Assuntos
Mieloma Múltiplo , Preparações Farmacêuticas , Proteínas Adaptadoras de Transdução de Sinal , Humanos , Fator de Transcrição Ikaros/genética , Fator de Transcrição Ikaros/metabolismo , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Mutação , Talidomida , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Despite an increasing number of approved therapies, multiple myeloma (MM) remains an incurable disease and only a small number of patients achieve prolonged disease control. Some genes have been linked with response to commonly used anti-MM compounds, including immunomodulators (IMiDs) and proteasome inhibitors (PIs). In this manuscript, we demonstrate an increased incidence of acquired proteasomal subunit mutations in relapsed MM compared to newly diagnosed disease, underpinning a potential role of point mutations in the clonal evolution of MM. Furthermore, we are first to present and functionally characterize four somatic PSMB5 mutations from primary MM cells identified in a patient under prolonged proteasome inhibition, with three of them affecting the PI-binding pocket S1. We confirm resistance induction through missense mutations not only to Bortezomib, but also, in variable extent, to the next-generation PIs Carfilzomib and Ixazomib. In addition, a negative impact on the proteasome activity is assessed, providing a potential explanation for later therapy-induced eradication of the affected tumor subclones in this patient.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais/genética , Resistencia a Medicamentos Antineoplásicos/genética , Mieloma Múltiplo/genética , Mutação , Recidiva Local de Neoplasia/genética , Complexo de Endopeptidases do Proteassoma/genética , Biomarcadores Tumorais/metabolismo , Compostos de Boro/administração & dosagem , Bortezomib/administração & dosagem , Estudos de Coortes , Feminino , Seguimentos , Regulação Neoplásica da Expressão Gênica , Glicina/administração & dosagem , Glicina/análogos & derivados , Humanos , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/patologia , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/patologia , Oligopeptídeos/administração & dosagem , Prognóstico , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Taxa de SobrevidaRESUMO
Ribosome synthesis is catalyzed by â¼200 assembly factors, which facilitate efficient production of mature ribosomes. Here, we determined the cryo-EM structure of a Saccharomyces cerevisiae nucleoplasmic pre-60S particle containing the dynein-related 550-kDa Rea1 AAA(+) ATPase and the Rix1 subcomplex. This particle differs from its preceding state, the early Arx1 particle, by two massive structural rearrangements: an â¼180° rotation of the 5S ribonucleoprotein complex and the central protuberance (CP) rRNA helices, and the removal of the 'foot' structure from the 3' end of the 5.8S rRNA. Progression from the Arx1 to the Rix1 particle was blocked by mutational perturbation of the Rix1-Rea1 interaction but not by a dominant-lethal Rea1 AAA(+) ATPase-ring mutant. After remodeling, the Rix1 subcomplex and Rea1 become suitably positioned to sense correct structural maturation of the CP, which allows unidirectional progression toward mature ribosomes.
Assuntos
Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Biogênese de Organelas , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/ultraestrutura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , ATPases Associadas a Diversas Atividades Celulares , Microscopia Crioeletrônica , Modelos Moleculares , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismoRESUMO
Eukaryotic ribosome biogenesis involves â¼200 assembly factors, but how these contribute to ribosome maturation is poorly understood. Here, we identify a network of factors on the nascent 60S subunit that actively remodels preribosome structure. At its hub is Rsa4, a direct substrate of the force-generating ATPase Rea1. We show that Rsa4 is connected to the central protuberance by binding to Rpl5 and to ribosomal RNA (rRNA) helix 89 of the nascent peptidyl transferase center (PTC) through Nsa2. Importantly, Nsa2 binds to helix 89 before relocation of helix 89 to the PTC. Structure-based mutations of these factors reveal the functional importance of their interactions for ribosome assembly. Thus, Rsa4 is held tightly in the preribosome and can serve as a "distribution box," transmitting remodeling energy from Rea1 into the developing ribosome. We suggest that a relay-like factor network coupled to a mechano-enzyme is strategically positioned to relocate rRNA elements during ribosome maturation.
Assuntos
RNA Ribossômico/genética , Proteínas Ribossômicas/genética , Subunidades Ribossômicas Maiores de Eucariotos/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , ATPases Associadas a Diversas Atividades Celulares , Adenosina Trifosfatases/genética , Sequência de Aminoácidos , Cristalografia por Raios X , Escherichia coli/genética , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Ribossômicas/ultraestrutura , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Alinhamento de SequênciaRESUMO
The biogenesis of secretory as well as transmembrane proteins requires the activity of the universally conserved protein-conducting channel (PCC), the Sec61 complex (SecY complex in bacteria). In eukaryotic cells the PCC is located in the membrane of the endoplasmic reticulum where it can bind to translating ribosomes for co-translational protein transport. The Sec complex consists of three subunits (Sec61α, ß and γ) and provides an aqueous environment for the translocation of hydrophilic peptides as well as a lateral opening in the Sec61α subunit that has been proposed to act as a gate for the membrane partitioning of hydrophobic domains. A plug helix and a so-called pore ring are believed to seal the PCC against ion flow and are proposed to rearrange for accommodation of translocating peptides. Several crystal and cryo-electron microscopy structures revealed different conformations of closed and partially open Sec61 and SecY complexes. However, in none of these samples has the translocation state been unambiguously defined biochemically. Here we present cryo-electron microscopy structures of ribosome-bound Sec61 complexes engaged in translocation or membrane insertion of nascent peptides. Our data show that a hydrophilic peptide can translocate through the Sec complex with an essentially closed lateral gate and an only slightly rearranged central channel. Membrane insertion of a hydrophobic domain seems to occur with the Sec complex opening the proposed lateral gate while rearranging the plug to maintain an ion permeability barrier. Taken together, we provide a structural model for the basic activities of the Sec61 complex as a protein-conducting channel.