RESUMO
Protein AMPylation by Fic domain-containing proteins (Fic proteins) is an ancient and conserved post-translational modification of mostly unexplored significance. Here we characterize the Caenorhabditis elegans Fic protein FIC-1 in vitro and in vivo. FIC-1 is an AMPylase that localizes to the nuclear surface and modifies core histones H2 and H3 as well as heat shock protein 70 family members and translation elongation factors. The three-dimensional structure of FIC-1 is similar to that of its human ortholog, HYPE, with 38% sequence identity. We identify a link between FIC-1-mediated AMPylation and susceptibility to the pathogen Pseudomonas aeruginosa, establishing a connection between AMPylation and innate immunity in C. elegans.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Proteínas de Ligação a DNA/genética , Nucleotidiltransferases/genética , Elongação Traducional da Cadeia Peptídica , Proteínas Recombinantes/genética , Animais , Caenorhabditis elegans/química , Caenorhabditis elegans/microbiologia , Proteínas de Transporte/química , Proteínas de Transporte/genética , Cristalografia por Raios X , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Resposta ao Choque Térmico/genética , Histonas/genética , Histonas/metabolismo , Humanos , Imunidade Inata/genética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Conformação Proteica , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidade , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
DEAD-box ATPases play crucial roles in guiding rRNA restructuring events during the biogenesis of large (60S) ribosomal subunits, but their precise molecular functions are currently unknown. In this study, we present cryo-EM reconstructions of nucleolar pre-60S intermediates that reveal an unexpected, alternate secondary structure within the nascent peptidyl-transferase-center (PTC). Our analysis of three sequential nucleolar pre-60S intermediates reveals that the DEAD-box ATPase Dbp10/DDX54 remodels this alternate base pairing and enables the formation of the rRNA junction that anchors the mature form of the universally conserved PTC A-loop. Post-catalysis, Dbp10 captures rRNA helix H61, initiating the concerted exchange of biogenesis factors during late nucleolar 60S maturation. Our findings show that Dbp10 activity is essential for the formation of the ribosome active site and reveal how this function is integrated with subsequent assembly steps to drive the biogenesis of the large ribosomal subunit.
Assuntos
RNA Helicases DEAD-box , Peptidil Transferases , Ribossomos , Proteínas de Saccharomyces cerevisiae , RNA Helicases DEAD-box/genética , Proteínas Ribossômicas/genética , Subunidades Ribossômicas Maiores de Eucariotos/química , Ribossomos/genética , Ribossomos/metabolismo , RNA Ribossômico/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
DEAD-box ATPases play crucial roles in guiding rRNA restructuring events during the biogenesis of large (60S) ribosomal subunits, but their precise molecular functions are currently unknown. In this study, we present cryo-EM reconstructions of nucleolar pre-60S intermediates that reveal an unexpected, alternate secondary structure within the nascent peptidyl-transferase-center (PTC). Our analysis of three sequential nucleolar pre-60S intermediates reveals that the DEAD-box ATPase Dbp10/DDX54 remodels this alternate base pairing and enables the formation of the rRNA junction that anchors the mature form of the universally conserved PTC A-loop. Post-catalysis, Dbp10 captures rRNA helix H61, initiating the concerted exchange of biogenesis factors during late nucleolar 60S maturation. Our findings show that Dbp10 activity is essential for the formation of the ribosome active site and reveal how this function is integrated with subsequent assembly steps to drive the biogenesis of the large ribosomal subunit.
RESUMO
Eukaryotic ribosome biogenesis is facilitated and regulated by numerous ribosome biogenesis factors (RBFs). High-resolution cryoelectron microscopy (cryo-EM) maps have defined the molecular interactions of RBFs during maturation, but many transient and dynamic interactions, particularly during early assembly, remain uncharacterized. Using quantitative proteomics and crosslinking coupled to mass spectrometry (XL-MS) data from an extensive set of pre-ribosomal particles, we derive a comprehensive and time-resolved interaction map of RBF engagement during 60S maturation. We localize 22 previously unmapped RBFs to specific biogenesis intermediates and validate our results by mapping the catalytic activity of the methyltransferases Bmt2 and Rcm1 to their predicted nucleolar 60S intermediates. Our analysis reveals the interaction sites for the RBFs Noc2 and Ecm1 and elucidates the interaction map and timing of 60S engagement by the DEAD-box ATPases Dbp9 and Dbp10. Our data provide a powerful resource for future studies of 60S ribosome biogenesis.
Assuntos
Microscopia Crioeletrônica , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Ribossomos/metabolismo , Nucléolo Celular/metabolismo , Microscopia Crioeletrônica/métodos , Modelos Moleculares , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
The nuclear position in eukaryotes is controlled by a nucleo-cytoskeletal network, critical in cell differentiation, division, and movement. Forces are transmitted through conserved Linker of Nucleoskeleton and Cytoskeleton (LINC) complexes that traverse the nuclear envelope and engage on either side of the membrane with diverse binding partners. Nesprin-2-giant (Nes2G), a LINC element in the outer nuclear membrane, connects to the actin directly as well as through FHOD1, a formin primarily involved in actin bundling. Here, we report the crystal structure of Nes2G bound to FHOD1 and show that the presumed G-binding domain of FHOD1 is rather a spectrin repeat (SR) binding enhancer for the neighboring FH3 domain. The structure reveals that SR binding by FHOD1 is likely not regulated by the diaphanous-autoregulatory domain helix of FHOD1. Finally, we establish that Nes1G also has one FHOD1 binding SR, indicating that these abundant, giant Nesprins have overlapping functions in actin-bundle recruitment for nuclear movement.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Proteínas Fetais/química , Proteínas Fetais/metabolismo , Forminas/química , Forminas/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Motivos de Aminoácidos , Animais , Cristalografia por Raios X , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Células HEK293 , Humanos , Camundongos , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/genética , Modelos Moleculares , Células NIH 3T3 , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Ligação Proteica , Conformação Proteica , Domínios ProteicosRESUMO
Linker of nucleoskeleton and cytoskeleton (LINC) complexes are molecular tethers that span the nuclear envelope (NE) and physically connect the nucleus to the cytoskeleton. They transmit mechanical force across the NE in processes such as nuclear anchorage, nuclear migration, and homologous chromosome pairing during meiosis. LINC complexes are composed of KASH proteins traversing the outer nuclear membrane, and SUN proteins crossing the inner nuclear membrane. Humans have several SUN- and KASH-containing proteins, yet what governs their proper engagement is poorly understood. To investigate this question, we solved high resolution crystal structures of human SUN2 in complex with the KASH-peptides of Nesprin3, Nesprin4, and KASH5. In comparison to the published structures of SUN2-KASH1/2 we observe alternative binding modes for these KASH peptides. While the core interactions between SUN and the C-terminal residues of the KASH peptide are similar in all five complexes, the extended KASH-peptide adopts at least two different conformations. The much-improved resolution allows for a more detailed analysis of other elements critical for KASH interaction, including the KASH-lid and the cation loop, and a possible self-locked state for unbound SUN. In summary, we observe distinct differences between the examined SUN-KASH complexes. These differences may have an important role in regulating the SUN-KASH network.
Assuntos
Proteínas de Ciclo Celular/ultraestrutura , Proteínas de Membrana/ultraestrutura , Proteínas dos Microfilamentos/ultraestrutura , Complexos Multiproteicos/ultraestrutura , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Nucléolo Celular/genética , Nucléolo Celular/ultraestrutura , Pareamento Cromossômico/genética , Cristalografia por Raios X , Citoesqueleto/genética , Citoesqueleto/ultraestrutura , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Meiose/genética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/genética , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Membrana Nuclear/genética , Membrana Nuclear/ultraestrutura , Matriz Nuclear/genética , Matriz Nuclear/ultraestrutura , Peptídeos/química , Peptídeos/genética , Conformação ProteicaRESUMO
Recombinant expression of proteins and their complexes is the routine laboratory procedure to generate pure reagents for biochemical and structural studies. Here we present the standard procedure developed in our lab for the production of milligram quantities of stoichiometric SUN-KASH complexes. The protocol was specifically developed for the purification of the periplasmic portion of LINC complexes.
Assuntos
Proteínas Nucleares/isolamento & purificação , Proteínas Recombinantes/isolamento & purificação , Cromatografia de Afinidade , Humanos , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Periplásmicas/genética , Proteínas Periplásmicas/isolamento & purificação , Proteólise , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Molecular tethers span the nuclear envelope to mechanically connect the cytoskeleton and nucleoskeleton. These bridge-like tethers, termed linkers of nucleoskeleton and cytoskeleton (LINC) complexes, consist of SUN proteins at the inner nuclear membrane and KASH proteins at the outer nuclear membrane. LINC complexes are central to a variety of cell activities including nuclear positioning and mechanotransduction, and LINC-related abnormalities are associated with a spectrum of tissue-specific diseases, termed laminopathies or envelopathies. Protocols used to study the biochemical and structural characteristics of core elements of SUN-KASH complexes are described here to facilitate further studies in this new field of cell biology.
Assuntos
Proteínas de Ciclo Celular/isolamento & purificação , Peptídeos e Proteínas de Sinalização Intracelular/isolamento & purificação , Proteínas de Membrana/isolamento & purificação , Proteínas Nucleares/isolamento & purificação , Sequência de Aminoácidos , Proteínas de Ciclo Celular/química , Cromatografia de Afinidade , Cromatografia em Gel , Cristalografia por Raios X , Escherichia coli , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Proteínas de Membrana/química , Dados de Sequência Molecular , Proteínas Nucleares/química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Estrutura Terciária de ProteínaRESUMO
The Caenorhabditis elegans SUN domain protein, UNC-84, functions in nuclear migration and anchorage in the soma. We discovered a novel role for UNC-84 in DNA damage repair and meiotic recombination. Loss of UNC-84 leads to defects in the loading and disassembly of the recombinase RAD-51. Similar to mutations in Fanconi anemia (FA) genes, unc-84 mutants and human cells depleted of Sun-1 are sensitive to DNA cross-linking agents, and sensitivity is rescued by the inactivation of nonhomologous end joining (NHEJ). UNC-84 also recruits FA nuclease FAN-1 to the nucleoplasm, suggesting that UNC-84 both alters the extent of repair by NHEJ and promotes the processing of cross-links by FAN-1. UNC-84 interacts with the KASH protein ZYG-12 for DNA damage repair. Furthermore, the microtubule network and interaction with the nucleoskeleton are important for repair, suggesting that a functional linker of nucleoskeleton and cytoskeleton (LINC) complex is required. We propose that LINC complexes serve a conserved role in DNA repair through both the inhibition of NHEJ and the promotion of homologous recombination at sites of chromosomal breaks.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Reparo do DNA por Junção de Extremidades , Recombinação Homóloga , Complexos Multiproteicos/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos da radiação , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Núcleo Celular/efeitos da radiação , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Cisplatino/farmacologia , Reagentes de Ligações Cruzadas/metabolismo , Dano ao DNA , Reparo do DNA por Junção de Extremidades/efeitos dos fármacos , Reparo do DNA por Junção de Extremidades/efeitos da radiação , Células Germinativas/citologia , Células Germinativas/efeitos dos fármacos , Células Germinativas/metabolismo , Células Germinativas/efeitos da radiação , Recombinação Homóloga/efeitos dos fármacos , Recombinação Homóloga/efeitos da radiação , Humanos , Hidroxiureia/farmacologia , Meiose/efeitos dos fármacos , Meiose/efeitos da radiação , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Microtúbulos/efeitos da radiação , Modelos Biológicos , Proteínas Nucleares/metabolismo , Polimerização/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/efeitos da radiação , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/efeitos da radiação , Radiação IonizanteRESUMO
El hidradenoma papilífero es una tumoración benigna, rara, generalmente localizada en la región vulvar o perineal siendo la región perianal una localización poco frecuente. Su asociación con enfermedades de transmisión sexual y el carcinoma ductal es controvertida; es importante realizar el estudio histopatológico de cualquier tumoración en esta zona. Se reporta el caso de una paciente de 49 años de edad, con un nódulo en la región perianal de 2 años de evolución; el estudio histopatológico confirmó el diagnóstico de hidradenoma papilífero. (AU)
The papilliferous hidradenoma is a benign, rare tumor, usually located in the vulvar or perineal region and the perianal region is a rare site. Its association with sexually transmitted diseases and ductal carcinoma is controversial. It is important to perform the histopathological study of any tumor in this area. The case of a 49-year-old patient with a nodule in the perianal region of 2 years of evolution is reported. The histopathological study confirmed the diagnosis of papilliferous hidradenoma.
Assuntos
Humanos , Feminino , Adulto , Pessoa de Meia-Idade , Idoso , Adenomas Tubulares de Glândulas Sudoríparas , Neoplasias das Glândulas Anais , Neoplasias , Acrospiroma , Neoplasias Epiteliais e Glandulares , Química AmbientalRESUMO
Sexual dimorphism during mammalian neural development seems to contribute to differential gender-related incidence in malformations of cortical development in both humans and rodents. Here we investigated the existence of differential gender-related susceptibility to learning and memory deficits and brain injury severity in mice submitted to a microgyria model. Newborn male and female C57BL/6 mice (P0) were submitted to a unilateral freezing lesion (FL) using a cooled steel probe, placed over the right midline anteroposterior plane. Mice were allowed to survive for 12-14 weeks and then were submitted to behavioral tasks and brain morphological analyses. Injured mice from both genders did not present gross locomotor alterations, and the freezing lesion resulted in similar brain damage in male and female mice. Additionally, a selective disruption in the short-term social recognition memory was observed in injured male mice while the long-term inhibitory avoidance memory was not affected by both the factors. These results indicate a reduced susceptibility of female to short-term social-memory deficits induced by neonatal model of microgyria in mice, suggesting that the cognitive deficits induced by freezing lesions in rodents may not be entirely related to the severity of brain injury.