RESUMEN
Parasitic diseases cause â¼ 500,000 deaths annually and remain a major challenge for therapeutic development. Using a rational design based approach, we developed peptide inhibitors with anti-parasitic activity that were derived from the sequences of parasite scaffold proteins LACK (Leishmania's receptor for activated C-kinase) and TRACK (Trypanosoma receptor for activated C-kinase). We hypothesized that sequences in LACK and TRACK that are conserved in the parasites, but not in the mammalian ortholog, RACK (Receptor for activated C-kinase), may be interaction sites for signaling proteins that are critical for the parasites' viability. One of these peptides exhibited leishmanicidal and trypanocidal activity in culture. Moreover, in infected mice, this peptide was also effective in reducing parasitemia and increasing survival without toxic effects. The identified peptide is a promising new anti-parasitic drug lead, as its unique features may limit toxicity and drug-resistance, thus overcoming central limitations of most anti-parasitic drugs.
Asunto(s)
Leishmania/efectos de los fármacos , Péptidos/síntesis química , Péptidos/farmacología , Proteínas Protozoarias/antagonistas & inhibidores , Receptores de Superficie Celular/antagonistas & inhibidores , Tripanocidas/farmacología , Trypanosoma/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Antígenos de Protozoos/química , Diseño de Fármacos , Leishmania/química , Leishmania/genética , Leishmaniasis/tratamiento farmacológico , Leishmaniasis/parasitología , Ratones , Parasitemia/tratamiento farmacológico , Péptidos/administración & dosificación , Proteínas Protozoarias/química , Receptores de Cinasa C Activada , Receptores de Superficie Celular/química , Alineación de Secuencia , Tripanocidas/administración & dosificación , Tripanocidas/química , Trypanosoma/genética , Tripanosomiasis/tratamiento farmacológico , Tripanosomiasis/parasitologíaRESUMEN
Linear consensus motifs are short contiguous sequences of residues within a protein that can form recognition modules for protein interaction or catalytic modification. Protein kinase specificity and the matching of kinases to substrates have been mostly defined by phosphorylation sites that occur in linear consensus motifs. However, phosphorylation can also occur within sequences that do not match known linear consensus motifs recognized by kinases and within flexible loops. We report the identification of Thr(253) in α-tubulin as a site that is phosphorylated by protein kinase C ßI (PKCßI). Thr(253) is not part of a linear PKC consensus motif. Instead, Thr(253) occurs within a region on the surface of α-tubulin that resembles a PKC phosphorylation site consensus motif formed by basic residues in different parts of the protein, which come together in the folded protein to form the recognition motif for PKCßI. Mutations of these basic residues decreased substrate phosphorylation, confirming the presence of this "structurally formed" consensus motif and its importance for the protein kinase-substrate interaction. Analysis of previously reported protein kinase A (PKA) and PKC substrates identified sites within structurally formed consensus motifs in many substrates of these two kinase families. Thus, the concept of consensus phosphorylation site motif needs to be expanded to include sites within these structurally formed consensus motifs.
Asunto(s)
Fosfotransferasas/química , Secuencias de Aminoácidos , Animales , Catálisis , Bovinos , Proteínas Quinasas Dependientes de AMP Cíclico/química , Proteínas Fluorescentes Verdes/química , Células HEK293 , Células HeLa , Humanos , Lisina/química , Simulación del Acoplamiento Molecular , Mutagénesis Sitio-Dirigida , Mutación , Fosforilación , Pliegue de Proteína , Proteína Quinasa C/química , Treonina/química , Tubulina (Proteína)/químicaRESUMEN
Protein kinase C (PKC) plays a key role in embryonic stem cell (ESC) proliferation, self-renewal, and differentiation. However, the function of specific PKC isoenzymes have yet to be determined. Of the PKCs expressed in undifferentiated ESCs, ßIPKC was the only isoenzyme abundantly expressed in the nuclei. To investigate the role of ßΙPKC in these cells, we employed a phosphoproteomics strategy and used two classical (cPKC) peptide modulators and one ßIPKC-specific inhibitor peptide. We identified 13 nuclear proteins that are direct or indirect ßΙPKC substrates in undifferentiated ESCs. These proteins are known to be involved in regulating transcription, splicing, and chromatin remodeling during proliferation and differentiation. Inhibiting ßΙPKC had no effect on DNA synthesis in undifferentiated ESCs. However, upon differentiation, many cells seized to express ßΙPKC and ßΙPKC was frequently found in the cytoplasm. Taken together, our results suggest that ßIPKC takes part in the processes that maintain ESCs in their undifferentiated state.