RESUMEN
SETD2 belongs to the family of histone methyltransferase proteins and has been associated with three nosologically distinct entities with different clinical and molecular features: Luscan-Lumish syndrome (LLS), intellectual developmental disorder, autosomal dominant 70 (MRD70), and Rabin-Pappas syndrome (RAPAS). LLS [MIM #616831] is an overgrowth disorder with multisystem involvement including intellectual disability, speech delay, autism spectrum disorder (ASD), macrocephaly, tall stature, and motor delay. RAPAS [MIM #6201551] is a recently reported multisystemic disorder characterized by severely impaired global and intellectual development, hypotonia, feeding difficulties with failure to thrive, microcephaly, and dysmorphic facial features. Other neurologic findings may include seizures, hearing loss, ophthalmologic defects, and brain imaging abnormalities. There is variable involvement of other organ systems, including skeletal, genitourinary, cardiac, and potentially endocrine. Three patients who carried the missense variant p.Arg1740Gln in SETD2 were reported with a moderately impaired intellectual disability, speech difficulties, and behavioral abnormalities. More variable findings included hypotonia and dysmorphic features. Due to the differences with the two previous phenotypes, this association was then named intellectual developmental disorder, autosomal dominant 70 [MIM 620157]. These three disorders seem to be allelic and are caused either by loss-of-function, gain-of-function, or missense variants in the SETD2 gene. Here we describe 18 new patients with variants in SETD2, most of them with the LLS phenotype, and reviewed 33 additional patients with variants in SETD2 that have been previously reported in the scientific literature. This article offers an expansion of the number of reported individuals with LLS and highlights the clinical features and the similarities and differences among the three phenotypes associated with SETD2.
Asunto(s)
Trastorno del Espectro Autista , Discapacidad Intelectual , Humanos , Trastorno del Espectro Autista/genética , Discapacidad Intelectual/genética , Hipotonía Muscular/genética , Fenotipo , SíndromeRESUMEN
Polyketide synthases (PKSs) are predominantly microbial biosynthetic enzymes. They assemble highly potent bioactive natural products from simple carboxylic acid precursors. The most versatile families of PKSs are organized as assembly lines of functional modules. Each module performs one round of precursor extension and optional modification, followed by directed transfer of the intermediate to the next module. While enzymatic domains and even modules of PKSs are well understood, the higher-order modular architecture of PKS assembly lines remains elusive. Here, we visualize a PKS bimodule core using cryo-electron microscopy and resolve a two-dimensional meshwork of the bimodule core formed by homotypic interactions between modules. The sheet-like organization provides the framework for efficient substrate transfer and for sequestration of trans-acting enzymes required for polyketide production.
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Productos Biológicos , Policétidos , Ácidos Carboxílicos , Microscopía por Crioelectrón , Sintasas Poliquetidas/químicaRESUMEN
The R2TP (RUVBL1-RUVBL2-RPAP3-PIH1D1) complex, in collaboration with heat shock protein 90 (HSP90), functions as a chaperone for the assembly and stability of protein complexes, including RNA polymerases, small nuclear ribonucleoprotein particles (snRNPs), and phosphatidylinositol 3-kinase (PI3K)-like kinases (PIKKs) such as TOR and SMG1. PIKK stabilization depends on an additional complex of TELO2, TTI1, and TTI2 (TTT), whose structure and function are poorly understood. The cryoelectron microscopy (cryo-EM) structure of the human R2TP-TTT complex, together with biochemical experiments, reveals the mechanism of TOR recruitment to the R2TP-TTT chaperone. The HEAT-repeat TTT complex binds the kinase domain of TOR, without blocking its activity, and delivers TOR to the R2TP chaperone. In addition, TTT regulates the R2TP chaperone by inhibiting RUVBL1-RUVBL2 ATPase activity and by modulating the conformation and interactions of the PIH1D1 and RPAP3 components of R2TP. Taken together, our results show how TTT couples the recruitment of TOR to R2TP with the regulation of this chaperone system.
Asunto(s)
Chaperonas Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatasas/metabolismo , Microscopía por Crioelectrón , Humanos , Modelos Moleculares , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Unión Proteica , Dominios Proteicos , Mapeo de Interacción de Proteínas , Saccharomyces cerevisiae/ultraestructura , Proteínas de Saccharomyces cerevisiae/ultraestructura , Relación Estructura-ActividadRESUMEN
The number of high-resolution structures of protein complexes obtained using cryo-electron microscopy (cryo-EM) is increasing rapidly. Cryo-EM maps of large macromolecular complexes frequently contain regions resolved at different resolution levels, and modeling atomic structures de novo can be difficult for domains determined at worse than 5 Å in the absence of atomic information from other structures. Here we describe the details and step-by-step decisions in the strategy we followed to model the RUVBL2-binding domain (RBD), a 14 kDa domain at the C-terminus of RNA Polymerase II associated protein 3 (RPAP3) for which atomic information was not available. Modeling was performed on a cryo-EM map at 4.0-5.5 Å resolution, integrating information from secondary structure predictions, homology modeling, restraints from cross-linked mass spectrometry, and molecular dynamics (MD) in AMBER. Here, we compare our model with the structure of RBD determined by NMR to evaluate our strategy. We also perform new MD simulations to describe important residues mediating the interaction of RBD with RUVBL2 and analyze their conservation in RBD homologous domains. Our approach and its evaluation can serve as an example to address the analysis of medium resolution regions in cryo-EM maps.
Asunto(s)
Simulación de Dinámica Molecular , Proteínas , Microscopía por Crioelectrón , Sustancias Macromoleculares , Conformación Proteica , Estructura Secundaria de ProteínaRESUMEN
The human R2TP complex (RUVBL1-RUVBL2-RPAP3-PIH1D1) is an HSP90 co-chaperone required for the maturation of several essential multiprotein complexes, including RNA polymerase II, small nucleolar ribonucleoproteins, and PIKK complexes such as mTORC1 and ATR-ATRIP. RUVBL1-RUVBL2 AAA-ATPases are also primary components of other essential complexes such as INO80 and Tip60 remodelers. Despite recent efforts, the molecular mechanisms regulating RUVBL1-RUVBL2 in these complexes remain elusive. Here, we report cryo-EM structures of R2TP and show how access to the nucleotide-binding site of RUVBL2 is coupled to binding of the client recruitment component of R2TP (PIH1D1) to its DII domain. This interaction induces conformational rearrangements that lead to the destabilization of an N-terminal segment of RUVBL2 that acts as a gatekeeper to nucleotide exchange. This mechanism couples protein-induced motions of the DII domains with accessibility of the nucleotide-binding site in RUVBL1-RUVBL2, and it is likely a general mechanism shared with other RUVBL1-RUVBL2-containing complexes.
Asunto(s)
ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas Portadoras/metabolismo , Microscopía por Crioelectrón/métodos , ADN Helicasas/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Sitios de Unión , Histidina/metabolismo , Humanos , Modelos Moleculares , Complejos Multiproteicos , Nucleótidos/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Dominios ProteicosRESUMEN
Cellular stability, assembly and activation of a growing list of macromolecular complexes require the action of HSP90 working in concert with the R2TP/Prefoldin-like (R2TP/PFDL) co-chaperone. RNA polymerase II, snoRNPs and complexes of PI3-kinase-like kinases, a family that includes the ATM, ATR, DNA-PKcs, TRAPP, SMG1 and mTOR proteins, are among the clients of the HSP90-R2TP system. Evidence links the R2TP/PFDL pathway with cancer, most likely because of the essential role in pathways commonly deregulated in cancer. R2TP forms the core of the co-cochaperone and orchestrates the recruitment of HSP90 and clients, whereas prefoldin and additional prefoldin-like proteins, including URI, associate with R2TP, but their function is still unclear. The mechanism by which R2TP/PFLD facilitates assembly and activation of such a variety of macromolecular complexes is poorly understood. Recent efforts in the structural characterization of R2TP have started to provide some mechanistic insights. We summarize recent structural findings, particularly how cryo-electron microscopy (cryo-EM) is contributing to our understanding of the architecture of the R2TP core complex. Structural differences discovered between yeast and human R2TP reveal unanticipated complexities of the metazoan R2TP complex, and opens new and interesting questions about how R2TP/PFLD works.
Asunto(s)
Chaperonas Moleculares/química , Animales , Microscopía por Crioelectrón , Proteínas HSP90 de Choque Térmico/química , Humanos , Neoplasias , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiaeRESUMEN
In the originally published version of this article, the affiliation details for Hugo Muñoz-Hernández, Carlos F. Rodríguez and Oscar Llorca incorrectly omitted 'Centro de Investigaciones Biológicas (CIB), Spanish National Research Council (CSIC), Ramiro de Maeztu 9, 28040 Madrid, Spain'. This has now been corrected in both the PDF and HTML versions of the Article.
RESUMEN
The R2TP/Prefoldin-like co-chaperone, in concert with HSP90, facilitates assembly and cellular stability of RNA polymerase II, and complexes of PI3-kinase-like kinases such as mTOR. However, the mechanism by which this occurs is poorly understood. Here we use cryo-EM and biochemical studies on the human R2TP core (RUVBL1-RUVBL2-RPAP3-PIH1D1) which reveal the distinctive role of RPAP3, distinguishing metazoan R2TP from the smaller yeast equivalent. RPAP3 spans both faces of a single RUVBL ring, providing an extended scaffold that recruits clients and provides a flexible tether for HSP90. A 3.6 Å cryo-EM structure reveals direct interaction of a C-terminal domain of RPAP3 and the ATPase domain of RUVBL2, necessary for human R2TP assembly but absent from yeast. The mobile TPR domains of RPAP3 map to the opposite face of the ring, associating with PIH1D1, which mediates client protein recruitment. Thus, RPAP3 provides a flexible platform for bringing HSP90 into proximity with diverse client proteins.
Asunto(s)
ATPasas Asociadas con Actividades Celulares Diversas/química , Proteínas Reguladoras de la Apoptosis/química , Proteínas Portadoras/química , ADN Helicasas/química , Proteínas HSP90 de Choque Térmico/química , Chaperonas Moleculares/química , ATPasas Asociadas con Actividades Celulares Diversas/genética , ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Sitios de Unión , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Clonación Molecular , Microscopía por Crioelectrón , ADN Helicasas/genética , ADN Helicasas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Proteínas HSP90 de Choque Térmico/genética , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Modelos Moleculares , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
The R2TP complex, comprising the Rvb1p-Rvb2p AAA-ATPases, Tah1p, and Pih1p in yeast, is a specialized Hsp90 co-chaperone required for the assembly and maturation of multi-subunit complexes. These include the small nucleolar ribonucleoproteins, RNA polymerase II, and complexes containing phosphatidylinositol-3-kinase-like kinases. The structure and stoichiometry of yeast R2TP and how it couples to Hsp90 are currently unknown. Here, we determine the 3D organization of yeast R2TP using sedimentation velocity analysis and cryo-electron microscopy. The 359-kDa complex comprises one Rvb1p/Rvb2p hetero-hexamer with domains II (DIIs) forming an open basket that accommodates a single copy of Tah1p-Pih1p. Tah1p-Pih1p binding to multiple DII domains regulates Rvb1p/Rvb2p ATPase activity. Using domain dissection and cross-linking mass spectrometry, we identified a unique region of Pih1p that is essential for interaction with Rvb1p/Rvb2p. These data provide a structural basis for understanding how R2TP couples an Hsp90 dimer to a diverse set of client proteins and complexes.
Asunto(s)
Adenosina Trifosfatasas/química , Proteínas HSP90 de Choque Térmico/química , Chaperonas Moleculares/química , Proteínas Nucleares/química , Proteínas de Saccharomyces cerevisiae/química , Factores de Transcripción/química , Adenosina Trifosfatasas/metabolismo , Sitios de Unión , Proteínas HSP90 de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Nucleares/metabolismo , Unión Proteica , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Microtubule-targeting agents (MTAs) are some of the clinically most successful anti-cancer drugs. Unfortunately, instances of multidrug resistances to MTA have been reported, which highlights the need for developing MTAs with different mechanistic properties. One less explored class of MTAs are [1,2,4]triazolo[1,5-a]pyrimidines (TPs). These cytotoxic compounds are microtubule-stabilizing agents that inexplicably bind to vinblastine binding site on tubulin, which is typically targeted by microtubule-destabilizing agents. Here we used cellular, biochemical, and structural biology approaches to address this apparent discrepancy. Our results establish TPs as vinca-site microtubule-stabilizing agents that promote longitudinal tubulin contacts in microtubules, in contrast to classical microtubule-stabilizing agents that primarily promote lateral contacts. Additionally we observe that TPs studied here are not affected by p-glycoprotein overexpression, and suggest that TPs are promising ligands against multidrug-resistant cancer cells.
Asunto(s)
Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Pirimidinas/farmacología , Triazoles/farmacología , Tubulina (Proteína)/metabolismo , Alcaloides de la Vinca/metabolismo , Sitios de Unión , Línea Celular Tumoral , Humanos , Ligandos , Modelos Moleculares , Multimerización de Proteína/efectos de los fármacos , Estructura Cuaternaria de Proteína , Tubulina (Proteína)/químicaRESUMEN
RuvBL1 and RuvBL2, also known as Pontin and Reptin, are AAA+ proteins essential in small nucleolar ribonucloprotein biogenesis, chromatin remodelling, nonsense-mediated messenger RNA decay and telomerase assembly, among other functions. They are homologous to prokaryotic RuvB, forming single- and double-hexameric rings; however, a DNA binding domain II (DII) is inserted within the AAA+ core. Despite their biological significance, questions remain regarding their structure. Here, we report cryo-electron microscopy structures of human double-ring RuvBL1-RuvBL2 complexes at â¼15 Å resolution. Significantly, we resolve two coexisting conformations, compact and stretched, by image classification techniques. Movements in DII domains drive these conformational transitions, extending the complex and regulating the exposure of DNA binding regions. DII domains connect with the AAA+ core and bind nucleic acids, suggesting that these conformational changes could impact the regulation of RuvBL1-RuvBL2 containing complexes. These findings resolve some of the controversies in the structure of RuvBL1-RuvBL2 by revealing a mechanism that extends the complex by adjustments in DII.