RESUMO
Liquid-liquid phase separation (LLPS) is involved in the formation of membraneless organelles (MLOs) associated with RNA processing. The RNA-binding protein TDP-43 is present in several MLOs, undergoes LLPS, and has been linked to the pathogenesis of amyotrophic lateral sclerosis (ALS). While some ALS-associated mutations in TDP-43 disrupt self-interaction and function, here we show that designed single mutations can enhance TDP-43 assembly and function via modulating helical structure. Using molecular simulation and NMR spectroscopy, we observe large structural changes upon dimerization of TDP-43. Two conserved glycine residues (G335 and G338) are potent inhibitors of helical extension and helix-helix interaction, which are removed in part by variants at these positions, including the ALS-associated G335D. Substitution to helix-enhancing alanine at either of these positions dramatically enhances phase separation in vitro and decreases fluidity of phase-separated TDP-43 reporter compartments in cells. Furthermore, G335A increases TDP-43 splicing function in a minigene assay. Therefore, the TDP-43 helical region serves as a short but uniquely tunable module where application of biophysical principles can precisely control assembly and function in cellular and synthetic biology applications of LLPS.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Conformação Proteica em alfa-Hélice , Esclerose Lateral Amiotrófica/genética , Proteínas de Ligação a DNA/genética , Escherichia coli/genética , Humanos , Espectroscopia de Ressonância Magnética , Simulação de Acoplamento Molecular , Mutação , Conformação Proteica , Domínios Proteicos , Domínios e Motivos de Interação entre Proteínas , Processamento de Proteína , Proteínas de Ligação a RNA/metabolismoRESUMO
TDP-43 is an RNA-binding protein active in splicing that concentrates into membraneless ribonucleoprotein granules and forms aggregates in amyotrophic lateral sclerosis (ALS) and Alzheimer's disease. Although best known for its predominantly disordered C-terminal domain which mediates ALS inclusions, TDP-43 has a globular N-terminal domain (NTD). Here, we show that TDP-43 NTD assembles into head-to-tail linear chains and that phosphomimetic substitution at S48 disrupts TDP-43 polymeric assembly, discourages liquid-liquid phase separation (LLPS) in vitro, fluidizes liquid-liquid phase separated nuclear TDP-43 reporter constructs in cells, and disrupts RNA splicing activity. Finally, we present the solution NMR structure of a head-to-tail NTD dimer comprised of two engineered variants that allow saturation of the native polymerization interface while disrupting higher-order polymerization. These data provide structural detail for the established mechanistic role of the well-folded TDP-43 NTD in splicing and link this function to LLPS. In addition, the fusion-tag solubilized, recombinant form of TDP-43 full-length protein developed here will enable future phase separation and in vitro biochemical assays on TDP-43 function and interactions that have been hampered in the past by TDP-43 aggregation.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Agregação Patológica de Proteínas/genética , Domínios Proteicos/genética , Splicing de RNA/genética , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Proteínas de Ligação a DNA/genética , Células HEK293 , Humanos , Polimerização , Polímeros/metabolismo , Agregação Patológica de Proteínas/patologiaRESUMO
The 62 kDa FG repeat domain of the nucleoporin Nsp1p forms a hydrogel-based, sieve-like permeability barrier that excludes inert macromolecules but allows rapid entry of nuclear transport receptors (NTRs). We found that the N-terminal part of this domain, which is characterized by Asn-rich inter-FG spacers, forms a tough hydrogel. The C-terminal part comprises charged inter-FG spacers, shows low gelation propensity on its own, but binds the N-terminal part and passivates the FG hydrogel against nonselective interactions. It was previously shown that a hydrophobic collapse involving Phe residues is required for FG hydrogel formation. Using solid-state NMR spectroscopy, we now identified two additional types of intragel interactions, namely, transient hydrophobic interactions between Phe and methyl side chains as well as intermolecular beta-sheets between the Asn-rich spacer regions. The latter appear to be the kinetically most stable structures within the FG hydrogel. They are also a central feature of neuronal inclusions formed by Asn/Gln-rich amyloid and prion proteins. The cohesive properties of FG repeats and the Asn/Gln-rich domain from the yeast prion Sup35p appear indeed so similar to each other that these two modules interact in trans. Our data, therefore, suggest a fully unexpected cellular function of such interchain beta-structures in maintaining the permeability barrier of nuclear pores. They provide an explanation for how contacts between FG repeats might gain the kinetic stability to suppress passive fluxes through nuclear pores and yet allow rapid NTR passage.
Assuntos
Amiloide/química , Hidrogéis/química , Complexo de Proteínas Formadoras de Poros Nucleares/química , Amiloide/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Ressonância Magnética Nuclear Biomolecular , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Fatores de Terminação de Peptídeos/química , Fatores de Terminação de Peptídeos/metabolismo , Estabilidade Proteica , Estrutura Secundária de Proteína , Sequências Repetitivas de Aminoácidos , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Cell homeostasis is perturbed when dramatic shifts in the external environment cause the physical-chemical properties inside the cell to change. Experimental approaches for dynamically monitoring these intracellular effects are currently lacking. Here, we leverage the environmental sensitivity and structural plasticity of intrinsically disordered protein regions (IDRs) to develop a FRET biosensor capable of monitoring rapid intracellular changes caused by osmotic stress. The biosensor, named SED1, utilizes the Arabidopsis intrinsically disordered AtLEA4-5 protein expressed in plants under water deficit. Computational modeling and in vitro studies reveal that SED1 is highly sensitive to macromolecular crowding. SED1 exhibits large and near-linear osmolarity-dependent changes in FRET inside living bacteria, yeast, plant, and human cells, demonstrating the broad utility of this tool for studying water-associated stress. This study demonstrates the remarkable ability of IDRs to sense the cellular environment across the tree of life and provides a blueprint for their use as environmentally-responsive molecular tools.
Assuntos
Proteínas de Arabidopsis/metabolismo , Técnicas Biossensoriais , Proteínas Intrinsicamente Desordenadas/metabolismo , Chaperonas Moleculares/metabolismo , Pressão Osmótica , Água/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Sítios de Ligação , Linhagem Celular Tumoral , Escherichia coli/genética , Escherichia coli/metabolismo , Transferência Ressonante de Energia de Fluorescência , Expressão Gênica , Humanos , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/genética , Cinética , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Concentração Osmolar , Osteoblastos/citologia , Osteoblastos/metabolismo , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , TermodinâmicaRESUMO
Intrinsically disordered regions (IDRs) are often fast-evolving protein domains of low sequence complexity that can drive phase transitions and are commonly found in many proteins associated with neurodegenerative diseases, including the RNA processing factor TDP43. Yet, how phase separation contributes to the physiological functions of TDP43 in cells remains enigmatic. Here, we combine systematic mutagenesis guided by evolutionary sequence analysis with a live-cell reporter assay of TDP43 phase dynamics to identify regularly-spaced hydrophobic motifs separated by flexible, hydrophilic segments in the IDR as a key determinant of TDP43 phase properties. This heuristic framework allows customization of the material properties of TDP43 condensates to determine effects on splicing function. Remarkably, even a mutant that fails to phase-separate at physiological concentrations can still efficiently mediate the splicing of a quantitative, single-cell splicing reporter and endogenous targets. This suggests that the ability of TDP43 to phase-separate is not essential for its splicing function.
Assuntos
Motivos de Aminoácidos/genética , Proteínas de Ligação a DNA/genética , Interações Hidrofóbicas e Hidrofílicas , Transição de Fase , Splicing de RNA/genética , Processamento Alternativo/genética , Proteínas de Ligação a DNA/metabolismo , Células HEK293 , Humanos , Mutagênese Sítio-DirigidaRESUMO
Eukaryotic cells contain membrane-less organelles, including nucleoli and stress granules, that behave like liquid droplets. Such endogenous condensates often have internal substructure, but how this is established in the absence of membrane encapsulation remains unclear. We find that the N- and C-terminal domains of TDP43, a heterogeneous nuclear ribonucleoprotein implicated in neurodegenerative diseases, are capable of driving the formation of sub-structured liquid droplets in vivo. These droplets contain dynamic internal "bubbles" of nucleoplasm, reminiscent of membrane-based multi-vesicular endosomes. A conserved sequence embedded within the intrinsically disordered region (IDR) of TDP43 promotes the formation of these multi-phase assemblies. Disease-causing point mutations in the IDR can change the propensity to form bubbles, protein dynamics within the phase, or phase-environment exchange rates. Our results show that a single IDR-containing protein can nucleate the assembly of compartmentalized liquid droplets approximating the morphological complexity of membrane-bound organelles.
Assuntos
Núcleo Celular/metabolismo , Grânulos Citoplasmáticos/metabolismo , Membranas/metabolismo , Animais , Linhagem Celular , Núcleo Celular/genética , Grânulos Citoplasmáticos/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endossomos/genética , Endossomos/metabolismo , Células HEK293 , Humanos , Mamíferos/genética , Mamíferos/metabolismo , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Mutação Puntual/genética , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismoRESUMO
Nuclear pore complexes (NPCs) conduct massive transport mediated by shuttling nuclear transport receptors (NTRs), while keeping nuclear and cytoplasmic contents separated. The NPC barrier in Xenopus relies primarily on the intrinsically disordered FG domain of Nup98. We now observed that Nup98 FG domains of mammals, lancelets, insects, nematodes, fungi, plants, amoebas, ciliates, and excavates spontaneously and rapidly phase-separate from dilute (submicromolar) aqueous solutions into characteristic 'FG particles'. This required neither sophisticated experimental conditions nor auxiliary eukaryotic factors. Instead, it occurred already during FG domain expression in bacteria. All Nup98 FG phases rejected inert macromolecules and yet allowed far larger NTR cargo complexes to rapidly enter. They even recapitulated the observations that large cargo-domains counteract NPC passage of NTRâ cargo complexes, while cargo shielding and increased NTRâ cargo surface-ratios override this inhibition. Their exquisite NPC-typical sorting selectivity and strong intrinsic assembly propensity suggest that Nup98 FG phases can form in authentic NPCs and indeed account for the permeability properties of the pore.