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

Wide-line H 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.

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.

A cyclin D1 intrinsically disordered domain accesses modified histone motifs to govern gene transcription.

The essential G-cyclin, CCND1, is frequently overexpressed in cancer, contributing to tumorigenesis by driving cell-cycle progression. D-type cyclins are rate-limiting regulators of G-S progression in mammalian cells via their ability to bind and activate CDK4 and CDK6. In addition, cyclin D1 conveys kinase-independent transcriptional functions of cyclin D1.

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.

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.

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.

Intrinsic protein disorder uncouples affinity from binding specificity.

Intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) of proteins often function by molecular recognition, in which they undergo induced folding. Based on prior generalizations, the idea prevails in the IDP field that due to the entropic penalty of induced folding, the major functional advantage associated with this binding mode is "uncoupling" specificity from binding strength. Nevertheless, both weaker binding and high specificity of IDPs/IDRs rest on limited experimental observations, making these assumptions more speculations than evidence-supported facts.

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

The 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.

Wide-Line NMR Melting Diagrams, Their Thermodynamic Interpretation, and Secondary Structure Predictions for A30P and E46K α-Synuclein.

Parkinson's disease is thought to be caused by aggregation of the intrinsically disordered protein, α-synuclein. Two amyloidogenic variants, A30P, and E46K familial mutants were investigated by wide-line H NMR spectrometry as a completion of our earlier work on wild-type and A53T α-synuclein (Bokor M. et al.

The Disordered EZH2 Loop: Atomic Level Characterization by H- and H-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.

Disordered-Ordered Protein Binary Classification by Circular Dichroism Spectroscopy.

Intrinsically disordered proteins lack a stable tertiary structure and form dynamic conformational ensembles due to their characteristic physicochemical properties and amino acid composition. They are abundant in nature and responsible for a large variety of cellular functions. While numerous bioinformatics tools have been developed for disorder prediction in the last decades, there is a need for experimental methods to verify the disordered state.

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 β-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.

Identification of Intrinsically Disordered Proteins and Regions in a Non-Model Insect Species (Hbn.).

Research in previous decades has shown that intrinsically disordered proteins (IDPs) and regions in proteins (IDRs) are as ubiquitous as highly ordered proteins. Despite this, research on IDPs and IDRs still has many gaps left to fill. Here, we present an approach that combines wet lab methods with bioinformatics tools to identify and analyze intrinsically disordered proteins in a non-model insect species that is cold-hardy.

Deep structural insights into RNA-binding disordered protein regions.

Recent efforts to identify RNA binding proteins in various organisms and cellular contexts have yielded a large collection of proteins that are capable of RNA binding in the absence of conventional RNA recognition domains. Many of the recently identified RNA interaction motifs fall into intrinsically disordered protein regions (IDRs). While the recognition mode and specificity of globular RNA binding elements have been thoroughly investigated and described, much less is known about the way IDRs can recognize their RNA partners.

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.

Protein-Protein Connections-Oligomer, Amyloid and Protein Complex-By Wide Line H NMR.

The amount of bonds between constituting parts of a protein aggregate were determined in wild type (WT) and A53T α-synuclein (αS) oligomers, amyloids and in the complex of thymosin-β-cytoplasmic domain of stabilin-2 (Tβ-stabilin CTD). A53T αS aggregates have more extensive βsheet contents reflected by constant regions at low potential barriers in difference (to monomers) melting diagrams (s). Energies of the intermolecular interactions and of secondary structures bonds, formed during polymerization, fall into the 5.

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

Secondary structure predictions of proteins were compared to experimental results by wide-line H NMR. IUPred2A was used to generate predictions of disordered protein or binding regions. Thymosin-β and the stabilin-2 cytoplasmic domain were found to be mainly disordered, in agreement with the experimental results.

A generic approach to study the kinetics of liquid-liquid phase separation under near-native conditions.

Understanding the kinetics, thermodynamics, and molecular mechanisms of liquid-liquid phase separation (LLPS) is of paramount importance in cell biology, requiring reproducible methods for studying often severely aggregation-prone proteins. Frequently applied approaches for inducing LLPS, such as dilution of the protein from an urea-containing solution or cleavage of its fused solubility tag, often lead to very different kinetic behaviors. Here we demonstrate that at carefully selected pH values proteins such as the low-complexity domain of hnRNPA2, TDP-43, and NUP98, or the stress protein ERD14, can be kept in solution and their LLPS can then be induced by a jump to native pH.

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

The potential barriers governing the motions of -synuclein (S) 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.

DisProt: intrinsic protein disorder annotation in 2020.

The Database of Protein Disorder (DisProt, URL: https://disprot.org) provides manually curated annotations of intrinsically disordered proteins from the literature. Here we report recent developments with DisProt (version 8), including the doubling of protein entries, a new disorder ontology, improvements of the annotation format and a completely new website.

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.