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1.
Proc Natl Acad Sci U S A ; 119(6)2022 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-35131851

RESUMO

For an efficacious vaccine immunogen, influenza hemagglutinin (HA) needs to maintain a stable quaternary structure, which is contrary to the inherently dynamic and metastable nature of class I fusion proteins. In this study, we stabilized HA with three substitutions within its pH-sensitive regions where the refolding starts. An X-ray structure reveals how these substitutions stabilize the intersubunit ß-sheet in the base and form an interprotomeric aliphatic layer across the stem while the native prefusion HA fold is retained. The identification of the stabilizing substitutions increases our understanding of how the pH sensitivity is structurally accomplished in HA and possibly other pH-sensitive class I fusion proteins. Our stabilization approach in combination with the occasional back mutation of rare amino acids to consensus results in well-expressing stable trimeric HAs. This repair and stabilization approach, which proves broadly applicable to all tested influenza A HAs of group 1 and 2, will improve the developability of influenza vaccines based on different types of platforms and formats and can potentially improve efficacy.


Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Hemaglutininas/genética , Aminoácidos/genética , Linhagem Celular , Humanos , Concentração de Íons de Hidrogênio , Vacinas contra Influenza/genética , Influenza Humana/virologia , Mutação/genética , Conformação Proteica em Folha beta/genética
2.
FASEB J ; 26(1): 93-103, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21940993

RESUMO

AHNAK is a 700-kDa protein involved in cytoarchitecture and calcium signaling. It is secondarily reduced in muscle of dysferlinopathy patients and accumulates in muscle of calpainopathy patients, both affected by a muscular dystrophy. AHNAK directly interacts with dysferlin. This interaction is lost on cleavage of AHNAK by the protease calpain 3, explaining the molecular observations in patients. Currently, little is known of AHNAK regulation. We describe the self-regulation of multiple mRNA transcripts emanating from the AHNAK locus in muscle cells. We show that the AHNAK gene consists of a 17-kb exon flanked by multiple small exons. This genetic structure is shared by AHNAK2 and Periaxin, which share a common ancestor. Two major AHNAK transcripts are differentially expressed during muscle differentiation that encode for a small (17-kDa) and a large (700-kDa) protein isoform. These proteins interact in the cytoplasm, but the small AHNAK is also present in the nucleus. During muscle differentiation the small AHNAK is strongly increased, thereby establishing a positive feedback loop to regulate mRNA splicing of its own locus. A small 17-kDa isoform of Periaxin similarly traffics between the cytoplasm and the nucleus to regulate mRNA splicing. Thus, AHNAK constitutes a novel mechanism in post-transcriptional control of gene expression.


Assuntos
Processamento Alternativo/fisiologia , Sinalização do Cálcio/fisiologia , Proteínas de Membrana/genética , Mioblastos Esqueléticos/fisiologia , Proteínas de Neoplasias/genética , Sequência de Aminoácidos , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Evolução Molecular , Retroalimentação Fisiológica/fisiologia , Regulação da Expressão Gênica/genética , Humanos , Proteínas de Membrana/metabolismo , Camundongos , Dados de Sequência Molecular , Mioblastos Esqueléticos/citologia , Proteínas de Neoplasias/metabolismo , Filogenia , Processamento Pós-Transcricional do RNA/fisiologia , RNA Mensageiro/genética
3.
Cell Rep ; 23(2): 584-595, 2018 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-29642014

RESUMO

The heavily glycosylated native-like envelope (Env) trimer of HIV-1 is expected to have low immunogenicity, whereas misfolded forms are often highly immunogenic. High-quality correctly folded Envs may therefore be critical for developing a vaccine that induces broadly neutralizing antibodies. Moreover, the high variability of Env may require immunizations with multiple Envs. Here, we report a universal strategy that provides for correctly folded Env trimers of high quality and yield through a repair-and-stabilize approach. In the repair stage, we utilized a consensus strategy that substituted rare strain-specific residues with more prevalent ones. The stabilization stage involved structure-based design and experimental assessment confirmed by crystallographic feedback. Regions important for the refolding of Env were targeted for stabilization. Notably, the α9-helix and an intersubunit ß sheet proved to be critical for trimer stability. Our approach provides a means to produce prefusion-closed Env trimers from diverse HIV-1 strains, a substantial advance for vaccine development.


Assuntos
HIV-1/metabolismo , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismo , Varredura Diferencial de Calorimetria , Cristalografia por Raios X , Anticorpos Anti-HIV/química , Anticorpos Anti-HIV/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Mutagênese Sítio-Dirigida , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Multimerização Proteica , Redobramento de Proteína , Estabilidade Proteica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Produtos do Gene env do Vírus da Imunodeficiência Humana/química , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética
4.
Nat Commun ; 6: 8143, 2015 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-26333350

RESUMO

Respiratory syncytial virus (RSV) causes acute lower respiratory tract infections and is the leading cause of infant hospitalizations. Recently, a promising vaccine antigen based on the RSV fusion protein (RSV F) stabilized in the native prefusion conformation has been described. Here we report alternative strategies to arrest RSV F in the prefusion conformation based on the prevention of hinge movements in the first refolding region and the elimination of proteolytic exposure of the fusion peptide. A limited number of unique mutations are identified that stabilize the prefusion conformation of RSV F and dramatically increase expression levels. This highly stable prefusion RSV F elicits neutralizing antibodies in cotton rats and induces complete protection against viral challenge. Moreover, the structural and biochemical analysis of the prefusion variants suggests a function for p27, the excised segment that precedes the fusion peptide in the polypeptide chain.


Assuntos
Anticorpos Neutralizantes/imunologia , Antígenos Virais/imunologia , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Vacinas contra Vírus Sincicial Respiratório/imunologia , Vírus Sinciciais Respiratórios/imunologia , Proteínas Virais de Fusão/imunologia , Animais , Antígenos Virais/genética , Western Blotting , Cristalização , Cristalografia por Raios X , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Camundongos , Microscopia Eletrônica , Mutação , Conformação Proteica , Infecções por Vírus Respiratório Sincicial/imunologia , Vírus Sinciciais Respiratórios/genética , Sigmodontinae , Proteínas Virais de Fusão/genética
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