Search Results (388)

The tardigrade Ramazzottius varieornatus exhibits extraordinary resilience to extreme environmental stresses, yet the functional diversity of its proteome remains largely unexplored. In this study, the structural and biochemical characterization of RvCA5, an atypical α-carbonic anhydrase (CA) identified in R. varieornatus, is presented. Expression analysis in E. coli revealed the spontaneous formation of a truncated RvCA5 species, which was confirmed to be unrelated to signal peptide cleavage. RvCA5 exhibited distinct structural features, including extended intrinsically disordered regions (IDRs) at both termini. Unlike canonical α-CAs, RvCA5 exhibited negligible CO₂ hydration activity, which was partially enhanced by the removal of the N-terminal IDR, suggesting that this region acts as a dynamic entropic barrier hindering substrate diffusion. RvCA5 possesses multiple surface-exposed reactive cysteine residues, resembling the redox-sensing human CA 3. Notably, consistent with a predicted nuclear localization signal, in silico modeling predicted that RvCA5 can bind DNA via a positively charged patch near the C-terminal IDR. The DNA-binding capability of RvCA5 was experimentally demonstrated by electrophoretic mobility shift assays. Collectively, these findings suggest that RvCA5 potentially functions as a redox-responsive transcriptional regulator. Full article

Triplexes (TRX) are a class of flipons that can form due to the interaction of RNA with B-DNA. While many proteins have been proposed to bind triplexes, structural models of these interactions do not exist. Here, I present AlphaFold V3 (AF3) models that reveal interactions between the high-mobility group protein B1 (HMGB1), HNRNPU (SAF-A), TP53, ARGONAUTE (AGO), and REL domain proteins. The TRXs result from the sequence-specific docking of RNAs to DNA via Hoogsteen base pairing. The RNA and DNA strands in apolar TRX are oriented in the opposite 5′ to 3′ direction, while copolar TRX have RNA and DNA strands pointing in the same 5′ to 3′ direction. TRXs can incorporate different RNA classes, including long noncoding RNAs (lncRNAs), short RNAs, such as miRNAs, piRNAs, and tRNAs, nascent RNA fragments, and non-canonical base triplets. Many pathways regulated by TRX formation have evolved to constrain retroelements (EREs), which are both an existential threat to the host and a source of genotypic variation. TRXs help set the boundaries of active chromatin, repressing the expression of most EREs, while depending on other flipons to modulate cellular programs. The TRXs help nucleate folding of intrinsically disordered proteins. Full article

Tau proteins are microtubule-associated proteins that regulate axonal structure, dynamics, and transport, and their dysregulation underlies several neurodegenerative diseases. The MAPT gene produces multiple tau isoforms through alternative splicing, including the high-molecular-weight isoform known as Big tau, which contains an insert of the large 4a exon of approximately 250 amino acids. Big tau is predominantly expressed in neurons of the peripheral nervous system (PNS), cranial motor nuclei, and select neurons of the central nervous system (CNS) such as the cerebellum and brainstem. Developmental expression studies indicate a switch from low-molecular-weight isoforms of tau to Big tau during axonal maturation, suggesting that Big tau optimizes cytoskeletal dynamics to accommodate long axonal projections. Comparative sequence and biophysical analyses show that the exon-4a insert is highly acidic, intrinsically disordered, and evolutionarily conserved in its length but not its primary sequence, implying a structural role. Emerging modeling and in vitro assays suggest that the extended projection domain provided by the exon-4a insert spatially and electrostatically shields the aggregation-prone PHF6 and PHF6* motifs in tau’s microtubule-binding domain, thereby reducing β-sheet driven aggregation. This mechanism may explain why tauopathies that involve aggregation of tau have little effect on the PNS and specific regions of the CNS such as the cerebellum, where Big tau predominates. Transcriptomic and proteomic data further suggest that alternative Big tau variants, including 4a-L, are expressed in certain cancerous tissues, indicating broader roles in cytoskeletal remodeling beyond neurons. Despite its putative anti-aggregation properties, the physiological regulation, interaction partners, and in vivo mechanisms of Big tau remain poorly defined. This review summarizes what is known about Big tau and what is missing toward a better understanding of how expansion via inclusion of exon 4a modifies tau’s structural and functional properties. Our purpose is to inspire future studies that could lead to novel therapeutic strategies to mitigate tau aggregation in neurodegenerative diseases. Full article

Thrombosis is a cardiovascular disease characterized by the pathological formation of a fibrin clot in blood vessels. Currently available fibrinolytic enzymes have some limitations, including severe side effects, high cost, short half-life, and low fibrin specificity. Proteins from microalgae and cyanobacteria have various biological effects and are emerging as promising sources for fibrinolytic enzymes. In this study, bioinformatics tools were used to evaluate the intrinsic disorder predisposition of microalgal fibrinolytic proteins, their capability to undergo liquid–liquid phase separation (LLPS), and the presence of disorder-based functional regions, and short linear motifs (SLiMs). Analysis revealed that these proteins are predominantly hydrophilic and exhibit acidic (pI 3.96–6.49) or basic (pI 8.05–11.0) isoelectric points. Most of them are expected to be moderately (61.4%) or highly disordered proteins (6.8%) and associated with LLPS, with nine proteins being predicted to behave as droplet drivers (i.e., being capable of spontaneous LLPS), and twenty-five proteins being expected to be droplet clients. These observations suggest that LLPS may be related to the regulation of the functionality of microalgal fibrinolytic proteins. The majority of these proteins belong to the blood coagulation inhibitor (disintegrin) 1 hit superfamily, which can inhibit fibrinogen binding to integrin receptors, preventing platelet aggregation. Furthermore, the SLiM-centered analysis indicated that the main motifs found in these proteins are MOD_GlcNHglycan and CLV_PCSK_SKI1_1, which can also play different roles in thrombolytic activity. Finally, Fisher and conservation analysis indicated that CLV_NRD_NRD_1, CLV_PCSK_FUR_1, CLV_PCSK_PC7_1, and MOD_Cter_Amidation motifs are enriched in intrinsically disordered regions (IDRs) of these proteins, showing significant conservation and suggesting compatibility with proteolytic activation and post-translational processing. These data provide important information regarding microalgal proteins with potential thrombolytic effects, which can be realized through protein–protein interactions mediated by SLiMs present in intrinsically disordered regions (IDRs). Additional analyses should be conducted to confirm these observations using experimental in vitro and in vivo approaches. Full article

The Keap1-Nrf2 signaling pathway is a central regulator of transcriptional responses to oxidative stress and is strongly linked to diverse pathologies, particularly cancer. In the cytoplasm, Keap1 (Kelch-like ECH-associated protein 1) promotes proteasomal degradation of Nrf2 (NF-E2–related factor 2). Oxidative stimuli disrupt the Keap1-Nrf2 interaction, facilitating Nrf2 nuclear accumulation and activation of antioxidant and detoxifying genes. Recent evidence suggests that Keap1 family proteins also enter the nucleus, bind chromatin, and regulate transcription, but the underlying mechanisms remain less understood. Here, we show that the Drosophila Keap1 ortholog, dKeap1, accumulates in the nucleus and gradually assembles stable nuclear foci in cells following oxidative treatment. FRAP analyses revealed reduced mobility of dKeap1 within these foci. Both the N-terminal (NTD) and C-terminal (CTD) domains of dKeap1 were required for foci formation. Two intrinsically disordered regions (IDRs) were identified within the CTD, and CTD-YFP fusion proteins readily formed condensates in vitro. Conversely, deletion of the Kelch domain resulted in robust cytoplasmic foci even under basal conditions, and in vitro assays also indicated that the Kelch domain suppresses dKeap1 condensate formation. Together, these findings reveal a novel molecular mechanism for the nuclear function of dKeap1, providing new insight into the broader roles of Keap1 factors in oxidative response, development, and disease. Full article

Intrinsically disordered regions enable transcription factors (TFs) to undergo structural changes upon ligand binding, facilitating the transduction of environmental signals into gene expression. In this study, we applied molecular modeling methods to explore the hypothesis that unstructured inter-domain and subdomain linkers in bacterial TFs can function as sensors for carbohydrate signaling molecules. We combined molecular dynamics simulations and carbohydrate docking to analyze six repressors with GntR-type DNA-binding domains, including UxuR, GntR and FarR from Escherichia coli, as well as AraR, NagR and YydK from Bacillus subtilis. Protein models obtained from different time points of the dynamic simulations were subjected to sequential carbohydrate docking. We found that the inter-domain linker of the UxuR monomer binds D-fructuronate, D-galacturonate, D-glucose, and D-glucuronate with an affinity comparable to nonspecific interactions. However, these ligands formed multimolecular clusters, a feature absent in the UxuR dimer, suggesting that protein dimerization may depend on linker occupancy by cellular carbohydrates. D-glucose interacted with linkers connecting subdomains of the LacI/GalR-type E-domains in GntR and AraR, forming hydrogen bonds that connected distant structural modules of the proteins, while in NagR, FarR and YydK, it bridged the inter-domain linkers and a β-sheet within the HutC-type E-domains. Hence, our results establish flexible linkers as pivotal metabolic sensors that directly integrate nutritional cues to alter gene expression in bacteria. Full article

Near-infrared organic photodetectors (NIR-OPDs) are emerging as versatile platforms for flexible and low-cost optical sensing, yet achieving high-performance in the NIR region remains difficult remains challenging due to intrinsic trade-offs at both the material and device levels, due to the inherent balance required among bandgap narrowing, exciton dissociation, charge transport, and dark-current suppression. This review provides a concise overview of OPD operating mechanisms and the performance metrics governing sensitivity and noise. We highlight recent molecular-engineering strategies—core fluorination, asymmetric π-bridge design, fused-ring rigidification, and polymer backbone/side-chain tuning—that effectively enhance intermolecular ordering, reduce energetic disorder, and extend NIR absorption. Progress in all-polymer detectors and ambipolar phototransistors further demonstrates improved stability and broadened detection capability. Additionally, emerging applications, including NIR communication, biosignal monitoring, flexible imaging, and biometric recognition, showcase the expanding utility of NIR-OPDs. Remaining challenges include pushing detection beyond 1200 nm, simplifying synthesis, and improving long-term stability. Overall, advances in low-bandgap molecular design and device engineering continue to accelerate the practical adoption of NIR-OPDs. Full article

Background: Intrinsically disordered regions (IDRs) within proteins often act as pivotal linkage units for the interaction of functional domains. The p53 tumor suppressor protein contains intrinsically disordered N-terminal and C-terminal domains (NTD and CTD), playing crucial regulatory roles in cellular processes. Furthermore, experimental approaches have encountered challenges in elucidating the structural regulation by the IDRs. Methods: In this work, we employed microsecond-scale molecular dynamics simulations to explore the allosteric regulation mechanism of the p53 DNA binding domain (DBD) induced by the CTD and the DNA binding. Subsequently, we integrated dynamic cross-correlation analysis with binding free energy calculations to evaluate the interaction between the CTD and DNA. Results: The free energy landscapes (FELs) were utilized to identify the conformational ensemble of the p53 DBD. The FELs revealed that the CTD enhances the allosteric regulatory mechanisms. Conclusions: Firstly, the conformation of DBD changes on the S6-S7 loop and L1 upon DNA binding. Then the CTD directly interacts with DNA and further regulates the allosteric network (involving the S6-S7 loop, L1 loop, S4, S10, H1, and H3) to promote the binding of DBD to DNA. The allosteric mechanisms presented in this work will provide new insights into the functional mechanisms of the p53 CTD and inform the rational design of p53-targeted drugs. Full article

Adult hippocampal neurogenesis (AHN) is a dynamic process that sustains neural plasticity and contributes to cognition, emotion, and stress resilience. While its functional significance in humans remains debated, growing evidence suggests that AHN plays an important role in health and disease. In this review, we summarize intrinsic and extrinsic factors that modulate AHN, with particular emphasis on hormones, behavior, diet, and their impact along the hippocampal dorsoventral axis, where baseline neurogenesis is higher dorsally, but ventral neurogenesis exhibits greater plasticity and sensitivity to modulatory systems. We highlight how cognitive stimulation, physical activity, and rewarding experiences preferentially enhance dorsal hippocampal neurogenesis, whereas chronic stress and glucocorticoids mainly impair neurogenesis in the ventral hippocampus. Nutritional influences such as caloric restriction, high-fat diets, vitamins, and polyphenols are also considered, with evidence for region-specific effects. We further examine the relevance of AHN alterations in neuropsychiatric diseases, such as major depressive disorder, schizophrenia, Alzheimer’s disease, and addiction, highlighting both common mechanisms and disorder-specific vulnerabilities. Collectively, current findings suggest that AHN serves as a converging pathway connecting lifestyle, neuroendocrine regulation, and psychiatric or neurodegenerative disease. Recognizing the dorsoventral specialization of AHN could refine mechanistic models of brain function and inform the development of targeted and distinct therapeutic strategies for cognitive and affective diseases. Full article

GC-rich sequences affect DNA replication, recombination and repair, as well as RNA transcription in vivo. Such sequences may also impede site-directed mutagenesis in vitro. P3a site-directed mutagenesis is a highly efficient method, but it has not been tested with plasmids possessing GC-rich sequences. Here we report that it is very efficient with a BRPF3 expression vector but unsuccessful with that for KAT2B. Because two GC-rich regions located within the synthetic CAG promoter and the KAT2B coding region may form guanine (G)-quadruplexes and hinder plasmid denaturation during PCR, we developed P3b site-specific mutagenesis, achieving an average efficiency of 97.5% in engineering ten KAT2B mutants. Importantly, deletion mutagenesis revealed that either of the two GC-rich regions is sufficient for rendering the plasmid incompatible with P3a mutagenesis. Consistent with this, only P3b mutagenesis worked efficiently with several widely used sgRNA/Cas9 expression vectors, which contain the CAG promoter, and with an expression vector for CDK13, which possesses an intrinsically disordered domain encoded by a GC-rich DNA fragment. Thus, this study highlights serious challenges posed by GC-rich sequences to site-directed mutagenesis and provides an effective remedy to address such challenges. The findings support that G-quadruplex formation is one mechanism whereby such sequences impede regular PCR-based mutagenesis methods. Full article

Valine-glutamine motif proteins (VQ), plant-specific transcriptional co-regulators harboring the conserved FxxhVQxhTG motif, play pivotal roles in coordinating plant stress adaptation through dynamic interactions with WRKY transcription factors (WRKY), mitogen-activated protein kinases (MAPKs) cascades, and hormone signaling pathways. Evolutionary analyses reveal the characteristics of their evolutionary protection and ancient origin, with lineage-specific expansion via genome duplication events. Structurally, compact genes lacking introns and the presence of intrinsic disordered regions (IDRs) facilitate rapid stress responses and versatile protein interactions. Functionally, VQ proteins orchestrate abiotic stress tolerance (e.g., drought, salinity, temperature extremes) by modulating reactive oxygen species (ROS) homeostasis, osmotic balance, and abscisic acid/salicylic acid (ABA/SA)-mediated signaling. Concurrently, they enhance biotic stress resistance via pathogen-responsive WRKY-VQ modules that regulate defense gene expression and hormone crosstalk. Despite advances, challenges persist in deciphering post-translational modifications, tissue-specific functions, and cross-stress integration mechanisms. Harnessing CRISPR-based editing and multi-omics approaches will accelerate the exploitation of VQ genes for developing climate-resilient crops. This review synthesizes the molecular architecture, evolutionary dynamics, and multifunctional regulatory networks of VQ proteins, providing a roadmap for their utilization in sustainable agriculture. Full article

Ataxin-2 (Atx2) is a general RNA-binding protein involved in processes such as RNA processing and metabolism in cells. Atx2 is also a polyglutamine (polyQ) tract-containing protein; its abnormal expansion can lead to protein aggregation associated with neurodegenerative diseases. Previous studies have shown that the C-terminal intrinsically disordered regions (c-IDRs) of Atx2 participate in its condensation and aggregation processes. To elucidate the role of polyQ expansion in biomolecular condensation and aggregation, we studied the N-terminal fragments of Atx2 (namely, Atx2-N317 and Atx2-N81) that preserve a polyQ tract and compared their molecular behaviors in cells to those of the full-length Atx2. We found that the molecular mobility of the N-terminal fragments decreases with the increasing length of polyQ, indicating that polyQ expansion promotes a gradual phase transition to an irreversible and insoluble state. Moreover, the molecular state and mobility of Atx2-N317 are not distinct from those of Atx2-N81, regardless of the presence of other domains, demonstrating that the polyQ tract is a direct and sufficient element for protein condensation and aggregation, while the Like Sm (LSm) and LSm-associated (LSmAD) domains and their interactions with RNA are not necessary for these processes. This result is also validated through the in vitro investigation of Atx2-N81 with different polyQ expansions. This study reveals that polyQ expansion controls the biomolecular condensation and aggregation of the N-terminal fragments of Atx2 and is thus thought to modulate the dynamic behaviors of the full-length protein as well, which is implicated in the pathological accumulation of Atx2 in cells. Full article

Eukaryotic phosphorylation of serine and threonine residues is a central regulatory mechanism in cell signalling, carried out by more than 500 kinases and a diverse array of phosphatases. Traditionally understood as a two-component system driven by writers (kinases) and erasers (phosphatases), this regulatory network is now appreciated to involve additional proteins that modulate or interpret phosphorylation-dependent changes. Among them, the 14-3-3 protein family has emerged as a prominent example due to its ability to bind phosphorylated serine/threonine motifs—typically located within intrinsically disordered regions—and influence the activity, stability, or localization of its partners. In this review, we discuss the importance, evolution, structure, and dynamics of 14-3-3 proteins, as well as their interactions with small molecules—both natural and designed—that bind to them. We highlight several underexplored aspects of their molecular behaviour, integrate recent discoveries, and emphasize how these insights contribute to a broader understanding of phosphorylation-dependent regulation across eukaryotes. Full article

Late Embryogenesis Abundant (LEA) proteins play vital roles in plant responses to abiotic stress and developmental regulation. Pineapple (Ananas comosus L.) is a major tropical fruit crop with high economic value, but its production is often threatened by cold stress, particularly in regions at the northern margin of its cultivation. Despite the recognized importance of LEA proteins in stress adaptation, their genomic landscape and functional characteristics in pineapple remain largely unexplored. In this study, 37 AcLEA genes were identified in the pineapple (Ananas comosus L.) genome and classified into six subfamilies, with LEA_2 being the largest. Most AcLEA proteins were predicted to be hydrophilic, thermally stable, and intrinsically disordered, consistent with typical LEA protein characteristics. Phylogenetic and collinearity analyses revealed species-specific expansion patterns, primarily driven by segmental duplication events. Most duplicated gene pairs shared similar exon–intron structures, motif compositions, and expression profiles, although several displayed signs of functional divergence based on distinct expression patterns, Ka/Ks ratios > 1, and motif differences. Promoter cis-element, transcription factor, and miRNA network predictions indicated that AcLEA genes are widely involved in stress responses as well as growth and development. Expression profiling showed that many AcLEA genes including AcLEA32, AcLEA7, AcLEA9, AcLEA30, AcLEA29, AcLEA33, and AcLEA18 were significantly upregulated under cold stress and declined upon stress removal, indicating a potential role in cold tolerance. Some AcLEA genes, such as AcLEA32 and AcLEA33, showed faster and stronger induction under cold stress in the cold-tolerant cultivar “Comte de Paris” (BL) compared to the sensitive cultivar “Tainong No. 20” (NN), suggesting that differential gene responsiveness may contribute to cultivar-specific cold tolerance. Additionally, most AcLEA genes exhibited distinct spatiotemporal expression patterns across floral organs and fruit at various developmental stages, suggesting their involvement in reproductive development. These findings provide a foundation for future functional studies and highlight candidate genes for improving cold resilience and developmental traits in pineapple through molecular breeding. Full article

We report the implementation of replica-averaged molecular dynamics in the UNRES coarse-grained model of polypeptide chains, with application to the restraints determined by nuclear magnetic resonance. The analytical ESCASA algorithm is used to estimate interproton distances from coarse-grained geometry. With synthetic restraints derived from two selected conformations of the L129–L153 loop of the Slr1183 protein from Synechocystis sp. (2KW5), the replica-averaged extension of UNRES retrieved the ensemble of conformations close to the parent structures, with residual content of those not similar to any of them, and comparable populations of both families. Tests with a small putatively multistate protein (PDB: 2LWA) and two proteins with disordered regions (2KW5 and 2KZN, respectively) run in multiplexed temperature replica exchange mode with replica averaging resulted in conformational ensembles that had fewer distance-restraint violations than those deposited in the Protein Data Bank. The ensembles obtained with replica averaging also had fewer distance-restraint violations than those obtained in our previous work, in which time-averaged restraints were implemented. The upgraded UNRES can be used in data-assisted simulations of multistate and intrinsically-disordered proteins and proteins with intrinsically disordered regions. Full article