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1.
Commun Biol ; 7(1): 968, 2024 Aug 09.
Artículo en Inglés | MEDLINE | ID: mdl-39122990

RESUMEN

Aggregation-prone-motifs (APRs) of proteins are short segments, which - as isolated peptides - form diverse amyloid-like crystals. We introduce two APRs - designed variants of the incretin mimetic Exendin-4 - that both display crystal-phase polymorphism. Crystallographic and spectroscopic analysis revealed that a single amino-acid substitution can greatly reduce topological variability: while LYIQWL can form both parallel and anti-parallel ß-sheets, LYIQNL selects only the former. We also found that the parallel/anti-parallel switch of LYIQWL can be induced by simply changing the crystallization temperature. One crystal form of LYIQNL was found to belong to the class 3 topology, an arrangement previously not encountered among proteinogenic systems. We also show that subtle environmental changes lead to crystalline assemblies with different topologies, but similar interfaces. Spectroscopic measurements showed that polymorphism is already apparent in the solution state. Our results suggest that the temperature-, sequence- and environmental sensitivity of physiological amyloids is reflected in assemblies of the APR segments, which, complete with the new class 3 crystal form, effectively sample all the originally proposed basic topologies of amyloid-like aggregates.


Asunto(s)
Amiloide , Solventes , Amiloide/química , Amiloide/genética , Amiloide/metabolismo , Solventes/química
2.
Nat Commun ; 14(1): 4621, 2023 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-37528104

RESUMEN

A large group of hormones are stored as amyloid fibrils in acidic secretion vesicles before they are released into the bloodstream and readopt their functional state. Here, we identify an evolutionarily conserved hexapeptide sequence as the major aggregation-prone region (APR) of gastrointestinal peptides of the glucagon family: xFxxWL. We determine nine polymorphic crystal structures of the APR segments of glucagon-like peptides 1 and 2, and exendin and its derivatives. We follow amyloid formation by CD, FTIR, ThT assays, and AFM. We propose that the pH-dependent changes of the protonation states of glutamate/aspartate residues of APRs initiate switching between the amyloid and the folded, monomeric forms of the hormones. We find that pH sensitivity diminishes in the absence of acidic gatekeepers and amyloid formation progresses over a broad pH range. Our results highlight the dual role of short aggregation core motifs in reversible amyloid formation and receptor binding.


Asunto(s)
Amiloide , Nanoestructuras , Amiloide/metabolismo , Péptidos/química , Proteínas Amiloidogénicas , Hormonas , Homeostasis , Nanoestructuras/química , Glucosa
3.
Bioconjug Chem ; 34(10): 1738-1753, 2023 10 18.
Artículo en Inglés | MEDLINE | ID: mdl-37606258

RESUMEN

The complex immunopathology ofMycobacterium tuberculosis(Mtb) is one of the main challenges in developing a novel vaccine against this pathogen, particularly regarding eliciting protection against both active and latent stages. Multistage vaccines, which contain antigens expressed in both phases, represent a promising strategy for addressing this issue, as testified by the tuberculosis vaccine clinical pipeline. Given this approach, we designed and characterized a multistage peptide-based vaccine platform containing CD4+ and CD8+ T cell epitopes previously validated for inducing a relevant T cell response against Mtb. After preliminary screening, CFP10 (32-39), GlfT2 (4-12), HBHA (185-194), and PPE15 (1-15) were selected as promising candidates, and we proved that the PM1 pool of these peptides triggered a T cell response in Mtb-sensitized human peripheral blood mononuclear cells (PBMCs). Taking advantage of the use of thiol-maleimide chemoselective ligation, we synthesized a multiepitope conjugate (Ac-CGHP). Our results showed a structure-activity relationship between the conjugation and a higher tendency to fold and assume an ordered secondary structure. Moreover, the palmitoylated conjugate (Pal-CGHP) comprising the same peptide antigens was associated with an enhanced cellular uptake in human and murine antigen-presenting cells and a better immunogenicity profile. Immunization study, conducted in BALB/c mice, showed that Pal-CGHP induced a significantly higher T cell proliferation and production of IFNγ and TNFα over PM1 formulated in the Sigma Adjuvant System.


Asunto(s)
Mycobacterium tuberculosis , Vacunas contra la Tuberculosis , Tuberculosis , Humanos , Animales , Ratones , Leucocitos Mononucleares , Antígenos Bacterianos , Linfocitos T CD4-Positivos , Tuberculosis/prevención & control , Linfocitos T CD8-positivos , Epítopos de Linfocito T , Péptidos
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