RESUMO
Nucleoli are multicomponent condensates defined by coexisting sub-phases. We identified distinct intrinsically disordered regions (IDRs), including acidic (D/E) tracts and K-blocks interspersed by E-rich regions, as defining features of nucleolar proteins. We show that the localization preferences of nucleolar proteins are determined by their IDRs and the types of RNA or DNA binding domains they encompass. In vitro reconstitutions and studies in cells showed how condensation, which combines binding and complex coacervation of nucleolar components, contributes to nucleolar organization. D/E tracts of nucleolar proteins contribute to lowering the pH of co-condensates formed with nucleolar RNAs in vitro. In cells, this sets up a pH gradient between nucleoli and the nucleoplasm. By contrast, juxta-nucleolar bodies, which have different macromolecular compositions, featuring protein IDRs with very different charge profiles, have pH values that are equivalent to or higher than the nucleoplasm. Our findings show that distinct compositional specificities generate distinct physicochemical properties for condensates.
Assuntos
Nucléolo Celular , Proteínas Nucleares , Força Próton-Motriz , Nucléolo Celular/química , Núcleo Celular/química , Proteínas Nucleares/química , RNA/metabolismo , Separação de Fases , Proteínas Intrinsicamente Desordenadas/química , Animais , Xenopus laevis , Oócitos/química , Oócitos/citologiaRESUMO
NLRP6 is important in host defense by inducing functional outcomes including inflammasome activation and interferon production. Here, we show that NLRP6 undergoes liquid-liquid phase separation (LLPS) upon interaction with double-stranded RNA (dsRNA) in vitro and in cells, and an intrinsically disordered poly-lysine sequence (K350-354) of NLRP6 is important for multivalent interactions, phase separation, and inflammasome activation. Nlrp6-deficient or Nlrp6K350-354A mutant mice show reduced inflammasome activation upon mouse hepatitis virus or rotavirus infection, and in steady state stimulated by intestinal microbiota, implicating NLRP6 LLPS in anti-microbial immunity. Recruitment of ASC via helical assembly solidifies NLRP6 condensates, and ASC further recruits and activates caspase-1. Lipoteichoic acid, a known NLRP6 ligand, also promotes NLRP6 LLPS, and DHX15, a helicase in NLRP6-induced interferon signaling, co-forms condensates with NLRP6 and dsRNA. Thus, LLPS of NLRP6 is a common response to ligand stimulation, which serves to direct NLRP6 to distinct functional outcomes depending on the cellular context.
Assuntos
Inflamassomos/metabolismo , Vírus de RNA/fisiologia , Receptores de Superfície Celular/metabolismo , Sequência de Aminoácidos , Animais , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Hepatócitos/virologia , Intestinos/virologia , Proteínas Intrinsicamente Desordenadas/química , Lipopolissacarídeos/metabolismo , Fígado/virologia , Camundongos , Polilisina/metabolismo , Ligação Proteica , RNA de Cadeia Dupla/metabolismo , Receptores de Superfície Celular/química , Transdução de Sinais , Ácidos Teicoicos/metabolismoRESUMO
Clustering of macromolecules is a fundamental cellular device underlying diverse biological processes that require high-avidity binding to effectors and substrates. Often, this involves a transition between diffuse and locally concentrated molecules akin to biophysical phase separation observable in vitro. One simple mechanistic paradigm underlying physiologically relevant phase transitions in cells is the reversible head-to-tail polymerization of hub proteins into filaments that are cross-linked by dimerization into dynamic three-dimensional molecular condensates. While many diverse folds and motifs can mediate dimerization, only two structurally distinct domains have been discovered so far to undergo head-to-tail polymerization, though these are widespread among all living kingdoms.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Substâncias Macromoleculares/metabolismo , Animais , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Substâncias Macromoleculares/química , Polimerização , Domínios Proteicos , Via de Sinalização WntRESUMO
In this review, we describe speculative ideas and early stage research concerning the flow of genetic information from the nuclear residence of genes to the disparate, cytoplasmic sites of protein synthesis. We propose that this process of information transfer is meticulously guided by transient structures formed from protein segments of low sequence complexity/intrinsic disorder. These low complexity domains are ubiquitously associated with regulatory proteins that control gene expression and RNA biogenesis, but they are also found in the central channel of nuclear pores, the nexus points of intermediate filament assembly, and the locations of action of other well-studied cellular proteins and pathways. Upon being organized into localized cellular positions via mechanisms utilizing properly folded protein domains, thereby facilitating elevated local concentration, certain low complexity domains adopt cross-ß interactions that are both structurally specific and labile to disassembly. These weakly tethered assemblies, we propose, are built to relay the passage of genetic information from one site to another within a cell, ensuring that the process is of extreme fidelity.
Assuntos
Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Modelos Biológicos , Animais , Grânulos Citoplasmáticos/genética , Grânulos Citoplasmáticos/metabolismo , Expressão Gênica , Transtornos Heredodegenerativos do Sistema Nervoso/genética , Transtornos Heredodegenerativos do Sistema Nervoso/metabolismo , Humanos , Hidrogéis , Proteínas Intrinsicamente Desordenadas/química , Modelos Moleculares , Mutação , Domínios e Motivos de Interação entre Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína FUS de Ligação a RNA/química , Proteína FUS de Ligação a RNA/genética , Proteína FUS de Ligação a RNA/metabolismoRESUMO
In eukaryotic cells, diverse stresses trigger coalescence of RNA-binding proteins into stress granules. In vitro, stress-granule-associated proteins can demix to form liquids, hydrogels, and other assemblies lacking fixed stoichiometry. Observing these phenomena has generally required conditions far removed from physiological stresses. We show that poly(A)-binding protein (Pab1 in yeast), a defining marker of stress granules, phase separates and forms hydrogels in vitro upon exposure to physiological stress conditions. Other RNA-binding proteins depend upon low-complexity regions (LCRs) or RNA for phase separation, whereas Pab1's LCR is not required for demixing, and RNA inhibits it. Based on unique evolutionary patterns, we create LCR mutations, which systematically tune its biophysical properties and Pab1 phase separation in vitro and in vivo. Mutations that impede phase separation reduce organism fitness during prolonged stress. Poly(A)-binding protein thus acts as a physiological stress sensor, exploiting phase separation to precisely mark stress onset, a broadly generalizable mechanism.
Assuntos
Grânulos Citoplasmáticos/metabolismo , Proteínas de Ligação a Poli(A)/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/fisiologia , Sequência de Aminoácidos , Grânulos Citoplasmáticos/química , Temperatura Alta , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Mutagênese , Proteínas de Ligação a Poli(A)/química , Proteínas de Ligação a Poli(A)/genética , Prolina/análise , Prolina/metabolismo , Domínios Proteicos , Ribonucleases/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Alinhamento de Sequência , Estresse FisiológicoRESUMO
In this issue of Molecular Cell, De La Cruz, Pradhan, Veettil et al.1 examine how selective partitioning of proteins via low-affinity IDR-dependent interactions may help regulate RNA polymerase II (RNA Pol II) function and identify sequence features that drive partitioning in cells.
Assuntos
RNA Polimerase II , Transcrição Gênica , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , Regulação da Expressão Gênica , Humanos , Ligação Proteica , Proteínas Intrinsicamente Desordenadas/metabolismo , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/genéticaRESUMO
Selective compartmentalization of cellular contents is fundamental to the regulation of biochemistry. Although membrane-bound organelles control composition by using a semi-permeable barrier, biomolecular condensates rely on interactions among constituents to determine composition. Condensates are formed by dynamic multivalent interactions, often involving intrinsically disordered regions (IDRs) of proteins, yet whether distinct compositions can arise from these dynamic interactions is not known. Here, by comparative analysis of proteins differentially partitioned by two different condensates, we find that distinct compositions arise through specific IDR-mediated interactions. The IDRs of differentially partitioned proteins are necessary and sufficient for selective partitioning. Distinct sequence features are required for IDRs to partition, and swapping these sequence features changes the specificity of partitioning. Swapping whole IDRs retargets proteins and their biochemical activity to different condensates. Our results demonstrate that IDR-mediated interactions can target proteins to specific condensates, enabling the spatial regulation of biochemistry within the cell.
Assuntos
Condensados Biomoleculares , Proteínas Intrinsicamente Desordenadas , Proteínas Intrinsicamente Desordenadas/metabolismo , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/genética , Condensados Biomoleculares/metabolismo , Condensados Biomoleculares/química , Ligação Proteica , Organelas/metabolismo , Humanos , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/químicaRESUMO
Transcriptional coregulators and transcription factors (TFs) contain intrinsically disordered regions (IDRs) that are critical for their association and function in gene regulation. More recently, IDRs have been shown to promote multivalent protein-protein interactions between coregulators and TFs to drive their association into condensates. By contrast, here we demonstrate how the IDR of the corepressor LSD1 excludes TF association, acting as a dynamic conformational switch that tunes repression of active cis-regulatory elements. Hydrogen-deuterium exchange shows that the LSD1 IDR interconverts between transient open and closed conformational states, the latter of which inhibits partitioning of the protein's structured domains with TF condensates. This autoinhibitory switch controls leukemic differentiation by modulating repression of active cis-regulatory elements bound by LSD1 and master hematopoietic TFs. Together, these studies unveil alternative mechanisms by which disordered regions and their dynamic crosstalk with structured regions can shape coregulator-TF interactions to control cis-regulatory landscapes and cell fate.
Assuntos
Elementos Facilitadores Genéticos , Histona Desmetilases , Histona Desmetilases/metabolismo , Histona Desmetilases/genética , Humanos , Proteínas Intrinsicamente Desordenadas/metabolismo , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/química , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Animais , Ligação Proteica , Camundongos , Diferenciação Celular , Inativação GênicaRESUMO
Complementary studies by Zhao et al.1 and Chen et al.2 reveal how an intrinsically disordered region in MED13 controls mutually exclusive binding of RNA Polymerase II and CDK8 kinase module to Mediator, switching Mediator and transcription activation on and off.
Assuntos
Quinase 8 Dependente de Ciclina , Complexo Mediador , RNA Polimerase II , Complexo Mediador/metabolismo , Complexo Mediador/genética , Complexo Mediador/química , Quinase 8 Dependente de Ciclina/metabolismo , Quinase 8 Dependente de Ciclina/genética , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , Humanos , Proteínas Intrinsicamente Desordenadas/metabolismo , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/genética , Ligação Proteica , Ativação TranscricionalRESUMO
The essential Mediator (MED) coactivator complex plays a well-understood role in regulation of basal transcription in all eukaryotes, but the mechanism underlying its role in activator-dependent transcription remains unknown. We investigated modulation of metazoan MED interaction with RNA polymerase II (RNA Pol II) by antagonistic effects of the MED26 subunit and the CDK8 kinase module (CKM). Biochemical analysis of CKM-MED showed that the CKM blocks binding of the RNA Pol II carboxy-terminal domain (CTD), preventing RNA Pol II interaction. This restriction is eliminated by nuclear receptor (NR) binding to CKM-MED, which enables CTD binding in a MED26-dependent manner. Cryoelectron microscopy (cryo-EM) and crosslinking-mass spectrometry (XL-MS) revealed that the structural basis for modulation of CTD interaction with MED relates to a large intrinsically disordered region (IDR) in CKM subunit MED13 that blocks MED26 and CTD interaction with MED but is repositioned upon NR binding. Hence, NRs can control transcription initiation by priming CKM-MED for MED26-dependent RNA Pol II interaction.
Assuntos
Microscopia Crioeletrônica , Quinase 8 Dependente de Ciclina , Complexo Mediador , Ligação Proteica , RNA Polimerase II , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , Complexo Mediador/metabolismo , Complexo Mediador/genética , Complexo Mediador/química , Humanos , Quinase 8 Dependente de Ciclina/metabolismo , Quinase 8 Dependente de Ciclina/genética , Animais , Proteínas Intrinsicamente Desordenadas/metabolismo , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/química , Sítios de Ligação , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Células HEK293 , Domínios e Motivos de Interação entre ProteínasRESUMO
The eukaryotic transcriptional Mediator comprises a large core (cMED) and a dissociable CDK8 kinase module (CKM). cMED recruits RNA polymerase II (RNA Pol II) and promotes pre-initiation complex formation in a manner repressed by the CKM through mechanisms presently unknown. Herein, we report cryoelectron microscopy structures of the complete human Mediator and its CKM. The CKM binds to multiple regions on cMED through both MED12 and MED13, including a large intrinsically disordered region (IDR) in the latter. MED12 and MED13 together anchor the CKM to the cMED hook, positioning CDK8 downstream and proximal to the transcription start site. Notably, the MED13 IDR obstructs the recruitment of RNA Pol II/MED26 onto cMED by direct occlusion of their respective binding sites, leading to functional repression of cMED-dependent transcription. Combined with biochemical and functional analyses, these structures provide a conserved mechanistic framework to explain the basis for CKM-mediated repression of cMED function.
Assuntos
Microscopia Crioeletrônica , Quinase 8 Dependente de Ciclina , Complexo Mediador , RNA Polimerase II , Humanos , Complexo Mediador/metabolismo , Complexo Mediador/genética , Complexo Mediador/química , Quinase 8 Dependente de Ciclina/metabolismo , Quinase 8 Dependente de Ciclina/genética , Quinase 8 Dependente de Ciclina/química , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/química , Sítios de Ligação , Ligação Proteica , Transcrição Gênica , Modelos Moleculares , Relação Estrutura-Atividade , Proteínas Intrinsicamente Desordenadas/metabolismo , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/genéticaRESUMO
Protein flexibility ranges from simple hinge movements to functional disorder. Around half of all human proteins contain apparently disordered regions with little 3D or functional information, and many of these proteins are associated with disease. Building on the evolutionary couplings approach previously successful in predicting 3D states of ordered proteins and RNA, we developed a method to predict the potential for ordered states for all apparently disordered proteins with sufficiently rich evolutionary information. The approach is highly accurate (79%) for residue interactions as tested in more than 60 known disordered regions captured in a bound or specific condition. Assessing the potential for structure of more than 1,000 apparently disordered regions of human proteins reveals a continuum of structural order with at least 50% with clear propensity for three- or two-dimensional states. Co-evolutionary constraints reveal hitherto unseen structures of functional importance in apparently disordered proteins.
Assuntos
Proteínas Intrinsicamente Desordenadas/química , Evolução Molecular Direcionada/métodos , Genômica , Humanos , Proteínas Intrinsicamente Desordenadas/genética , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteoma/química , Proteoma/genéticaRESUMO
A large segment of the proteome consists of disordered regions, yet in most cases, little is known about their mechanisms and functions. What are the roles of protein disorder in cell biology, and how do intrinsically disordered proteins function? These are the questions Cell's Robert Kruger posed to Madan Babu, Julie Forman-Kay, and Richard Kriwacki. Annotated excerpts from this conversation are presented below, and the full conversation is available with the article online. PAPERCLIP.
Assuntos
Proteínas Intrinsicamente Desordenadas , Proteoma , Motivos de Aminoácidos , Animais , Biologia Celular , Biologia Computacional , Humanos , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/fisiologia , Processamento de Proteína Pós-Traducional , Proteoma/química , Proteoma/genética , Proteoma/fisiologiaRESUMO
Prions are a paradigm-shifting mechanism of inheritance in which phenotypes are encoded by self-templating protein conformations rather than nucleic acids. Here, we examine the breadth of protein-based inheritance across the yeast proteome by assessing the ability of nearly every open reading frame (ORF; â¼5,300 ORFs) to induce heritable traits. Transient overexpression of nearly 50 proteins created traits that remained heritable long after their expression returned to normal. These traits were beneficial, had prion-like patterns of inheritance, were common in wild yeasts, and could be transmitted to naive cells with protein alone. Most inducing proteins were not known prions and did not form amyloid. Instead, they are highly enriched in nucleic acid binding proteins with large intrinsically disordered domains that have been widely conserved across evolution. Thus, our data establish a common type of protein-based inheritance through which intrinsically disordered proteins can drive the emergence of new traits and adaptive opportunities.
Assuntos
Proteínas Intrinsicamente Desordenadas/metabolismo , Característica Quantitativa Herdável , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Amiloide/metabolismo , Evolução Molecular , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/genética , Fases de Leitura Aberta , Príons/química , Príons/metabolismo , Proteoma , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Intrinsically disordered proteins and regions (collectively, IDRs) are pervasive across proteomes in all kingdoms of life, help to shape biological functions and are involved in numerous diseases. IDRs populate a diverse set of transiently formed structures and defy conventional sequence-structure-function relationships1. Developments in protein science have made it possible to predict the three-dimensional structures of folded proteins at the proteome scale2. By contrast, there is a lack of knowledge about the conformational properties of IDRs, partly because the sequences of disordered proteins are poorly conserved and also because only a few of these proteins have been characterized experimentally. The inability to predict structural properties of IDRs across the proteome has limited our understanding of the functional roles of IDRs and how evolution shapes them. As a supplement to previous structural studies of individual IDRs3, we developed an efficient molecular model to generate conformational ensembles of IDRs and thereby to predict their conformational properties from sequences4,5. Here we use this model to simulate nearly all of the IDRs in the human proteome. Examining conformational ensembles of 28,058 IDRs, we show how chain compaction is correlated with cellular function and localization. We provide insights into how sequence features relate to chain compaction and, using a machine-learning model trained on our simulation data, show the conservation of conformational properties across orthologues. Our results recapitulate observations from previous studies of individual protein systems and exemplify how to link-at the proteome scale-conformational ensembles with cellular function and localization, amino acid sequence, evolutionary conservation and disease variants. Our freely available database of conformational properties will encourage further experimental investigation and enable the generation of hypotheses about the biological roles and evolution of IDRs.
Assuntos
Proteínas Intrinsicamente Desordenadas , Modelos Moleculares , Conformação Proteica , Proteoma , Humanos , Sequência de Aminoácidos , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Proteoma/química , Proteoma/metabolismo , Relação Estrutura-Atividade , Evolução Molecular , Doença/genéticaRESUMO
Progression through the cell cycle is controlled by regulated and abrupt changes in phosphorylation1. Mitotic entry is initiated by increased phosphorylation of mitotic proteins, a process driven by kinases2, whereas mitotic exit is achieved by counteracting dephosphorylation, a process driven by phosphatases, especially PP2A:B553. Although the role of kinases in mitotic entry is well established, recent data have shown that mitosis is only successfully initiated when the counterbalancing phosphatases are also inhibited4. Inhibition of PP2A:B55 is achieved by the intrinsically disordered proteins ARPP195,6 and FAM122A7. Despite their critical roles in mitosis, the mechanisms by which they achieve PP2A:B55 inhibition is unknown. Here, we report the single-particle cryo-electron microscopy structures of PP2A:B55 bound to phosphorylated ARPP19 and FAM122A. Consistent with our complementary NMR spectroscopy studies, both intrinsically disordered proteins bind PP2A:B55, but do so in highly distinct manners, leveraging multiple distinct binding sites on B55. Our extensive structural, biophysical and biochemical data explain how substrates and inhibitors are recruited to PP2A:B55 and provide a molecular roadmap for the development of therapeutic interventions for PP2A:B55-related diseases.
Assuntos
Microscopia Crioeletrônica , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Intrinsicamente Desordenadas , Fosfoproteínas , Proteína Fosfatase 2 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Proteínas Intrinsicamente Desordenadas/ultraestrutura , Mitose , Ressonância Magnética Nuclear Biomolecular , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Fosfoproteínas/ultraestrutura , Fosforilação , Proteína Fosfatase 2/química , Proteína Fosfatase 2/metabolismo , Proteína Fosfatase 2/ultraestruturaRESUMO
Plants exposed to incidences of excessive temperatures activate heat-stress responses to cope with the physiological challenge and stimulate long-term acclimation1,2. The mechanism that senses cellular temperature for inducing thermotolerance is still unclear3. Here we show that TWA1 is a temperature-sensing transcriptional co-regulator that is needed for basal and acquired thermotolerance in Arabidopsis thaliana. At elevated temperatures, TWA1 changes its conformation and allows physical interaction with JASMONATE-ASSOCIATED MYC-LIKE (JAM) transcription factors and TOPLESS (TPL) and TOPLESS-RELATED (TPR) proteins for repressor complex assembly. TWA1 is a predicted intrinsically disordered protein that has a key thermosensory role functioning through an amino-terminal highly variable region. At elevated temperatures, TWA1 accumulates in nuclear subdomains, and physical interactions with JAM2 and TPL appear to be restricted to these nuclear subdomains. The transcriptional upregulation of the heat shock transcription factor A2 (HSFA2) and heat shock proteins depended on TWA1, and TWA1 orthologues provided different temperature thresholds, consistent with the sensor function in early signalling of heat stress. The identification of the plant thermosensors offers a molecular tool for adjusting thermal acclimation responses of crops by breeding and biotechnology, and a sensitive temperature switch for thermogenetics.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas Intrinsicamente Desordenadas , Temperatura , Sensação Térmica , Termotolerância , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/fisiologia , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Núcleo Celular/metabolismo , Regulação da Expressão Gênica de Plantas , Fatores de Transcrição de Choque Térmico/metabolismo , Fatores de Transcrição de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Proteínas de Choque Térmico/genética , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Sensação Térmica/genética , Sensação Térmica/fisiologia , Termotolerância/genética , Termotolerância/fisiologia , Fatores de Transcrição/metabolismo , Transdução de SinaisRESUMO
HIV can infect non-dividing cells because the viral capsid can overcome the selective barrier of the nuclear pore complex and deliver the genome directly into the nucleus1,2. Remarkably, the intact HIV capsid is more than 1,000 times larger than the size limit prescribed by the diffusion barrier of the nuclear pore3. This barrier in the central channel of the nuclear pore is composed of intrinsically disordered nucleoporin domains enriched in phenylalanine-glycine (FG) dipeptides. Through multivalent FG interactions, cellular karyopherins and their bound cargoes solubilize in this phase to drive nucleocytoplasmic transport4. By performing an in vitro dissection of the nuclear pore complex, we show that a pocket on the surface of the HIV capsid similarly interacts with FG motifs from multiple nucleoporins and that this interaction licences capsids to penetrate FG-nucleoporin condensates. This karyopherin mimicry model addresses a key conceptual challenge for the role of the HIV capsid in nuclear entry and offers an explanation as to how an exogenous entity much larger than any known cellular cargo may be able to non-destructively breach the nuclear envelope.
Assuntos
Proteínas do Capsídeo , Glicina , HIV , Carioferinas , Mimetismo Molecular , Complexo de Proteínas Formadoras de Poros Nucleares , Poro Nuclear , Fenilalanina , Humanos , Transporte Ativo do Núcleo Celular , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Difusão , Dipeptídeos/química , Dipeptídeos/metabolismo , Glicina/metabolismo , HIV/química , HIV/metabolismo , Técnicas In Vitro , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Carioferinas/metabolismo , Poro Nuclear/química , Poro Nuclear/metabolismo , Poro Nuclear/virologia , Complexo de Proteínas Formadoras de Poros Nucleares/química , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Permeabilidade , Fenilalanina/metabolismo , Solubilidade , Internalização do Vírus , Capsídeo/química , Capsídeo/metabolismoRESUMO
Intrinsically disordered proteins (IDPs) and IDP regions fail to form a stable structure, yet they exhibit biological activities. Their mobile flexibility and structural instability are encoded by their amino acid sequences. They recognize proteins, nucleic acids, and other types of partners; they accelerate interactions and chemical reactions between bound partners; and they help accommodate posttranslational modifications, alternative splicing, protein fusions, and insertions or deletions. Overall, IDP-associated biological activities complement those of structured proteins. Recently, there has been an explosion of studies on IDP regions and their functions, yet the discovery and investigation of these proteins have a long, mostly ignored history. Along with recent discoveries, we present several early examples and the mechanisms by which IDPs contribute to function, which we hope will encourage comprehensive discussion of IDPs and IDP regions in biochemistry textbooks. Finally, we propose future directions for IDP research.
Assuntos
Proteínas Intrinsicamente Desordenadas/química , Animais , Calcineurina/química , Caseínas/química , Biologia Computacional , Espectroscopia de Ressonância de Spin Eletrônica , Fibrina/química , Fibrinogênio/química , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Fosvitina/química , Ligação Proteica , Mapeamento de Interação de Proteínas , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Espalhamento de Radiação , Solubilidade , Tripsina/química , Tripsinogênio/química , Difração de Raios XRESUMO
Proteins and nucleic acids can phase-separate in the cell to form concentrated biomolecular condensates1-4. The functions of condensates span many length scales: they modulate interactions and chemical reactions at the molecular scale5, organize biochemical processes at the mesoscale6 and compartmentalize cells4. Understanding the underlying mechanisms of these processes will require detailed knowledge of the rich dynamics across these scales7. The mesoscopic dynamics of biomolecular condensates have been extensively characterized8, but their behaviour at the molecular scale has remained more elusive. Here, as an example of biomolecular phase separation, we study complex coacervates of two highly and oppositely charged disordered human proteins9. Their dense phase is 1,000 times more concentrated than the dilute phase, and the resulting percolated interaction network10 leads to a bulk viscosity 300 times greater than that of water. However, single-molecule spectroscopy optimized for measurements within individual droplets reveals that at the molecular scale, the disordered proteins remain exceedingly dynamic, with their chain configurations interconverting on submicrosecond timescales. Massive all-atom molecular dynamics simulations reproduce the experimental observations and explain this apparent discrepancy: the underlying interactions between individual charged side chains are short-lived and exchange on a pico- to nanosecond timescale. Our results indicate that, despite the high macroscopic viscosity of phase-separated systems, local biomolecular rearrangements required for efficient reactions at the molecular scale can remain rapid.