Search Results (361)

Phase separation processes facilitate the formation of membrane-less organelles and involve interactions within structured domains and intrinsically disordered regions (IDRs) in protein sequences. The literature suggests that the involvement of proteins in phase separation can be predicted from their sequences, leading to the development of over 30 computational predictors. We focused on intrinsic disorder due to its fundamental role in related diseases, and because recent analysis has shown that phase separation can be accurately predicted for structured proteins. We evaluated eight representative amino acid-level predictors of phase separation, capable of identifying phase-separating IDRs, using a well-annotated, low-similarity test dataset under two complementary evaluation scenarios. Several methods generate accurate predictions in the easier scenario that includes both structured and disordered sequences. However, we demonstrate that modern disorder predictors perform equally well in this scenario by effectively differentiating phase-separating IDRs from structured regions. In the second, more challenging scenario—considering only predictions in disordered regions—disorder predictors underperform, and most phase separation predictors produce only modestly accurate results. Moreover, some predictors are broadly biased to classify disordered residues as phase-separating, which results in low predictive performance in this scenario. Finally, we recommend PSPHunter as the most accurate tool for identifying phase-separating IDRs in both scenarios. Full article

Fanconi anemia (FA) is a DNA repair deficiency disorder associated with genomic and chromosomal instability and a high cancer risk. In a small percentage of cases, FA is caused by biallelic pathogenic variants (PVs) in the BRCA2/FANCD1 gene, defining the FA-D1 subtype. Experimental and epidemiologic data indicate that the complete absence of BRCA2 is incompatible with viability. Therefore, cells from individuals affected with FA caused by biallelic BRCA2 PVs must have a residual BRCA2 function. This activity may be maintained through hypomorphic missense mutations, translation termination–reinitiation associated with a translational stop mutation, or other non-canonical or uncommon translation initiation and elongation events. In some cases, however, residual BRCA2 function is provided by alternatively or aberrantly spliced BRCA2 transcripts. Here, we review and debate aspects of the contribution of splicing in the 5′ segment to BRCA2 functions in the context of PVs affecting this largely intrinsically disordered protein region, with a focus on recent findings in individuals with FA-D1. In this Perspective, we also discuss some of the broader biological implications and open questions that arise from considering 5′-terminal BRCA2 splicing in light of old and new findings from FA-D1 patients and beyond. Full article

Intrinsically disordered regions (IDRs), defined as protein segments lacking stable tertiary structures, are ubiquitously present in the human proteome and enriched with disease-associated mutations. IDRs harbor molecular recognition features (MoRFs) and post-translational modification sites (e.g., phosphorylation), enabling dynamic intermolecular interactions through conformational plasticity. Furthermore, IDRs drive liquid–liquid phase separation (LLPS) of biomacromolecules via multivalent interactions such as electrostatic attraction and pi–pi interactions, generating biomolecular condensates that are essential throughout the cellular lifecycle. These condensates separate intracellular space, forming a physical barrier to avoid interference between other molecules, thereby improving reaction specificity and efficiency. As a dynamically equilibrated process, LLPS formation and maintenance are regulated by multiple factors, endowing the condensates with rapid responsiveness to environmental cues and functional versatility in modulating diverse signaling cascades. Consequently, disruption of LLPS homeostasis can derail its associated biological processes, ultimately contributing to disease pathogenesis. Moreover, precisely because liquid–liquid phase separation (LLPS) is co-regulated by multiple factors, it may provide novel insights into the pathogenic mechanisms of disorders such as autism spectrum disorder (ASD), which result from the cumulative effects of multiple etiological factors. Full article

This comprehensive review examines FINCHES (Force field-based Interaction Network for Characterizing Heterotypic and Entropic Sequences). This groundbreaking computational framework enables the rapid, sequence-based prediction of intermolecular interactions in intrinsically disordered regions (IDRs) without the need for molecular simulations. The document provides detailed comparisons with other computational methods, including their mathematical foundations, specific applications, and experimental validations. We explore both the potential for advancing our understanding of disordered protein function and the inherent challenges in computationally modeling these dynamic biological systems. Additionally, we discuss computational assessment tools for interface prediction in molecular complexes, providing a comprehensive framework for evaluating IDR interaction predictions. Full article

PDE11A is a little-studied phosphodiesterase sub-family that breaks down cAMP/cGMP, with the PDE11A4 isoform enriched in the memory-related hippocampal formation. Age-related increases in PDE11A expression occur in human and rodent hippocampus and cause age-related cognitive decline of social memories. Interestingly, age-related increases in PDE11A4 protein ectopically accumulate in spherical clusters that group together in the brain to form linear filamentous patterns termed “PDE11A4 ghost axons”. The biophysical/physiochemical mechanisms underlying this age-related clustering are not known. Here, we determine if age-related clustering of PDE11A4 reflects liquid–liquid phase separation (LLPS; biomolecular condensation), and if PDE11A inhibitors can reverse this LLPS. We show human and mouse PDE11A4 exhibit several LLPS-promoting sequence features, including intrinsically disordered regions, non-covalent pi–pi interactions, and prion-like domains that were particularly enriched in the N-terminal regulatory region. Further, multiple bioinformatic tools predict PDE11A4 undergoes LLPS. Consistent with these predictions, aging-like PDE11A4 clusters in HT22 hippocampal neuronal cells were membraneless spherical droplets that progressively fuse over time in a concentration-dependent manner. Deletion of the N-terminal intrinsically disordered region prevented PDE11A4 LLPS despite equal protein expression between WT and mutant constructs. 1,6-hexanediol, along with tadalafil and BC11-38 that inhibit PDE11A4, reversed PDE11A4 LLPS in HT22 hippocampal neuronal cells. Interestingly, PDE11A4 inhibitors reverse PDE11A4 LLPS independently of increasing cAMP/cGMP levels via catalytic inhibition. Importantly, orally dosed tadalafil reduced PDE11A4 ghost axons in old mouse ventral hippocampus by 50%. Thus, PDE11A4 exhibits the four defining criteria of LLPS, and PDE11A inhibitors reverse this age-related phenotype both in vitro and in vivo. Full article

The human coagulation pathway orchestrates a complex series of events vital for maintaining vascular integrity, in which the intrinsic pathway plays a pivotal role in amplifying and propagating the coagulation response. Dysregulation of this pathway can lead to various bleeding disorders and thrombotic complications, posing significant health risks. In this pathway, the activation of Factor (F) X zymogen is catalyzed by the FVIIIa-FIXa binary complex, but knowledge about this is still incomplete. Understanding the structural and functional intricacies of the FVIIIa-FIXa-FX (zymogen) complex is imperative for unraveling the molecular mechanisms underlying coagulation regulation and guiding the development of targeted therapeutic interventions. In this study, utilizing Alphafold-Multimer and molecular dynamics (MD) simulations, we provide insights into factor interactions within the ternary complex and propose novel functional mechanisms contributing to the functional defects inflicted by their cross-reactive material (CRM) positive mutations. The amino acid residue replacement impairs the coagulation function by interfering with structure elements, including the following: (1) a knot-like structure between Arg-562 of FVIIIa’s 558-Loop (residue 555–571) and the 333-Loop of FIXa (residue 333–346) contributes to FVIIIa-FIXa binding; (2) the a2 region of FVIIIa (residue 716–740) opens the lid of active site (FIXa’s 266-Loop, residue 256–270) and facilitates substrate binding; (3) the activation peptide (AP) of FX zymogen (residue 143–194) not only assists in the activation of itself but also adheres the interface of the three factors like a double-sided tape. Our work provides novel insights for the pathogenesis of a number of reported clinical CRM-positive mutations and may lay the groundwork for the structure-based development of therapeutic interventions. Full article

Nipah virus (NiV), a highly lethal zoonotic pathogen causing encephalitis and respiratory diseases with mortality rates up to 40–70%, faces research limitations due to its strict biosafety level 4 (BSL-4) containment requirements, hindering antiviral development. To address this, we generated two NiV minigenome replicons (Fluc- and EGFP-based) expressed via helper plasmids encoding viral N, P, and L proteins, enabling replication studies under BSL-2 conditions. The minigenome replicon recapitulated the cytoplasmic inclusion body (IB) formation observed in live NiV infections. We further demonstrated that IB assembly is driven by liquid–liquid phase separation (LLPS), with biochemical analyses identifying the C-terminal N core domain of the N protein, as well as N₀ and XD domains and the intrinsically disordered region (IDR) of the P protein, as essential structural determinants for LLPS-mediated IB biogenesis. The targeted siRNA silencing of the 5′ and 3′ untranslated regions (UTRs) significantly reduced replicon-derived mRNA levels, validating the regulatory roles of these regions. Importantly, the minigenome replicon demonstrated sensitivity to type I/II/III interferons and antivirals (remdesivir, azvudine, molnupiravir), establishing its utility for drug screening. This study provides a safe and efficient platform for investigating NiV replication mechanisms and accelerating therapeutic development, circumventing the constraints of BSL-4 facilities while preserving key virological features. Full article

Intrinsically disordered proteins (IDPs), such as tau, beta-amyloid (Aβ), and alpha-synuclein (αSyn), are prone to misfolding, resulting in pathological aggregation and propagation that drive neurodegenerative diseases, including Alzheimer’s disease (AD), frontotemporal dementia (FTD), and Parkinson’s disease (PD). Misfolded IDPs are prone to aggregate into oligomers and fibrils, exacerbating disease progression by disrupting cellular functions in the central nervous system, triggering neuroinflammation and neurodegeneration. Furthermore, aggregated IDPs exhibit prion-like behavior, acting as seeds that are released into the extracellular space, taken up by neighboring cells, and have a propagating pathology across different regions of the brain. Conventional inhibitors, such as small molecules, peptides, and antibodies, face challenges in stability and blood–brain barrier penetration, limiting their efficacy. In recent years, nanotechnology-based strategies, such as multifunctional nanoplatforms or nanoparticles, have emerged as promising tools to address these challenges. These nanoplatforms leverage tailored designs to prevent or remodel the aggregation of IDPs and reduce associated neurotoxicity. This review discusses recent advances in nanoplatforms designed to target tau, Aβ, and αSyn aggregation, with a focus on their roles in reducing neuroinflammation and neurodegeneration. We examine critical aspects of nanoplatform design, including the choice of material backbone and targeting moieties, which influence interactions with IDPs. We also highlight key mechanisms including the interaction between nanoplatforms and IDPs to inhibit their aggregation, redirect aggregation cascade towards nontoxic, off-pathway species, and disrupt fibrillar structures into soluble forms. We further outline future directions for enhancing IDP clearance, achieving spatiotemporal control, and improving cell-specific targeting. These nanomedicine strategies offer compelling paths forward for developing more effective and targeted therapies for neurodegenerative diseases. Full article

Mild starvation due to low concentrations of an inhibitor of glycolysis, 2-deoxy-D-glucose, activates AMP-activated protein kinase (AMPK) and lysine-specific demethylase 2A (KDM2A) to reduce rRNA transcription and cell proliferation in breast cancer cells. However, the mechanisms of how AMPK regulates KDM2A are unknown. Here, we found that PHD finger protein 8 (PHF8) interacted with KDM2A and contributed to the reduction in rRNA transcription and cell proliferation by 2-deoxy-D-glucose in a breast cancer cell line, MCF-7. We analyzed how KDM2A bound PHF8 in detail and found that PHF8 interacted with KDM2A via two regions of KDM2A. One of the regions contained an intrinsically disordered region (IDR). IDRs can show rapidly switchable protein–protein interactions. Deletion of the PHF8-binding region activated KDM2A to reduce rRNA transcription, and 2-deoxy-D-glucose reduced the interaction between PHF8 and the KDM2A fragment containing the PHF8-binding region. A 2-deoxy-D-glucose or AMPK activator dephosphorylated KDM2A at Ser731, which is located on the N-terminal side of the PHF8-binding region. Replacement of Ser731 by Ala decreased binding of PHF8 to the KDM2A fragment that contains the PHF8-binding region and Ser731 and reduced rRNA transcription and cell proliferation. These results suggest that the mode of interaction between KDM2A and PHF8 is regulated via dephosphorylation of KDM2A through AMPK to control rRNA transcription, and control of the phosphorylation state of Ser731 would be a novel target for breast cancer therapy. Full article

Intrinsic disorder refers to protein regions that lack a fixed three−dimensional structure under physiological conditions, enabling conformational plasticity. This flexibility allows for diverse functions, including transient interactions, signaling, and phase separation via disorder-to-order transitions upon binding. Our study focused on investigating the role of intrinsic disorder and liquid−liquid phase separation (LLPS) in the human acrosome, a sperm-specific organelle essential for fertilization. Using computational prediction models, network analysis, Structural Classification of Proteins (SCOP) functional assessments, and Gene Ontology, we analyzed 250 proteins within the acrosomal proteome. Our bioinformatic analysis yielded 97 proteins with high levels (>30%) of structural disorder. Further analysis of functional enrichment identified associations between disordered regions overlapping with SCOP domains and critical acrosomal processes, including vesicle trafficking, membrane fusion, and enzymatic activation. Examples of disordered SCOP domains include the PLC-like phosphodiesterase domain, the t-SNARE domain, and the P-domain of calnexin/calreticulin. Protein–protein interaction networks revealed acrosomal proteins as hubs in tightly interconnected systems, emphasizing their functional importance. LLPS propensity modeling determined that over 30% of these proteins are high-probability LLPS drivers (>60%), underscoring their role in dynamic compartmentalization. Proteins such as myristoylated alanine-rich C-kinase substrate and nuclear transition protein 2 exhibited both high LLPS propensities and high levels of structural disorder. A significant relationship (p < 0.0001, R² = 0.649) was observed between the level of intrinsic disorder and LLPS propensity, showing the role of disorder in facilitating phase separation. Overall, these findings provide insights into how intrinsic disorder and LLPS contribute to the structural adaptability and functional precision required for fertilization, with implications for understanding disorders associated with the human acrosome reaction. Full article

ALOG (Arabidopsis LSH1 and Oryza G1) proteins constitute a plant-specific family of transcription factors that play crucial roles in lateral organ development across land plants. Initially identified through forward genetic studies of Arabidopsis LSH1 and rice G1 proteins, ALOG family members have since been functionally characterized in various plant species. However, research focusing on the characteristics and expression patterns of all ALOG family members in rice remains relatively limited. In this study, we systematically characterized OsALOG family genes in rice. Compared to other genes in rice and Arabidopsis, the ALOG family genes have a relatively simple structure. The alignment of OsALOG amino acid sequences and analysis of disorder predictions reveal that all members possess conserved ALOG domains, while the conservation of intrinsically disordered regions (IDRs) is relatively low. Four amino acids—alanine, glycine, proline, and serine—are significantly enriched in the IDRs of each ALOG protein. Synteny analysis indicates that most OsALOG genes have undergone considerable divergence compared to their counterparts in Arabidopsis. Bioinformatic analysis of cis-regulatory elements predicts that OsALOG family genes contain elements responsive to ABA, light, and methyl jasmonate, although the abundance and composition of these elements vary among different members. The expression patterns associated with the rice floral development of OsALOG genes can be broadly categorized into two types; however, even within the same type, differences in expression levels, as well as the initiation time and duration of expression, were observed. These results provide a comprehensive understanding of the structural characteristics and expression patterns of OsALOG members in rice. Full article

Hypothermia (HT) is used clinically for neonatal hypoxic–ischemic encephalopathy (HIE); however, the brain protection is incomplete and selective regional vulnerability and lifelong consequences remain. Refractory damage and impairment with HT cooling/rewarming could result from unchecked or altered persisting cell death and proteinopathy. We tested two hypotheses: (1) HT modifies neurodegeneration type, and (2) intrinsically disordered proteins (IDPs) and encephalopathy cause toxic conformer protein (TCP) proteinopathy neonatally. We studied postmortem human neonatal HIE cases with or without therapeutic HT, neonatal piglets subjected to global hypoxia-ischemia (HI) with and without HT or combinations of HI and quinolinic acid (QA) excitotoxicity surviving for 29–96 h to 14 days, and human oligodendrocytes and neurons exposed to QA for cell models. In human and piglet encephalopathies with normothermia, the neuropathology by hematoxylin and eosin staining was similar; necrotic cell degeneration predominated. With HT, neurodegeneration morphology shifted to apoptosis-necrosis hybrid and apoptotic forms in human HIE, while neurons in HI piglets were unshifting and protected robustly. Oligomers and putative TCPs of α-synuclein (αSyn), nitrated-Syn and aggregated αSyn, misfolded/oxidized superoxide dismutase-1 (SOD1), and prion protein (PrP) were detected with highly specific antibodies by immunohistochemistry, immunofluorescence, and immunoblotting. αSyn and SOD1 TCPs were seen in human HIE brains regardless of HT treatment. αSyn and SOD1 TCPs were detected as early as 29 h after injury in piglets and QA-injured human oligodendrocytes and neurons in culture. Cell immunophenotyping by immunofluorescence showed αSyn detected with antibodies to aggregated/oligomerized protein; nitrated-Syn accumulated in neurons, sometimes appearing as focal dendritic aggregations. Co-localization also showed aberrant αSyn accumulating in presynaptic terminals. Proteinase K-resistant PrP accumulated in ischemic Purkinje cells, and their target regions had PrP-positive neuritic plaque-like pathology. Immunofluorescence revealed misfolded/oxidized SOD1 in neurons, axons, astrocytes, and oligodendrocytes. HT attenuated TCP formation in piglets. We conclude that HT differentially affects brain damage in humans and piglets. HT shifts neuronal cell death to other forms in human while blocking ischemic necrosis in piglet for sustained protection. HI and excitotoxicity also acutely induce formation of TCPs and prion-like proteins from IDPs globally throughout the brain in gray matter and white matter. HT attenuates proteinopathy in piglets but seemingly not in humans. Shifting of cell death type and aberrant toxic protein formation could explain the selective system vulnerability, connectome spreading, and persistent damage seen in neonatal HIE leading to lifelong consequences even after HT treatment. Full article

Human topoisomerase IIα and IIβ regulate DNA topology and knots in chromosomes during crucial cellular processes, making these enzymes common targets for anticancer drugs. However, selective inhibition of topoisomerase IIα (TOP2A) is desired to decrease adverse effects, which may be mediated by topoisomerase IIβ (TOP2B). The main region of difference between the two isoforms is the intrinsically disordered C-terminal domain (CTD), which is being studied as a target for selective inhibition. Our previous work examined several regions within the CTD to determine whether those regions impact biochemical function. In this current study, we designed and constructed four TOP2A mutants with amino acid substitutions in the CTD, which were then assessed for impact on biochemical activity. V1482D exhibited increased levels of relaxation, while both V1482D and K1520I exhibited increased levels of decatenation. No major impact on DNA cleavage or binding were observed with any of the mutants. The isolated impact of the changes on relaxation and decatenation supports the concept that the CTD can affect one aspect of the enzyme’s function in an isolated manner, which was seen in our previous study. Taken together, these results suggest that modification of specific positions within the CTD affects substrate selection. These results are mapped onto the CTD for consideration of potential regions to target for inhibition of TOP2A. Full article

The sustainable production of carbon-free fuels such as hydrogen is an important goal in the search for alternative energy sources. Herein, we report a peptide-based system for light-driven hydrogen evolution from water under neutral conditions. The M1 peptide is an ABC triblock polymer featuring two coiled-coil alpha-helical regions flanking a water-soluble, polyanionic, intrinsically disordered region. M1 formed a hydrogel at high concentrations upon binding to cobalt protoporphyrin IX. This process is driven by the terminal regions, which coordinate the metalloporphyrin through histidine residues and form helical oligomers interconnected by flexible, intrinsically disordered regions, resulting in network formation. Co-M1 catalyzes hydrogen production upon irradiation in the presence of a photosensitizer and a sacrificial electron donor; the activity of Co-M1 is eight times higher than that of free Co-PPIX. Full article

Human viral infections remain a significant global health challenge, contributing to a substantial number of cancer cases worldwide. Among them, infections with oncoviruses such as hepatitis B virus (HBV) and hepatitis C virus (HCV) are key drivers of hepatocellular carcinoma (HCC). Despite the availability of an effective HBV vaccine since the 1980s, millions remain chronically infected due to the persistence of covalently closed circular DNA (cccDNA) as a reservoir in hepatocytes. Current antiviral therapies, including nucleos(t)ide analogs and interferon, effectively suppress viral replication but fail to eliminate cccDNA, underscoring the urgent need for innovative therapeutic strategies. Direct-acting antiviral agents (DAAs), which have revolutionized HCV treatment with high cure rates, offer a promising model for HBV therapy. A particularly attractive target is the intrinsically disordered region (IDR) of the HBx protein, which regulates cccDNA transcription, viral replication, and oncogenesis by interacting with key host proteins. DAAs targeting these interactions could inhibit viral persistence, suppress oncogenic signaling, and overcome treatment resistance. This review highlights the potential of HBx-directed DAAs to complement existing therapies, offering renewed hope for a functional HBV cure and reduced cancer risk. Full article