastly accelerates chemical interactions between them by their colocalization. Here we propose that the complex works by random movements of a “stochastic machine,” not by coordinated conformational changes. This noncovalent, modular assembly process allows the various molecular machine components to be used in multiple processes.
Scaffold proteins; molecular machines; signaling; protein complexes
Hepatitis C virus (HCV) infection represents a worldwide health threat that still needs efficient protective vaccine and/or effective drug. The traditional medicine, such as camel milk, is heavily used by the large sector of HCV patients to control the infection due to the high cost of the available standard therapy. Camel milk contains lactoferrin, which plays an important and multifunctional role in innate immunity and specific host defense against microbial infection. Continuing the analysis of the effectiveness of camel lactoferrin against HCV, the current study aimed to separate and purify the native N- and C-lobes from the proteolytically cleaved camel lactoferrin (cLF) and to compare their in vitro activities against the HCV infection in Huh7.5 cells in order to determine the most active domain.
Lactoferrin and its digested N- and C-lobes were purified by Mono S 5/50 GL column and Superdex 200 5/150 column. The purified proteins were assessed through three venues: 1. To inhibit intracellular replication, HCV infected cells were treated with the proteins at different concentrations and time intervals; 2. The proteins were directly incubated with the viral particles (neutralization) and then such neutralized viruses were used to infect cells; 3. The cells were protected with proteins before exposure to the virus. The antiviral potentials of the cLf and its lobes were determined using three techniques: 1. RT-nested PCR, 2. Real-time PCR, and 3. Flow cytometry.
N- and C-lobes were purified in two consecutive steps; using Mono-S and Superdex 200 columns. The molecular mass of N- and C-lobes was about 40 kDa. cLF and its lobes could prevent HCV entry into Huh 7.5 cells with activity reached 100% through direct interaction with the virus. The inhibition of intracellular viral replication by N-lobe is 2-fold and 3-fold more effective than that of the cLF and C-lobe, respectively.
Generated native N- and C-lobes from camel lactoferrin demonstrated a range of noticeably different potentials against HCV cellular infectivity. The anti-HCV activities were sorted as N-lobe > cLf > C-lobe.
Camel lactoferrin; Proteolytic digestion; Purification; N- and C-lobes; Huh7.5 cells; Anti-HCV
Hypothetical proteins comprise roughly half of the predicted gene complement of Toxoplasma gondii and Plasmodium falciparum and represent the largest class of uniquely functioning proteins in these parasites. Following the idea that functional relationships can be informed by the timing of gene expression, we devised a strategy to identify the core set of apicomplexan cell division cycling genes with important roles in parasite division, which includes many uncharacterized proteins. We assembled an expanded list of orthologs from the T. gondii and P. falciparum genome sequences (2781 putative orthologs), compared their mRNA profiles during synchronous replication, and sorted the resulting set of dual cell cycle regulated orthologs (744 total) into protein pairs conserved across many eukaryotic families versus those unique to the Apicomplexa. The analysis identified more than 100 ortholog gene pairs with unknown function in T. gondii and P. falciparum that displayed co-conserved mRNA abundance, dynamics of cyclical expression and similar peak timing that spanned the complete division cycle in each parasite. The unknown cyclical mRNAs encoded a diverse set of proteins with a wide range of mass and showed a remarkable conservation in the internal organization of ordered versus disordered structural domains. A representative sample of cyclical unknown genes (16 total) was epitope tagged in T. gondii tachyzoites yielding the discovery of new protein constituents of the parasite inner membrane complex, key mitotic structures and invasion organelles. These results demonstrate the utility of using gene expression timing and dynamic profile to identify proteins with unique roles in Apicomplexa biology.
The large-conductance Ca2+-activated K+ (BK) channel is broadly expressed in various mammalian cells and tissues such as neurons, skeletal and smooth muscles, exocrine cells, and sensory cells of the inner ear. Previous studies suggest that BK channels are promiscuous binders involved in a multitude of protein-protein interactions. To gain a better understanding of the potential mechanisms underlying BK interactions, we analyzed the abundance, distribution, and potential mechanisms of intrinsic disorder in 27 BK channel variants from mouse cochlea, 104 previously reported BK-associated proteins (BKAPS) from cytoplasmic and membrane/cytoskeletal regions, plus BK β- and γ-subunits. Disorder was evaluated using the MFDp algorithm, which is a consensus-based predictor that provides a strong and competitive predictive quality and PONDR, which can determine long intrinsically disordered regions (IDRs). Disorder-based binding sites or molecular recognition features (MoRFs) were found using MoRFpred and ANCHOR. BKAP functions were categorized based on Gene Ontology (GO) terms. The analyses revealed that the BK variants contain a number of IDRs. Intrinsic disorder is also common in BKAPs, of which ∼5% are completely disordered. However, intrinsic disorder is very differently distributed within BK and its partners. Approximately 65% of the disordered segments in BK channels are long (IDRs) (>50 residues), whereas >60% of the disordered segments in BKAPs are short IDRs that range in length from 4 to 30 residues. Both α and γ subunits showed various amounts of disorder as did hub proteins of the BK interactome. Our analyses suggest that intrinsic disorder is important for the function of BK and its BKAPs. Long IDRs in BK are engaged in protein-protein and protein-ligand interactions, contain multiple post-translational modification sites, and are subjected to alternative splicing. The disordered structure of BK and its BKAPs suggests one of the underlying mechanisms of their interaction.
Toxoplasma gondii is an obligate intracellular parasite of the phylum Apicomplexa, which includes a number of species of medical and veterinary importance. Inhibitors of lysine deacetylases (KDACs) exhibit potent antiparasitic activity, suggesting that interference with lysine acetylation pathways hold promise for future drug targeting. Using high resolution LC-MS/MS to identify parasite peptides enriched by immunopurification with acetyl-lysine antibody, we recently produced an acetylome of the proliferative intracellular stage of Toxoplasma. In this study, we used similar approaches to greatly expand the Toxoplasma acetylome by identifying acetylated proteins in non-replicating extracellular tachyzoites. The functional breakdown of acetylated proteins in extracellular parasites is similar to intracellular parasites, with an enrichment of proteins involved in metabolism, translation, and chromatin biology. Altogether, we have now detected over 700 acetylation sites on a wide variety of parasite proteins of diverse function in multiple subcellular compartments. We found 96 proteins uniquely acetylated in intracellular parasites, 216 uniquely acetylated in extracellular parasites, and 177 proteins acetylated in both states. Our findings suggest that dramatic changes occur at the proteomic level as tachyzoites transition from the intracellular to extracellular environment, similar to reports documenting significant changes in gene expression during this transition. The expanded dataset also allowed a thorough analysis of the degree of protein intrinsic disorder surrounding lysine residues targeted for this post-translational modification. These analyses indicate that acetylated lysines in proteins from extracellular and intracellular tachyzoites are largely located within similar local environments, and that lysine acetylation preferentially occurs in intrinsically disordered or flexible regions.
parasite; proteomics; acetylation; lysine; Apicomplexa; tachyzoite
Proteins of the p53 family are expressed in vertebrates and in some invertebrate species. The main function of these proteins is to control and regulate cell cycle in response to various cellular signals, and therefore to control the organism’s development. The regulatory functions of the p53 family members originate mostly from their highly-conserved and well-structured DNA-binding domains. Many human diseases (including various types of cancer) are related to the missense mutations within this domain. The ordered DNA-binding domains of the p53 family members are surrounded by functionally important intrinsically disordered regions. In this study, substitution rates and propensities in different regions of p53 were analyzed. The analyses revealed that the ordered DNA-binding domain is conserved, whereas disordered regions are characterized by high sequence diversity. This diversity was reflected both in the number of substitutions and in the types of substitutions to which each amino acid was prone. These results support the existence of a positive correlation between protein intrinsic disorder and sequence divergence during the evolutionary process. This higher sequence divergence provides strong support for the existence of disordered regions in p53 in vivo for if they were structured, they would evolve at similar rates as the rest of the protein.
intrinsically disordered proteins; protein evolution; protein-protein interactions; protein-DNA interaction; p53 family
Intrinsically disordered proteins (IDPs) and proteins with long disordered regions are highly abundant in various proteomes. Despite their lack of well-defined ordered structure, these proteins and regions are frequently involved in crucial biological processes. Although in recent years these proteins have attracted the attention of many researchers, IDPs represent a significant challenge for structural characterization since these proteins can impact many of the processes in the structure determination pipeline. Here we investigate the effects of IDPs on the structure determination process and the utility of disorder prediction in selecting and improving proteins for structural characterization. Examination of the extent of intrinsic disorder in existing crystal structures found that relatively few protein crystal structures contain extensive regions of intrinsic disorder. Although intrinsic disorder is not the only cause of crystallization failures and many structured proteins cannot be crystallized, filtering out highly disordered proteins from structure-determination target lists is still likely to be cost effective. Therefore it is desirable to avoid highly disordered proteins from structure-determination target lists and we show that disorder prediction can be applied effectively to enrich structure determination pipelines with proteins more likely to yield crystal structures. For structural investigation of specific proteins, disorder prediction can be used to improve targets for structure determination. Finally, a framework for considering intrinsic disorder in the structure determination pipeline is proposed.
Proteomics; Structural genomics; Structural proteomics; Intrinsically disordered protein
A novel coronavirus, MERS-CoV (NCoV, HCoV-EMC/2012), originating from the Middle-East, has been discovered. Incoming data reveal that the virus is highly virulent to humans. A model that categorizes coronaviuses according to the hardness of their shells was developed before the discovery of MERS-CoV. Using protein intrinsic disorder prediction, coronaviruses were categorized into three groups that can be linked to the levels of oral-fecal and respiratory transmission regardless of genetic proximity. Using this model, MERS-CoV is placed into disorder group C, which consists of coronaviruses that have relatively hard inner and outer shells. The members of this group are likely to persist in the environment for a longer period of time and possess the highest oral-fecal components but relatively low respiratory transmission components. Oral-urine and saliva transmission are also highly possible since both require harder protective shells. Results show that disorder prediction can be used as a tool that suggests clues to look for in further epidemiological investigations.
The goal of pE-DB (http://pedb.vib.be) is to serve as an openly accessible database for the deposition of structural ensembles of intrinsically disordered proteins (IDPs) and of denatured proteins based on nuclear magnetic resonance spectroscopy, small-angle X-ray scattering and other data measured in solution. Owing to the inherent flexibility of IDPs, solution techniques are particularly appropriate for characterizing their biophysical properties, and structural ensembles in agreement with these data provide a convenient tool for describing the underlying conformational sampling. Database entries consist of (i) primary experimental data with descriptions of the acquisition methods and algorithms used for the ensemble calculations, and (ii) the structural ensembles consistent with these data, provided as a set of models in a Protein Data Bank format. PE-DB is open for submissions from the community, and is intended as a forum for disseminating the structural ensembles and the methodologies used to generate them. While the need to represent the IDP structures is clear, methods for determining and evaluating the structural ensembles are still evolving. The availability of the pE-DB database is expected to promote the development of new modeling methods and leads to a better understanding of how function arises from disordered states.
The identification of intrinsically disordered proteins (IDPs) among the targets that fail to form satisfactory crystal structures in the Protein Structure Initiative represent a key to reducing the costs and time for determining three-dimensional structures of proteins. To help in this endeavor, several Protein Structure Initiative Centers were asked to send samples of both crystallizable proteins and proteins that failed to crystallize. The abundance of intrinsic disorder in these proteins was evaluated via computational analysis using Predictors of Natural Disordered Regions (PONDR®) and the potential cleavage sites and corresponding fragments were determined. Then, the target proteins were analyzed for intrinsic disorder by their resistance to limited proteolysis. The rates of tryptic digestion of sample target proteins were compared to those of lysozyme/myoglobin, apo-myoglobin and α-casein as standards of ordered, partially disordered and completely disordered proteins, respectively. At the next stage, the protein samples were subjected to both far-UV and near-UV circular dichroism (CD) analysis. For most of the samples, a good agreement between CD data, predictions of disorder and the rates of limited tryptic digestion was established. Further experimentation is being performed on a smaller subset of these samples in order to obtain more detailed information on the ordered/disordered nature of the proteins.
Intrinsically disordered proteins; protein disorder prediction; Protein Structure Initiative; limited proteolysis
Parkinson’s disease (PD) is a slowly progressive movement disorder that results from the loss of dopaminergic neurons in the substantia nigra, a small area of cells in the mid-brain. PD is a multifactorial disorder with unknown etiology, in which both genetic and environmental factors play important roles. Substantial evidence links α-synuclein, a small highly conserved presynaptic protein with unknown function, to both familial and sporadic PD. Rare familial cases of PD are associated with missense point mutations in α-synuclein, or with the hyper-expression of the wild type protein due to its gene duplication/triplication. Furthermore, α-synuclein was identified as the major component of amyloid fibrils found in Lewy body and Lewy neurites, the characteristic proteinaceous deposits that are the diagnostic hallmarks of PD. α-Synuclein is abundant in various regions of the brain and has two closely related homologs, β-synuclein and γ-synuclein. When isolated in solution, the protein is intrinsically disordered, but in the presence of lipid surfaces α-synuclein adopts a highly helical structure that is believed to mediate its normal function(s). A number of different conformational states of α-synuclein have been observed. Besides the membrane-bound form, other critical conformations include a partially-folded state that is a key intermediate in aggregation and fibrillation, various oligomeric species, and fibrillar and amorphous aggregates. A number of intrinsic and extrinsic factors that either accelerate or inhibit the rate of α-synuclein aggregation and fibrillation in vitro are known. There is a strong correlation between the conformation of α-synuclein (induced by various factors) and its rate of fibrillation. The aggregation process appears to be branched, with one pathway leading to fibrils and another to oligomeric intermediates that may ultimately form amorphous deposits. The molecular basis of Parkinson’s disease appears to be tightly coupled to the aggregation of α-synuclein and the factors that affect its conformation. This review focuses on the contributions of Prof. Anthony L. Fink to the field and presents some recent developments in this exciting area.
α-Synuclein; synucleinopathies; aggregation; amyloid; fibril; neurodegeneration; intrinsically disordered protein; NMR; partially folded intermediate
Earlier computational and bioinformatics analysis of several large protein datasets across 28 species showed that proteins involved in regulation and execution of programmed cell death (PCD) possess substantial amounts of intrinsic disorder. Based on the comprehensive analysis of these datasets by a wide array of modern bioinformatics tools it was concluded that disordered regions of PCD-related proteins are involved in a multitude of biological functions and interactions with various partners, possess numerous posttranslational modification sites, and have specific evolutionary patterns (Peng
et al. 2013). This study extends our previous work by providing information on the intrinsic disorder status of some of the major players of the three major PCD pathways: apoptosis, autophagy, and necroptosis. We also present a detailed description of the disorder status and interactomes of selected proteins that are involved in the p53-mediated apoptotic signaling pathways.
Aggregation of tau protein in the brain is associated with a class of neurodegenerative diseases known as tauopathies. FK506 binding protein 51 kDa (FKBP51, encoded by FKBP5) forms a mature chaperone complex with Hsp90 that prevents tau degradation. In this study, we have shown that tau levels are reduced throughout the brains of Fkbp5–/– mice. Recombinant FKBP51 and Hsp90 synergized to block tau clearance through the proteasome, resulting in tau oligomerization. Overexpression of FKBP51 in a tau transgenic mouse model revealed that FKBP51 preserved the species of tau that have been linked to Alzheimer’s disease (AD) pathogenesis, blocked amyloid formation, and decreased tangle load in the brain. Alterations in tau turnover and aggregate structure corresponded with enhanced neurotoxicity in mice. In human brains, FKBP51 levels increased relative to age and AD, corresponding with demethylation of the regulatory regions in the FKBP5 gene. We also found that higher FKBP51 levels were associated with AD progression. Our data support a model in which age-associated increases in FKBP51 levels and its interaction with Hsp90 promote neurotoxic tau accumulation. Strategies aimed at attenuating FKBP51 levels or its interaction with Hsp90 have the potential to be therapeutically relevant for AD and other tauopathies.
Previous studies revealed that pesticides interact with α-synuclein and accelerate the rate of fibrillation. These results are consistent with the prevailing hypothesis that the direct interaction of α-synuclein with pesticides is one of many suspected factors leading to α-synuclein fibrillation and ultimately to Parkinson’s disease. In this study, the biophysical properties and fibrillation kinetics of α-synuclein in the presence of rotenone were investigated and, more specifically, the effects of rotenone on the early-stage misfolded forms of α-synuclein were considered. The thioflavine T (ThT) fluorescence assay studies provide evidence that early-phase misfolded α-synuclein forms are affected by rotenone and that the fibrillation process is accelerated. Further characterization by attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) shows that rotenone increases the amount of ordered secondary structure in this intrinsically disordered protein. Morphological characterization by transmission electron microscopy (TEM) and atomic force microscopy (AFM) provide visualization of the differences in the aggregated α-synuclein species developing during the early kinetics of the fibrillation process in the absence and presence of rotenone. We believe that these data provide useful information for a better understanding of the molecular basis of rotenone-induced misfolding and aggregation of α-synuclein.
α-Synuclein; Parkinson’s disease; environmental toxin; misfolding; fibrillation; intrinsically disordered protein; pesticide; agrochemical; rotenone
The abundance and potential functional roles of intrinsically disordered regions in aquaporin-4, Kir4.1, a dystrophin isoforms Dp71, α-1 syntrophin, and α-dystrobrevin; i.e., proteins constituting the functional core of the astrocytic dystrophin-associated protein complex (DAPC), are analyzed by a wealth of computational tools. The correlation between protein intrinsic disorder, single nucleotide polymorphisms (SNPs) and protein function is also studied together with the peculiarities of structural and functional conservation of these proteins. Our study revealed that the DAPC members are typical hybrid proteins that contain both ordered and intrinsically disordered regions. Both ordered and disordered regions are important for the stabilization of this complex. Many disordered binding regions of these five proteins are highly conserved among vertebrates. Conserved eukaryotic linear motifs and molecular recognition features found in the disordered regions of five protein constituting DAPC likely enhance protein-protein interactions that are required for the cellular functions of this complex. Curiously, the disorder-based binding regions are rarely affected by SNPs suggesting that these regions are crucial for the biological functions of their corresponding proteins.
We analyzed the applicability of high-resolution 2H-HMR spectroscopy for the analysis of microbe metabolism in samples of mitochondrion isolated from rat liver and from aqueous extracts of homogenates of rat liver and other organs and tissues in the presence of high D2O contents. Such analysis is possible due to the fast microbe adaptation to life in the heavy water. It is also shown that some enzymatic processes typical for the intact cells are preserved in the homogenized tissue preparations. The microbial and cellular metabolic processes can be differentiated via the strategic use of cell poisons and antibiotics.
Microbe adaptation; Microbial metabolism; 2H-HMR spectroscopy; High resolution NMR; Heavy water
IDPs, while structurally poor, are functionally rich by virtue of their flexibility and modularity. However, how mutations in IDPs elicit diseases, remain elusive. Herein, we have identified tumor suppressor PTEN as an intrinsically disordered protein (IDP) and elucidated the molecular principles by which its intrinsically disordered region (IDR) at the carboxyl-terminus (C-tail) executes its functions. Post-translational modifications, conserved eukaryotic linear motifs and molecular recognition features present in the C-tail IDR enhance PTEN's protein-protein interactions that are required for its myriad cellular functions. PTEN primary and secondary interactomes are also enriched in IDPs, most being cancer related, revealing that PTEN functions emanate from and are nucleated by the C-tail IDR, which form pliable network-hubs. Together, PTEN higher order functional networks operate via multiple IDP-IDP interactions facilitated by its C-tail IDR. Targeting PTEN IDR and its interaction hubs emerges as a new paradigm for treatment of PTEN related pathologies.
Targeted therapy for cancer, which is specifically directed toward the cancer without any potential for effects outside of controlling the tumor, is a gold standard for treatment. Ewing’s sarcoma contains the potential target EWS-FLI1, as a result of a pathognomonic chromosomal translocation. The EWS-FLI1 fusion protein includes the EWS domain, a potent transcriptional activator alongside the highly conserved FLI1 ets DNA-binding domain. Because of the combination of these domains, the EWS-FLI1 fusion protein acts as an aberrant transcription factor whose expression results in cellular transformation. EWS-FLI1 functions by binding to normal cellular protein partners in transcription and splicing, similar to how a virus would corrupt normal cellular machinery for virion production. Therefore, understanding the protein-protein interactions of EWS-FLI1 and the pathways that are regulated by these partnerships will inform both oncogenesis and therapeutics. This review describes the known protein partners and transcriptional targets of EWS-FLI1, while proposing strategies for exploiting these partnerships with targeted therapy.
Alternative splicing is critical for generating complex proteomes in response to extracellular signals. Nuclear receptors including estrogen receptor alpha (ERα) and their ligands promote alternative splicing. The endogenous targets of ERα:estradiol (E2)-mediated alternative splicing and the influence of extracellular kinases that phosphorylate ERα on E2-induced splicing are unknown.
MCF-7 and its anti-estrogen derivatives were used for the majority of the assays. CD44 mini gene was used to measure the effect of E2 and AKT on alternative splicing. ExonHit array analysis was performed to identify E2 and AKT-regulated endogenous alternatively spliced apoptosis-related genes. Quantitative reverse transcription polymerase chain reaction was performed to verify alternative splicing. ERα binding to alternatively spliced genes was verified by chromatin immunoprecipitation assay. Bromodeoxyuridine incorporation-ELISA and Annexin V labeling assays were done to measure cell proliferation and apoptosis, respectively.
We identified the targets of E2-induced alternative splicing and deconstructed some of the mechanisms surrounding E2-induced splicing by combining splice array with ERα cistrome and gene expression array. E2-induced alternatively spliced genes fall into at least two subgroups: coupled to E2-regulated transcription and ERα binding to the gene without an effect on rate of transcription. Further, AKT, which phosphorylates both ERα and splicing factors, influenced ERα:E2 dependent splicing in a gene-specific manner. Genes that are alternatively spliced include FAS/CD95, FGFR2, and AXIN-1. E2 increased the expression of FGFR2 C1 isoform but reduced C3 isoform at mRNA level. E2-induced alternative splicing of FAS and FGFR2 in MCF-7 cells correlated with resistance to FAS activation-induced apoptosis and response to keratinocyte growth factor (KGF), respectively. Resistance of MCF-7 breast cancer cells to the anti-estrogen tamoxifen was associated with ERα-dependent overexpression of FGFR2, whereas resistance to fulvestrant was associated with ERα-dependent isoform switching, which correlated with altered response to KGF.
E2 may partly alter cellular proteome through alternative splicing uncoupled to its effects on transcription initiation and aberration in E2-induced alternative splicing events may influence response to anti-estrogens.
Intrinsically disordered proteins (IDPs) are a relatively recently defined class of proteins which, under native conditions, lack a unique tertiary structure whilst maintaining essential biological functions. Functional classification of IDPs have implicated such proteins as being involved in various physiological processes including transcription and translation regulation, signal transduction and protein modification. Actinidia DRM1 (Ade DORMANCY ASSOCIATED GENE 1), represents a robust dormancy marker whose mRNA transcript expression exhibits a strong inverse correlation with the onset of growth following periods of physiological dormancy. Bioinformatic analyses suggest that DRM1 is plant specific and highly conserved at both the nucleotide and protein levels. It is predicted to be an intrinsically disordered protein with two distinct highly conserved domains. Several Actinidia DRM1 homologues, which align into two distinct Actinidia-specific families, Type I and Type II, have been identified. No candidates for the Arabidopsis DRM1-Homologue (AtDRM2) an additional family member, has been identified in Actinidia.
Protein structure is commonly regarded to be conserved and to dictate function. Most proteins rely on conformational flexibility to some degree. Are regions that convey conformational flexibility conserved over evolutionary time? Can changes in conformational flexibility alter protein function? Here, the evolutionary dynamics of structurally ordered and disordered (flexible) regions are investigated genome-wide in flaviviruses, revealing that the amount and location of structural disorder fluctuates highly among related proteins. Some regions are prone to shift between structured and flexible states. Increased evolutionary dynamics of structural disorder is observed for some lineages but not in others. Lineage-specific transitions of this kind could alter the conformational ensemble accessible to the same protein in different species, causing a functional change, even if the predominant function remains conserved. Thus, rapid evolutionary dynamics of structural disorder is a potential driving force for phenotypic divergence among flaviviruses.
conformational flexibility; comparative genomics; structural disorder; flavivirus; divergence