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1.
J Biosci ; 452020.
Artigo em Inglês | MEDLINE | ID: mdl-32385219

RESUMO

COVID-19 is an emerging infectious disease that has turned into a pandemic. It spreads through droplet transmission of the new coronavirus SARS-CoV-2. It is an RNA virus displaying a spike protein as the major surface protein with significant sequence similarity to SARS-CoV which causes severe acute respiratory syndrome. The receptor binding domain of the spike protein interacts with the human angiotensin converting enzyme 2 and is considered as the antigenic determinant for stimulating an immune response. While multiple candidate vaccines are currently under different stages of development, there are no known therapeutic interventions at the moment. This review describes the key genetic features that are being considered for generating vaccine candidates by employing innovative technologies. It also highlights the global efforts being undertaken to deliver vaccines for COVID-19 through unprecedented international cooperation and future challenges post development.


Assuntos
Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , Vacinas Virais/imunologia , Animais , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Desenho de Fármacos , Humanos , Pandemias/prevenção & controle , Pneumonia Viral/imunologia , Pneumonia Viral/prevenção & controle , Domínios Proteicos , Subunidades Proteicas , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas de DNA , Vacinas Virais/química
2.
Mol Pharmacol ; 97(5): 336-350, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32111699

RESUMO

Positive allosteric modulators (PAMs) of AMPA receptors boost cognitive performance in preclinical and clinical studies. Their therapeutic window is narrow, however, and clinical application will likely only occur if greater discrimination in activity is achieved. Toward that end, we compared the modulatory activity of two PAMs recently considered as clinical candidates, LY451395 (mibampator) and PF-04958242/BIIB104, on recombinant and native AMPA receptors (AMPARs). We found that the principle molecular determinant that shaped modulatory activity of both PAMs on deactivation (recombinant) and decay (synaptic) of AMPARs was the auxiliary protein incorporated into the receptor complexes. AMPARs containing the stargazin/γ2 transmembrane AMPAR regulatory protein (TARP) were slowed to a >10-fold degree by both PAMs as compared with those incorporating γ8 TARP. Neither subunit composition nor flip/flop splice variation had substantive effect. Similarly, stargazin/γ2-containing mossy fiber EPSCs in cerebellar granule neurons were slowed to a ∼5-fold greater degree than EPSCs in hippocampal CA1 pyramidal cell neurons, which express the γ8 TARP. LY451395 exhibited greater efficacy than BIIB104 at both synapses. These studies provide insight into the receptor constituents that determine efficacy of sulfonamide PAMs. We conclude that compounds that discriminate between AMPARs complexed with distinct TARPs, and particularly those with lower stargazin/γ2 efficacy such as BIIB104, could act as viable procognitive therapeutics. SIGNIFICANCE STATEMENT: Positive allosteric modulators (PAMs) of AMPA receptors enhance cognitive function in a variety of preclinical models. A clearer understanding of the critical determinants of PAM activity could yield critical insight into pathways to maximize their therapeutic index. Here we show that auxiliary proteins for AMPARs play a major, but thus far underappreciated, role in shaping recombinant and neuronal AMPAR modulation by two clinical candidate PAMs. These data will inform both clinical outcomes as well as future rational development of new modulators.


Assuntos
Proteínas de Membrana/metabolismo , Receptores de AMPA/metabolismo , Regulação Alostérica/efeitos dos fármacos , Animais , Compostos de Bifenilo/farmacologia , Cerebelo/metabolismo , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Feminino , Células HEK293 , Hipocampo/metabolismo , Humanos , Masculino , Camundongos Endogâmicos C57BL , Multimerização Proteica/efeitos dos fármacos , Subunidades Proteicas/metabolismo , Pirimidinas/farmacologia , Sulfonamidas/farmacologia , Sinapses/efeitos dos fármacos , Sinapses/metabolismo , Resultado do Tratamento , Triazóis/farmacologia
3.
BMC Med Genet ; 21(1): 51, 2020 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-32171285

RESUMO

INTRODUCTION: The AP4B1 gene encodes a subunit of adaptor protein complex-4 (AP4), a component of intracellular transportation of proteins which plays important roles in neurons. Bi-allelic mutations in AP4B1 cause autosomal recessive spastic paraplegia-47(SPG47). CASE PRESENTATION: Here we present a Chinese patient with spastic tetraplegia, moderate psychomotor development delay and febrile seizures plus. Brain MRIs showed dilated supratentorial ventricle, thin posterior and splenium part of corpus callosum. The patient had little progress through medical treatments and rehabilitating regimens. Whole exome sequencing identified novel compound heterozygous truncating variants c.1207C > T (p.Gln403*) and c.52_53delAC (p.Cys18Glnfs*7) in AP4B1 gene. Causal mutations in AP4B1 have been reported in 29 individuals from 22 families so far, most of which are homozygous mutations. CONCLUSIONS: Our study enriched the genetic and phenotypic spectrum of SPG47. Early discovery, diagnosis and proper treatment on the conditions generally increase chances of improvement on the quality of life for patients.


Assuntos
Proteínas de Ligação a DNA/genética , Transtornos Psicomotores/genética , Quadriplegia/genética , Proteínas de Ligação a RNA/genética , Convulsões Febris/genética , Grupo com Ancestrais do Continente Asiático , Criança , China , Códon sem Sentido , Heterozigoto , Humanos , Masculino , Fenótipo , Subunidades Proteicas/genética , Transtornos Psicomotores/complicações , Quadriplegia/complicações , Convulsões Febris/complicações , Sequenciamento Completo do Exoma
4.
Nature ; 579(7799): 448-451, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32188943

RESUMO

Chromatin-remodelling complexes of the SWI/SNF family function in the formation of nucleosome-depleted, transcriptionally active promoter regions (NDRs)1,2. In the yeast Saccharomyces cerevisiae, the essential SWI/SNF complex RSC3 contains 16 subunits, including the ATP-dependent DNA translocase Sth14,5. RSC removes nucleosomes from promoter regions6,7 and positions the specialized +1 and -1 nucleosomes that flank NDRs8,9. Here we present the cryo-electron microscopy structure of RSC in complex with a nucleosome substrate. The structure reveals that RSC forms five protein modules and suggests key features of the remodelling mechanism. The body module serves as a scaffold for the four flexible modules that we call DNA-interacting, ATPase, arm and actin-related protein (ARP) modules. The DNA-interacting module binds extra-nucleosomal DNA and is involved in the recognition of promoter DNA elements8,10,11 that influence RSC functionality12. The ATPase and arm modules sandwich the nucleosome disc with the Snf2 ATP-coupling (SnAC) domain and the finger helix, respectively. The translocase motor of the ATPase module engages with the edge of the nucleosome at superhelical location +2. The mobile ARP module may modulate translocase-nucleosome interactions to regulate RSC activity5. The RSC-nucleosome structure provides a basis for understanding NDR formation and the structure and function of human SWI/SNF complexes that are frequently mutated in cancer13.


Assuntos
Microscopia Crioeletrônica , Complexos Multiproteicos/química , Complexos Multiproteicos/ultraestrutura , Nucleossomos/metabolismo , Nucleossomos/ultraestrutura , Saccharomyces cerevisiae/química , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Adenosina Trifosfatases/ultraestrutura , Sequência de Aminoácidos , Animais , Transporte Biológico , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/ultraestrutura , Drosophila melanogaster , Humanos , Camundongos , Modelos Moleculares , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas Nucleares/ultraestrutura , Nucleossomos/química , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Xenopus laevis
5.
Nat Struct Mol Biol ; 27(3): 288-296, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32123390

RESUMO

The iota toxin produced by Clostridium perfringens type E is a binary toxin comprising two independent polypeptides: Ia, an ADP-ribosyltransferase, and Ib, which is involved in cell binding and translocation of Ia across the cell membrane. Here we report cryo-EM structures of the translocation channel Ib-pore and its complex with Ia. The high-resolution Ib-pore structure demonstrates a similar structural framework to that of the catalytic ϕ-clamp of the anthrax protective antigen pore. However, the Ia-bound Ib-pore structure shows a unique binding mode of Ia: one Ia binds to the Ib-pore, and the Ia amino-terminal domain forms multiple weak interactions with two additional Ib-pore constriction sites. Furthermore, Ib-binding induces tilting and partial unfolding of the Ia N-terminal α-helix, permitting its extension to the ϕ-clamp gate. This new mechanism of N-terminal unfolding is crucial for protein translocation.


Assuntos
ADP Ribose Transferases/química , Antígenos de Bactérias/química , Toxinas Bacterianas/química , Clostridium perfringens/química , Subunidades Proteicas/química , ADP Ribose Transferases/genética , ADP Ribose Transferases/metabolismo , Sequência de Aminoácidos , Antígenos de Bactérias/genética , Antígenos de Bactérias/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Sítios de Ligação , Clonagem Molecular , Clostridium perfringens/genética , Clostridium perfringens/metabolismo , Clostridium perfringens/patogenicidade , Microscopia Crioeletrônica , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Transporte Proteico , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato
6.
Nat Commun ; 11(1): 610, 2020 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-32001694

RESUMO

NAD(P)H dehydrogenase-like (NDH) complex NDH-1L of cyanobacteria plays a crucial role in cyclic electron flow (CEF) around photosystem I and respiration processes. NDH-1L couples the electron transport from ferredoxin (Fd) to plastoquinone (PQ) and proton pumping from cytoplasm to the lumen that drives the ATP production. NDH-1L-dependent CEF increases the ATP/NADPH ratio, and is therefore pivotal for oxygenic phototrophs to function under stress. Here we report two structures of NDH-1L from Thermosynechococcus elongatus BP-1, in complex with one Fd and an endogenous PQ, respectively. Our structures represent the complete model of cyanobacterial NDH-1L, revealing the binding manner of NDH-1L with Fd and PQ, as well as the structural elements crucial for proper functioning of the NDH-1L complex. Together, our data provides deep insights into the electron transport from Fd to PQ, and its coupling with proton translocation in NDH-1L.


Assuntos
Complexo I de Transporte de Elétrons/química , NADPH Desidrogenase/química , Fotossíntese , Thermus/enzimologia , Sítios de Ligação , Carotenoides/química , Membrana Celular/química , Transporte de Elétrons , Complexo I de Transporte de Elétrons/ultraestrutura , Ferredoxinas/química , Ferredoxinas/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Lipídeos/química , Modelos Moleculares , NADPH Desidrogenase/ultraestrutura , Plastoquinona/química , Plastoquinona/metabolismo , Domínios Proteicos , Subunidades Proteicas/química , Homologia Estrutural de Proteína
7.
PLoS Biol ; 18(2): e3000507, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32092071

RESUMO

The enzyme methyl-coenzyme M reductase (MCR) plays an important role in mediating global levels of methane by catalyzing a reversible reaction that leads to the production or consumption of this potent greenhouse gas in methanogenic and methanotrophic archaea. In methanogenic archaea, the alpha subunit of MCR (McrA) typically contains four to six posttranslationally modified amino acids near the active site. Recent studies have identified enzymes performing two of these modifications (thioglycine and 5-[S]-methylarginine), yet little is known about the formation and function of the remaining posttranslationally modified residues. Here, we provide in vivo evidence that a dedicated S-adenosylmethionine-dependent methyltransferase encoded by a gene we designated methylcysteine modification (mcmA) is responsible for formation of S-methylcysteine in Methanosarcina acetivorans McrA. Phenotypic analysis of mutants incapable of cysteine methylation suggests that the S-methylcysteine residue might play a role in adaption to mesophilic conditions. To examine the interactions between the S-methylcysteine residue and the previously characterized thioglycine, 5-(S)-methylarginine modifications, we generated M. acetivorans mutants lacking the three known modification genes in all possible combinations. Phenotypic analyses revealed complex, physiologically relevant interactions between the modified residues, which alter the thermal stability of MCR in a combinatorial fashion that is not readily predictable from the phenotypes of single mutants. High-resolution crystal structures of inactive MCR lacking the modified amino acids were indistinguishable from the fully modified enzyme, suggesting that interactions between the posttranslationally modified residues do not exert a major influence on the static structure of the enzyme but rather serve to fine-tune the activity and efficiency of MCR.


Assuntos
Aminoácidos/metabolismo , Methanosarcina/enzimologia , Oxirredutases/química , Oxirredutases/metabolismo , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Domínio Catalítico , Methanosarcina/genética , Methanosarcina/crescimento & desenvolvimento , Methanosarcina/metabolismo , Metilação , Metiltransferases/genética , Metiltransferases/metabolismo , Modelos Moleculares , Mutação , Óperon , Oxirredutases/genética , Fenótipo , Processamento de Proteína Pós-Traducional/genética , Subunidades Proteicas , Temperatura
8.
PLoS One ; 15(2): e0228029, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32045419

RESUMO

Influenza A (H1N1) pdm09 virus emerged in North America in 2009 and has been established as a seasonal strain in humans. After an antigenic stasis of about six years, new antigenically distinct variants of the virus emerged globally in 2016 necessitating a change in the vaccine formulation for the first time in 2017. Herein, we analyzed thirty-eight HA sequences of influenza A (H1N1) pdm09 strains isolated in Kenya during 2015-2018 seasons, to evaluate their antigenic and molecular properties based on the HA1 sub-unit. Our analyses revealed that the A (H1N1) pdm09 strains that circulated in Kenya during this period belonged to genetic clade 6B, subclade 6B.1 and 6B.2. The Kenyan 2015 and 2016 isolates differed from the vaccine strain A/California/07/2009 at nine and fourteen antigenic sites in the HA1 respectively. Further, those isolated in 2017 and 2018 correspondingly varied from A/Michigan/45/2015 vaccine strain at three and fifteen antigenic sites. The predicted vaccine efficacy of A/California/07/2009 against Kenyan 2015/2016 was estimated to be 32.4% while A/Michigan/45/2015 showed estimated vaccine efficacies of 39.6% - 41.8% and 32.4% - 42.1% against Kenyan 2017 and 2018 strains, respectively. Hemagglutination-inhibition (HAI) assay using ferret post-infection reference antiserum showed that the titers for the Kenyan 2015/2016 isolates were 2-8-fold lower compared to the vaccine strain. Overall, our results suggest the A (H1N1) pdm09 viruses that circulated in Kenya during 2015/2016 influenza seasons were antigenic variants of the recommended vaccine strains, denoting sub-optimal vaccine efficacy. Additionally, data generated point to a swiftly evolving influenza A (H1N1) pdm09 virus in recent post pandemic era, underscoring the need for sustained surveillance coupled with molecular and antigenic analyses, to inform appropriate and timely influenza vaccine update.


Assuntos
Antígenos Virais/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A Subtipo H1N1/classificação , Vírus da Influenza A Subtipo H1N1/imunologia , Filogenia , Subunidades Proteicas/imunologia , Sequência de Aminoácidos , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Vacinas contra Influenza/imunologia , Quênia , Homologia de Sequência de Aminoácidos , Organização Mundial da Saúde
9.
Cell Physiol Biochem ; 54(2): 211-229, 2020 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-32100973

RESUMO

BACKGROUND/AIMS: Mitochondrial ATP synthase, in addition to being involved in ATP synthesis, is involved in permeability transition pore (PTP) formation, which precedes apoptosis in mammalian cells and programmed cell death in yeast. Mutations in genes encoding ATP synthase subunits cause neuromuscular disorders and have been identified in cancer samples. PTP is also involved in pathology. We previously found that in Saccharomyces cerevisiae, two mutations in ATP synthase subunit a (atp6-P163S and atp6-K90E, equivalent to those detected in prostate and thyroid cancer samples, respectively) in the OM45-GFP background affected ROS and calcium homeostasis and delayed yeast PTP (yPTP) induction upon calcium treatment by modulating the dynamics of ATP synthase dimer/oligomer formation. The Om45 protein is a component of the porin complex, which is equivalent to mammalian VDAC. We aimed to investigate yPTP function in atp6-P163S and atp6-K90E mutants lacking the e and g dimerization subunits of ATP synthase. METHODS: Triple mutants with the atp6-P163S or atp6-K90E mutation, the OM45-GFP gene and deletion of the TIM11 gene encoding subunit e were constructed by crossing and tetrad dissection. In spores capable of growing, the original atp6 mutations reverted to wild type, and two compensatory mutations, namely, atp6-C33S-T215C, were selected. The effects of these mutations on cellular physiology, mitochondrial morphology, bioenergetics and permeability transition (PT) were analyzed by fluorescence and electron microscopy, mitochondrial respiration, ATP synthase activity, calcium retention capacity and swelling assays. RESULTS: The atp6-C33S-T215C mutations in the OM45-GFP background led to delayed growth at elevated temperature on both fermentative and respiratory media and increased sensitivity to high calcium ions concentration or hydrogen peroxide in the medium. The ATP synthase activity was reduced by approximately 50% and mitochondrial network was hyperfused in these cells grown at elevated temperature. The atp6-C33S-T215C stabilized ATP synthase dimers and restored the yPTP properties in Tim11∆ cells. In OM45-GFP cells, in which Tim11 is present, these mutations increased the fraction of swollen mitochondria by up to 85% vs 60% in the wild type, although the time required for calcium release doubled. CONCLUSION: ATP synthase subunit e is essential in the S. cerevisiae atp6-P163S and atp6-K90E mutants. In addition to subunits e and g, subunit a is critical for yPTP induction and conduction. The increased yPTP conduction decrease the S. cerevisiae cell fitness.


Assuntos
Proteínas de Transporte da Membrana Mitocondrial/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Cálcio/metabolismo , Cobre/farmacologia , DNA Mitocondrial/metabolismo , Dimerização , Peróxido de Hidrogênio/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/efeitos dos fármacos , ATPases Mitocondriais Próton-Translocadoras/química , ATPases Mitocondriais Próton-Translocadoras/genética , Mutagênese , Estrutura Quaternária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Temperatura
10.
Zoolog Sci ; 37(1): 7-13, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32068369

RESUMO

The outer dynein arm-docking complex (ODA-DC), which was first identified in the green alga Chlamydomonas reinhardtii, is a protein complex that mediates the binding of axonemal dynein and doublet microtubules. To gain a better understanding of the evolutionary conservation and functional diversity of the ODA-DC, we knocked down a homolog of DC2, a major subunit of the ODA-DC, in the planarian Schmidtea mediterranea. Planaria are carnivorous flatworms that move by beating cilia on their ventral surface against a secreted mucus layer. These organisms have recently been used for cilia research because knockdown of flatworm genes by RNA interference (RNAi) is readily achieved through feeding with double-stranded RNA (dsRNA). Lack of DC2 in S. mediterranea caused several defects in cilia, including low beat frequency, decreased ciliary density, and a reduction in ciliary length. The loss of DC2 function C. reinhardtii mutant oda1 shows slow jerky swimming, but has two flagella of almost normal length. These data suggest that the function of a DC2 homolog in S. mediterranea cilia may be somewhat different from DC2 in C. reinhardtii flagella.


Assuntos
Dineínas do Axonema/metabolismo , Cílios/patologia , Planárias/metabolismo , Sequência de Aminoácidos , Animais , Dineínas do Axonema/genética , Cílios/genética , Cílios/metabolismo , Cílios/ultraestrutura , Flagelos , Microscopia Eletrônica de Transmissão , Movimento , Planárias/genética , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Interferência de RNA
11.
Nature ; 579(7797): 146-151, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32076272

RESUMO

Photosynthetic organisms have developed various light-harvesting systems to adapt to their environments1. Phycobilisomes are large light-harvesting protein complexes found in cyanobacteria and red algae2-4, although how the energies of the chromophores within these complexes are modulated by their environment is unclear. Here we report the cryo-electron microscopy structure of a 14.7-megadalton phycobilisome with a hemiellipsoidal shape from the red alga Porphyridium purpureum. Within this complex we determine the structures of 706 protein subunits, including 528 phycoerythrin, 72 phycocyanin, 46 allophycocyanin and 60 linker proteins. In addition, 1,598 chromophores are resolved comprising 1,430 phycoerythrobilin, 48 phycourobilin and 120 phycocyanobilin molecules. The markedly improved resolution of our structure compared with that of the phycobilisome of Griffithsia pacifica5 enabled us to build an accurate atomic model of the P. purpureum phycobilisome system. The model reveals how the linker proteins affect the microenvironment of the chromophores, and suggests that interactions of the aromatic amino acids of the linker proteins with the chromophores may be a key factor in fine-tuning the energy states of the chromophores to ensure the efficient unidirectional transfer of energy.


Assuntos
Microscopia Crioeletrônica , Transferência de Energia , Ficobilissomas/química , Ficobilissomas/ultraestrutura , Porphyridium/química , Porphyridium/ultraestrutura , Proteínas de Algas/química , Proteínas de Algas/metabolismo , Proteínas de Algas/ultraestrutura , Modelos Moleculares , Fotossíntese , Ficobilinas/química , Ficobilinas/metabolismo , Ficobilissomas/metabolismo , Conformação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Rodófitas/química , Rodófitas/ultraestrutura
12.
Nat Struct Mol Biol ; 27(3): 229-232, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32066962

RESUMO

Maf1 is a conserved inhibitor of RNA polymerase III (Pol III) that influences phenotypes ranging from metabolic efficiency to lifespan. Here, we present a 3.3-Å-resolution cryo-EM structure of yeast Maf1 bound to Pol III, establishing that Maf1 sequesters Pol III elements involved in transcription initiation and binds the mobile C34 winged helix 2 domain, sealing off the active site. The Maf1 binding site overlaps with that of TFIIIB in the preinitiation complex.


Assuntos
RNA Polimerase III/química , Proteínas Repressoras/química , Proteínas de Saccharomyces cerevisiae/química , Fator de Transcrição TFIIIB/química , Fatores de Transcrição/química , Transcrição Genética , Sequência de Aminoácidos , Sítios de Ligação , Clonagem Molecular , Microscopia Crioeletrônica , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RNA Polimerase III/genética , RNA Polimerase III/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Fator de Transcrição TFIIIB/genética , Fator de Transcrição TFIIIB/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
13.
Proc Natl Acad Sci U S A ; 117(2): 872-876, 2020 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-31888984

RESUMO

Virtually all proton-pumping terminal respiratory oxygen reductases are members of the heme-copper oxidoreductase superfamily. Most of these enzymes use reduced cytochrome c as a source of electrons, but a group of enzymes have evolved to directly oxidize membrane-bound quinols, usually menaquinol or ubiquinol. All of the quinol oxidases have an additional transmembrane helix (TM0) in subunit I that is not present in the related cytochrome c oxidases. The current work reports the 3.6-Å-resolution X-ray structure of the cytochrome aa 3 -600 menaquinol oxidase from Bacillus subtilis containing 1 equivalent of menaquinone. The structure shows that TM0 forms part of a cleft to accommodate the menaquinol-7 substrate. Crystals which have been soaked with the quinol-analog inhibitor HQNO (N-oxo-2-heptyl-4-hydroxyquinoline) or 3-iodo-HQNO reveal a single binding site where the inhibitor forms hydrogen bonds to amino acid residues shown previously by spectroscopic methods to interact with the semiquinone state of menaquinone, a catalytic intermediate.


Assuntos
Bacillus subtilis/metabolismo , Cobre/química , Complexo IV da Cadeia de Transporte de Elétrons/química , Heme/química , Hidroquinonas/química , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Grupo dos Citocromos b/química , Transporte de Elétrons , Ligação de Hidrogênio , Modelos Moleculares , Naftóis/metabolismo , Oxirredutases , Conformação Proteica , Subunidades Proteicas/química , Bombas de Próton/química , Bombas de Próton/metabolismo , Terpenos/metabolismo , Vitamina K 2/análogos & derivados , Vitamina K 2/química
14.
Adv Exp Med Biol ; 1217: 9-31, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31898219

RESUMO

Cullin-RING ubiquitin ligases (CRLs) represent the largest superfamily of multi-subunit E3s conserved in all eukaryotes. Soon after the discovery of these important ubiquitin ligase machineries, structural studies have made tremendous contributions to our understanding of their functions. Identification of the key components of CRLs by early studies raised immediate questions as to how these multi-subunit complexes assemble to promote the polyubiquitination of substrates. Specifically, how do the CRL subunits interact with each other to form a versatile E3 platform? How do they recognize specific substrates? How are the CRL-substrate interactions regulated in response to upstream signals? How are the CRL E3s themselves activated and deactivated, and how are substrate receptor subunits of CRLs exchanged in the cell? Even though we might not yet have complete answers to these questions, extensive structural analyses of CRL complexes in the past two decades have begun to unveil the themes and variations of CRL biology. In this chapter we will discuss both classic and emerging structures that help elucidate the overall architecture of CRLs, their substrate recognition modes, and regulatory mechanism of CRLs by NEDD8 modification.


Assuntos
Proteínas Culina/química , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Proteínas Culina/metabolismo , Humanos , Proteína NEDD8/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Especificidade por Substrato , Ubiquitina/metabolismo , Ubiquitinação
15.
Nature ; 577(7792): 695-700, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31969708

RESUMO

Increased cardiac contractility during the fight-or-flight response is caused by ß-adrenergic augmentation of CaV1.2 voltage-gated calcium channels1-4. However, this augmentation persists in transgenic murine hearts expressing mutant CaV1.2 α1C and ß subunits that can no longer be phosphorylated by protein kinase A-an essential downstream mediator of ß-adrenergic signalling-suggesting that non-channel factors are also required. Here we identify the mechanism by which ß-adrenergic agonists stimulate voltage-gated calcium channels. We express α1C or ß2B subunits conjugated to ascorbate peroxidase5 in mouse hearts, and use multiplexed quantitative proteomics6,7 to track hundreds of proteins in the proximity of CaV1.2. We observe that the calcium-channel inhibitor Rad8,9, a monomeric G protein, is enriched in the CaV1.2 microenvironment but is depleted during ß-adrenergic stimulation. Phosphorylation by protein kinase A of specific serine residues on Rad decreases its affinity for ß subunits and relieves constitutive inhibition of CaV1.2, observed as an increase in channel open probability. Expression of Rad or its homologue Rem in HEK293T cells also imparts stimulation of CaV1.3 and CaV2.2 by protein kinase A, revealing an evolutionarily conserved mechanism that confers adrenergic modulation upon voltage-gated calcium channels.


Assuntos
Canais de Cálcio Tipo L/metabolismo , Proteômica , Receptores Adrenérgicos beta/metabolismo , Animais , Canais de Cálcio Tipo L/química , Canais de Cálcio Tipo N/metabolismo , Microambiente Celular , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Feminino , Células HEK293 , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Humanos , Masculino , Camundongos , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Miocárdio/metabolismo , Fosforilação , Domínios Proteicos , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Transdução de Sinais , Proteínas ras/química , Proteínas ras/metabolismo
16.
Nat Commun ; 11(1): 512, 2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31980605

RESUMO

Mechanisms for human sinoatrial node (SAN) dysfunction are poorly understood and whether human SAN excitability requires voltage-gated sodium channels (Nav) remains controversial. Here, we report that neuronal (n)Nav blockade and selective nNav1.6 blockade during high-resolution optical mapping in explanted human hearts depress intranodal SAN conduction, which worsens during autonomic stimulation and overdrive suppression to conduction failure. Partial cardiac (c)Nav blockade further impairs automaticity and intranodal conduction, leading to beat-to-beat variability and reentry. Multiple nNav transcripts are higher in SAN vs atria; heterogeneous alterations of several isoforms, specifically nNav1.6, are associated with heart failure and chronic alcohol consumption. In silico simulations of Nav distributions suggest that INa is essential for SAN conduction, especially in fibrotic failing hearts. Our results reveal that not only cNav but nNav are also integral for preventing disease-induced failure in human SAN intranodal conduction. Disease-impaired nNav may underlie patient-specific SAN dysfunctions and should be considered to treat arrhythmias.


Assuntos
Arritmias Cardíacas/fisiopatologia , Sistema de Condução Cardíaco/fisiopatologia , Neurônios/metabolismo , Nó Sinoatrial/fisiopatologia , Canais de Sódio/metabolismo , Potenciais de Ação/fisiologia , Adulto , Idoso , Alcoolismo/genética , Arritmias Cardíacas/genética , Doença Crônica , Simulação por Computador , Feminino , Átrios do Coração/metabolismo , Átrios do Coração/fisiopatologia , Sistema de Condução Cardíaco/metabolismo , Insuficiência Cardíaca/genética , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Cardiovasculares , Imagem Óptica , Subunidades Proteicas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Nó Sinoatrial/metabolismo , Canais de Sódio/genética , Estresse Fisiológico , Adulto Jovem
17.
Nat Commun ; 11(1): 501, 2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31980633

RESUMO

Centromeres are defined by a self-propagating chromatin structure based on stable inheritance of CENP-A containing nucleosomes. Here, we present a genetic screen coupled to pulse-chase labeling that allow us to identify proteins selectively involved in deposition of nascent CENP-A or in long-term transmission of chromatin-bound CENP-A. These include factors with known roles in DNA replication, repair, chromatin modification, and transcription, revealing a broad set of chromatin regulators that impact on CENP-A dynamics. We further identify the SUMO-protease SENP6 as a key factor, not only controlling CENP-A stability but virtually the entire centromere and kinetochore. Loss of SENP6 results in hyper-SUMOylation of CENP-C and CENP-I but not CENP-A itself. SENP6 activity is required throughout the cell cycle, suggesting that a dynamic SUMO cycle underlies a continuous surveillance of the centromere complex that in turn ensures stable transmission of CENP-A chromatin.


Assuntos
Centrômero/metabolismo , Cromatina/metabolismo , Cisteína Endopeptidases/metabolismo , Testes Genéticos , Biocatálise , Ciclo Celular , Proteína Centromérica A/metabolismo , Genótipo , Células HeLa , Humanos , Cinetocoros/metabolismo , Subunidades Proteicas/metabolismo , Proteólise , Sumoilação
18.
Nat Commun ; 11(1): 477, 2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31980598

RESUMO

Proteins are targeted to the proteasome by the attachment of ubiquitin chains, which are markedly varied in structure. Three proteasome subunits-Rpn10, Rpn13, and Rpn1-can recognize ubiquitin chains. Here we report that proteins with single chains of K48-linked ubiquitin are targeted for degradation almost exclusively through binding to Rpn10. Rpn1 can act as a co-receptor with Rpn10 for K63 chains and for certain other chain types. Differences in targeting do not correlate with chain affinity to receptors. Surprisingly, in steady-state assays Rpn13 retarded degradation of various single-chain substrates. Substrates with multiple short ubiquitin chains can be presented for degradation by any of the known receptors, whereas those targeted to the proteasome through a ubiquitin-like domain are degraded most efficiently when bound by Rpn13 or Rpn1. Thus, the proteasome provides an unexpectedly versatile binding platform that can recognize substrates targeted for degradation by ubiquitin chains differing greatly in length and topology.


Assuntos
Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina/química , Ubiquitina/metabolismo , Sítios de Ligação , Cinética , Modelos Moleculares , Complexo de Endopeptidases do Proteassoma/genética , Subunidades Proteicas , Proteólise , Proteínas de Saccharomyces cerevisiae/genética , Especificidade por Substrato , Ubiquitina/genética
19.
Biochim Biophys Acta Bioenerg ; 1861(3): 148153, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31935361

RESUMO

Complex I is the largest and most intricate redox-driven proton pump of the respiratory chain. The structure of bacterial and mitochondrial complex I has been determined by X-ray crystallography and cryo-EM at increasing resolution. The recent cryo-EM structures of the complex I-like NDH complex and membrane bound hydrogenase open a new and more comprehensive perspective on the complex I superfamily. Functional studies and molecular modeling approaches have greatly advanced our understanding of the catalytic cycle of complex I. However, the molecular mechanism by which energy is extracted from the redox reaction and utilized to drive proton translocation is unresolved and a matter of ongoing debate. Here, we review progress in structure determination and functional characterization of complex I and discuss current mechanistic models.


Assuntos
Complexo I de Transporte de Elétrons/química , Complexo I de Transporte de Elétrons/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Oxirredução , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Prótons , Ubiquinona/química , Ubiquinona/metabolismo
20.
Nature ; 577(7792): 717-720, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31969703

RESUMO

Gene transcription by RNA polymerase II is regulated by activator proteins that recruit the coactivator complexes SAGA (Spt-Ada-Gcn5-acetyltransferase)1,2 and transcription factor IID (TFIID)2-4. SAGA is required for all regulated transcription5 and is conserved among eukaryotes6. SAGA contains four modules7-9: the activator-binding Tra1 module, the core module, the histone acetyltransferase (HAT) module and the histone deubiquitination (DUB) module. Previous studies provided partial structures10-14, but the structure of the central core module is unknown. Here we present the cryo-electron microscopy structure of SAGA from the yeast Saccharomyces cerevisiae and resolve the core module at 3.3 Å resolution. The core module consists of subunits Taf5, Sgf73 and Spt20, and a histone octamer-like fold. The octamer-like fold comprises the heterodimers Taf6-Taf9, Taf10-Spt7 and Taf12-Ada1, and two histone-fold domains in Spt3. Spt3 and the adjacent subunit Spt8 interact with the TATA box-binding protein (TBP)2,7,15-17. The octamer-like fold and its TBP-interacting region are similar in TFIID, whereas Taf5 and the Taf6 HEAT domain adopt distinct conformations. Taf12 and Spt20 form flexible connections to the Tra1 module, whereas Sgf73 tethers the DUB module. Binding of a nucleosome to SAGA displaces the HAT and DUB modules from the core-module surface, allowing the DUB module to bind one face of an ubiquitinated nucleosome.


Assuntos
Microscopia Crioeletrônica , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Saccharomyces cerevisiae , Transativadores/química , Transativadores/ultraestrutura , Transcrição Genética , Regulação Fúngica da Expressão Gênica , Histona Acetiltransferases/química , Histona Acetiltransferases/metabolismo , Histona Acetiltransferases/ultraestrutura , Histonas/metabolismo , Modelos Moleculares , Nucleossomos/química , Nucleossomos/metabolismo , Nucleossomos/ultraestrutura , Ligação Proteica , Domínios Proteicos , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteína de Ligação a TATA-Box/química , Proteína de Ligação a TATA-Box/metabolismo , Transativadores/metabolismo , Fator de Transcrição TFIID/metabolismo , Ubiquitinação
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