RESUMO
In his commentary in this issue of Molecular Cell,1 Struhl reasons that the term "intrinsically disordered regions" represents a vague and confusing concept for protein function. However, the term "intrinsically disordered" highlights the important physicochemical characteristic of conformational heterogeneity. Thus, "intrinsically disordered" is the counterpart to the term "folded, " with neither term having specific functional implications.
Assuntos
Proteínas Intrinsicamente Desordenadas , Proteínas Intrinsicamente Desordenadas/metabolismo , Conformação ProteicaRESUMO
The nucleolus and other ribonucleoprotein (RNP) bodies are membrane-less organelles that appear to assemble through phase separation of their molecular components. However, many such RNP bodies contain internal subcompartments, and the mechanism of their formation remains unclear. Here, we combine in vivo and in vitro studies, together with computational modeling, to show that subcompartments within the nucleolus represent distinct, coexisting liquid phases. Consistent with their in vivo immiscibility, purified nucleolar proteins phase separate into droplets containing distinct non-coalescing phases that are remarkably similar to nucleoli in vivo. This layered droplet organization is caused by differences in the biophysical properties of the phases-particularly droplet surface tension-which arises from sequence-encoded features of their macromolecular components. These results suggest that phase separation can give rise to multilayered liquids that may facilitate sequential RNA processing reactions in a variety of RNP bodies. PAPERCLIP.
Assuntos
Nucléolo Celular/química , Animais , Caenorhabditis elegans , Células Cultivadas , Proteínas Cromossômicas não Histona/análise , Intestinos/química , Intestinos/citologia , Mamíferos , Proteínas Nucleares/análise , Nucleofosmina , Oócitos/química , Oócitos/citologia , Processamento Pós-Transcricional do RNA , Ribonucleoproteínas/metabolismo , Xenopus laevisRESUMO
Expansion of a hexanucleotide repeat GGGGCC (G4C2) in C9ORF72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Transcripts carrying (G4C2) expansions undergo unconventional, non-ATG-dependent translation, generating toxic dipeptide repeat (DPR) proteins thought to contribute to disease. Here, we identify the interactome of all DPRs and find that arginine-containing DPRs, polyGly-Arg (GR) and polyPro-Arg (PR), interact with RNA-binding proteins and proteins with low complexity sequence domains (LCDs) that often mediate the assembly of membrane-less organelles. Indeed, most GR/PR interactors are components of membrane-less organelles such as nucleoli, the nuclear pore complex and stress granules. Genetic analysis in Drosophila demonstrated the functional relevance of these interactions to DPR toxicity. Furthermore, we show that GR and PR altered phase separation of LCD-containing proteins, insinuating into their liquid assemblies and changing their material properties, resulting in perturbed dynamics and/or functions of multiple membrane-less organelles.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Dipeptídeos/metabolismo , Demência Frontotemporal/metabolismo , Proteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Esclerose Lateral Amiotrófica/genética , Animais , Proteína C9orf72 , Nucléolo Celular/metabolismo , Grânulos Citoplasmáticos/metabolismo , Expansão das Repetições de DNA , Dipeptídeos/genética , Drosophila melanogaster/genética , Demência Frontotemporal/genética , Humanos , Membranas Intracelulares/metabolismo , Poro Nuclear/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Proteínas/genéticaRESUMO
T-lineage acute lymphoblastic leukaemia (T-ALL) is a high-risk tumour1 that has eluded comprehensive genomic characterization, which is partly due to the high frequency of noncoding genomic alterations that result in oncogene deregulation2,3. Here we report an integrated analysis of genome and transcriptome sequencing of tumour and remission samples from more than 1,300 uniformly treated children with T-ALL, coupled with epigenomic and single-cell analyses of malignant and normal T cell precursors. This approach identified 15 subtypes with distinct genomic drivers, gene expression patterns, developmental states and outcomes. Analyses of chromatin topology revealed multiple mechanisms of enhancer deregulation that involve enhancers and genes in a subtype-specific manner, thereby demonstrating widespread involvement of the noncoding genome. We show that the immunophenotypically described, high-risk entity of early T cell precursor ALL is superseded by a broader category of 'early T cell precursor-like' leukaemia. This category has a variable immunophenotype and diverse genomic alterations of a core set of genes that encode regulators of hematopoietic stem cell development. Using multivariable outcome models, we show that genetic subtypes, driver and concomitant genetic alterations independently predict treatment failure and survival. These findings provide a roadmap for the classification, risk stratification and mechanistic understanding of this disease.
RESUMO
Ribosome biogenesis takes place in the nucleolus, a nuclear membrane-less organelle. Although well studied, it remains unknown how nascent ribosomal subunits separate from the central chromatin compartment and move to the outer granular component, where maturation occurs. We find that the Schizosaccharomyces pombe nucleophosmin-like protein Fkbp39 localizes to rDNA sites encoding the 60S subunit rRNA, and this localization contributes to its specific association with nascent 60S subunits. Fkbp39 dissociates from chromatin to bind nascent 60S subunits, causing the latter to partition away from chromatin and from nascent 40S subunits through liquid-liquid phase separation. In vivo, Fkbp39 binding directs the translocation of nascent 60S subunits toward the nucleophosmin-rich granular component. This process increases the efficiency of 60S subunit assembly, facilitating the incorporation of 60S RNA domain III. Thus, chromatin localization determines the specificity of nucleophosmin in sorting nascent ribosomal subunits and coordinates their movement into specialized assembly compartments within the nucleolus.
Assuntos
Cromatina , Schizosaccharomyces , Cromatina/genética , Nucleofosmina , Nucléolo Celular/genética , Membrana Nuclear , Schizosaccharomyces/genética , Ribossomos/genéticaRESUMO
Repeat expansion in the C9orf72 gene is the most common cause of the neurodegenerative disorder amyotrophic lateral sclerosis (C9-ALS) and is linked to the unconventional translation of five dipeptide-repeat polypeptides (DPRs). The two enriched in arginine, poly(GR) and poly(PR), infiltrate liquid-like nucleoli, co-localize with the nucleolar protein nucleophosmin (NPM1), and alter the phase separation behavior of NPM1 in vitro. Here, we show that poly(PR) DPRs bind tightly to a long acidic tract within the intrinsically disordered region of NPM1, altering its phase separation with nucleolar partners to the extreme of forming large, soluble complexes that cause droplet dissolution in vitro. In cells, poly(PR) DPRs disperse NPM1 from nucleoli and entrap rRNA in static condensates in a DPR-length-dependent manner. We propose that R-rich DPR toxicity involves disrupting the role of phase separation by NPM1 in organizing ribosomal proteins and RNAs within the nucleolus.
Assuntos
Esclerose Lateral Amiotrófica/genética , Proteína C9orf72/genética , Proteínas Nucleares/genética , Sequências Repetitivas de Aminoácidos/genética , Esclerose Lateral Amiotrófica/patologia , Arginina/genética , Nucléolo Celular/química , Nucléolo Celular/genética , Dipeptídeos/genética , Humanos , Nucleofosmina , Peptídeos/genética , Poli A/genética , RNA Ribossômico/genéticaRESUMO
ABSTRACT: Inotuzumab ozogamicin (InO) is an antibody-drug conjugate that delivers calicheamicin to CD22-expressing cells. In a retrospective cohort of InO-treated patients with B-cell acute lymphoblastic leukemia, we sought to understand the genomic determinants of the response and resistance to InO. Pre- and post-InO-treated patient samples were analyzed by whole genome, exome, and/or transcriptome sequencing. Acquired CD22 mutations were observed in 11% (3/27) of post-InO-relapsed tumor samples, but not in refractory samples (0/16). There were multiple CD22 mutations per sample and the mechanisms of CD22 escape included epitope loss (protein truncation and destabilization) and epitope alteration. Two CD22 mutant cases were post-InO hyper-mutators resulting from error-prone DNA damage repair (nonhomologous/alternative end-joining repair, or mismatch repair deficiency), suggesting that hypermutation drove escape from CD22-directed therapy. CD22-mutant relapses occurred after InO and subsequent hematopoietic stem cell transplantation (HSCT), suggesting that InO eliminated the predominant clones, leaving subclones with acquired CD22 mutations that conferred resistance to InO and subsequently expanded. Acquired loss-of-function mutations in TP53, ATM, and CDKN2A were observed, consistent with a compromise of the G1/S DNA damage checkpoint as a mechanism for evading InO-induced apoptosis. Genome-wide CRISPR/Cas9 screening of cell lines identified DNTT (terminal deoxynucleotidyl transferase) loss as a marker of InO resistance. In conclusion, genetic alterations modulating CD22 expression and DNA damage response influence InO efficacy. Our findings highlight the importance of defining the basis of CD22 escape and eradication of residual disease before HSCT. The identified mechanisms of escape from CD22-targeted therapy extend beyond antigen loss and provide opportunities to improve therapeutic approaches and overcome resistance. These trials were registered at www.ClinicalTrials.gov as NCT01134575, NCT01371630, and NCT03441061.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Inotuzumab Ozogamicina , Leucemia-Linfoma Linfoblástico de Células Precursoras B , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico , Humanos , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/genética , Resistencia a Medicamentos Antineoplásicos/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Feminino , Mutação , Masculino , Antineoplásicos Imunológicos/uso terapêutico , Antineoplásicos Imunológicos/farmacologia , Adulto , Pessoa de Meia-Idade , Estudos Retrospectivos , AdolescenteRESUMO
Intracellular bodies such as nucleoli, Cajal bodies and various signalling assemblies represent membraneless organelles, or condensates, that form via liquid-liquid phase separation (LLPS)1,2. Biomolecular interactions-particularly homotypic interactions mediated by self-associating intrinsically disordered protein regions-are thought to underlie the thermodynamic driving forces for LLPS, forming condensates that can facilitate the assembly and processing of biochemically active complexes, such as ribosomal subunits within the nucleolus. Simplified model systems3-6 have led to the concept that a single fixed saturation concentration is a defining feature of endogenous LLPS7-9, and has been suggested as a mechanism for intracellular concentration buffering2,7,8,10. However, the assumption of a fixed saturation concentration remains largely untested within living cells, in which the richly multicomponent nature of condensates could complicate this simple picture. Here we show that heterotypic multicomponent interactions dominate endogenous LLPS, and give rise to nucleoli and other condensates that do not exhibit a fixed saturation concentration. As the concentration of individual components is varied, their partition coefficients change in a manner that can be used to determine the thermodynamic free energies that underlie LLPS. We find that heterotypic interactions among protein and RNA components stabilize various archetypal intracellular condensates-including the nucleolus, Cajal bodies, stress granules and P-bodies-implying that the composition of condensates is finely tuned by the thermodynamics of the underlying biomolecular interaction network. In the context of RNA-processing condensates such as the nucleolus, this manifests in the selective exclusion of fully assembled ribonucleoprotein complexes, providing a thermodynamic basis for vectorial ribosomal RNA flux out of the nucleolus. This methodology is conceptually straightforward and readily implemented, and can be broadly used to extract thermodynamic parameters from microscopy images. These approaches pave the way for a deeper understanding of the thermodynamics of multicomponent intracellular phase behaviour and its interplay with the nonequilibrium activity that is characteristic of endogenous condensates.
Assuntos
Espaço Intracelular/química , Espaço Intracelular/metabolismo , Organelas/química , Organelas/metabolismo , Termodinâmica , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Nucléolo Celular/química , Nucléolo Celular/metabolismo , Corpos Enovelados/química , Corpos Enovelados/metabolismo , Grânulos Citoplasmáticos/química , Grânulos Citoplasmáticos/metabolismo , DNA Helicases/deficiência , Células HeLa , Humanos , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Nucleofosmina , Transição de Fase , Proteínas de Ligação a Poli-ADP-Ribose/deficiência , RNA Helicases/deficiência , Proteínas com Motivo de Reconhecimento de RNA/deficiência , RNA Ribossômico/química , RNA Ribossômico/metabolismo , Proteínas de Ligação a RNA , Ribossomos/química , Ribossomos/metabolismoRESUMO
The cytosolic fraction of the tumor suppressor p53 activates the apoptotic effector protein BAX to trigger apoptosis. Here we report that p53 activates BAX through a mechanism different from that associated with activation by BH3 only proteins (BIM and BID). We observed that cis-trans isomerization of proline 47 (Pro47) within p53, an inherently rare molecular event, was required for BAX activation. The prolyl isomerase Pin1 enhanced p53-dependent BAX activation by catalyzing cis-trans interconversion of p53 Pro47. Our results reveal a signaling mechanism whereby proline cis-trans isomerization in one protein triggers conformational and functional changes in a downstream signaling partner. Activation of BAX through the concerted action of cytosolic p53 and Pin1 may integrate cell stress signals to induce a direct apoptotic response.
Assuntos
Apoptose , Peptidilprolil Isomerase/fisiologia , Proteína Supressora de Tumor p53/metabolismo , Proteína X Associada a bcl-2/metabolismo , Linhagem Celular Tumoral , Humanos , Cinética , Peptidilprolil Isomerase de Interação com NIMA , Prolina/química , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estereoisomerismo , Proteína Supressora de Tumor p53/química , Proteína X Associada a bcl-2/químicaRESUMO
Intrinsically disordered regions (IDRs) of proteins often regulate function upon post-translational modification (PTM) through interactions with folded domains. An IDR linking two α-helices (α1-α2) of the antiapoptotic protein Bcl-xL experiences several PTMs that reduce antiapoptotic activity. Here, we report that PTMs within the α1-α2 IDR promote its interaction with the folded core of Bcl-xL that inhibits the proapoptotic activity of two types of regulatory targets, BH3-only proteins and p53. This autoregulation utilizes an allosteric pathway whereby, in one direction, the IDR induces a direct displacement of p53 from Bcl-xL coupled to allosteric displacement of simultaneously bound BH3-only partners. This pathway operates in the opposite direction when the BH3-only protein PUMA binds to the BH3 binding groove of Bcl-xL, directly displacing other bound BH3-only proteins, and allosterically remodels the distal site, displacing p53. Our findings show how an IDR enhances functional versatility through PTM-dependent allosteric regulation of a folded protein domain.
Assuntos
Apoptose , Regulação da Expressão Gênica , Proteínas Intrinsicamente Desordenadas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteína bcl-X/metabolismo , Sítio Alostérico , Sítios de Ligação , Humanos , Proteínas Intrinsicamente Desordenadas/genética , Cinética , Mutação , Ligação Proteica , Domínios Proteicos , Dobramento de Proteína , Processamento de Proteína Pós-Traducional , Estrutura Secundária de Proteína , Transdução de Sinais , Proteína bcl-X/genéticaRESUMO
The global dimensions and amplitudes of conformational fluctuations of intrinsically disordered proteins are governed, in part, by the linear segregation versus clustering of oppositely charged residues within the primary sequence. Ion mobility-mass spectrometry (IM-MS) affords unique advantages for probing the conformational consequences of the linear patterning of oppositely charged residues because it measures and separates proteins electrosprayed from solution on the basis of charge and shape. Here, we use IM-MS to measure the conformational consequences of charge patterning on the C-terminal intrinsically disordered region (p27 IDR) of the cell cycle inhibitory protein p27Kip1. We report the range of charge states and accompanying collisional cross section distributions for wild-type p27 IDR and two variants with identical amino acid compositions, κ14 and κ56, distinguished by the extent of linear mixing versus segregation of oppositely charged residues. Wild-type p27 IDR (κ31) and κ14, where the oppositely charged residues are more evenly distributed, exhibit a broad distribution of charge states. This is concordant with high degrees of conformational heterogeneity in solution. By contrast, κ56 with linear segregation of oppositely charged residues leads to limited conformational heterogeneity and a narrow distribution of charged states. Gas-phase molecular dynamics simulations demonstrate that the interplay between chain solvation and intrachain interactions (self-solvation) leads to conformational distributions that are modulated by salt concentration, with the wild-type sequence showing the most sensitivity to changes in salt concentration. These results suggest that the charge patterning within the wild-type p27 IDR may be optimized to sample both highly solvated and self-solvated conformational states.
Assuntos
Proteínas Intrinsicamente Desordenadas/química , Espectrometria de Massas , Inibidor de Quinase Dependente de Ciclina p27/química , Simulação de Dinâmica Molecular , Conformação ProteicaRESUMO
Peptide motifs embedded within intrinsically disordered regions (IDRs) of proteins are often the sites of posttranslational modifications that control cell-signaling pathways. How do IDR sequences modulate the functionalities of motifs? We answer this question using the polyampholytic C-terminal IDR of the cell cycle inhibitory protein p27(Kip1) (p27). Phosphorylation of Thr-187 (T187) within the p27 IDR controls entry into S phase of the cell division cycle. Additionally, the conformational properties of polyampholytic sequences are predicted to be influenced by the linear patterning of oppositely charged residues. Therefore, we designed sequence variants of the p27 IDR to alter charge patterning outside the primary substrate motif containing T187. Computer simulations and biophysical measurements confirm predictions regarding the impact of charge patterning on the global dimensions of IDRs. Through functional studies, we uncover cryptic sequence features within the p27 IDR that influence the efficiency of T187 phosphorylation. Specifically, we find a positive correlation between T187 phosphorylation efficiency and the weighted net charge per residue of an auxiliary motif. We also find that accumulation of positive charges within the auxiliary motif can diminish the efficiency of T187 phosphorylation because this increases the likelihood of long-range intra-IDR interactions that involve both the primary and auxiliary motifs and inhibit their contributions to function. Importantly, our findings suggest that the cryptic sequence features of the WT p27 IDR negatively regulate T187 phosphorylation signaling. Our approaches provide a generalizable strategy for uncovering the influence of sequence contexts on the functionalities of primary motifs in other IDRs.
Assuntos
Ciclo Celular/fisiologia , Inibidor de Quinase Dependente de Ciclina p27/fisiologia , Transdução de Sinais/fisiologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Inibidor de Quinase Dependente de Ciclina p27/química , Humanos , Fosforilação , Conformação ProteicaRESUMO
Intrinsically disordered proteins have been reported to undergo disorder-to-order transitions upon binding to their partners in the cell. The extent of the ordering upon binding and the lack of order prior to binding is difficult to visualize with classical structure determination methods. Binding of p27 to the Cdk2/cyclinâ A complex is accompanied by partial folding of p27 in the KID domain, with the retention of dynamic behavior for function, particularly in the C-terminal half of the protein. Herein, native ion mobility mass spectrometry (IM-MS) is employed to measure the intrinsic dynamic properties of p27, both in isolation and within the trimeric complex with Cdk2/cyclinâ A. The trimeric Cdk2/cyclinâ A/p27-KID complex possesses significant structural heterogeneity compared to Cdk2/cyclinâ A. These findings support the formation of a fuzzy complex in which both the N- and C-termini of p27 interact with Cdk2/cyclinâ A in multiple, closely associated states.
Assuntos
Ciclina A/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Ciclina A/química , Quinase 2 Dependente de Ciclina/química , Inibidor de Quinase Dependente de Ciclina p27/química , Humanos , Proteínas Intrinsicamente Desordenadas/metabolismo , Espectrometria de Mobilidade Iônica , Espectrometria de Massas , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Mapas de Interação de Proteínas , Multimerização ProteicaRESUMO
During apoptotic cell death, cellular stress signals converge at the mitochondria to induce mitochondrial outer-membrane permeabilization (MOMP) through B cell lymphoma-2 (BCL-2) family proteins and their effectors. BCL-2 proteins function through protein-protein interactions, the mechanisms and structural aspects of which are only now being uncovered. Recently, the elucidation of the dynamic features underlying their function has highlighted their structural plasticity and the consequent complex thermodynamic landscape governing their protein-protein interactions. These studies show that canonical interactions involve a conserved, hydrophobic groove, whereas non-canonical interactions function allosterically outside the groove. We review the latest structural advances in understanding the interactions and functions of mammalian BCL-2 family members, and discuss new opportunities to modulate these proteins in health and disease.
Assuntos
Apoptose/fisiologia , Membranas Mitocondriais/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2 , Animais , Humanos , Permeabilidade , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismoRESUMO
Understanding signaling and other complex biological processes requires elucidating the critical roles of intrinsically disordered proteins (IDPs) and regions (IDRs), which represent â¼30% of the proteome and enable unique regulatory mechanisms. In this review, we describe the structural heterogeneity of disordered proteins that underpins these mechanisms and the latest progress in obtaining structural descriptions of conformational ensembles of disordered proteins that are needed for linking structure and dynamics to function. We describe the diverse interactions of IDPs that can have unusual characteristics such as "ultrasensitivity" and "regulated folding and unfolding". We also summarize the mounting data showing that large-scale assembly and protein phase separation occurs within a variety of signaling complexes and cellular structures. In addition, we discuss efforts to therapeutically target disordered proteins with small molecules. Overall, we interpret the remodeling of disordered state ensembles due to binding and post-translational modifications within an expanded framework for allostery that provides significant insights into how disordered proteins transmit biological information.
Assuntos
Proteínas Intrinsicamente Desordenadas/metabolismo , Transdução de Sinais/fisiologia , Regulação Alostérica , Proteínas Intrinsicamente Desordenadas/química , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Desdobramento de Proteína , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismoRESUMO
Early T-cell precursor acute lymphoblastic leukaemia (ETP ALL) is an aggressive malignancy of unknown genetic basis. We performed whole-genome sequencing of 12 ETP ALL cases and assessed the frequency of the identified somatic mutations in 94 T-cell acute lymphoblastic leukaemia cases. ETP ALL was characterized by activating mutations in genes regulating cytokine receptor and RAS signalling (67% of cases; NRAS, KRAS, FLT3, IL7R, JAK3, JAK1, SH2B3 and BRAF), inactivating lesions disrupting haematopoietic development (58%; GATA3, ETV6, RUNX1, IKZF1 and EP300) and histone-modifying genes (48%; EZH2, EED, SUZ12, SETD2 and EP300). We also identified new targets of recurrent mutation including DNM2, ECT2L and RELN. The mutational spectrum is similar to myeloid tumours, and moreover, the global transcriptional profile of ETP ALL was similar to that of normal and myeloid leukaemia haematopoietic stem cells. These findings suggest that addition of myeloid-directed therapies might improve the poor outcome of ETP ALL.
Assuntos
Predisposição Genética para Doença/genética , Mutação/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Idade de Início , Criança , Variações do Número de Cópias de DNA/genética , Genes ras/genética , Genoma Humano/genética , Genômica , Hematopoese/genética , Histonas/metabolismo , Humanos , Janus Quinases/genética , Janus Quinases/metabolismo , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Dados de Sequência Molecular , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Receptores de Interleucina-7/genética , Proteína Reelina , Análise de Sequência de DNA , Transdução de Sinais/genética , Células-Tronco/metabolismo , Células-Tronco/patologia , Linfócitos T/metabolismo , Linfócitos T/patologia , Translocação Genética/genéticaRESUMO
In ubiquitin-like protein (UBL) cascades, a thioester-linked E2â¼UBL complex typically interacts with an E3 enzyme for UBL transfer to the target. Here we demonstrate a variant mechanism, whereby the E2 Ubc12 functions with two E3s, Hrt1 and Dcn1, for ligation of the UBL Rub1 to Cdc53's WHB subdomain. Hrt1 functions like a conventional RING E3, with its N terminus recruiting Cdc53 and C-terminal RING activating Ubc12â¼Rub1. Dcn1's "potentiating neddylation" domain (Dcn1(P)) acts as an additional E3, reducing nonspecific Hrt1-mediated Ubc12â¼Rub1 discharge and directing Ubc12's active site to Cdc53. Crystal structures of Dcn1(P)-Cdc53(WHB) and Ubc12 allow modeling of a catalytic complex, supported by mutational data. We propose that Dcn1's interactions with both Cdc53 and Ubc12 would restrict the otherwise flexible Hrt1 RING-bound Ubc12â¼Rub1 to a catalytically competent orientation. Our data reveal mechanisms by which two E3s function synergistically to promote UBL transfer from one E2 to a target.
Assuntos
Proteínas Culina/metabolismo , Proteínas Ligases SKP Culina F-Box/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinas/metabolismo , Cristalografia por Raios X , Proteínas Culina/química , Proteínas Culina/genética , Modelos Moleculares , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Estrutura Terciária de Proteína , Proteínas Ligases SKP Culina F-Box/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinas/química , Ubiquitinas/genéticaRESUMO
Intrinsically disordered proteins (IDPs) have roles in myriad biological processes and numerous human diseases. However, kinetic and amplitude information regarding their ground-state conformational fluctuations has remained elusive. We demonstrate using nuclear magnetic resonance (NMR)-based relaxation dispersion that the D2 domain of p27Kip1, a prototypical IDP, samples multiple discrete, rapidly exchanging conformational states. By combining NMR with mutagenesis and small-angle X-ray scattering (SAXS), we show that these states involve aromatic residue clustering through long-range hydrophobic interactions. Theoretical studies have proposed that small molecules bind promiscuously to IDPs, causing expansion of their conformational landscapes. However, on the basis of previous NMR-based screening results, we show here that compound binding only shifts the populations of states that existed within the ground state of apo p27-D2 without changing the barriers between states. Our results provide atomic resolution insight into how a small molecule binds an IDP and emphasize the need to examine motions on the low microsecond time scale when probing these types of interactions.
Assuntos
Proteínas Intrinsicamente Desordenadas/química , Bibliotecas de Moléculas Pequenas/química , Conformação Molecular , Ressonância Magnética Nuclear BiomolecularRESUMO
Nucleophosmin (NPM1) is a multifunctional phospho-protein with critical roles in ribosome biogenesis, tumor suppression, and nucleolar stress response. Here we show that the N-terminal oligomerization domain of NPM1 (Npm-N) exhibits structural polymorphism by populating conformational states ranging from a highly ordered, folded pentamer to a highly disordered monomer. The monomer-pentamer equilibrium is modulated by posttranslational modification and protein binding. Phosphorylation drives the equilibrium in favor of monomeric forms, and this effect can be reversed by Npm-N binding to its interaction partners. We have identified a short, arginine-rich linear motif in NPM1 binding partners that mediates Npm-N oligomerization. We propose that the diverse functional repertoire associated with NPM1 is controlled through a regulated unfolding mechanism signaled through posttranslational modifications and intermolecular interactions.
Assuntos
Biopolímeros/química , Proteínas Nucleares/química , Sequência de Aminoácidos , Biopolímeros/metabolismo , Cromatografia em Gel , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Eletroforese em Gel de Poliacrilamida Nativa , Ressonância Magnética Nuclear Biomolecular , Proteínas Nucleares/metabolismo , Nucleofosmina , Fosforilação , Ligação Proteica , Conformação ProteicaRESUMO
The kinase inhibitory domain of the cell cycle regulatory protein p27Kip1 (p27) was nuclear spin hyperpolarized using dissolution dynamic nuclear polarization (D-DNP). While intrinsically disordered in isolation, p27 adopts secondary structural motifs, including an α-helical structure, upon binding to cyclin-dependent kinase 2 (Cdk2)/cyclin A. The sensitivity gains obtained with hyperpolarization enable the real-time observation of 13 Câ NMR signals during p27 folding upon binding to Cdk2/cyclin A on a time scale of several seconds. Time-dependent intensity changes are dependent on the extent of folding and binding, as manifested in differential spin relaxation. The analysis of signal decay rates suggests the existence of a partially folded p27 intermediate during the timescale of the D-DNP NMR experiment.