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1.
EMBO J ; 41(23): e112787, 2022 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-36314692

RESUMO

In bacteria, N-terminal signal peptides mark proteins for transport across the plasma membrane. A recent study by Smets et al (2022) followed the folding of a pair of structural twins to shed light on how evolution has optimised the secretory process.


Assuntos
Sinais Direcionadores de Proteínas , Proteínas , Transporte Proteico , Membrana Celular , Dobramento de Proteína
2.
Proc Natl Acad Sci U S A ; 118(44)2021 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-34654739

RESUMO

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in over 100 million infections and millions of deaths. Effective vaccines remain the best hope of curtailing SARS-CoV-2 transmission, morbidity, and mortality. The vaccines in current use require cold storage and sophisticated manufacturing capacity, which complicates their distribution, especially in less developed countries. We report the development of a candidate SARS-CoV-2 vaccine that is purely protein based and directly targets antigen-presenting cells. It consists of the SARS-CoV-2 Spike receptor-binding domain (SpikeRBD) fused to an alpaca-derived nanobody that recognizes class II major histocompatibility complex antigens (VHHMHCII). This vaccine elicits robust humoral and cellular immunity against SARS-CoV-2 and its variants. Both young and aged mice immunized with two doses of VHHMHCII-SpikeRBD elicit high-titer binding and neutralizing antibodies. Immunization also induces strong cellular immunity, including a robust CD8 T cell response. VHHMHCII-SpikeRBD is stable for at least 7 d at room temperature and can be lyophilized without loss of efficacy.


Assuntos
Vacinas contra COVID-19/imunologia , Vacinas contra COVID-19/farmacologia , COVID-19/imunologia , COVID-19/prevenção & controle , Pandemias , SARS-CoV-2/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Neutralizantes/biossíntese , Anticorpos Antivirais/biossíntese , Células Apresentadoras de Antígenos/imunologia , Linfócitos T CD8-Positivos/imunologia , COVID-19/epidemiologia , Vacinas contra COVID-19/administração & dosagem , Camelídeos Americanos/imunologia , Feminino , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Imunidade Celular , Imunidade Humoral , Imunização Secundária , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pandemias/prevenção & controle , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , SARS-CoV-2/genética , Anticorpos de Domínio Único/administração & dosagem , Anticorpos de Domínio Único/imunologia , Glicoproteína da Espícula de Coronavírus/administração & dosagem , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia
3.
J Biol Chem ; 297(3): 100991, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34419450

RESUMO

Fic domain-containing AMP transferases (fic AMPylases) are conserved enzymes that catalyze the covalent transfer of AMP to proteins. This posttranslational modification regulates the function of several proteins, including the ER-resident chaperone Grp78/BiP. Here we introduce a mouse FICD (mFICD) AMPylase knockout mouse model to study fic AMPylase function in vertebrates. We find that mFICD deficiency is well tolerated in unstressed mice. We also show that mFICD-deficient mouse embryonic fibroblasts are depleted of AMPylated proteins. mFICD deletion alters protein synthesis and secretion in splenocytes, including that of IgM, an antibody secreted early during infections, and the proinflammatory cytokine IL-1ß, without affecting the unfolded protein response. Finally, we demonstrate that visual nonspatial short-term learning is stronger in old mFICD-/- mice than in wild-type controls while other measures of cognition, memory, and learning are unaffected. Together, our results suggest a role for mFICD in adaptive immunity and neuronal plasticity in vivo.


Assuntos
Citocinas/metabolismo , Aprendizagem , Transferases/metabolismo , Percepção Visual , Animais , Células Cultivadas , Chaperona BiP do Retículo Endoplasmático , Camundongos , Camundongos Knockout
4.
Traffic ; 17(6): 615-38, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26947578

RESUMO

The biophysical rules that govern folding of small, single-domain proteins in dilute solutions are now quite well understood. The mechanisms underlying co-translational folding of multidomain and membrane-spanning proteins in complex cellular environments are often less clear. The endoplasmic reticulum (ER) produces a plethora of membrane and secretory proteins, which must fold and assemble correctly before ER exit - if these processes fail, misfolded species accumulate in the ER or are degraded. The ER differs from other cellular organelles in terms of the physicochemical environment and the variety of ER-specific protein modifications. Here, we review chaperone-assisted co- and post-translational folding and assembly in the ER and underline the influence of protein modifications on these processes. We emphasize how method development has helped advance the field by allowing researchers to monitor the progression of folding as it occurs inside living cells, while at the same time probing the intricate relationship between protein modifications during folding.


Assuntos
Retículo Endoplasmático/metabolismo , Dobramento de Proteína , Processamento de Proteína Pós-Traducional , Animais , Glicosilação , Humanos , Chaperonas Moleculares/metabolismo
5.
Cell Rep ; 36(9): 109646, 2021 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-34469718

RESUMO

Removal of the membrane-tethering signal peptides that target secretory proteins to the endoplasmic reticulum is a prerequisite for proper folding. While generally thought to be removed co-translationally, we report two additional post-targeting functions for the HIV-1 gp120 signal peptide, which remains attached until gp120 folding triggers its removal. First, the signal peptide improves folding fidelity by enhancing conformational plasticity of gp120 by driving disulfide isomerization through a redox-active cysteine. Simultaneously, the signal peptide delays folding by tethering the N terminus to the membrane, until assembly with the C terminus. Second, its carefully timed cleavage represents intramolecular quality control and ensures release of (only) natively folded gp120. Postponed cleavage and the redox-active cysteine are both highly conserved and important for viral fitness. Considering the ∼15% proteins with signal peptides and the frequency of N-to-C contacts in protein structures, these regulatory roles of signal peptides are bound to be more common in secretory-protein biogenesis.


Assuntos
Proteína gp120 do Envelope de HIV/metabolismo , Proteína gp160 do Envelope de HIV/metabolismo , HIV-1/metabolismo , Processamento de Proteína Pós-Traducional , Cisteína , Células HEK293 , Proteína gp120 do Envelope de HIV/genética , Proteína gp160 do Envelope de HIV/genética , HIV-1/genética , HIV-1/crescimento & desenvolvimento , Células HeLa , Humanos , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Sinais Direcionadores de Proteínas , Estabilidade Proteica , Relação Estrutura-Atividade , Carga Viral , Replicação Viral
6.
Nat Biomed Eng ; 5(11): 1389-1401, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34127819

RESUMO

The association of autoimmune diseases with particular allellic products of the class-II major histocompatibility complex (MHCII) region implicates the presentation of the offending self-antigens to T cells. Because antigen-presenting cells are tolerogenic when they encounter an antigen under non-inflammatory conditions, the manipulation of antigen presentation may induce antigen-specific tolerance. Here, we show that, in mouse models of experimental autoimmune encephalomyelitis, type 1 diabetes and rheumatoid arthritis, the systemic administration of a single dose of nanobodies that recognize MHCII molecules and conjugated to the relevant self-antigen under non-inflammatory conditions confers long-lasting protection against these diseases. Moreover, co-administration of a nanobody-antigen adduct and the glucocorticoid dexamethasone, conjugated to the nanobody via a cleavable linker, halted the progression of established experimental autoimmune encephalomyelitis in symptomatic mice and alleviated their symptoms. This approach may represent a means of treating autoimmune conditions.


Assuntos
Encefalomielite Autoimune Experimental , Tolerância Imunológica , Animais , Autoantígenos , Histocompatibilidade , Complexo Principal de Histocompatibilidade , Camundongos
7.
J Vis Exp ; (144)2019 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-30829321

RESUMO

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and the degradation of target proteins in live cells. By using short (pulse) radiolabeling times (<30 min) and tightly controlled chase times, it is possible to label only a small fraction of the total protein pool and follow its folding. When combined with nonreducing/reducing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoprecipitation with (conformation-specific) antibodies, folding processes can be examined in great detail. This system has been used to analyze the folding of proteins with a huge variation in properties such as soluble proteins, single and multi-pass transmembrane proteins, heavily N- and O-glycosylated proteins, and proteins with and without extensive disulfide bonding. Pulse-chase methods are the basis of kinetic studies into a range of additional features, including co- and posttranslational modifications, oligomerization, and polymerization, essentially allowing the analysis of a protein from birth to death. Pulse-chase studies on protein folding are complementary with other biochemical and biophysical methods for studying proteins in vitro by providing increased temporal resolution and physiological information. The methods as described within this paper are adapted easily to study the folding of almost any protein that can be expressed in mammalian or insect-cell systems.


Assuntos
Dobramento de Proteína , Poluentes Radioativos/efeitos adversos , Movimento Celular , Transfecção
8.
Mol Immunol ; 114: 513-523, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31518855

RESUMO

A substantial fraction of eukaryotic proteins is folded and modified in the endoplasmic reticulum (ER) prior to export and secretion. Proteins that enter the ER but fail to fold correctly must be degraded, mostly in a process termed ER-associated degradation (ERAD). Both protein folding in the ER and ERAD are essential for proper immune function. Several E2 and E3 enzymes localize to the ER and are essential for various aspects of ERAD, but their functions and regulation are incompletely understood. Here we identify and characterize single domain antibody fragments derived from the variable domain of alpaca heavy chain-only antibodies (VHHs or nanobodies) that bind to the ER-localized E2 UBC6e, an enzyme implicated in ERAD. One such VHH, VHH05 recognizes a 14 residue stretch and enhances the rate of E1-catalyzed ubiquitin E2 loading in vitroand interferes with phosphorylation of UBC6e in response to cell stress. Identification of the peptide epitope recognized by VHH05 places it outside the E2 catalytic core, close to the position of activation-induced phosphorylation on Ser184. Our data thus suggests a site involved in allosteric regulation of UBC6e's activity. This VHH should be useful not only to dissect the participation of UBC6e in ERAD and in response to cell stress, but also as a high affinity epitope tag-specific reagent of more general utility.


Assuntos
Epitopos/imunologia , Peptídeos/imunologia , Anticorpos de Domínio Único/imunologia , Enzimas de Conjugação de Ubiquitina/imunologia , Anticorpos/imunologia , Linhagem Celular Tumoral , Células Cultivadas , Degradação Associada com o Retículo Endoplasmático/imunologia , Células HeLa , Humanos , Células K562 , Fosforilação/imunologia , Ubiquitina/imunologia , Ubiquitina-Proteína Ligases/imunologia
9.
Elife ; 62017 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-28753126

RESUMO

Like all other secretory proteins, the HIV-1 envelope glycoprotein gp160 is targeted to the endoplasmic reticulum (ER) by its signal peptide during synthesis. Proper gp160 folding in the ER requires core glycosylation, disulfide-bond formation and proline isomerization. Signal-peptide cleavage occurs only late after gp160 chain termination and is dependent on folding of the soluble subunit gp120 to a near-native conformation. We here detail the mechanism by which co-translational signal-peptide cleavage is prevented. Conserved residues from the signal peptide and residues downstream of the canonical cleavage site form an extended alpha-helix in the ER membrane, which covers the cleavage site, thus preventing cleavage. A point mutation in the signal peptide breaks the alpha helix allowing co-translational cleavage. We demonstrate that postponed cleavage of gp160 enhances functional folding of the molecule. The change to early cleavage results in decreased viral fitness compared to wild-type HIV.


Assuntos
Proteína gp160 do Envelope de HIV/química , Proteína gp160 do Envelope de HIV/metabolismo , HIV-1/fisiologia , Dobramento de Proteína , Sinais Direcionadores de Proteínas , Linhagem Celular , Humanos , Conformação Proteica , Transporte Proteico , Proteólise
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