BeStSel: webserver for secondary structure and fold prediction for protein CD spectroscopy.

Circular dichroism (CD) spectroscopy is widely used to characterize the secondary structure composition of proteins. To derive accurate and detailed structural information from the CD spectra, we have developed the Beta Structure Selection (BeStSel) method (PNAS, 112, E3095), which can handle the spectral diversity of ß-structured proteins. The BeStSel webserver provides this method with useful accessories to the community with the main goal to analyze single or multiple protein CD spectra. Uniquely, BeStSel provides information on eight secondary structure components including parallel ß-structure and antiparallel ß-sheets with three different groups of twist. It overperforms any available method in accuracy and information content, moreover, it is capable of predicting the protein fold down to the topology/homology level of the CATH classification. A new module of the webserver helps to distinguish intrinsically disordered proteins by their CD spectrum. Secondary structure calculation for uploaded PDB files will help the experimental verification of protein MD and in silico modelling using CD spectroscopy. The server also calculates extinction coefficients from the primary sequence for CD users to determine the accurate protein concentrations which is a prerequisite for reliable secondary structure determination. The BeStSel server can be freely accessed at https://bestsel.elte.hu.

Absence of Scaffold Protein Tks4 Disrupts Several Signaling Pathways in Colon Cancer Cells.

Tks4 is a large scaffold protein in the EGFR signal transduction pathway that is involved in several cellular processes, such as cellular motility, reactive oxygen species-dependent processes, and embryonic development. It is also implicated in a rare developmental disorder, Frank-ter Haar syndrome. Loss of Tks4 resulted in the induction of an EMT-like process, with increased motility and overexpression of EMT markers in colorectal carcinoma cells. In this work, we explored the broader effects of deletion of Tks4 on the gene expression pattern of HCT116 colorectal carcinoma cells by transcriptome sequencing of wild-type and Tks4 knockout (KO) cells. We identified several protein coding genes with altered mRNA levels in the Tks4 KO cell line, as well as a set of long non-coding RNAs, and confirmed these changes with quantitative PCR on a selected set of genes. Our results show a significant perturbation of gene expression upon the deletion of Tks4, suggesting the involvement of different signal transduction pathways over the well-known EGFR signaling.

In Vivo and In Vitro Characterization of the RNA Binding Capacity of SETD1A (KMT2F).

For several histone lysine methyltransferases (HKMTs), RNA binding has been already shown to be a functionally relevant feature, but detailed information on the RNA interactome of these proteins is not always known. Of the six human KMT2 proteins responsible for the methylation of the H3K4 residue, two-SETD1A and SETD1B-contain RNA recognition domains (RRMs). Here we investigated the RNA binding capacity of SETD1A and identified a broad range of interacting RNAs within HEK293T cells. Our analysis revealed that similar to yeast Set1, SETD1A is also capable of binding several coding and non-coding RNAs, including RNA species related to RNA processing. We also show direct RNA binding activity of the individual RRM domain in vitro, which is in contrast with the RRM domain found in yeast Set1. Structural modeling revealed important details on the possible RNA recognition mode of SETD1A and highlighted some fundamental differences between SETD1A and Set1, explaining the differences in the RNA binding capacity of their respective RRMs.

PhaSePro: the database of proteins driving liquid-liquid phase separation.

Membraneless organelles (MOs) are dynamic liquid condensates that host a variety of specific cellular processes, such as ribosome biogenesis or RNA degradation. MOs form through liquid-liquid phase separation (LLPS), a process that relies on multivalent weak interactions of the constituent proteins and other macromolecules. Since the first discoveries of certain proteins being able to drive LLPS, it emerged as a general mechanism for the effective organization of cellular space that is exploited in all kingdoms of life. While numerous experimental studies report novel cases, the computational identification of LLPS drivers is lagging behind, and many open questions remain about the sequence determinants, composition, regulation and biological relevance of the resulting condensates. Our limited ability to overcome these issues is largely due to the lack of a dedicated LLPS database. Therefore, here we introduce PhaSePro (https://phasepro.elte.hu), an openly accessible, comprehensive, manually curated database of experimentally validated LLPS driver proteins/protein regions. It not only provides a wealth of information on such systems, but improves the standardization of data by introducing novel LLPS-specific controlled vocabularies. PhaSePro can be accessed through an appealing, user-friendly interface and thus has definite potential to become the central resource in this dynamically developing field.

The Disordered EZH2 Loop: Atomic Level Characterization by 1HN- and 1Hα-Detected NMR Approaches, Interaction with the Long Noncoding HOTAIR RNA.

The 96-residue-long loop of EZH2 is proposed to play a role in the interaction with long non-coding RNAs (lncRNAs) and to contribute to EZH2 recruitment to the chromatin. However, molecular details of RNA recognition have not been described so far. Cellular studies have suggested that phosphorylation of the Thr345 residue localized in this loop influences RNA binding; however, no mechanistic explanation has been offered. To address these issues, a systematic NMR study was performed. As the 1HN-detected NMR approach presents many challenges under physiological conditions, our earlier developed, as well as improved, 1Hα-detected experiments were used. As a result of the successful resonance assignment, the obtained chemical shift values indicate the highly disordered nature of the EZH2 loop, with some nascent helical tendency in the Ser407-Ser412 region. Further investigations conducted on the phosphomimetic mutant EZH2T345D showed that the mutation has only a local effect, and that the loop remains disordered. On the other hand, the mutation influences the cis/trans Pro346 equilibrium. Interactions of both the wild-type and the phosphomimetic mutant with the lncRNA HOTAIR140 (1-140 nt) highlight that the Thr367-Ser375 region is affected. This segment does not resemble any of the previously reported RNA-binding motifs, therefore the identified binding region is unique. As no structural changes occur in the EZH2 loop upon RNA binding, we can consider the protein-RNA interaction as a "fuzzy" complex.

Functional Characterization of the N-Terminal Disordered Region of the piggyBac Transposase.

The piggyBac DNA transposon is an active element initially isolated from the cabbage looper moth, but members of this superfamily are also present in most eukaryotic evolutionary lineages. The functionally important regions of the transposase are well described. There is an RNase H-like fold containing the DDD motif responsible for the catalytic DNA cleavage and joining reactions and a C-terminal cysteine-rich domain important for interaction with the transposon DNA. However, the protein also contains a ~100 amino acid long N-terminal disordered region (NTDR) whose function is currently unknown. Here we show that deletion of the NTDR significantly impairs piggyBac transposition, although the extent of decrease is strongly cell-type specific. Moreover, replacing the NTDR with scrambled but similarly disordered sequences did not rescue transposase activity, indicating the importance of sequence conservation. Cell-based transposon excision and integration assays reveal that the excision step is more severely affected by NTDR deletion. Finally, bioinformatic analyses indicated that the NTDR is specific for the piggyBac superfamily and is also present in domesticated, transposase-derived proteins incapable of catalyzing transposition. Our results indicate an essential role of the NTDR in the "fine-tuning" of transposition and its significance in the functions of piggyBac-originated co-opted genes.

Secondary Structures of Proteins: A Comparison of Models and Experimental Results.

Secondary structure predictions of proteins were compared to experimental results by wide-line 1H NMR. IUPred2A was used to generate predictions of disordered protein or binding regions. Thymosin-ß4 and the stabilin-2 cytoplasmic domain were found to be mainly disordered, in agreement with the experimental results. α-Synuclein variants were predicted to be disordered, as in the experiments, but the A53T mutant showed less predicted disorder, in contrast with the wide-line 1H NMR result. A disordered binding site was found for thymosin-ß4, whereas the stabilin-2 cytoplasmic domain was indicated as such in its entire length. The last third of the α-synuclein variant's sequence was a disordered binding site. Thymosin-ß4 and the stabilin-2 cytoplasmic domain contained only coils and helices according to five secondary structure prediction methods (SPIDER3-SPOT-1D, PSRSM, MUFold-SSW, Porter 5, and RaptorX). ß-Sheets are present in α-synucleins, and they extend to more amino acid residues in the A53T mutant according to the predictions. The latter is verified by experiments. The comparison of the predictions with the experiments suggests that helical parts are buried.

Cellular Chaperone Function of Intrinsically Disordered Dehydrin ERD14.

Disordered plant chaperones play key roles in helping plants survive in harsh conditions, and they are indispensable for seeds to remain viable. Aside from well-known and thoroughly characterized globular chaperone proteins, there are a number of intrinsically disordered proteins (IDPs) that can also serve as highly effective protecting agents in the cells. One of the largest groups of disordered chaperones is the group of dehydrins, proteins that are expressed at high levels under different abiotic stress conditions, such as drought, high temperature, or osmotic stress. Dehydrins are characterized by the presence of different conserved sequence motifs that also serve as the basis for their categorization. Despite their accepted importance, the exact role and relevance of the conserved regions have not yet been formally addressed. Here, we explored the involvement of each conserved segment in the protective function of the intrinsically disordered stress protein (IDSP) A. thaliana's Early Response to Dehydration (ERD14). We show that segments that are directly involved in partner binding, and others that are not, are equally necessary for proper function and that cellular protection emerges from the balanced interplay of different regions of ERD14.

WT and A53T α-Synuclein Systems: Melting Diagram and Its New Interpretation.

The potential barriers governing the motions of α-synuclein (αS) variants' hydration water, especially energetics of them, is in the focus of the work. The thermodynamical approach yielded essential information about distributions and heights of the potential barriers. The proteins' structural disorder was measured by ratios of heterogeneous water-binding interfaces. They showed the αS monomers, oligomers and amyloids to possess secondary structural elements, although monomers are intrinsically disordered. Despite their disordered nature, monomers have 33% secondary structure, and therefore they are more compact than a random coil. At the lowest potential barriers with mobile hydration water, monomers are already functional, a monolayer of mobile hydration water is surrounding them. Monomers realize all possible hydrogen bonds with the solvent water. αS oligomers and amyloids have half of the mobile hydration water amount than monomers because aggregation involves less mobile hydration. The solvent-accessible surface of the oligomers is ordered or homogenous in its interactions with water to 66%. As a contrast, αS amyloids are disordered or heterogeneous to 75% of their solvent accessible surface and both wild type and A53T amyloids show identical, low-level hydration. Mobile water molecules in the first hydration shell of amyloids are the weakest bound compared to other forms.

DisProt 7.0: a major update of the database of disordered proteins.

The Database of Protein Disorder (DisProt, URL: www.disprot.org) has been significantly updated and upgraded since its last major renewal in 2007. The current release holds information on more than 800 entries of IDPs/IDRs, i.e. intrinsically disordered proteins or regions that exist and function without a well-defined three-dimensional structure. We have re-curated previous entries to purge DisProt from conflicting cases, and also upgraded the functional classification scheme to reflect continuous advance in the field in the past 10 years or so. We define IDPs as proteins that are disordered along their entire sequence, i.e. entirely lack structural elements, and IDRs as regions that are at least five consecutive residues without well-defined structure. We base our assessment of disorder strictly on experimental evidence, such as X-ray crystallography and nuclear magnetic resonance (primary techniques) and a broad range of other experimental approaches (secondary techniques). Confident and ambiguous annotations are highlighted separately. DisProt 7.0 presents classified knowledge regarding the experimental characterization and functional annotations of IDPs/IDRs, and is intended to provide an invaluable resource for the research community for a better understanding structural disorder and for developing better computational tools for studying disordered proteins.

Intrinsically Disordered Linkers Impart Processivity on Enzymes by Spatial Confinement of Binding Domains.

(1) Background: Processivity is common among enzymes and mechanochemical motors that synthesize, degrade, modify or move along polymeric substrates, such as DNA, RNA, polysaccharides or proteins. Processive enzymes can make multiple rounds of modification without releasing the substrate/partner, making their operation extremely effective and economical. The molecular mechanism of processivity is rather well understood in cases when the enzyme structurally confines the substrate, such as the DNA replication factor PCNA, and also when ATP energy is used to confine the succession of molecular events, such as with mechanochemical motors. Processivity may also result from the kinetic bias of binding imposed by spatial confinement of two binding elements connected by an intrinsically disordered (ID) linker. (2) Method: By statistical physical modeling, we show that this arrangement results in processive systems, in which the linker ensures an optimized effective concentration around novel binding site(s), favoring rebinding over full release of the polymeric partner. (3) Results: By analyzing 12 such proteins, such as cellulase, and RNAse-H, we illustrate that in these proteins linker length and flexibility, and the kinetic parameters of binding elements, are fine-tuned for optimizing processivity. We also report a conservation of structural disorder, special amino acid composition of linkers, and the correlation of their length with step size. (4) Conclusion: These observations suggest a unique type of entropic chain function of ID proteins, that may impart functional advantages on diverse enzymes in a variety of biological contexts.

Molecular Motions and Interactions in Aqueous Solutions of Thymosin-ß4 , Stabilin C-Terminal Domain (CTD) and Their 1:1 Complex Studied by 1 H†NMR Spectroscopy.

Wide-line 1 H NMR measurements were extended and all results were reinterpreted in a new thermodynamics-based approach to study aqueous solutions of thymosin-ß4 (Tß4 ), stabilin C-terminal domain (CTD) and their 1:1 complex. The energy distributions of the potential barriers, which control motion of protein-bound water molecules, were determined. Heterogeneous and homogeneous regions were found at the protein-water interface. The measure of heterogeneity gives a quantitative value for the portion of disordered parts in the protein. Ordered structural elements were found extending up to 20 % of the whole proteins. About 40 % of the binding sites of free Tß4 become involved in bonds holding the complex together. The complex has the most heterogeneous solvent accessible surface (SAS) in terms of protein-water interactions. The complex is more disordered than Tß4 or stabilin CTD. The greater SAS area of the complex is interpreted as a clear sign of its open structure.

Disordered Regions of Mixed Lineage Leukemia 4 (MLL4) Protein Are Capable of RNA Binding.

Long non-coding RNAs (lncRNAs) are emerging as important regulators of cellular processes and are extensively involved in the development of different cancers; including leukemias. As one of the accepted methods of lncRNA function is affecting chromatin structure; lncRNA binding has been shown for different chromatin modifiers. Histone lysine methyltransferases (HKMTs) are also subject of lncRNA regulation as demonstrated for example in the case of Polycomb Repressive Complex 2 (PRC2). Mixed Lineage Leukemia (MLL) proteins that catalyze the methylation of H3K4 have been implicated in several different cancers; yet many details of their regulation and targeting remain elusive. In this work we explored the RNA binding capability of two; so far uncharacterized regions of MLL4; with the aim of shedding light to the existence of possible regulatory lncRNA interactions of the protein. We demonstrated that both regions; one that contains a predicted RNA binding sequence and one that does not; are capable of binding to different RNA constructs in vitro. To our knowledge, these findings are the first to indicate that an MLL protein itself is capable of lncRNA binding.

The role of structural disorder in cell cycle regulation, related clinical proteomics, disease development and drug targeting.

Understanding the molecular mechanisms of the regulation of cell cycle is a central issue in molecular cell biology, due to its fundamental role in the existence of cells. The regulatory circuits that make decisions on when a cell should divide are very complex and particularly subtly balanced in eukaryotes, in which the harmony of many different cells in an organism is essential for life. Several hundred proteins are involved in these processes, and a great deal of studies attests that most of them have functionally relevant intrinsic structural disorder. Structural disorder imparts many functional advantages on these proteins, and we discuss it in detail that it is involved in all key steps from signaling through the cell membrane to regulating transcription of proteins that execute timely responses to an ever-changing environment.

Structural disorder and local order of hNopp140.

Human nucleolar phosphoprotein p140 (hNopp 140) is a highly phosphorylated protein inhibitor of casein kinase 2 (CK2). As in the case of many kinase-inhibitor systems, the inhibitor has been described to belong to the family of intrinsically disordered proteins (IDPs), which often utilize transient structural elements to bind their cognate enzyme. Here we investigated the structural status of this protein both to provide distinct lines of evidence for its disorder and to point out its transient structure potentially involved in interactions and also its tendency to aggregate. Structural disorder of hNopp140 is apparent by its anomalous electrophoretic mobility, protease sensitivity, heat stability, hydrodynamic behavior on size-exclusion chromatography, (1)H NMR spectrum and differential scanning calorimetry scan. hNopp140 has a significant tendency to aggregate and the change of its circular dichroism spectrum in the presence of 0-80% TFE suggests a tendency to form local helical structures. Wide-line NMR measurements suggest the overall disordered character of the protein. In all, our data suggest that this protein falls into the pre-molten globule state of IDPs, with a significant tendency to become ordered in the presence of its partner as demonstrated in the presence of transcription factor IIB (TFIIB).

General Characterization of Properties of Ordered and Disordered Proteins by Wide-Line 1H NMR.

Wide-line 1H NMR is an efficient spectroscopic method to determine the disorder tendency of a protein. It directly measures the properties of the hydration shell of proteins, delivering exact and measurable values of their disorder/order content. A comparison is performed between several globular and disordered proteins. The common properties of the subzero mobile hydration water of these two groups were investigated. The amount of the mobile hydration water and the shape of the melting diagram at subzero temperatures together provide a possibility to distinguish globular proteins from disordered proteins. The shape of the melting diagram also gives information about the presence of secondary structural elements. The disordered and globular protein regions' fundamentally different structures are reflected in their melting diagrams, allowing one to directly determine the level of disorder in a specific protein structure. Intrinsically disordered proteins bind water more strongly than globular proteins, which is shown by the somewhat higher temperature values where mobile hydration water first appears but with a significantly higher heterogeneity in the energy distributions of protein-water interactions.

Morphological Changes Induced by TKS4 Deficiency Can Be Reversed by EZH2 Inhibition in Colorectal Carcinoma Cells.

BACKGROUND: The scaffold protein tyrosine kinase substrate 4 (TKS4) undergoes tyrosine phosphorylation by the epidermal growth factor receptor (EGFR) pathway via Src kinase. The TKS4 deficiency in humans is responsible for the manifestation of a genetic disorder known as Frank-Ter Haar syndrome (FTHS). Based on our earlier investigation, the absence of TKS4 triggers migration, invasion, and epithelial-mesenchymal transition (EMT)-like phenomena while concurrently suppressing cell proliferation in HCT116 colorectal carcinoma cells. This indicates that TKS4 may play a unique role in the progression of cancer. In this study, we demonstrated that the enhancer of zeste homolog 2 (EZH2) and the histone methyltransferase of polycomb repressive complex 2 (PRC2) are involved in the migration, invasion, and EMT-like changes in TKS4-deficient cells (KO). EZH2 is responsible for the maintenance of the trimethylated lysine 27 on histone H3 (H3K27me3). METHODS: We performed transcriptome sequencing, chromatin immunoprecipitation, protein and RNA quantitative studies, cell mobility, invasion, and proliferation studies combined with/without the EZH2 activity inhibitor 3-deazanoplanocine (DZNep). RESULTS: We detected an elevation of global H3K27me3 levels in the TKS4 KO cells, which could be reduced with treatment with DZNep, an EZH2 inhibitor. Inhibition of EZH2 activity reversed the phenotypic effects of the knockout of TKS4, reducing the migration speed and wound healing capacity of the cells as well as decreasing the invasion capacity, while the decrease in cell proliferation became stronger. In addition, inhibition of EZH2 activity also reversed most epithelial and mesenchymal markers. We investigated the wider impact of TKS4 deletion on the gene expression profile of colorectal cancer cells using transcriptome sequencing of wild-type and TKS4 knockout cells, particularly before and after treatment with DZNep. Additionally, we observed changes in the expression of several protein-coding genes and long non-coding RNAs that showed a recovery in expression levels following EZH2 inhibition. CONCLUSIONS: Our results indicate that the removal of TKS4 causes a notable disruption in the gene expression pattern, leading to the disruption of several signal transduction pathways. Inhibiting the activity of EZH2 can restore most of these transcriptomics and phenotypic effects in colorectal carcinoma cells.

KMT2D preferentially binds mRNAs of the genes it regulates, suggesting a role in RNA processing.

Histone lysine methyltransferases (HKMTs) perform vital roles in cellular life by controlling gene expression programs through the posttranslational modification of histone tails. Since many of them are intimately involved in the development of different diseases, including several cancers, understanding the molecular mechanisms that control their target recognition and activity is vital for the treatment and prevention of such conditions. RNA binding has been shown to be an important regulatory factor in the function of several HKMTs, such as the yeast Set1 and the human Ezh2. Moreover, many HKMTs are capable of RNA binding in the absence of a canonical RNA binding domain. Here, we explored the RNA binding capacity of KMT2D, one of the major H3K4 monomethyl transferases in enhancers, using RNA immunoprecipitation followed by sequencing. We identified a broad range of coding and non-coding RNAs associated with KMT2D and confirmed their binding through RNA immunoprecipitation and quantitative PCR. We also showed that a separated RNA binding region within KMT2D is capable of binding a similar RNA pool, but differences in the binding specificity indicate the existence of other regulatory elements in the sequence of KMT2D. Analysis of the bound mRNAs revealed that KMT2D preferentially binds co-transcriptionally to the mRNAs of the genes under its control, while also interacting with super enhancer- and splicing-related non-coding RNAs. These observations, together with the nuclear colocalization of KMT2D with differentially phosphorylated forms of RNA Polymerase II suggest a so far unexplored role of KMT2D in the RNA processing of the nascent transcripts.