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1.
Int J Mol Sci ; 22(17)2021 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-34502139

RESUMO

SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) is the causative agent of the COVID19 pandemic. The SARS-CoV-2 genome encodes for a small accessory protein termed Orf9b, which targets the mitochondrial outer membrane protein TOM70 in infected cells. TOM70 is involved in a signaling cascade that ultimately leads to the induction of type I interferons (IFN-I). This cascade depends on the recruitment of Hsp90-bound proteins to the N-terminal domain of TOM70. Binding of Orf9b to TOM70 decreases the expression of IFN-I; however, the underlying mechanism remains elusive. We show that the binding of Orf9b to TOM70 inhibits the recruitment of Hsp90 and chaperone-associated proteins. We characterized the binding site of Orf9b within the C-terminal domain of TOM70 and found that a serine in position 53 of Orf9b and a glutamate in position 477 of TOM70 are crucial for the association of both proteins. A phosphomimetic variant Orf9bS53E showed drastically reduced binding to TOM70 and did not inhibit Hsp90 recruitment, suggesting that Orf9b-TOM70 complex formation is regulated by phosphorylation. Eventually, we identified the N-terminal TPR domain of TOM70 as a second binding site for Orf9b, which indicates a so far unobserved contribution of chaperones in the mitochondrial targeting of the viral protein.


Assuntos
COVID-19/transmissão , Proteínas do Nucleocapsídeo de Coronavírus/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , SARS-CoV-2/patogenicidade , Animais , Sítios de Ligação/genética , COVID-19/imunologia , COVID-19/virologia , Chlorocebus aethiops , Proteínas do Nucleocapsídeo de Coronavírus/genética , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , Proteínas do Nucleocapsídeo de Coronavírus/isolamento & purificação , Humanos , Interferon Tipo I/imunologia , Interferon Tipo I/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/isolamento & purificação , Mutação , Fosfoproteínas/genética , Fosfoproteínas/imunologia , Fosfoproteínas/isolamento & purificação , Fosfoproteínas/metabolismo , Fosforilação , Ligação Proteica/genética , Ligação Proteica/imunologia , Domínios Proteicos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Células Vero
2.
Nat Commun ; 12(1): 5215, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34471122

RESUMO

Achieving sufficient worldwide vaccination coverage against SARS-CoV-2 will require additional approaches to currently approved viral vector and mRNA vaccines. Subunit vaccines may have distinct advantages when immunizing vulnerable individuals, children and pregnant women. Here, we present a new generation of subunit vaccines targeting viral antigens to CD40-expressing antigen-presenting cells. We demonstrate that targeting the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein to CD40 (αCD40.RBD) induces significant levels of specific T and B cells, with long-term memory phenotypes, in a humanized mouse model. Additionally, we demonstrate that a single dose of the αCD40.RBD vaccine, injected without adjuvant, is sufficient to boost a rapid increase in neutralizing antibodies in convalescent non-human primates (NHPs) exposed six months previously to SARS-CoV-2. Vaccine-elicited antibodies cross-neutralize different SARS-CoV-2 variants, including D614G, B1.1.7 and to a lesser extent B1.351. Such vaccination significantly improves protection against a new high-dose virulent challenge versus that in non-vaccinated convalescent animals.


Assuntos
Antígenos CD40/imunologia , Vacinas contra COVID-19/imunologia , COVID-19/prevenção & controle , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Linfócitos B/imunologia , Convalescença , Humanos , Macaca , Camundongos , Mutação , Domínios Proteicos , Reinfecção/prevenção & controle , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Linfócitos T/imunologia , Vacinação , Vacinas de Subunidades/imunologia
3.
Nat Commun ; 12(1): 5107, 2021 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-34429425

RESUMO

The ability to design a protein to bind specifically to a target RNA enables numerous applications, with the modular architecture of the PUF domain lending itself to new RNA-binding specificities. For each repeat of the Pumilio-1 PUF domain, we generate a library that contains the 8,000 possible combinations of amino acid substitutions at residues critical for RNA contact. We carry out yeast three-hybrid selections with each library against the RNA recognition sequence for Pumilio-1, with any possible base present at the position recognized by the randomized repeat. We use sequencing to score the binding of each variant, identifying many variants with highly repeat-specific interactions. From these data, we generate an RNA binding code specific to each repeat and base. We use this code to design PUF domains against 16 RNAs, and find that some of these domains recognize RNAs with two, three or four changes from the wild type sequence.


Assuntos
Ligação Proteica , Domínios Proteicos , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Modelos Moleculares , Conformação Proteica , RNA/química , Motivos de Ligação ao RNA , Proteínas de Ligação a RNA/química , Saccharomyces cerevisiae/metabolismo
4.
Int J Mol Sci ; 22(16)2021 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-34445220

RESUMO

Recently, the quest for the mythical fountain of youth has produced extensive research programs that aim to extend the healthy lifespan of humans. Despite advances in our understanding of the aging process, the surprisingly extended lifespan and cancer resistance of some animal species remain unexplained. The p53 protein plays a crucial role in tumor suppression, tissue homeostasis, and aging. Long-lived, cancer-free African elephants have 20 copies of the TP53 gene, including 19 retrogenes (38 alleles), which are partially active, whereas humans possess only one copy of TP53 and have an estimated cancer mortality rate of 11-25%. The mechanism through which p53 contributes to the resolution of the Peto's paradox in Animalia remains vague. Thus, in this work, we took advantage of the available datasets and inspected the p53 amino acid sequence of phylogenetically related organisms that show variations in their lifespans. We discovered new correlations between specific amino acid deviations in p53 and the lifespans across different animal species. We found that species with extended lifespans have certain characteristic amino acid substitutions in the p53 DNA-binding domain that alter its function, as depicted from the Phenotypic Annotation of p53 Mutations, using the PROVEAN tool or SWISS-MODEL workflow. In addition, the loop 2 region of the human p53 DNA-binding domain was identified as the longest region that was associated with longevity. The 3D model revealed variations in the loop 2 structure in long-lived species when compared with human p53. Our findings show a direct association between specific amino acid residues in p53 protein, changes in p53 functionality, and the extended animal lifespan, and further highlight the importance of p53 protein in aging.


Assuntos
Bases de Dados Genéticas , Dosagem de Genes , Longevidade , Modelos Moleculares , Animais , Domínios Proteicos , Estrutura Secundária de Proteína , Especificidade da Espécie , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
5.
Int J Mol Sci ; 22(16)2021 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-34445230

RESUMO

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is one of the best studied enzymes. It is crucial for photosynthesis, and thus for all of biosphere's productivity. There are four isoforms of this enzyme, differing by amino acid sequence composition and quaternary structure. However, there is still a group of organisms, dinoflagellates, single-cell eukaryotes, that are confirmed to possess Rubisco, but no successful purification of the enzyme of such origin, and hence a generation of a crystal structure was reported to date. Here, we are using in silico tools to generate the possible structure of Rubisco from a dinoflagellate representative, Symbiodinium sp. We selected two templates: Rubisco from Rhodospirillum rubrum and Rhodopseudomonas palustris. Both enzymes are the so-called form II Rubiscos, but the first is exclusively a homodimer, while the second one forms homo-hexamers. Obtained models show no differences in amino acids crucial for Rubisco activity. The variation was found at two closely located inserts in the C-terminal domain, of which one extends a helix and the other forms a loop. These inserts most probably do not play a direct role in the enzyme's activity, but may be responsible for interaction with an unknown protein partner, possibly a regulator or a chaperone. Analysis of the possible oligomerization interface indicated that Symbiodinium sp. Rubisco most likely forms a trimer of homodimers, not just a homodimer. This hypothesis was empowered by calculation of binding energies. Additionally, we found that the protein of study is significantly richer in cysteine residues, which may be the cause for its activity loss shortly after cell lysis. Furthermore, we evaluated the influence of the loop insert, identified exclusively in the Symbiodinium sp. protein, on the functionality of the recombinantly expressed R. rubrum Rubisco. All these findings shed new light onto dinoflagellate Rubisco and may help in future obtainment of a native, active enzyme.


Assuntos
Multimerização Proteica , Rhodospirillum rubrum/enzimologia , Ribulose-Bifosfato Carboxilase/química , Domínios Proteicos , Rhodospirillum rubrum/genética , Ribulose-Bifosfato Carboxilase/genética
6.
Int J Mol Sci ; 22(16)2021 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-34445396

RESUMO

Dicers are multidomain proteins, usually comprising an amino-terminal putative helicase domain, a DUF283 domain (domain of unknown function), a PAZ domain, two RNase III domains (RNase IIIa and RNase IIIb) and a dsRNA-binding domain. Dicer homologs play an important role in the biogenesis of small regulatory RNAs by cleaving single-stranded precursors adopting stem-loop structures (pre-miRNAs) and double-strand RNAs into short RNA duplexes containing functional microRNAs or small interfering RNAs, respectively. Growing evidence shows that apart from the canonical role, Dicer proteins can serve a number of other functions. For example, results of our previous studies showed that human Dicer (hDicer), presumably through its DUF283 domain, can facilitate hybridization between two complementary RNAs, thus, acting as a nucleic acid annealer. Here, to test this assumption, we prepared a hDicer deletion variant lacking the amino acid residues 625-752 corresponding to the DUF283 domain. The respective 128-amino acid fragment of hDicer was earlier demonstrated to accelerate base-pairing between two complementary RNAs in vitro. We show that the ΔDUF(625-752) hDicer variant loses the potential to facilitate RNA-RNA base pairing, which strongly proves our hypothesis about the importance of the DUF283 domain for the RNA-RNA annealing activity of hDicer. Interestingly, the in vitro biochemical characterization of the obtained deletion variant reveals that it displays different RNA cleavage properties depending on the pre-miRNA substrate.


Assuntos
RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/metabolismo , RNA/metabolismo , Ribonuclease III/química , Ribonuclease III/metabolismo , Deleção de Sequência , Pareamento de Bases , RNA Helicases DEAD-box/genética , Células HEK293 , Humanos , Modelos Moleculares , Conformação de Ácido Nucleico , Conformação Proteica , Domínios Proteicos , RNA/química , Ribonuclease III/genética
7.
Int J Mol Sci ; 22(16)2021 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-34445398

RESUMO

Gibberellins (GAs) are an important group of phytohormones associated with diverse growth and developmental processes, including cell elongation, seed germination, and secondary growth. Recent genomic and genetic analyses have advanced our knowledge of GA signaling pathways and related genes in model plant species. However, functional genomics analyses of GA signaling pathways in Panax ginseng, a perennial herb, have rarely been carried out, despite its well-known economical and medicinal importance. Here, we conducted functional characterization of GA receptors and investigated their physiological roles in the secondary growth of P. ginseng storage roots. We found that the physiological and genetic functions of P. ginseng gibberellin-insensitive dwarf1s (PgGID1s) have been evolutionarily conserved. Additionally, the essential domains and residues in the primary protein structure for interaction with active GAs and DELLA proteins are well-conserved. Overexpression of PgGID1s in Arabidopsis completely restored the GA deficient phenotype of the Arabidopsis gid1a gid1c (atgid1a/c) double mutant. Exogenous GA treatment greatly enhanced the secondary growth of tap roots; however, paclobutrazol (PCZ), a GA biosynthetic inhibitor, reduced root growth in P. ginseng. Transcriptome profiling of P. ginseng roots revealed that GA-induced root secondary growth is closely associated with cell wall biogenesis, the cell cycle, the jasmonic acid (JA) response, and nitrate assimilation, suggesting that a transcriptional network regulate root secondary growth in P. ginseng. These results provide novel insights into the mechanism controlling secondary root growth in P. ginseng.


Assuntos
Perfilação da Expressão Gênica/métodos , Giberelinas/farmacologia , Panax/crescimento & desenvolvimento , Receptores de Superfície Celular/genética , Evolução Molecular , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Mutação com Perda de Função , Panax/genética , Proteínas de Plantas/genética , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Domínios Proteicos , Receptores de Superfície Celular/química , Análise de Sequência de RNA , Transdução de Sinais/efeitos dos fármacos , Triazóis/farmacologia
8.
mBio ; 12(4): e0159021, 2021 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-34340537

RESUMO

Selective pressures drive adaptive changes in the coronavirus spike proteins directing virus-cell entry. These changes are concentrated in the amino-terminal domains (NTDs) and the receptor-binding domains (RBDs) of complex modular spike protein trimers. The impact of this hypervariability on virus entry is often unclear, particularly with respect to sarbecovirus NTD variations. Therefore, we constructed indels and substitutions within hypervariable NTD regions and used severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus-like particles and quantitative virus-cell entry assays to elucidate spike structures controlling this initial infection stage. We identified NTD variations that increased SARS-CoV-2 spike protein-mediated membrane fusion and cell entry. Increased cell entry correlated with greater presentation of RBDs to ACE2 receptors. This revealed a significant allosteric effect, in that changes within the NTDs can orient RBDs for effective virus-cell binding. Yet, those NTD changes elevating receptor binding and membrane fusion also reduced interdomain associations, leaving spikes on virus-like particles susceptible to irreversible inactivation. These findings parallel those obtained decades ago, in which comparisons of murine coronavirus spike protein variants established inverse relationships between membrane fusion potential and virus stability. Considerable hypervariability in the SARS-CoV-2 spike protein NTDs also appear to be driven by counterbalancing pressures for effective virus-cell entry and durable extracellular virus infectivity. These forces may selectively amplify SARS-CoV-2 variants of concern. IMPORTANCE Adaptive changes that increase SARS-CoV-2 transmissibility may expand and prolong the coronavirus disease 2019 (COVID-19) pandemic. Transmission requires metastable and dynamic spike proteins that bind viruses to cells and catalyze virus-cell membrane fusion. Using newly developed assays reflecting these two essential steps in virus-cell entry, we focused on adaptive changes in SARS-CoV-2 spike proteins and found that deletions in amino-terminal domains reset spike protein metastability, rendering viruses less stable yet more poised to respond to cellular factors that prompt entry and subsequent infection. The results identify adjustable control features that balance extracellular virus stability with facile virus dynamics during cell entry. These equilibrating elements warrant attention when monitoring the evolution of pandemic coronaviruses.


Assuntos
COVID-19/transmissão , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Internalização do Vírus , Enzima de Conversão de Angiotensina 2/metabolismo , COVID-19/patologia , Linhagem Celular Tumoral , Células HEK293 , Células HeLa , Humanos , Fusão de Membrana/fisiologia , Domínios Proteicos/fisiologia , Receptores Virais/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo
9.
J Mol Graph Model ; 108: 107997, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34343818

RESUMO

One of the most important challenges in the battle against contagious SARS-CoV-2 is subtle identification of the virus pathogenesis. The broad range of COVID-19 clinical manifestations may indicate diversity of virus-host cells. Amongst key manifestations, especially in severe COVID-19 patients, reduction and/or exhaustion of lymphocytes, monocytes, basophils, and dendritic cells are seen.; therefore, it is required to recognize that how the virus infects the cells. Interestingly, angiotensin-converting enzyme 2 (ACE2) as the well-known receptor of SARS-CoV-2 is low or non-expressed in these cells. Using computational approach, several receptor candidates including leukocyte surface molecules and chemokine receptors that expressed in most lineages of immune cells were evaluated as the feasible receptor of spike receptor-binding domain (RBD) of SARS-CoV-2. The results revealed the higher binding affinity of CD26, CD2, CD56, CD7, CCR9, CD150, CD4, CD50, XCR1 and CD106 compared to ACE2. However, the modes of binding and amino acids involved in the interactions with the RBD domain of spike were various. Overall, the affinity of immune receptor candidates in binding to SARS-CoV-2 RBD may offer insight into the recognition of novel therapeutic targets in association with COVID-19.


Assuntos
COVID-19 , Glicoproteína da Espícula de Coronavírus , Sítios de Ligação , Humanos , Ligação Proteica , Domínios Proteicos , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/metabolismo
10.
Nucleic Acids Res ; 49(15): 8822-8835, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34352100

RESUMO

The catalytic subunit of SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) contains two active sites that catalyze nucleotidyl-monophosphate transfer (NMPylation). Mechanistic studies and drug discovery have focused on RNA synthesis by the highly conserved RdRp. The second active site, which resides in a Nidovirus RdRp-Associated Nucleotidyl transferase (NiRAN) domain, is poorly characterized, but both catalytic reactions are essential for viral replication. One study showed that NiRAN transfers NMP to the first residue of RNA-binding protein nsp9; another reported a structure of nsp9 containing two additional N-terminal residues bound to the NiRAN active site but observed NMP transfer to RNA instead. We show that SARS-CoV-2 RdRp NMPylates the native but not the extended nsp9. Substitutions of the invariant NiRAN residues abolish NMPylation, whereas substitution of a catalytic RdRp Asp residue does not. NMPylation can utilize diverse nucleotide triphosphates, including remdesivir triphosphate, is reversible in the presence of pyrophosphate, and is inhibited by nucleotide analogs and bisphosphonates, suggesting a path for rational design of NiRAN inhibitors. We reconcile these and existing findings using a new model in which nsp9 remodels both active sites to alternately support initiation of RNA synthesis by RdRp or subsequent capping of the product RNA by the NiRAN domain.


Assuntos
Nidovirales/enzimologia , Nucleotídeos/metabolismo , Domínios Proteicos , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/metabolismo , SARS-CoV-2/enzimologia , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Coenzimas/metabolismo , RNA-Polimerase RNA-Dependente de Coronavírus/metabolismo , Difosfatos/farmacologia , Difosfonatos/farmacologia , Guanosina Trifosfato/metabolismo , Manganês , Modelos Moleculares , Nidovirales/química , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Uridina Trifosfato/metabolismo
11.
Int J Mol Sci ; 22(15)2021 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-34360882

RESUMO

The human natural killer (HNK-1) carbohydrate plays important roles during nervous system development, regeneration after trauma and synaptic plasticity. Four proteins have been identified as receptors for HNK-1: the laminin adhesion molecule, high-mobility group box 1 and 2 (also called amphoterin) and cadherin 2 (also called N-cadherin). Because of HNK-1's importance, we asked whether additional receptors for HNK-1 exist and whether the four identified proteins share any similarity in their primary structures. A set of 40,000 sequences homologous to the known HNK-1 receptors was selected and used for large-scale sequence alignments and motif searches. Although there are conserved regions and highly conserved sites within each of these protein families, there was no sequence similarity or conserved sequence motifs found to be shared by all families. Since HNK-1 receptors have not been compared regarding binding constants and since it is not known whether the sulfated or non-sulfated part of HKN-1 represents the structurally crucial ligand, the receptors are more heterogeneous in primary structure than anticipated, possibly involving different receptor or ligand regions. We thus conclude that the primary protein structure may not be the sole determinant for a bona fide HNK-1 receptor, rendering receptor structure more complex than originally assumed.


Assuntos
Antígenos CD57/metabolismo , Caderinas/metabolismo , Proteína HMGB1/metabolismo , Proteína HMGB2/metabolismo , Laminina/metabolismo , Oligossacarídeos/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Antígenos CD57/química , Caderinas/química , Proteína HMGB1/química , Proteína HMGB2/química , Humanos , Laminina/química , Ligantes , Regeneração Nervosa/fisiologia , Plasticidade Neuronal/fisiologia , Oligossacarídeos/química , Ligação Proteica , Domínios Proteicos
12.
Int J Mol Sci ; 22(15)2021 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-34360888

RESUMO

Osteoarthritis (OA) is a degenerative joint disease characterized by irreversible cartilage damage, inflammation and altered chondrocyte phenotype. Transforming growth factor-ß (TGF-ß) signaling via SMAD2/3 is crucial for blocking hypertrophy. The post-translational modifications of these SMAD proteins in the linker domain regulate their function and these can be triggered by inflammation through the activation of kinases or phosphatases. Therefore, we investigated if OA-related inflammation affects TGF-ß signaling via SMAD2/3 linker-modifications in chondrocytes. We found that both Interleukin (IL)-1ß and OA-synovium conditioned medium negated SMAD2/3 transcriptional activity in chondrocytes. This inhibition of TGF-ß signaling was enhanced if SMAD3 could not be phosphorylated on Ser213 in the linker region and the inhibition by IL-1ß was less if the SMAD3 linker could not be phosphorylated at Ser204. Our study shows evidence that inflammation inhibits SMAD2/3 signaling in chondrocytes via SMAD linker (de)-phosphorylation. The involvement of linker region modifications may represent a new therapeutic target for OA.


Assuntos
Condrócitos/metabolismo , Condrócitos/patologia , Osteoartrite/metabolismo , Transdução de Sinais/genética , Proteína Smad2/química , Proteína Smad2/metabolismo , Proteína Smad3/química , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Adulto , Animais , Bovinos , Linhagem Celular Tumoral , Humanos , Hipertrofia/metabolismo , Inflamação/metabolismo , Interleucina-1beta/farmacologia , Osteoartrite/genética , Osteoartrite/patologia , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Domínios Proteicos/efeitos dos fármacos , Proteínas Recombinantes/farmacologia , Transdução de Sinais/efeitos dos fármacos , Proteína Smad2/genética , Proteína Smad3/genética , Membrana Sinovial/metabolismo , Transfecção , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/farmacologia
13.
Proc Natl Acad Sci U S A ; 118(34)2021 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-34373319

RESUMO

Atomic structures of several proteins from the coronavirus family are still partial or unavailable. A possible reason for this gap is the instability of these proteins outside of the cellular context, thereby prompting the use of in-cell approaches. In situ cross-linking and mass spectrometry (in situ CLMS) can provide information on the structures of such proteins as they occur in the intact cell. Here, we applied targeted in situ CLMS to structurally probe Nsp1, Nsp2, and nucleocapsid (N) proteins from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and obtained cross-link sets with an average density of one cross-link per 20 residues. We then employed integrative modeling that computationally combined the cross-linking data with domain structures to determine full-length atomic models. For the Nsp2, the cross-links report on a complex topology with long-range interactions. Integrative modeling with structural prediction of individual domains by the AlphaFold2 system allowed us to generate a single consistent all-atom model of the full-length Nsp2. The model reveals three putative metal binding sites and suggests a role for Nsp2 in zinc regulation within the replication-transcription complex. For the N protein, we identified multiple intra- and interdomain cross-links. Our integrative model of the N dimer demonstrates that it can accommodate three single RNA strands simultaneously, both stereochemically and electrostatically. For the Nsp1, cross-links with the 40S ribosome were highly consistent with recent cryogenic electron microscopy structures. These results highlight the importance of cellular context for the structural probing of recalcitrant proteins and demonstrate the effectiveness of targeted in situ CLMS and integrative modeling.


Assuntos
Modelos Moleculares , SARS-CoV-2/química , Proteínas Virais/química , Reagentes para Ligações Cruzadas/química , Células HEK293 , Humanos , Espectrometria de Massas , Domínios Proteicos
14.
Cells ; 10(7)2021 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-34359983

RESUMO

Recently emerged severe acute respiratory syndrome coronavirus (SARS-CoV)-1 and -2 initiate virus infection by binding of their spike glycoprotein with the cell-surface receptor angiotensin-converting enzyme 2 (ACE2) and enter into the host cells mainly via the clathrin-mediated endocytosis pathway. However, the internalization process post attachment with the receptor is not clear for both SARS-CoV-1 and -2. Understanding the cellular factor/s or pathways used by these CoVs for internalization might provide insights into viral pathogenesis, transmission, and development of novel therapeutics. Here, we demonstrated that the cytoplasmic tail of ACE2 is not essential for the entry of SARS-CoV-1 and -2 by using bioinformatics, mutational, confocal imaging, and pseudotyped SARS-CoVs infection studies. ACE2 cytoplasmic domain (cytACE2) contains a conserved internalization motif and eight putative phosphorylation sites. Complete cytoplasmic domain deleted ACE2 (∆cytACE2) was properly synthesized and presented on the surface of HEK293T and BHK21 cells like wtACE2. The SARS-CoVs S1 or RBD of spike protein binds and colocalizes with the receptors followed by internalization into the host cells. Moreover, pseudotyped SARS-CoVs entered into wtACE2- and ∆cytACE2-transfected cells but not into dipeptidyl peptidase 4 (DPP4)-expressing cells. Their entry was significantly inhibited by treatment with dynasore, a dynamin inhibitor, and NH4Cl, an endosomal acidification inhibitor. Furthermore, SARS-CoV antibodies and the soluble form of ACE2-treated pseudotyped SARS-CoVs were unable to enter the wtACE2 and ∆cytACE2-expressing cells. Altogether, our data show that ACE2 cytoplasmic domain signaling is not essential for the entry of SARS-CoV-1 and -2 and that SARS-CoVs entry might be mediated via known/unknown host factor/s.


Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , COVID-19/metabolismo , SARS-CoV-2/fisiologia , Transdução de Sinais , Internalização do Vírus , Enzima de Conversão de Angiotensina 2/química , Animais , Chlorocebus aethiops , Células HEK293 , Humanos , Domínios Proteicos , Células Vero
15.
Nat Commun ; 12(1): 4909, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34389707

RESUMO

In bacteria, trans-translation is the main rescue system, freeing ribosomes stalled on defective messenger RNAs. This mechanism is driven by small protein B (SmpB) and transfer-messenger RNA (tmRNA), a hybrid RNA known to have both a tRNA-like and an mRNA-like domain. Here we present four cryo-EM structures of the ribosome during trans-translation at resolutions from 3.0 to 3.4 Å. These include the high-resolution structure of the whole pre-accommodated state, as well as structures of the accommodated state, the translocated state, and a translocation intermediate. Together, they shed light on the movements of the tmRNA-SmpB complex in the ribosome, from its delivery by the elongation factor EF-Tu to its passage through the ribosomal A and P sites after the opening of the B1 bridges. Additionally, we describe the interactions between the tmRNA-SmpB complex and the ribosome. These explain why the process does not interfere with canonical translation.


Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Biossíntese de Proteínas/genética , RNA Bacteriano/genética , Proteínas de Ligação a RNA/genética , Ribossomos/genética , Sítios de Ligação/genética , Microscopia Crioeletrônica , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Domínios Proteicos , RNA Bacteriano/química , RNA Bacteriano/metabolismo , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Transferência/química , RNA de Transferência/genética , RNA de Transferência/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Ribossomos/metabolismo , Ribossomos/ultraestrutura
16.
Nat Commun ; 12(1): 5083, 2021 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-34426577

RESUMO

AMPA receptor (AMPAR) abundance and positioning at excitatory synapses regulates the strength of transmission. Changes in AMPAR localisation can enact synaptic plasticity, allowing long-term information storage, and is therefore tightly controlled. Multiple mechanisms regulating AMPAR synaptic anchoring have been described, but with limited coherence or comparison between reports, our understanding of this process is unclear. Here, combining synaptic recordings from mouse hippocampal slices and super-resolution imaging in dissociated cultures, we compare the contributions of three AMPAR interaction domains controlling transmission at hippocampal CA1 synapses. We show that the AMPAR C-termini play only a modulatory role, whereas the extracellular N-terminal domain (NTD) and PDZ interactions of the auxiliary subunit TARP γ8 are both crucial, and each is sufficient to maintain transmission. Our data support a model in which γ8 accumulates AMPARs at the postsynaptic density, where the NTD further tunes their positioning. This interplay between cytosolic (TARP γ8) and synaptic cleft (NTD) interactions provides versatility to regulate synaptic transmission and plasticity.


Assuntos
Região CA1 Hipocampal/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Receptores de AMPA/metabolismo , Sinapses/metabolismo , Sequência de Aminoácidos , Animais , Corantes Fluorescentes/metabolismo , Imageamento Tridimensional , Camundongos Endogâmicos C57BL , Modelos Neurológicos , Mutação/genética , Plasticidade Neuronal , Neurônios/metabolismo , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , Receptores de AMPA/química , Transmissão Sináptica
17.
Nat Commun ; 12(1): 5082, 2021 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-34426584

RESUMO

Gram-positive bacteria can resist large mechanical perturbations during their invasion and colonization by secreting various surface proteins with intramolecular isopeptide or ester bonds. Compared to isopeptide bonds, ester bonds are prone to hydrolysis. It remains elusive whether ester bonds can completely block mechanical extension similarly to isopeptide bonds, or whether ester bonds dissipate mechanical energy by bond rupture. Here, we show that an ester-bond containing stalk domain of Cpe0147 is inextensible even at forces > 2 nN. The ester bond locks the structure to a partially unfolded conformation, in which the ester bond remains largely water inaccessible. This allows the ester bond to withstand considerable mechanical forces and in turn prevent complete protein unfolding. However, the protecting effect might be reduced at non-physiological basic pHs or low calcium concentrations due to destabilizing the protein structures. Inspired by this design principle, we engineer a disulfide mutant resistant to mechanical unfolding under reducing conditions.


Assuntos
Proteínas de Bactérias/química , Ésteres/química , Fenômenos Mecânicos , Dissulfetos/química , Microscopia de Força Atômica , Simulação de Dinâmica Molecular , Proteínas Mutantes/química , Domínios Proteicos , Água/química
18.
Avian Dis ; 65(3): 364-372, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34427409

RESUMO

We previously reported that recombinant Newcastle disease virus LaSota (rLS) expressing infectious bronchitis virus (IBV) Arkansas (Ark)-type trimeric spike (S) ectodomain (Se; rLS/ArkSe) provides suboptimal protection against IBV challenge. We have now developed rLS expressing chicken granulocyte-macrophage colony-stimulating factor (GMCSF) and IBV Ark Se in an attempt to enhance vaccine effectiveness. In the current study, we first compared protection conferred by vaccination with rLS/ArkSe and rLS/ArkSe.GMCSF. Vaccinated chickens were challenged with virulent Ark, and protection was determined by clinical signs, viral load, and tracheal histomorphometry. Results showed that coexpression of GMCSF and the Se from rLS significantly reduced tracheal viral load and tracheal lesions compared with chickens vaccinated with rLS/ArkSe. In a second experiment, we evaluated enhancement of cross-protection of a Massachusetts (Mass) attenuated vaccine by priming or boosting with rLS/ArkSe.GMCSF. Vaccinated chickens were challenged with Ark, and protection was evaluated. Results show that priming or boosting with the recombinant virus significantly increased cross-protection conferred by Mass against Ark virulent challenge. Greater reductions of viral loads in both trachea and lachrymal fluids were observed in chickens primed with rLS/ArkSe.GMCSF and boosted with Mass. Consistently, Ark Se antibody levels measured with recombinant Ark Se protein-coated ELISA plates 14 days after boost were significantly higher in these chickens. Unexpectedly, the inverse vaccination scheme, that is, priming with Mass and boosting with the recombinant vaccine, proved somewhat less effective. We concluded that a prime and boost strategy by using rLS/ArkSe.GMCSF and the worldwide ubiquitous Mass attenuated vaccine provides enhanced cross-protection. Thus, rLS/GMCSF coexpressing the Se of regionally relevant IBV serotypes could be used in combination with live Mass to protect against regionally circulating IBV variant strains.


Assuntos
Infecções por Coronavirus/veterinária , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Vírus da Bronquite Infecciosa/imunologia , Vírus da Doença de Newcastle/genética , Doenças das Aves Domésticas/prevenção & controle , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Anticorpos Antivirais/imunologia , Galinhas/genética , Galinhas/imunologia , Galinhas/virologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Infecções por Coronavirus/virologia , Proteção Cruzada , Expressão Gênica , Fator Estimulador de Colônias de Granulócitos e Macrófagos/administração & dosagem , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Vírus da Bronquite Infecciosa/química , Vírus da Bronquite Infecciosa/genética , Vírus da Bronquite Infecciosa/fisiologia , Vírus da Doença de Newcastle/metabolismo , Doenças das Aves Domésticas/imunologia , Doenças das Aves Domésticas/virologia , Domínios Proteicos , Glicoproteína da Espícula de Coronavírus/administração & dosagem , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Traqueia/imunologia , Traqueia/virologia , Vacinação , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , Carga Viral
19.
Int J Mol Sci ; 22(16)2021 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-34445741

RESUMO

(1) Background: coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been linked to hematological dysfunctions, but there are little experimental data that explain this. Spike (S) and Nucleoprotein (N) proteins have been putatively associated with these dysfunctions. In this work, we analyzed the recruitment of hemoglobin (Hb) and other metabolites (hemin and protoporphyrin IX-PpIX) by SARS-Cov2 proteins using different approaches. (2) Methods: shotgun proteomics (LC-MS/MS) after affinity column adsorption identified hemin-binding SARS-CoV-2 proteins. The parallel synthesis of the peptides technique was used to study the interaction of the receptor bind domain (RBD) and N-terminal domain (NTD) of the S protein with Hb and in silico analysis to identify the binding motifs of the N protein. The plaque assay was used to investigate the inhibitory effect of Hb and the metabolites hemin and PpIX on virus adsorption and replication in Vero cells. (3) Results: the proteomic analysis by LC-MS/MS identified the S, N, M, Nsp3, and Nsp7 as putative hemin-binding proteins. Six short sequences in the RBD and 11 in the NTD of the spike were identified by microarray of peptides to interact with Hb and tree motifs in the N protein by in silico analysis to bind with heme. An inhibitory effect in vitro of Hb, hemin, and PpIX at different levels was observed. Strikingly, free Hb at 1mM suppressed viral replication (99%), and its interaction with SARS-CoV-2 was localized into the RBD region of the spike protein. (4) Conclusions: in this study, we identified that (at least) five proteins (S, N, M, Nsp3, and Nsp7) of SARS-CoV-2 recruit Hb/metabolites. The motifs of the RDB of SARS-CoV-2 spike, which binds Hb, and the sites of the heme bind-N protein were disclosed. In addition, these compounds and PpIX block the virus's adsorption and replication. Furthermore, we also identified heme-binding motifs and interaction with hemin in N protein and other structural (S and M) and non-structural (Nsp3 and Nsp7) proteins.


Assuntos
COVID-19/etiologia , Hemoglobinas/metabolismo , SARS-CoV-2/metabolismo , Proteínas não Estruturais Virais/metabolismo , Proteínas Estruturais Virais/metabolismo , COVID-19/sangue , Hemina/metabolismo , Hemoglobinas/ultraestrutura , Humanos , Simulação de Acoplamento Molecular , Ligação Proteica , Domínios Proteicos , Proteômica , Protoporfirinas/metabolismo , SARS-CoV-2/patogenicidade , Proteínas não Estruturais Virais/ultraestrutura , Proteínas Estruturais Virais/ultraestrutura , Ligação Viral , Replicação Viral
20.
Science ; 373(6555): 700-704, 2021 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-34353956

RESUMO

Gag, the primary structural protein of HIV-1, is recruited to the plasma membrane for virus assembly by its matrix (MA) domain. Gag is subsequently cleaved into its component domains, causing structural maturation to repurpose the virion for cell entry. We determined the structure and arrangement of MA within immature and mature HIV-1 through cryo-electron tomography. We found that MA rearranges between two different hexameric lattices upon maturation. In mature HIV-1, a lipid extends out of the membrane to bind with a pocket in MA. Our data suggest that proteolytic maturation of HIV-1 not only assembles the viral capsid surrounding the genome but also repurposes the membrane-bound MA lattice for an entry or postentry function and results in the partial removal of up to 2500 lipids from the viral membrane.


Assuntos
Antígenos HIV/química , Antígenos HIV/metabolismo , HIV-1/química , HIV-1/fisiologia , Envelope Viral/metabolismo , Produtos do Gene gag do Vírus da Imunodeficiência Humana/química , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Capsídeo/química , Capsídeo/fisiologia , Tomografia com Microscopia Eletrônica , HIV-1/ultraestrutura , Bicamadas Lipídicas , Lipídeos de Membrana/metabolismo , Modelos Moleculares , Conformação Proteica , Domínios Proteicos , Estrutura Secundária de Proteína , Envelope Viral/química , Envelope Viral/ultraestrutura , Vírion/química , Vírion/fisiologia , Vírion/ultraestrutura , Montagem de Vírus , Produtos do Gene env do Vírus da Imunodeficiência Humana/química , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismo
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