Structural Adaptation of Secondary p53 Binding Sites on MDM2 and MDMX.

The thermodynamics of secondary p53 binding sites on MDM2 and MDMX were evaluated using p53 peptides containing residues 16-29, 17-35, and 1-73. All the peptides had large, negative heat capacity (ΔCp), consistent with the burial of p53 residues F19, W23, and L26 in the primary binding sites of MDM2 and MDMX. MDMX has a higher affinity and more negative ΔCp than MDM2 for p5317-35, which is due to MDMX stabilization and not additional interactions with the secondary binding site. ΔCp measurements show binding to the secondary site is inhibited by the disordered tails of MDM2 for WT p53 but not a more helical mutant where proline 27 is changed to alanine. This result is supported by all-atom molecular dynamics simulations showing that p53 residues 30-35 turn away from the disordered tails of MDM2 in P27A17-35 and make direct contact with this region in p5317-35. Molecular dynamics simulations also suggest that an intramolecular methionine-aromatic motif found in both MDM2 and MDMX structurally adapts to support multiple p53 binding modes with the secondary site. ΔCp measurements also show that tighter binding of the P27A mutant to MDM2 and MDMX is due to increased helicity, which reduces the energetic penalty associated with coupled folding and binding. Our results will facilitate the design of selective p53 inhibitors for MDM2 and MDMX.

The Proteomic Analysis of Cancer-Related Alterations in the Human Unfoldome.

Many proteins lack stable 3D structures. These intrinsically disordered proteins (IDPs) or hybrid proteins containing ordered domains with intrinsically disordered protein regions (IDPRs) often carry out regulatory functions related to molecular recognition and signal transduction. IDPs/IDPRs constitute a substantial portion of the human proteome and are termed "the unfoldome". Herein, we probe the human breast cancer unfoldome and investigate relations between IDPs and key disease genes and pathways. We utilized bottom-up proteomics, MudPIT (Multidimensional Protein Identification Technology), to profile differentially expressed IDPs in human normal (MCF-10A) and breast cancer (BT-549) cell lines. Overall, we identified 2271 protein groups in the unfoldome of normal and cancer proteomes, with 148 IDPs found to be significantly differentially expressed in cancer cells. Further analysis produced annotations of 140 IDPs, which were then classified to GO (Gene Ontology) categories and pathways. In total, 65% (91 of 140) IDPs were related to various diseases, and 20% (28 of 140) mapped to cancer terms. A substantial portion of the differentially expressed IDPs contained disordered regions, confirmed by in silico characterization. Overall, our analyses suggest high levels of interactivity in the human cancer unfoldome and a prevalence of moderately and highly disordered proteins in the network.

The distal-proximal relationships among the human moonlighting proteins: Evolutionary hotspots and Darwinian checkpoints.

Moonlighting proteins, known for their ability to perform multiple, often unrelated functions within a single polypeptide chain, challenge the traditional "one gene, one protein, one function" paradigm. As organisms evolved, their genomes remained relatively stable in size, but the introduction of post-translational modifications and sub-strategies like protein promiscuity and intrinsic disorder enabled multifunctionality. Enzymes, in particular, exemplify this phenomenon, engaging in unrelated processes alongside their primary catalytic roles. This study employs a systematic, quantitative informatics approach to shed light on human moonlighting protein sequences. Phylogenetic analyses of human moonlighting proteins are presented, elucidating the distal-proximal relationships among these proteins based on sequence-derived quantitative features. The findings unveil the captivating world of human moonlighting proteins, urging further investigations in the emerging field of moonlighting proteomics, with the potential for significant contributions to our understanding of multifunctional proteins and their roles in diverse cellular processes and diseases.

Intrinsic disorder in PRAME and its role in uveal melanoma.

INTRODUCTION: The PReferentially expressed Antigen in MElanoma (PRAME) protein has been shown to be an independent biomarker for increased risk of metastasis in Class 1 uveal melanomas (UM). Intrinsically disordered proteins and regions of proteins (IDPs/IDPRs) are proteins that do not have a well-defined three-dimensional structure and have been linked to neoplastic development. Our study aimed to evaluate the presence of intrinsic disorder in PRAME and the role these structureless regions have in PRAME( +) Class 1 UM. METHODS: A bioinformatics study to characterize PRAME's propensity for the intrinsic disorder. We first used the AlphaFold tool to qualitatively assess the protein structure of PRAME. Then we used the Compositional Profiler and a set of per-residue intrinsic disorder predictors to quantify the intrinsic disorder. The Database of Disordered Protein Prediction (D2P2) platform, IUPred, FuzDrop, fIDPnn, AUCpred, SPOT-Disorder2, and metapredict V2 allowed us to evaluate the potential functional disorder of PRAME. Additionally, we used the Search Tool for the Retrieval of Interacting Genes (STRING) to analyze PRAME's potential interactions with other proteins. RESULTS: Our structural analysis showed that PRAME contains intrinsically disordered protein regions (IDPRs), which are structureless and flexible. We found that PRAME is significantly enriched with serine (p-value < 0.05), a disorder-promoting amino acid. PRAME was found to have an average disorder score of 16.49% (i.e., moderately disordered) across six per-residue intrinsic disorder predictors. Our IUPred analysis revealed the presence of disorder-to-order transition (DOT) regions in PRAME near the C-terminus of the protein (residues 475-509). The D2P2 platform predicted a region from approximately 140 and 175 to be highly concentrated with post-translational modifications (PTMs). FuzDrop predicted the PTM hot spot of PRAME to be a droplet-promoting region and an aggregation hotspot. Finally, our analysis using the STRING tool revealed that PRAME has significantly more interactions with other proteins than expected for randomly selected proteins of the same size, with the ability to interact with 84 different partners (STRING analysis result: p-value < 1.0 × 10-16; model confidence: 0.400). CONCLUSION: Our study revealed that PRAME has IDPRs that are possibly linked to its functionality in the context of Class 1 UM. The regions of functionality (i.e., DOT regions, PTM sites, droplet-promoting regions, and aggregation hotspots) are localized to regions of high levels of disorder. PRAME has a complex protein-protein interaction (PPI) network that may be secondary to the structureless features of the polypeptide. Our findings contribute to our understanding of UM and suggest that IDPRs and DOT regions in PRAME may be targeted in developing new therapies for this aggressive cancer. Video Abstract.

Chaotic aging: intrinsically disordered proteins in aging-related processes.

The development of aging is associated with the disruption of key cellular processes manifested as well-established hallmarks of aging. Intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) have no stable tertiary structure that provide them a power to be configurable hubs in signaling cascades and regulate many processes, potentially including those related to aging. There is a need to clarify the roles of IDPs/IDRs in aging. The dataset of 1702 aging-related proteins was collected from established aging databases and experimental studies. There is a noticeable presence of IDPs/IDRs, accounting for about 36% of the aging-related dataset, which is however less than the disorder content of the whole human proteome (about 40%). A Gene Ontology analysis of the used here aging proteome reveals an abundance of IDPs/IDRs in one-third of aging-associated processes, especially in genome regulation. Signaling pathways associated with aging also contain IDPs/IDRs on different hierarchical levels, revealing the importance of "structure-function continuum" in aging. Protein-protein interaction network analysis showed that IDPs present in different clusters associated with different aging hallmarks. Protein cluster with IDPs enrichment has simultaneously high liquid-liquid phase separation (LLPS) probability, "nuclear" localization and DNA-associated functions, related to aging hallmarks: genomic instability, telomere attrition, epigenetic alterations, and stem cells exhaustion. Intrinsic disorder, LLPS, and aggregation propensity should be considered as features that could be markers of pathogenic proteins. Overall, our analyses indicate that IDPs/IDRs play significant roles in aging-associated processes, particularly in the regulation of DNA functioning. IDP aggregation, which can lead to loss of function and toxicity, could be critically harmful to the cell. A structure-based analysis of aging and the identification of proteins that are particularly susceptible to disturbances can enhance our understanding of the molecular mechanisms of aging and open up new avenues for slowing it down.

Intrinsic Disorder in the Human Tear Proteome.

Purpose: We aimed to characterize the proteome of human tears and assess for the presence of intrinsically disordered proteins (IDPs). IDPs, despite lacking a rigid three-dimensional structure, maintain biological functionality and could shed light on the molecular interactions within tears. Methods: We analyzed a dataset of 1475 proteins identified in the tear film of three healthy subjects. We employed several computational tools, including the Compositional Profiler, Rapid Intrinsic Disorder Analysis Online, Search Tool for the Retrieval of Interacting Genes, and Database of Disordered Protein Predictors to evaluate the intrinsic disorder, protein interactions, and functional characterization of the disordered regions within this proteome. Results: Our analysis showed a notable inclination toward intrinsic disorder. Two out of 10 order-promoting residues and five out of 10 disorder-promoting residues were found enriched. Using the Predictor of Natural Disordered Regions (PONDR) VSL2 output, 95% of these proteins were classified as highly or moderately disordered. We revealed an extensive protein-protein interaction network with significant interaction enrichment. The most disordered proteins exhibited higher disorder binding sites and diverse posttranslational modifications compared to the most ordered ones. Conclusions: To the best of our knowledge, our study is the first comprehensive analysis of intrinsic disorder in the human tear film proteome, and it revealed an abundance of IDPs and their role in protein function and interaction networks. These findings suggest that variations in the intrinsic disorder of a tear film could be impacted by systemic and ocular conditions, offering promising avenues for disease biomarker identification and drug target development. Further research is needed to understand the implications of these findings in human health and disease.

EGFR TKI resistance in lung cancer cells using RNA sequencing and analytical bioinformatics tools.

Epidermal Growth Factor Receptor (EGFR) signaling and EGFR mutations play key roles in cancer pathogenesis, particularly in the development of drug resistance. For the ∼20% of all non-small cell lung cancer (NSCLC) patients that harbor an activating mutation, EGFR tyrosine kinase inhibitors (TKIs) provide initial clinical responses. However, long-term efficacy is not possible due to acquired drug resistance. Despite a gradually increasing knowledge of the mechanisms underpinning the development of resistance in tumors, there has been very little success in overcoming it and it is probable that many additional mechanisms are still unknown. Herein, publicly available RNASeq (RNA sequencing) datasets comparing lung cancer cell lines treated with EGFR TKIs until resistance developed with their corresponding parental cells and protein array data from our own EGFR TKI treated xenograft tumors, were analyzed for differential gene expression, with the intent to investigate the potential mechanisms of drug resistance to EGFR TKIs. Pathway analysis, as well as structural disorder analysis of proteins in these pathways, revealed several key proteins, including DUSP1, DUSP6, GAB2, and FOS, that could be targeted using novel combination therapies to overcome EGFR TKI resistance in lung cancer.

Rapid prediction and analysis of protein intrinsic disorder.

Protein intrinsic disorder is found in all kingdoms of life and is known to underpin numerous physiological and pathological processes. Computational methods play an important role in characterizing and identifying intrinsically disordered proteins and protein regions. Herein, we present a new high-efficiency web-based disorder predictor named Rapid Intrinsic Disorder Analysis Online (RIDAO) that is designed to facilitate the application of protein intrinsic disorder analysis in genome-scale structural bioinformatics and comparative genomics/proteomics. RIDAO integrates six established disorder predictors into a single, unified platform that reproduces the results of individual predictors with near-perfect fidelity. To demonstrate the potential applications, we construct a test set containing more than one million sequences from one hundred organisms comprising over 420 million residues. Using this test set, we compare the efficiency and accessibility (i.e., ease of use) of RIDAO to five well-known and popular disorder predictors, namely: AUCpreD, IUPred3, metapredict V2, flDPnn, and SPOT-Disorder2. We show that RIDAO yields per-residue predictions at a rate two to six orders of magnitude greater than the other predictors and completely processes the test set in under an hour. RIDAO can be accessed free of charge at https://ridao.app.

Lighting up Nobel Prize-winning studies with protein intrinsic disorder.

Intrinsically disordered proteins and regions (IDPs and IDRs) and their importance in biology are becoming increasingly recognized in biology, biochemistry, molecular biology and chemistry textbooks, as well as in current protein science and structural biology curricula. We argue that the sequence → dynamic conformational ensemble → function principle is of equal importance as the classical sequence → structure → function paradigm. To highlight this point, we describe the IDPs and/or IDRs behind the discoveries associated with 17 Nobel Prizes, 11 in Physiology or Medicine and 6 in Chemistry. The Nobel Laureates themselves did not always mention that the proteins underlying the phenomena investigated in their award-winning studies are in fact IDPs or contain IDRs. In several cases, IDP- or IDR-based molecular functions have been elucidated, while in other instances, it is recognized that the respective protein(s) contain IDRs, but the specific IDR-based molecular functions have yet to be determined. To highlight the importance of IDPs and IDRs as general principle in biology, we present here illustrative examples of IDPs/IDRs in Nobel Prize-winning mechanisms and processes.

The importance of accessory protein variants in the pathogenicity of SARS-CoV-2.

The coronavirus disease 2019 (COVID-19) is caused by the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS- CoV-2) with an estimated fatality rate of less than 1%. The SARS-CoV-2 accessory proteins ORF3a, ORF6, ORF7a, ORF7b, ORF8, and ORF10 possess putative functions to manipulate host immune mechanisms. These involve interferons, which appear as a consensus function, immune signaling receptor NLRP3 (NLR family pyrin domain-containing 3) inflammasome, and inflammatory cytokines such as interleukin 1ß (IL-1ß) and are critical in COVID-19 pathology. Outspread variations of each of the six accessory proteins were observed across six continents of all complete SARS-CoV-2 proteomes based on the data reported before November 2020. A decreasing order of percentage of unique variations in the accessory proteins was determined as ORF3a > ORF8 > ORF7a > ORF6 > ORF10 > ORF7b across all continents. The highest and lowest unique variations of ORF3a were observed in South America and Oceania, respectively. These findings suggest that the wide variations in accessory proteins seem to affect the pathogenicity of SARS-CoV-2.

Tudor staphylococcal nuclease is a docking platform for stress granule components and is essential for SnRK1 activation in Arabidopsis.

Tudor staphylococcal nuclease (TSN; also known as Tudor-SN, p100, or SND1) is a multifunctional, evolutionarily conserved regulator of gene expression, exhibiting cytoprotective activity in animals and plants and oncogenic activity in mammals. During stress, TSN stably associates with stress granules (SGs), in a poorly understood process. Here, we show that in the model plant Arabidopsis thaliana, TSN is an intrinsically disordered protein (IDP) acting as a scaffold for a large pool of other IDPs, enriched for conserved stress granule components as well as novel or plant-specific SG-localized proteins. While approximately 30% of TSN interactors are recruited to stress granules de novo upon stress perception, 70% form a protein-protein interaction network present before the onset of stress. Finally, we demonstrate that TSN and stress granule formation promote heat-induced activation of the evolutionarily conserved energy-sensing SNF1-related protein kinase 1 (SnRK1), the plant orthologue of mammalian AMP-activated protein kinase (AMPK). Our results establish TSN as a docking platform for stress granule proteins, with an important role in stress signalling.

Isolation and Characterization of Human Colon Adenocarcinoma Stem-Like Cells Based on the Endogenous Expression of the Stem Markers.

BACKGROUND: Cancer stem cells' (CSCs) self-maintenance is regulated via the pluripotency pathways promoting the most aggressive tumor phenotype. This study aimed to use the activity of these pathways for the CSCs' subpopulation enrichment and separating cells characterized by the OCT4 and SOX2 expression. METHODS: To select and analyze CSCs, we used the SORE6x lentiviral reporter plasmid for viral transduction of colon adenocarcinoma cells. Additionally, we assessed cell chemoresistance, clonogenic, invasive and migratory activity and the data of mRNA-seq and intrinsic disorder predisposition protein analysis (IDPPA). RESULTS: We obtained the line of CSC-like cells selected on the basis of the expression of the OCT4 and SOX2 stem cell factors. The enriched CSC-like subpopulation had increased chemoresistance as well as clonogenic and migration activities. The bioinformatic analysis of mRNA seq data identified the up-regulation of pluripotency, development, drug resistance and phototransduction pathways, and the downregulation of pathways related to proliferation, cell cycle, aging, and differentiation. IDPPA indicated that CSC-like cells are predisposed to increased intrinsic protein disorder. CONCLUSION: The use of the SORE6x reporter construct for CSCs enrichment allows us to obtain CSC-like population that can be used as a model to search for the new prognostic factors and potential therapeutic targets for colon cancer treatment.

A unique view of SARS-CoV-2 through the lens of ORF8 protein.

Immune evasion is one of the unique characteristics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) attributed to its ORF8 protein. This protein modulates the adaptive host immunity through down-regulation of MHC-1 (Major Histocompatibility Complex) molecules and innate immune responses by surpassing the host's interferon-mediated antiviral response. To understand the host's immune perspective in reference to the ORF8 protein, a comprehensive study of the ORF8 protein and mutations possessed by it have been performed. Chemical and structural properties of ORF8 proteins from different hosts, such as human, bat, and pangolin, suggest that the ORF8 of SARS-CoV-2 is much closer to ORF8 of Bat RaTG13-CoV than to that of Pangolin-CoV. Eighty-seven mutations across unique variants of ORF8 in SARS-CoV-2 can be grouped into four classes based on their predicted effects (Hussain et al., 2021) [1]. Based on the geo-locations and timescale of sample collection, a possible flow of mutations was built. Furthermore, conclusive flows of amalgamation of mutations were found upon sequence similarity analyses and consideration of the amino acid conservation phylogenies. Therefore, this study seeks to highlight the uniqueness of the rapidly evolving SARS-CoV-2 through the ORF8.

Intrinsic Disorder in Human Proteins Encoded by Core Duplicon Gene Families.

Segmental duplications (i.e., highly homologous DNA fragments greater than 1 kb in length that are present within a genome at more than one site) are typically found in genome regions that are prone to rearrangements. A noticeable fraction of the human genome (∼5%) includes segmental duplications (or duplicons) that are assumed to play a number of vital roles in human evolution, human-specific adaptation, and genomic instability. Despite their importance for crucial events such as synaptogenesis, neuronal migration, and neocortical expansion, these segmental duplications continue to be rather poorly characterized. Of particular interest are the core duplicon gene (CDG) families, which are replicates sharing common "core" DNA among the randomly attached pieces and which expand along single chromosomes and might harbor newly acquired protein domains. Another important feature of proteins encoded by CDG families is their multifunctionality. Although it seems that these proteins might possess many characteristic features of intrinsically disordered proteins, to the best of our knowledge, a systematic investigation of the intrinsic disorder predisposition of the proteins encoded by core duplicon gene families has not been conducted yet. To fill this gap and to determine the degree to which these proteins might be affected by intrinsic disorder, we analyzed a set of human proteins encoded by the members of 10 core duplicon gene families, such as NBPF, RGPD, GUSBP, PMS2P, SPATA31, TRIM51, GOLGA8, NPIP, TBC1D3, and LRRC37. Our analysis revealed that the vast majority of these proteins are highly disordered, with their disordered regions often being utilized as means for the protein-protein interactions and/or targeted for numerous posttranslational modifications of different nature.

Intrinsic disorder in protein sense-antisense recognition.

In addition to the well-established sense-antisense complementarity abundantly present in the nucleic acid world and serving as a basic principle of the specific double-helical structure of DNA, production of mRNA, and genetic code-based biosynthesis of proteins, sense-antisense complementarity is also present in proteins, where sense and antisense peptides were shown to interact with each other with increased probability. In nucleic acids, sense-antisense complementarity is achieved via the Watson-Crick complementarity of the base pairs or nucleotide pairing. In proteins, the complementarity between sense and antisense peptides depends on a specific hydropathic pattern, where codons for hydrophilic and hydrophobic amino acids in a sense peptide are complemented by the codons for hydrophobic and hydrophilic amino acids in its antisense counterpart. We are showing here that in addition to this pattern of the complementary hydrophobicity, sense and antisense peptides are characterized by the complementary order-disorder patterns and show complementarity in sequence distribution of their disorder-based interaction sites. We also discuss how this order-disorder complementarity can be related to protein evolution.

Hydration and Dispersion Forces in Hydroxypropylcellulose Phase Behavior.

Many-body polarization and hydration forces can strongly affect the equilibrium structure and energetics of mixed phases. Accurately reproducing both forces presents a challenge to force field models because it requires balancing hydrogen bonding at short range with many-body orientational order and dispersive attraction at long range. This work reports the first comparison of experimental measurements of the pressure-area isotherm for hydroxypropylcellulose (HPC) against molecular dynamics results with four different force field models-united-atom, all-atom (OPLS and CHARMM), and Drude oscillator models. All force fields exhibit the experimentally determined, exponentially shaped repulsive force at short range. Above a critical temperature of about 40 °C and a lattice spacing of around 14 Å, HPC experiments show a reversible, heat-induced polymer aggregation into an ordered phase driven by loss of water. The nonpolarizable force fields do not display the critical point and instead show biphasic behavior at all temperatures tested. This indicates net attractive forces at intermediate lattice spacings. In contrast, the Drude polarizable force field shows positive osmotic pressure and a single, homogeneous phase over all temperatures and spacings tested. Analysis of structural data from our simulations provides several clues to help interpret these findings. Although all force fields show similar water-water hydrogen bond numbers in the mixed phase, the polarizable model predicts that water-HPC hydrogen bonds are much more favorable than HPC-HPC hydrogen bonds when polymers are dispersed. At high density, water is driven out and replaced by HPC-HPC hydrogen bonds. The polarizable force field shows that both effects have a stronger dependence on polymer density than any of the nonpolarizable models. Our observations support the conclusion that hydration forces are coupled to the polymer coordination number by local, structural waters and that long-range dispersive attraction is overestimated by pairwise additive models.