Search Results (127)

Theories on the early steps of the HIV-1 life cycle have been radically revised over the past five years. The long-held assumption that the capsid fully disassembles in the cytoplasm has given way to a more nuanced view: Cytoplasmic disassembly does occur and, in several myeloid systems, is increasingly linked to cytosolic cDNA sensing and to abortive infection. However, a substantial fraction of intact or nearly intact capsid cores instead traverse the nuclear pore complex (NPC) and, upon interacting with the host factor CPSF6, induce liquid–liquid phase separation. This leads to the formation of biomolecular condensates, termed HIV-1 membraneless organelles (HIV-1-MLOs), which subsequently merge with nuclear speckles (NSs). In this Perspective we read these condensates along five interlocking axes. First, the virus drives the host phase separation of cleavage and polyadenylation specificity factor 6 (CPSF6), which quickly fuses with another MLO: the NS composed of the speckle scaffold factors, SON and SRRM2. Second, the resulting condensate behaves as a catalytic site that concentrates the reverse-transcription machinery and thereby promotes integration of the viral DNA into speckle-associated chromatin (SPADs). Third, the same compartment is the final layer of a stratified programme of innate immune evasion, shielding nascent double-stranded DNA from cGAS–STING after cytoplasmic restriction factors and sensors have been outmanoeuvred. Fourth, although demonstrated only in vitro, stable HIV-1-MLOs can maintain the viral RNA genome in the presence of a reverse-transcription inhibitor. Upon removal of the inhibitor, reverse transcription resumes, mirroring, to some extent, the situation in individuals undergoing interruption of antiretroviral therapy and suggesting that these structures may act as a pre-integration reservoir. Fifth, and still largely unexplored, the sanctuary has a pharmacological dimension: anatomical lymphoid compartments, and possibly the condensate itself through selective small-molecule partitioning, may limit antiretroviral drug access. We situate HIV-1-MLOs within the convergent condensate strategies of SARS-CoV-2 and other viruses, and we discuss the clinical, diagnostic, therapeutic, and vaccine implications, including capsid inhibitors as “block-and-expose” tools. Full article

In this work, charge characteristics of submicron heterogeneities (SMH) spontaneously formed in aqueous solutions of various biomolecules: seven amino acids of various types (nonpolar glycine, polar serine, hydrophobic valine, aromatic phenylalanine, sulfur-containing methionine, glutamic acid and basic arginine), ATP, monosaccharide glucose and disaccharide sucrose were studied. The isoelectric points of the SMH in the amino acid solutions determined turned out to be in the pH range from 2.4 to 4, being shifted to the acidic region relative to the isoelectric points of the amino acids themselves (except for glutamic acid). The zeta potential of the SMH was measured in solutions of all the biomolecules under conditions close to the intracellular environment at pH = 7 and basic K⁺ ion content 150 mM. The zeta potential appeared to be negative in all cases. Using these values of the zeta potential, the concentration of OH-anions inside the SMH was estimated, and the pH values corresponding to this concentration turned out to be in the range of 7–10. Since the cell cytosol is an aqueous solution of various biomolecules, SMH must also form inside cells. An analogy is drawn between SMH and membraneless organelles, many of which have been discovered recently. The presence of compact regions with alkaline pH inside the cell is a fundamentally new factor in cell biology, which may have important consequences. Full article

Liquid–liquid phase separation (LLPS) of RNA drives the formation of membraneless organelles, and its dysregulation is closely linked to major human diseases, including cancer, neurodegenerative disorders, and various rare genetic diseases. Current strategies for modulating LLPS often require the introduction of exogenous molecules or specific genetic modifications. Here, coarse-grained molecular dynamics (CGMD) simulations suggest that terahertz (THz) oscillatory fields may influence the condensation of pathogenic G4C2 RNA repeats under the simulated conditions. Oscillatory fields at 10 THz and 37.3 THz are shown to effectively counteract salt-induced condensate dissolution. At the molecular level, THz oscillatory fields are associated with reduced phosphate–sodium contacts at low ionic strengths and with faster water diffusion in the hydration layer at higher salt levels. These changes correlate with an increase in stable intermolecular contacts and a more compact RNA state, suggesting a field-driven shift in the balance of interactions. These findings provide a conceptual and mechanistic basis for understanding how oscillatory fields may influence biomolecular condensation, establishing a microscopic framework for using external variable fields to manipulate biomolecular assemblies. Full article

Ubiquitin-specific protease 7 (USP7/HAUSP) is one of the most studied deubiquitinating enzymes and plays a crucial role in regulating numerous cellular processes, making it a promising therapeutic target. In the nucleus, USP7 partially colocalizes with PML nuclear bodies (PML-NB)—multifunctional membraneless organelles involved in post-translational modifications and protein complexes assembly. The molecular basis and functional significance of this association remain uncharacterized. In this study, comparison of USP7 and PML interactomes revealed a significant overlap of 166 shared proteins. Functional enrichment analysis showed that USP7 and PML may operate within a common molecular context related to transcriptional regulation, chromatin remodeling, and DNA damage responses. Furthermore, these processes are also linked to cellular senescence and human aging (CellAge and GenAge databases). Focused analysis of overlaps between the USP7 interactome and core PML-NB proteins identified 61 proteins forming a dense “small-world” network. Most are prone to liquid–liquid phase separation, are intrinsically disordered, and serve as substrates for SUMOylation or ubiquitination. These findings not only expand our understanding of the molecular functions of USP7 but also highlight PML-NB as an important cellular context for investigating mechanisms associated with USP7 activity. Full article

Long non-coding RNAs (lncRNAs) and RNA–protein complexes (RNPs) are increasingly recognized as central to the regulatory complexity of modern eukaryotes. This review proposes that the remarkable diversity of eukaryotic systems arises from the long-term integration of ancient RNA/RNP mechanisms, layered with innovations introduced by successive symbioses. We outline four interconnected levels of symbiosis contributing to this process: (1) molecular symbiosis, involving dynamic assemblies of RNAs, proteins, and membraneless organelles (MLOs); (2) genome symbiosis, driven by the expansion of non-coding and repetitive DNA; (3) intracellular symbiosis, initiated by mitochondria acquisition; and (4) intercellular symbiosis, rooted in the cellular cooperation that enables multicellularity. We highlight lncRNAs and intrinsically disordered proteins (IDPs) as versatile mediators that interweave interactions across scales, predominantly within phase-separated condensates. Building upon these multi-level processes, we propose the framework of integrated symbiotic pleiotropy—a concept where molecular components acquire layered functional roles as a direct consequence of successive symbiotic acquisitions. This paradigm unites information layering, functional moonlighting, molecular tinkering, and exaptation into a coherent trajectory for eukaryotic evolution. Full article

Cellular survival and adaptability depend on the dynamic regulation of proteins—the central actors of biological systems. Through mechanisms such as post-translational modifications, protein turnover, and the formation of membraneless organelles, cells can sense and respond to a variety of stressors. Recent advances in artificial intelligence and chemical biology have provided powerful tools to study and manipulate these processes, paving the way for novel therapeutic strategies in cancer. This review explores how cells “tame” their proteome in response to stress by coordinating protein synthesis, modification, degradation, and structural organization to maintain functional resilience. Full article

Stress granules (SGs) are dynamic, membraneless organelles that form in response to stress and play pivotal roles in translational control, RNA metabolism, and cell survival. In cancer, SGs are increasingly recognized as central mediators of therapy resistance, enabling malignant cells to evade apoptosis, reprogram metabolism, and modulate immune responses. Understanding the mechanistic and clinical insights into SG kinetics in healthy versus cancer cells holds significant potential for targeting them in precision oncology. This review integrates current knowledge on how chemotherapeutic agents, oncogenic signaling pathways, and tumor microenvironmental stressors promote SG formation, as well as evidence of altered SG kinetics across tumor types. We further highlight how the upregulation of SG components within the tumor microenvironment shapes cancer cell behavior and adaptability, and how crosstalk between SGs and other biomolecular condensates could contribute to resistance. Finally, we discuss emerging therapeutic strategies targeting SGs, including kinase inhibitors and modulators of SG dynamics, and propose that SGs represent tractable vulnerabilities in precision oncology. By bridging mechanistic insights with clinical implications, this review positions SGs as a promising frontier in overcoming cancer therapy resistance. Full article

Liquid–liquid phase separation (LLPS) is a fundamental biophysical process in which proteins and nucleic acids dynamically demix from the cellular milieu to form membraneless organelles (MLO) with liquid-like properties. Environmental cues, such as light, temperature fluctuations, and pathogen interactions, induce LLPS of fungal proteins with intrinsically disordered regions (IDRs) or multimerization domains, thereby regulating fungal hyphal growth, sexual reproduction, pathogenesis, and adaptation. Recently, LLPS has emerged as a powerful tool for biomolecular research, innovative biotechnological application, biosynthesis and metabolic engineering. This review focuses on the current advances in environmental cue-triggered fungal condensates assembled by LLPS, with a focus on their roles in regulating the fungal physical biology and cellular processes including transcription, RNA modification, translation, posttranslational modification process (PTM), transport, and stress response. It further discusses the strategies of engineering synthetic biomolecular condensates in microbial cell factories to enhance production and metabolic efficiency. Full article

Liquid–liquid phase separation (LLPS) in cell biology has revolutionized our understanding of how cells organize biochemical reactions and structures through dynamic, membraneless organelles (MLOs). In neurons, LLPS-driven processes are particularly important for regulating synaptic plasticity, RNA metabolism, and responses to environmental stressors. Over the past decade, LLPS has gained increasing attention in neurobiology as a framework to interpret altered synaptic functions in various neurodevelopmental disorders (NDDs). These diseases comprise a diverse spectrum of clinical and pathological symptoms (e.g., global developmental delay, impaired cognitive and mental functions, as well as social withdrawal). Recent studies have highlighted how mutations in proteins containing intrinsically disordered regions (IDRs)—key drivers of LLPS—can alter condensate properties, resulting in persistent or defective MLO formation. These aberrant assemblies may disrupt RNA transport, splicing, and translation in developing neurons, thereby contributing to disorder pathology. IDRs are known to be enriched in membraneless components, such as stress granules, nuclear paraspeckles, and P-bodies, where they play crucial role in the formation, maintenance, and function of protein–RNA networks. This review explores the role of stress-induced MLOs in the nervous system, the molecular principles governing their formation, and how their dysfunction bridges the gap between environmental stress responses and neurodevelopmental impairment. Full article

Cellular organization relies on both membrane-bound organelles and membraneless biomolecular condensates formed through liquid–liquid phase separation. Recent discoveries reveal intricate coupling between lipid membrane organization and condensate assembly, reshaping our understanding of cellular compartmentalization. This review synthesizes multidisciplinary research using advanced techniques including super-resolution microscopy, fluorescence recovery after photobleaching, and in vitro reconstitution to examine lipid-condensate interactions. Lipid membranes serve as nucleation platforms that reduce critical concentrations for condensate formation by orders of magnitude through membrane anchoring and thermodynamic coupling, creating specialized microenvironments that substantially enhance enzymatic activities. Key regulatory mechanisms include phosphorylation-driven assembly and disassembly, membrane composition effects from cholesterol content and fatty acid saturation, and environmental factors such as calcium and pH. These interactions drive signal transduction through receptor clustering, membrane trafficking via organized domains, and stress responses through protective condensate formation. Dysregulation of lipid-condensate coupling, including aberrant phase transitions and membrane dysfunction, underlies metabolic disorders and neurodegenerative diseases. This coupling represents a fundamental organizing principle with significant therapeutic potential. Current challenges include developing quantitative methods for characterizing condensate dynamics in complex cellular environments and translating molecular mechanisms into clinical applications. Future progress requires interdisciplinary approaches combining advanced experimental techniques, computational modeling, and standardized protocols to advance both fundamental understanding and therapeutic innovations. Full article

Promyelocytic leukemia nuclear bodies (PML-NBs) are dynamic membrane-less organelles (MLOs) located in the nucleus that serve as regulatory hubs for multiple cellular processes. This review examines current understanding of PML-NB structure, assembly mechanisms, and their diverse functional roles. We discuss how PML-NBs interact with chromatin to influence gene expression, regulate transcription factors, and participate in protein quality control. The review highlights their critical functions in tumor suppression, particularly in acute promyelocytic leukemia, and their role in intrinsic antiviral defense against various pathogens. Despite significant advances in the field, key questions remain regarding the mechanistic triggers of PML-NB formation and their common roles across different pathologies. Further elucidation of these aspects may provide valuable insights for developing therapeutic approaches targeting the PML-NB axis in disease treatment. Full article

Many intrinsically disordered proteins (IDPs) are known to undergo liquid–liquid phase separation (LLPS), which is a physical process that drives the formation of biomolecular condensates and membraneless organelles in biological cells. Molecular dynamics (MD) simulations provide valuable tools to explore both the molecular mechanisms of LLPS and the physical properties of biomolecular condensates. However, a direct comparison of MD simulation results with phase diagrams obtained experimentally is normally prevented not only by the high computational costs of simulating large biomacromolecular systems on sufficient timescales but also by conceptual challenges. Specifically, there currently seems to be no standard or unambiguous method of defining and determining volumes occupied by coexisting phases at the nanoscale, with typically no more than a few hundred biomacromolecules in the simulation box. The goal of this work is to fill in this gap in the methodology. Focusing on $\alpha$-synuclein as a model IDP, we test and compare three methods for determining the molecular density of protein nanodroplets, or clusters, generated in MD simulations or using other molecular modeling approaches. Two of the methods are based on approximating nanodroplets with homogeneous spheres and ellipsoids, respectively. The third method, which is expected to yield the most physically accurate results, is based on the SPACEBALL algorithm, with optimized, cluster-specific radii for volume probes. Our results contribute to the construction of accurate phase diagrams on the basis of MD simulations of IDP systems. Full article

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

Outer-membrane vesicles (OMVs), naturally secreted by Gram-negative bacteria, have gained recognition as a versatile platform for the development of next-generation vaccines. OMVs are essential contributors to bacterial pathogenesis, horizontal gene transfer, cellular communication, the maintenance of bacterial fitness, and quorum sensing. Their intrinsic immunogenicity, adjuvant properties, and scalability establish OMVs as potent tools for combating infectious diseases and cancer. Recent advancements in genetic engineering and biotechnology have further expanded the utility of OMVs, enabling the incorporation of multiple epitopes and antigens from diverse pathogens. These developments address critical challenges such as antigenic variability and co-infections, offering broader immune coverage and cost-effective solutions. This review explores the unique structural and immunological properties of OMVs, emphasizing their capacity to elicit robust immune responses. It critically examines established and emerging engineering strategies, including the genetic engineering of surface-displayed antigens, surface conjugation, glycoengineering, nanoparticle-based OMV engineering, hybrid OMVs, and in situ OMV production, among others. Furthermore, recent advancements in preclinical research on OMV-based vaccines, including synthetic OMVs, OMV-based nanorobots, and nanodiscs, as well as emerging isolation and purification methods, are discussed. Lastly, future directions are proposed, highlighting the potential integration of synthetic biology techniques to accelerate research on OMV engineering. Full article

Coacervate is a form of liquid–liquid phase separation (LLPS) in which a solution containing one or more charged components spontaneously separates into two immiscible liquid phases. Due to their ability to mimic membraneless cellular environments and their high biocompatibility, coacervates have found broad applications across various fields of life sciences. This review provides a comprehensive overview of recent advances in biomolecule-based coacervation for biotechnological and biomedical applications. Encapsulation via biomolecule-based coacervation enables high encapsulation efficiency, enhanced stability, and the sustained release of cargos. In the field of tissue engineering, coacervates not only support cell adhesion and proliferation but also serve as printable bioinks with tunable rheological properties for 3D bioprinting. Moreover, biomolecule-based coacervates have been utilized to mimic membraneless organelles, serving as experimental models to understand the origin of life or investigate the mechanisms of biochemical compartmentalization. This review discusses the mechanisms of coacervation induced by various types of biomolecules, evaluates their respective advantages and limitations in applied contexts, and outlines future research directions. Given their modularity and biocompatibility, biomolecule-based coacervates are expected to play a pivotal role in next-generation therapeutic development and the construction of controlled tissue microenvironments, especially when integrated with emerging technologies. Full article