RESUMEN
Iron-sulfur (Fe-S) clusters are essential protein cofactors whose biosynthetic defects lead to severe diseases among which is Friedreich's ataxia caused by impaired expression of frataxin (FXN). Fe-S clusters are biosynthesized on the scaffold protein ISCU, with cysteine desulfurase NFS1 providing sulfur as persulfide and ferredoxin FDX2 supplying electrons, in a process stimulated by FXN but not clearly understood. Here, we report the breakdown of this process, made possible by removing a zinc ion in ISCU that hinders iron insertion and promotes non-physiological Fe-S cluster synthesis from free sulfide in vitro. By binding zinc-free ISCU, iron drives persulfide uptake from NFS1 and allows persulfide reduction into sulfide by FDX2, thereby coordinating sulfide production with its availability to generate Fe-S clusters. FXN stimulates the whole process by accelerating persulfide transfer. We propose that this reconstitution recapitulates physiological conditions which provides a model for Fe-S cluster biosynthesis, clarifies the roles of FDX2 and FXN and may help develop Friedreich's ataxia therapies.
Asunto(s)
Ferredoxinas/metabolismo , Proteínas de Unión a Hierro/metabolismo , Proteínas Hierro-Azufre/metabolismo , Sulfuros/metabolismo , Liasas de Carbono-Azufre/metabolismo , Ferredoxinas/aislamiento & purificación , Ataxia de Friedreich/patología , Hierro/metabolismo , Proteínas de Unión a Hierro/aislamiento & purificación , Proteínas Hierro-Azufre/química , Proteínas Hierro-Azufre/genética , Mutagénesis Sitio-Dirigida , Resonancia Magnética Nuclear Biomolecular , Oxidación-Reducción , Espectroscopía de Protones por Resonancia Magnética , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Zinc/metabolismo , FrataxinaRESUMEN
Addition of small molecule Retro-1 has been described to enhance antisense and splice switching oligonucleotides. With the aim of assessing the effect of covalently linking Retro-1 to the biologically active oligonucleotide, three different derivatives of Retro-1 were prepared that incorporated a phosphoramidite group, a thiol or a 1,3-diene, respectively. Retro-1â»oligonucleotide conjugates were assembled both on-resin (coupling of the phosphoramidite) and from reactions in solution (Michael-type thiol-maleimide reaction and Diels-Alder cycloaddition). Splice switching assays with the resulting conjugates showed that they were active but that they provided little advantage over the unconjugated oligonucleotide in the well-known HeLa Luc705 reporter system.
Asunto(s)
Oligonucleótidos/síntesis química , Oligonucleótidos/farmacología , Línea Celular Tumoral , Técnicas de Química Sintética , Cromatografía Líquida de Alta Presión , Humanos , Espectrometría de Masas , Estructura Molecular , Oligonucleótidos/química , Empalme del ARN/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
The cysteine-cyclopentenedione reaction can be combined with the copper(i)-catalyzed azide-alkyne cycloaddition provided that the former is carried out first. If not, the azide and the cyclopentenedione undergo a 1,3-dipolar cycloaddition, which furnishes triazole-containing compounds and products resulting from nitrogen loss. Both of these products were fully characterized. Attempts to obtain either of them as the main compound or to drive the reaction nearly to completion were unsuccessful, which points to the azide-cyclopentenedione reaction as not being useful for bioconjugation.
RESUMEN
Unprotected linear peptides containing N-terminal cysteines and another cysteine residue can be simultaneously cyclized and derivatized using 2,2-disubstituted cyclopentenediones. High yields of cyclic peptide conjugates may be obtained in short reaction times using only a slight excess of the cyclopentenedione moiety under TEMPO catalysis and in the presence of LiCl.
Asunto(s)
Péptidos/química , Ciclización , Cisteína , Estructura MolecularRESUMEN
The outcome of the Michael-type reaction between thiols and 2,2-disubstituted cyclopentenediones varies depending on the thiol. Stable compounds with two fused rings were formed upon reaction with 1,2-aminothiols (such as N-terminal cysteines in peptides). Other thiols gave reversibly Michael-type adducts that were in equilibrium with the starting materials. This differential reactivity allows differently placed cysteines to be distinguished and has been exploited to prepare bioconjugates incorporating two or three different moieties.