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1.
Science ; 373(6552): 306-315, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34437148

RESUMO

Mammalian SWI/SNF (mSWI/SNF) adenosine triphosphate-dependent chromatin remodelers modulate genomic architecture and gene expression and are frequently mutated in disease. However, the specific chromatin features that govern their nucleosome binding and remodeling activities remain unknown. We subjected endogenously purified mSWI/SNF complexes and their constituent assembly modules to a diverse library of DNA-barcoded mononucleosomes, performing more than 25,000 binding and remodeling measurements. Here, we define histone modification-, variant-, and mutation-specific effects, alone and in combination, on mSWI/SNF activities and chromatin interactions. Further, we identify the combinatorial contributions of complex module components, reader domains, and nucleosome engagement properties to the localization of complexes to selectively permissive chromatin states. These findings uncover principles that shape the genomic binding and activity of a major chromatin remodeler complex family.


Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Nucleossomos/metabolismo , Fatores de Transcrição/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas Cromossômicas não Histona/química , Código das Histonas , Histonas/química , Histonas/metabolismo , Humanos , Modelos Moleculares , Complexos Multiproteicos/metabolismo , Mutação , Nucleossomos/química , Ligação Proteica , Domínios Proteicos , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Fatores de Transcrição/química
2.
Science ; 373(6558): 998-1004, 2021 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-34446601

RESUMO

In eukaryotic cells, half of all proteins function as subunits within multiprotein complexes. Imbalanced synthesis of subunits leads to unassembled intermediates that must be degraded to minimize cellular toxicity. Here, we found that excess PSMC5, a subunit of the proteasome base, was targeted for degradation by the HERC1 ubiquitin ligase in mammalian cells. HERC1 identified unassembled PSMC5 by its cognate assembly chaperone PAAF1. Because PAAF1 only dissociates after assembly, HERC1 could also engage later assembly intermediates such as the PSMC4-PSMC5-PAAF1 complex. A missense mutant of HERC1 that causes neurodegeneration in mice was impaired in the recognition and ubiquitination of the PSMC5-PAAF1 complex. Thus, proteasome assembly factors can serve as adaptors for ubiquitin ligases to facilitate elimination of unassembled intermediates and maintain protein homeostasis.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Calmodulina/metabolismo , Humanos , Células MCF-7 , Camundongos , Mutação , Mutação de Sentido Incorreto , Doenças Neurodegenerativas/genética , Mutação Puntual , Domínios e Motivos de Interação entre Proteínas , Subunidades Proteicas/metabolismo , Proteólise , Proteínas Proto-Oncogênicas/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
3.
Science ; 373(6556)2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34385370

RESUMO

The neurotransmitter acetylcholine (ACh) acts in part through a family of nicotinic ACh receptors (nAChRs), which mediate diverse physiological processes including muscle contraction, neurotransmission, and sensory transduction. Pharmacologically, nAChRs are responsible for tobacco addiction and are targeted by medicines for hypertension and dementia. Nicotinic AChRs were the first ion channels to be isolated. Recent studies have identified molecules that control nAChR biogenesis, trafficking, and function. These nAChR accessories include protein and chemical chaperones as well as auxiliary subunits. Whereas some factors act on many nAChRs, others are receptor specific. Discovery of these regulatory mechanisms is transforming nAChR research in cells and tissues ranging from central neurons to spinal ganglia to cochlear hair cells. Nicotinic AChR-specific accessories also enable drug discovery on high-confidence targets for psychiatric, neurological, and auditory disorders.


Assuntos
Chaperonas Moleculares/metabolismo , Neurônios/metabolismo , Proteínas/metabolismo , Receptores Nicotínicos/metabolismo , Animais , Membrana Celular/metabolismo , Descoberta de Drogas , Retículo Endoplasmático/metabolismo , Humanos , Ligantes , Músculo Esquelético/metabolismo , Neurofarmacologia , Nicotina/metabolismo , Subunidades Proteicas/metabolismo , Receptores Nicotínicos/química
4.
Nat Commun ; 12(1): 4835, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34376679

RESUMO

F-ATP synthase is a leading candidate as the mitochondrial permeability transition pore (PTP) but the mechanism(s) leading to channel formation remain undefined. Here, to shed light on the structural requirements for PTP formation, we test cells ablated for g, OSCP and b subunits, and ρ0 cells lacking subunits a and A6L. Δg cells (that also lack subunit e) do not show PTP channel opening in intact cells or patch-clamped mitoplasts unless atractylate is added. Δb and ΔOSCP cells display currents insensitive to cyclosporin A but inhibited by bongkrekate, suggesting that the adenine nucleotide translocator (ANT) can contribute to channel formation in the absence of an assembled F-ATP synthase. Mitoplasts from ρ0 mitochondria display PTP currents indistinguishable from their wild-type counterparts. In this work, we show that peripheral stalk subunits are essential to turn the F-ATP synthase into the PTP and that the ANT provides mitochondria with a distinct permeability pathway.


Assuntos
Cálcio/metabolismo , Mitocôndrias/metabolismo , Poro de Transição de Permeabilidade Mitocondrial/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Cálcio/farmacologia , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Linhagem Celular Tumoral , Células HeLa , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/fisiologia , Mitocôndrias/efeitos dos fármacos , ATPases Mitocondriais Próton-Translocadoras/genética , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Ionóforos de Próton/farmacologia
5.
Zoolog Sci ; 38(4): 305-310, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34342950

RESUMO

To clarify the molecular mechanism of prevention of entry into diapause in Bombyx mori by HCl treatment, we biochemically analyzed calcineurin regulatory B subunit (CNB) in diapause eggs treated with HCl solution. Our previous studies revealed that HCl treatment causes Ca2+ to efflux from diapause eggs. Therefore, we attempted to analyze CNB, which is known to associate with Ca2+. The gene expression level of CNB was increased by HCl treatment and the changes of the gene expression were almost the same as that in the non-diapause eggs. As for diapause eggs, almost no gene expression of CNB was confirmed except just after oviposition. In the assay for phosphorylation by protein kinase CK2, recombinant CNB (rCNB) was phosphorylated in vitro. Additionally, a Ca2+ binding assay indicated that rCNB shows affinity for Ca2+. The distribution of CNB was investigated with an immunohistochemical technique using antiserum against rCNB in diapause eggs and HCl-treated diapause eggs. CNB was localized in serosa cells and yolk cells in both eggs. These data may suggest that CNB is activated by intracellular Ca2+ or efflux Ca2+ resulting from HCl treatment, and that it plays a role in the molecular mechanisms of artificial diapause prevention or the breaking of diapause in the silkworm.


Assuntos
Bombyx/fisiologia , Calcineurina/metabolismo , Diapausa , Proteínas de Insetos/metabolismo , Subunidades Proteicas/metabolismo , Animais , Bombyx/efeitos dos fármacos , Bombyx/genética , Calcineurina/química , Calcineurina/genética , Cálcio/metabolismo , Diapausa/efeitos dos fármacos , Regulação da Expressão Gênica , Ácido Clorídrico/farmacologia , Imuno-Histoquímica , Proteínas de Insetos/química , Proteínas de Insetos/genética , Óvulo/metabolismo , Fosforilação , Análise Serial de Proteínas , Subunidades Proteicas/química , Subunidades Proteicas/genética , Distribuição Tecidual
6.
J Am Chem Soc ; 143(33): 13205-13211, 2021 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-34375093

RESUMO

The receptor binding and proteolysis of Spike of SARS-CoV-2 release its S2 subunit to rearrange and catalyze viral-cell fusion. This deploys the fusion peptide for insertion into the cell membranes targeted. We show that this fusion peptide transforms from intrinsic disorder in solution into a wedge-shaped structure inserted in bilayered micelles, according to chemical shifts, 15N NMR relaxation, and NOEs. The globular fold of three helices contrasts the open, extended forms of this region observed in the electron density of compact prefusion states. In the hydrophobic, narrow end of the wedge, helices 1 and 2 contact the fatty acyl chains of phospholipids, according to NOEs and proximity to a nitroxide spin label deep in the membrane mimic. The polar end of the wedge may engage and displace lipid head groups and bind Ca2+ ions for membrane fusion. Polar helix 3 protrudes from the bilayer where it might be accessible to antibodies.


Assuntos
Micelas , Peptídeos/metabolismo , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/química , COVID-19/patologia , COVID-19/virologia , Humanos , Interações Hidrofóbicas e Hidrofílicas , Peptídeos/química , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , SARS-CoV-2/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/metabolismo
7.
J Am Chem Soc ; 143(33): 12930-12934, 2021 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-34398611

RESUMO

The main protease from SARS-CoV-2 is a homodimer. Yet, a recent 0.1-ms-long molecular dynamics simulation performed by D. E. Shaw's research group shows that it readily undergoes a symmetry-breaking event on passing from the solid state to aqueous solution. As a result, the subunits present distinct conformations of the binding pocket. By analyzing this long simulation, we uncover a previously unrecognized role of water molecules in triggering the transition. Interestingly, each subunit presents a different collection of long-lived water molecules. Enhanced sampling simulations performed here, along with machine learning approaches, further establish that the transition to the asymmetric state is essentially irreversible.


Assuntos
SARS-CoV-2/enzimologia , Proteínas da Matriz Viral/química , Água/química , COVID-19/patologia , COVID-19/virologia , Cristalografia por Raios X , Humanos , Ligação de Hidrogênio , Simulação de Dinâmica Molecular , Estrutura Quaternária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , SARS-CoV-2/isolamento & purificação , Proteínas da Matriz Viral/metabolismo
8.
Microbiol Spectr ; 9(1): e0003021, 2021 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-34346753

RESUMO

Monitoring and strategic response to variants in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) represent a considerable challenge in the current pandemic and for future viral outbreaks. Mutations/deletions of the virion's prefusion Spike protein may have significant impact on vaccines and therapeutics that utilize this key structural protein in their mitigation strategies. In this study, we have demonstrated how dominant energetic landscape mappings ("glue points") based on ab inito all-atom force fields coupled with phylogenetic sequence alignment information can identify key residue mutations and deletions associated with variants. We also found several examples of excellent homology of stabilizing residue glue points across the lineages of betacoronavirus Spike proteins that we have called "sequence homologous glue points." SARS-CoV-2 demonstrates the least number of stabilizing glue points associated with interchain interactions among Down-state protomers across lineages. Additionally, we computationally studied variants among the trimeric Spike protein of SARS-CoV-2 using all-atom molecular dynamics to ascertain structural and energetic changes among variants. We examined both a theoretically based triple mutant and the UK or B.1.1.7 variant. For the theoretical triple mutant, we demonstrated through alanine substitutions that three key residues could cause the transition of Down-to-Up protomer states, where the transition is characterized by the "arm" length of the receptor-binding domain (RBD) rather than the hinge angle. For the B.1.1.7 variant, we demonstrated the critical importance of mutations D614G and N501Y on the structure and binding, respectively, of the Spike protein. We note that these same two key mutations are also found in the South African B.1.351 variant. IMPORTANCE Viral variants represent a major challenge to monitoring viral outbreaks and formulating strategic health care responses. Variants represent transmitting viruses that have specific mutations and deletions associated with their genome. In the case of SARS-CoV-2 and other related viruses (betacoronaviruses), many of these mutations and deletions are associated with the Spike protein that the virus uses to infect cells. Here, we have analyzed both SARS-CoV-2 variants and related viruses, such as Middle Eastern respiratory syndrome coronavirus (MERS-CoV), in order to understand not only differences, but also key similarities between them. Understanding similarities can be as important as differences in determining key functional features of a class of viruses, such as the betacoronaviruses. We have used both phylogenetic analysis, which traces genetic similarities and differences, along with independent biophysics analysis, which adds function or behavior, in order to determine possible functional differences and hence possible transmission and infection differences among variants and lineages.


Assuntos
Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Sequência de Bases , COVID-19/virologia , Humanos , Simulação de Dinâmica Molecular , Mutação , Filogenia , Ligação Proteica , Conformação Proteica , SARS-CoV-2/classificação , Alinhamento de Sequência , Glicoproteína da Espícula de Coronavírus/classificação , Reino Unido
9.
Int J Mol Sci ; 22(16)2021 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-34445747

RESUMO

SARS-CoV-2 primarily infects epithelial airway cells that express the host entry receptor angiotensin-converting enzyme 2 (ACE2), which binds to the S1 spike protein on the surface of the virus. To delineate the impact of S1 spike protein interaction with the ACE2 receptor, we incubated the S1 spike protein with human pulmonary arterial endothelial cells (HPAEC). HPAEC treatment with the S1 spike protein caused disruption of endothelial barrier function, increased levels of numerous inflammatory molecules (VCAM-1, ICAM-1, IL-1ß, CCL5, CXCL10), elevated mitochondrial reactive oxygen species (ROS), and a mild rise in glycolytic reserve capacity. Because low oxygen tension (hypoxia) is associated with severe cases of COVID-19, we also evaluated treatment with hemoglobin (HbA) as a potential countermeasure in hypoxic and normal oxygen environments in analyses with the S1 spike protein. We found hypoxia downregulated the expression of the ACE2 receptor and increased the critical oxygen homeostatic signaling protein, hypoxia-inducible factor (HIF-1α); however, treatment of the cells with HbA yielded no apparent change in the levels of ACE2 or HIF-1α. Use of quantitative proteomics revealed that S1 spike protein-treated cells have few differentially regulated proteins in hypoxic conditions, consistent with the finding that ACE2 serves as the host viral receptor and is reduced in hypoxia. However, in normoxic conditions, we found perturbed abundance of proteins in signaling pathways related to lysosomes, extracellular matrix receptor interaction, focal adhesion, and pyrimidine metabolism. We conclude that the spike protein alone without the rest of the viral components is sufficient to elicit cell signaling in HPAEC, and that treatment with HbA failed to reverse the vast majority of these spike protein-induced changes.


Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , COVID-19/patologia , Células Endoteliais/metabolismo , Hemoglobinas/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , COVID-19/virologia , Hipóxia Celular , Sobrevivência Celular , Células Cultivadas , Células Endoteliais/virologia , Endotélio Vascular/citologia , Endotélio Vascular/patologia , Humanos , Subunidades Proteicas/metabolismo , Artéria Pulmonar/citologia , Artéria Pulmonar/patologia , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes/metabolismo , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidade
10.
Nat Commun ; 12(1): 4754, 2021 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-34362932

RESUMO

Chaperonins are homo- or hetero-oligomeric complexes that use ATP binding and hydrolysis to facilitate protein folding. ATP hydrolysis exhibits both positive and negative cooperativity. The mechanism by which chaperonins coordinate ATP utilization in their multiple subunits remains unclear. Here we use cryoEM to study ATP binding in the homo-oligomeric archaeal chaperonin from Methanococcus maripaludis (MmCpn), consisting of two stacked rings composed of eight identical subunits each. Using a series of image classification steps, we obtained different structural snapshots of individual chaperonins undergoing the nucleotide binding process. We identified nucleotide-bound and free states of individual subunits in each chaperonin, allowing us to determine the ATP occupancy state of each MmCpn particle. We observe distinctive tertiary and quaternary structures reflecting variations in nucleotide occupancy and subunit conformations in each chaperonin complex. Detailed analysis of the nucleotide distribution in each MmCpn complex indicates that individual ATP binding events occur in a statistically random manner for MmCpn, both within and across the rings. Our findings illustrate the power of cryoEM to characterize a biochemical property of multi-subunit ligand binding cooperativity at the individual particle level.


Assuntos
Trifosfato de Adenosina/metabolismo , Microscopia Crioeletrônica , Chaperoninas do Grupo II/química , Chaperoninas do Grupo II/metabolismo , Chaperoninas/metabolismo , Hidrólise , Mathanococcus/metabolismo , Modelos Moleculares , Conformação Proteica , Dobramento de Proteína , Subunidades Proteicas/metabolismo
11.
Int J Mol Sci ; 22(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34360989

RESUMO

Increasing evidence suggests that elderly people with dementia are vulnerable to the development of severe coronavirus disease 2019 (COVID-19). In Alzheimer's disease (AD), the major form of dementia, ß-amyloid (Aß) levels in the blood are increased; however, the impact of elevated Aß levels on the progression of COVID-19 remains largely unknown. Here, our findings demonstrate that Aß1-42, but not Aß1-40, bound to various viral proteins with a preferentially high affinity for the spike protein S1 subunit (S1) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the viral receptor, angiotensin-converting enzyme 2 (ACE2). These bindings were mainly through the C-terminal residues of Aß1-42. Furthermore, Aß1-42 strengthened the binding of the S1 of SARS-CoV-2 to ACE2 and increased the viral entry and production of IL-6 in a SARS-CoV-2 pseudovirus infection model. Intriguingly, data from a surrogate mouse model with intravenous inoculation of Aß1-42 show that the clearance of Aß1-42 in the blood was dampened in the presence of the extracellular domain of the spike protein trimers of SARS-CoV-2, whose effects can be prevented by a novel anti-Aß antibody. In conclusion, these findings suggest that the binding of Aß1-42 to the S1 of SARS-CoV-2 and ACE2 may have a negative impact on the course and severity of SARS-CoV-2 infection. Further investigations are warranted to elucidate the underlying mechanisms and examine whether reducing the level of Aß1-42 in the blood is beneficial to the fight against COVID-19 and AD.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Enzima de Conversão de Angiotensina 2/metabolismo , Fragmentos de Peptídeos/metabolismo , SARS-CoV-2/enzimologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Células A549 , Doença de Alzheimer/complicações , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/química , Animais , COVID-19/complicações , COVID-19/metabolismo , Chlorocebus aethiops , Humanos , Interleucina-6/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fragmentos de Peptídeos/química , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Células Vero , Internalização do Vírus
12.
Nat Commun ; 12(1): 5065, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34417452

RESUMO

The widespread UbiD enzyme family utilises the prFMN cofactor to achieve reversible decarboxylation of acrylic and (hetero)aromatic compounds. The reaction with acrylic compounds based on reversible 1,3-dipolar cycloaddition between substrate and prFMN occurs within the confines of the active site. In contrast, during aromatic acid decarboxylation, substantial rearrangement of the substrate aromatic moiety associated with covalent catalysis presents a molecular dynamic challenge. Here we determine the crystal structures of the multi-subunit vanillic acid decarboxylase VdcCD. We demonstrate that the small VdcD subunit acts as an allosteric activator of the UbiD-like VdcC. Comparison of distinct VdcCD structures reveals domain motion of the prFMN-binding domain directly affects active site architecture. Docking of substrate and prFMN-adduct species reveals active site reorganisation coupled to domain motion supports rearrangement of the substrate aromatic moiety. Together with kinetic solvent viscosity effects, this establishes prFMN covalent catalysis of aromatic (de)carboxylation is afforded by UbiD dynamics.


Assuntos
Carboxiliases/química , Carboxiliases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Biocatálise , Domínio Catalítico , Reação de Cicloadição , Descarboxilação , Mononucleotídeo de Flavina/metabolismo , Cinética , Modelos Moleculares , Oxigênio/farmacologia , Domínios Proteicos , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Solventes , Relação Estrutura-Atividade , Especificidade por Substrato , Viscosidade
13.
Int J Mol Sci ; 22(12)2021 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-34204767

RESUMO

Increased airway wall thickness and remodeling of bronchial mucosa are characteristic of asthma and may arise from altered integrin signaling on airway cells. Here, we analyzed the expression of ß1-subfamily integrins on blood and airway cells (flow cytometry), inflammatory biomarkers in serum and bronchoalveolar lavage, reticular basement membrane (RBM) thickness and collagen deposits in the mucosa (histology), and airway geometry (CT-imaging) in 92 asthma patients (persistent airflow limitation subtype: n = 47) and 36 controls. Persistent airflow limitation was associated with type-2 inflammation, elevated soluble α2 integrin chain, and changes in the bronchial wall geometry. Both subtypes of asthma showed thicker RBM than control, but collagen deposition and epithelial α1 and α2 integrins staining were similar. Type-I collagen accumulation and RBM thickness were inversely related to the epithelial expression of the α2 integrin chain. Expression of α2ß1 integrin on T-cells and eosinophils was not altered in asthma. Collagen I deposits were, however, more abundant in patients with lower α2ß1 integrin on blood and airway CD8+ T-cells. Thicker airway walls in CT were associated with lower α2 integrin chain on blood CD4+ T-cells and airway eosinophils. Our data suggest that α2ß1 integrin on inflammatory and epithelial cells may protect against airway remodeling advancement in asthma.


Assuntos
Asma/metabolismo , Asma/patologia , Progressão da Doença , Integrina alfa2beta1/metabolismo , Pulmão/patologia , Substâncias Protetoras/metabolismo , Adulto , Idoso , Remodelação das Vias Aéreas , Asma/sangue , Asma/imunologia , Membrana Basal/patologia , Brônquios/diagnóstico por imagem , Brônquios/patologia , Brônquios/fisiopatologia , Lavagem Broncoalveolar , Feminino , Humanos , Inflamação/patologia , Pulmão/diagnóstico por imagem , Pulmão/fisiopatologia , Masculino , Pessoa de Meia-Idade , Membrana Mucosa/patologia , Subunidades Proteicas/metabolismo , Ventilação Pulmonar , Solubilidade , Linfócitos T/metabolismo , Tomografia Computadorizada por Raios X
14.
Int J Mol Sci ; 22(12)2021 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-34208315

RESUMO

Onset and progression of Alzheimer's disease (AD) pathophysiology differs between brain regions. The neocortex, for example, is a brain region that is affected very early during AD. NMDA receptors (NMDARs) are involved in mediating amyloid beta (Aß) toxicity. NMDAR expression, on the other hand, can be affected by Aß. We tested whether the high vulnerability of neocortical neurons for Aß-toxicity may result from specific NMDAR expression profiles or from a particular regulation of NMDAR expression by Aß. Electrophysiological analyses suggested that pyramidal cells of 6-months-old wildtype mice express mostly GluN1/GluN2A NMDARs. While synaptic NMDAR-mediated currents are unaltered in 5xFAD mice, extrasynaptic NMDARs seem to contain GluN1/GluN2A and GluN1/GluN2A/GluN2B. We used conditional GluN1 and GluN2B knockout mice to investigate whether NMDARs contribute to Aß-toxicity. Spine number was decreased in pyramidal cells of 5xFAD mice and increased in neurons with 3-week virus-mediated Aß-overexpression. NMDARs were required for both Aß-mediated changes in spine number and functional synapses. Thus, our study gives novel insights into the Aß-mediated regulation of NMDAR expression and the role of NMDARs in Aß pathophysiology in the somatosensory cortex.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Espinhas Dendríticas/metabolismo , Neocórtex/metabolismo , Neurônios/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapses/metabolismo , Doença de Alzheimer , Animais , Potenciais Pós-Sinápticos Excitadores , Camundongos Transgênicos , Subunidades Proteicas/metabolismo , Células Piramidais/metabolismo , Córtex Somatossensorial/metabolismo
15.
J Cell Sci ; 134(13)2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34228795

RESUMO

Neurodevelopmental disorders (NDDs), including intellectual disability (ID), autism and schizophrenia, have high socioeconomic impact, yet poorly understood etiologies. A recent surge of large-scale genome or exome sequencing studies has identified a multitude of mostly de novo mutations in subunits of the protein phosphatase 2A (PP2A) holoenzyme that are strongly associated with NDDs. PP2A is responsible for at least 50% of total Ser/Thr dephosphorylation in most cell types and is predominantly found as trimeric holoenzymes composed of catalytic (C), scaffolding (A) and variable regulatory (B) subunits. PP2A can exist in nearly 100 different subunit combinations in mammalian cells, dictating distinct localizations, substrates and regulatory mechanisms. PP2A is well established as a regulator of cell division, growth, and differentiation, and the roles of PP2A in cancer and various neurodegenerative disorders, such as Alzheimer's disease, have been reviewed in detail. This Review summarizes and discusses recent reports on NDDs associated with mutations of PP2A subunits and PP2A-associated proteins. We also discuss the potential impact of these mutations on the structure and function of the PP2A holoenzymes and the etiology of NDDs.


Assuntos
Deficiência Intelectual , Proteína Fosfatase 2 , Animais , Humanos , Deficiência Intelectual/genética , Mutação , Fosforilação , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , Subunidades Proteicas/metabolismo
16.
Nat Commun ; 12(1): 4621, 2021 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-34330928

RESUMO

Cytochromes bd are ubiquitous amongst prokaryotes including many human-pathogenic bacteria. Such complexes are targets for the development of antimicrobial drugs. However, an understanding of the relationship between the structure and functional mechanisms of these oxidases is incomplete. Here, we have determined the 2.8 Å structure of Mycobacterium smegmatis cytochrome bd by single-particle cryo-electron microscopy. This bd oxidase consists of two subunits CydA and CydB, that adopt a pseudo two-fold symmetrical arrangement. The structural topology of its Q-loop domain, whose function is to bind the substrate, quinol, is significantly different compared to the C-terminal region reported for cytochromes bd from Geobacillus thermodenitrificans (G. th) and Escherichia coli (E. coli). In addition, we have identified two potential oxygen access channels in the structure and shown that similar tunnels also exist in G. th and E. coli cytochromes bd. This study provides insights to develop a framework for the rational design of antituberculosis compounds that block the oxygen access channels of this oxidase.


Assuntos
Proteínas de Bactérias/ultraestrutura , Microscopia Crioeletrônica/métodos , Grupo dos Citocromos b/ultraestrutura , Complexo de Proteínas da Cadeia de Transporte de Elétrons/ultraestrutura , Mycobacterium smegmatis/enzimologia , Oxirredutases/ultraestrutura , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Grupo dos Citocromos b/química , Grupo dos Citocromos b/metabolismo , Transporte de Elétrons , Complexo de Proteínas da Cadeia de Transporte de Elétrons/química , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Heme/química , Heme/metabolismo , Modelos Moleculares , Mycobacterium smegmatis/genética , Oxirredutases/química , Oxirredutases/metabolismo , Oxigênio/metabolismo , Conformação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Especificidade por Substrato
17.
Nucleic Acids Res ; 49(13): 7732-7739, 2021 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-34181731

RESUMO

Bacteriophage ΦKZ (PhiKZ) is the archetype of a family of massive bacterial viruses. It is considered to have therapeutic potential as its host, Pseudomonas aeruginosa, is an opportunistic, intrinsically antibiotic resistant, pathogen that kills tens of thousands worldwide each year. ΦKZ is an incredibly interesting virus, expressing many systems that the host already possesses. On infection, it forms a 'nucleus', erecting a barrier around its genome to exclude host endonucleases and CRISPR-Cas systems. ΦKZ infection is independent of the host transcriptional apparatus. It expresses two different multi-subunit RNA polymerases (RNAPs): the virion RNAP (vRNAP) is injected with the viral DNA during infection to transcribe early genes, including those encoding the non-virion RNAP (nvRNAP), which transcribes all further genes. ΦKZ nvRNAP is formed by four polypeptides thought to represent homologues of the eubacterial ß/ß' subunits, and a fifth with unclear homology, but essential for transcription. We have resolved the structure of ΦKZ nvRNAP to better than 3.0 Å, shedding light on its assembly, homology, and the biological role of the fifth subunit: it is an embedded, integral member of the complex, the position, structural homology and biochemical role of which imply that it has evolved from an ancestral homologue to σ-factor.


Assuntos
RNA Polimerases Dirigidas por DNA/química , Fagos de Pseudomonas/enzimologia , Proteínas Virais/química , Microscopia Crioeletrônica , RNA Polimerases Dirigidas por DNA/metabolismo , Modelos Moleculares , Regiões Promotoras Genéticas , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Proteínas Virais/metabolismo
18.
Development ; 148(11)2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-34129030

RESUMO

We describe a previously unreported macroscopic Arabidopsis organ, the cantil, named for its 'cantilever' function of holding the pedicel at a distance from the stem. Cantil development is strongest at the first nodes after the vegetative to reproductive inflorescence transition; cantil magnitude and frequency decrease acropetally. Cantils develop in wild-type Arabidopsis accessions (e.g. Col-0, Ws and Di-G) as a consequence of delayed flowering in short days; cantil formation is observed in long days when flowering is delayed by null mutation of the floral regulator FLOWERING LOCUS T. The receptor-like kinase ERECTA is a global positive regulator of cantil formation; therefore, cantils never form in the Arabidopsis strain Ler. ERECTA functions genetically upstream of heterotrimeric G proteins. Cantil expressivity is repressed by the specific heterotrimeric complex subunits GPA1, AGB1 and AGG3, which also play independent roles: GPA1 suppresses distal spurs at cantil termini, while AGB1 and AGG3 suppress ectopic epidermal rippling. These G protein mutant traits are recapitulated in long-day flowering gpa1-3 ft-10 plants, demonstrating that cantils, spurs and ectopic rippling occur as a function of delayed phase transition, rather than as a function of photoperiod per se.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Benzilatos/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Piperidinas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Flores/genética , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/genética , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas Heterotriméricas de Ligação ao GTP/genética , Mutação com Perda de Função , Fenótipo , Fotoperíodo , Plantas Geneticamente Modificadas/metabolismo , Subunidades Proteicas/metabolismo , Proteínas Serina-Treonina Quinases/genética , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo
19.
Int J Mol Sci ; 22(9)2021 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-34063002

RESUMO

Cone Dystrophy with Supernormal Rod Response (CDSRR) is a rare autosomal recessive disorder leading to severe visual impairment in humans, but little is known about its unique pathophysiology. We have previously shown that CDSRR is caused by mutations in the KCNV2 (Potassium Voltage-Gated Channel Modifier Subfamily V Member 2) gene encoding the Kv8.2 subunit, a modulatory subunit of voltage-gated potassium (Kv) channels. In a recent study, we validated a novel mouse model of Kv8.2 deficiency at a late stage of the disease and showed that it replicates the human electroretinogram (ERG) phenotype. In this current study, we focused our investigation on young adult retinas to look for early markers of disease and evaluate their effect on retinal morphology, electrophysiology and immune response in both the Kv8.2 knockout (KO) mouse and in the Kv2.1 KO mouse, the obligate partner of Kv8.2 in functional retinal Kv channels. By evaluating the severity of retinal dystrophy in these KO models, we demonstrated that retinas of Kv KO mice have significantly higher apoptotic cells, a thinner outer nuclear cell layer and increased activated microglia cells in the subretinal space. Our results indicate that in the murine retina, the loss of Kv8.2 subunits contributes to early cellular and physiological changes leading to retinal dysfunction. These results could have potential implications in the early management of CDSRR despite its relatively nonprogressive nature in humans.


Assuntos
Envelhecimento/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Subunidades Proteicas/metabolismo , Retina/citologia , Retina/metabolismo , Canais de Potássio Shab/metabolismo , Animais , Morte Celular , Eletrorretinografia , Gliose/patologia , Imunidade , Camundongos Knockout , Microglia/patologia , Visão Noturna , Retina/fisiologia
20.
Methods Enzymol ; 653: 377-404, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34099180

RESUMO

Most membrane proteins, and ion channels in particular, assemble to multimeric biological complexes. This starts with the quarternary structure and continues with the recruitment of auxiliary subunits and oligomerization or clustering of the complexes. While the quarternary structure is best determined by atomic-scale structures, stoichiometry of heteromers and dynamic changes in the assembly cannot necessarily be investigated with structural methods. Here, single subunit counting has proven a powerful method to study the composition of these complexes. Single subunit counting uses the irreversible photodestruction of fluorescent tags as means to directly count a labeled subunit and thereby derive the composition of the assemblies. In this chapter, we discuss single subunit counting and its limitations. We present alternative methods and provide a detailed protocol for recording and analysis of single subunit counting data.


Assuntos
Canais Iônicos , Subunidades Proteicas/metabolismo
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