RESUMO
Peptide aptamers are combinatorial recognition molecules that consist of a constant scaffold protein displaying a doubly constrained variable peptide loop. They bind specifically target proteins and interfere with their function. We have built a peptide aptamer library in a lentiviral expression system to isolate aptamers that inhibit cell proliferation in vitro. Using one of the isolated aptamers (R5G42) as a bait protein, we have performed yeast two-hybrid screening of cDNA libraries and identified calcineurin A as a target protein candidate. R5G42 bound calcineurin A in vitro and stimulated its phosphatase activity. When expressed transiently in human cells, R5G42 induced the dephosphorylation of BAD. We have identified an antiproliferative peptide aptamer that binds calcineurin and stimulates its activity. The use of this ligand may help elucidate the still elusive structural mechanisms of activation and inhibition of calcineurin. Our work illustrates the power of phenotypic screening of combinatorial protein libraries to interrogate the proteome and chart molecular regulatory networks.
Assuntos
Aptâmeros de Peptídeos/farmacologia , Calcineurina/biossíntese , Proliferação de Células/efeitos dos fármacos , Animais , Aptâmeros de Peptídeos/genética , Linhagem Celular Tumoral , Vetores Genéticos , Humanos , Biblioteca de Peptídeos , Monoéster Fosfórico Hidrolases/metabolismo , Ratos , Vírus da Imunodeficiência Símia/genética , Técnicas do Sistema de Duplo-Híbrido , Regulação para CimaRESUMO
A major limitation of current lentiviral vectors (LVs) is their inability to govern efficient gene transfer into quiescent cells, such as human CD34(+) cells, that reside in the G(0) phase of the cell cycle and that are highly enriched in hematopoietic stem cells. This hampers their application for gene therapy of hematopoietic cells. Here, we designed novel LVs that overcome this restriction by displaying "early-acting cytokines" on their surface. Display of thrombopoietin, stem cell factor, or both cytokines on the LV surface allowed efficient gene delivery into quiescent cord blood CD34(+) cells. Moreover, these surface-engineered LVs preferentially transduced and promoted survival of resting CD34(+) cells rather than cycling cells. Finally, and most importantly, these novel LVs allowed superior gene transfer in the most immature CD34(+) cells as compared to conventional LVs, even when the latter vectors were used to transduce cells in the presence of recombinant cytokines. This was demonstrated by their capacity to promote selective transduction of CD34(+) cell in in vitro derived long-term culture-initiating cell (LTC-IC) colonies and of long-term NOD/SCID repopulating cells (SRCs) in vivo.
Assuntos
Antígenos CD34 , Terapia Genética , Células-Tronco Hematopoéticas , Lentivirus , Fator de Células-Tronco , Trombopoetina , Animais , Antígenos CD34/metabolismo , Transplante de Células-Tronco de Sangue do Cordão Umbilical , Terapia Genética/métodos , Células-Tronco Hematopoéticas/fisiologia , Humanos , Lentivirus/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Fase de Repouso do Ciclo Celular/fisiologia , Fator de Células-Tronco/genética , Fator de Células-Tronco/metabolismo , Trombopoetina/genética , Trombopoetina/metabolismo , Transdução Genética/métodos , Transplante HeterólogoRESUMO
We recently demonstrated that the product of the HERV-W env gene, a retroviral envelope protein also dubbed syncytin, is a highly fusogenic membrane glycoprotein inducing the formation of syncytia on interaction with the type D mammalian retrovirus receptor. In addition, the detection of HERV-W Env protein (Env-W) expression in placental tissue sections led us to propose a role for this fusogenic glycoprotein in placenta formation. To evaluate this hypothesis, we analyzed the involvement of Env-W in the differentiation of primary cultures of human villous cytotrophoblasts that spontaneously differentiate by cell fusion into syncytiotrophoblasts in vitro. First, we observed that HERV-W env mRNA and glycoprotein expression are colinear with primary cytotrophoblast differentiation and with expression of human chorionic gonadotropin (hCG), a marker of syncytiotrophoblast formation. Second, we observed that in vitro stimulation of trophoblast cell fusion and differentiation by cyclic AMP is also associated with a concomitant increase in HERV-W env and hCG mRNA and protein expression. Finally, by using specific antisense oligonucleotides, we demonstrated that inhibition of Env-W protein expression leads to a decrease of trophoblast fusion and differentiation, with the secretion of hCG in culture medium of antisense oligonucleotide-treated cells being decreased by fivefold. Taken together, these results strongly support a direct role for Env-W in human trophoblast cell fusion and differentiation.
