RESUMEN
The pro-apoptotic BH3-only protein Bim has a major role in hematopoietic homeostasis, particularly in the lymphocyte compartment, where it strongly affects immune function. The three major Bim isoforms (Bim(EL), Bim(L) and Bim(S)) are generated by alternative splicing. Bim(EL), the most abundant isoform, contains a unique sequence that has been reported to be the target of phosphorylation by several MAP kinases. In particular, Erk1/2 has been shown to interact with Bim(EL) through the DEF2 domain of Bim(EL) and specifically phosphorylate this isoform, thereby targeting it for ubiquitination and proteasomal degradation. To examine the physiological importance of this mechanism of regulation and of the alternative splicing of Bim, we have generated several Bim knock-in mouse strains and analyzed their hematopoietic system. Although mutation in the DEF2 domain reduces Bim(EL) degradation in some circumstances, this mutation did not significantly increase Bim's pro-apoptotic activity in vivo nor impact on the homeostasis of the hematopoietic system. We also show that Bim(EL) and Bim(L) are interchangeable, and that Bim(S) is dispensable for the function of Bim. Hence, we conclude that physiological regulation of Bim relies on mechanisms independent of its alternative splicing or the Erk-dependent phosphorylation of Bim(EL).
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas de la Membrana/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Empalme Alternativo , Animales , Apoptosis , Proteínas Reguladoras de la Apoptosis/química , Proteínas Reguladoras de la Apoptosis/genética , Proteína 11 Similar a Bcl2 , Técnicas de Sustitución del Gen , Hematopoyesis/fisiología , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Ratones , Fosforilación , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Timocitos/metabolismoRESUMEN
This study examined the incidence of human herpes virus-6 (HHV-6) and human cytomegalovirus (HCMV) infections that are potentially transmitted to haematopoietic stem cells (HSC) transplant recipients via bone marrow (BM) or umbilical cord blood (UCB). Bone marrow progenitor cells were collected from 30 allogenic BM donors. UCB HSC were collected from 34 subjects. The extracted DNA was then processed using nested polymerase chain reaction (nPCR) technique. HCMV and HHV-6 serological status were determined by enzyme immunoassay (EIA). Nested PCR identified HCMV in 22 (73%) of 30 samples of BM progenitor cells but in only eight (23.5%) of 34 samples of UBC HSC ( P = 0.001). HHV-6 DNA was detected in 11 (36.6%) of 30 BM progenitor cells and in only one (2.9%) of 34 UBC cells ( P = 0.002). Both HHV-6 and HCMV infections were determined in nine (26.5%) of 34 bone marrow samples. The results indicate that, the risk of HCMV and HHV-6 via BM progenitor cells is higher than transmission by UCB cells ( P= 0.04).
Asunto(s)
Médula Ósea/virología , Citomegalovirus/aislamiento & purificación , Sangre Fetal/virología , Células Madre Hematopoyéticas/virología , Herpesvirus Humano 6/aislamiento & purificación , Células Progenitoras Mieloides/virología , Adulto , Anticuerpos Antivirales/sangre , Citomegalovirus/genética , Infecciones por Citomegalovirus , ADN Viral/genética , Femenino , Herpesvirus Humano 6/genética , Humanos , Técnicas para Inmunoenzimas , Incidencia , Masculino , Reacción en Cadena de la Polimerasa , Infecciones por Roseolovirus/epidemiologíaRESUMEN
We have recently demonstrated that ablation of one or both alleles of the proapoptotic gene Bim prevents the polycystic kidney disease (PKD) that develops in mice deficient for the prosurvival protein Bcl-2. The aim of the present study was to investigate whether loss of Bim or Bcl-2 could influence the disease in the PKD1del34/del34 mutant mice, a model of autosomal dominant PKD. PKD1del34/del34 mice were intercrossed with Bim-deficient mice and Bcl-2+/- mice to generate double mutants. Loss of Bim does not prevent the development of PKD in PKD1del34/del34 mice. On the C57BL/6 genetic background, most older PKD1del34/+ mice do not develop PKD, but present with liver cysts. Surprisingly, loss of Bim completely prevented liver cysts formation in PKD1del34/+ mice. Loss of one Bcl-2 allele did not influence the PKD1del34 phenotype significantly. We conclude that loss of PKD1 and loss of Bcl-2 elicit PKD through distinct mechanisms.
Asunto(s)
Enfermedades Renales Poliquísticas/fisiopatología , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Canales Catiónicos TRPP/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/fisiología , Proteína 11 Similar a Bcl2 , Femenino , Genotipo , Riñón/metabolismo , Riñón/patología , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Enfermedades Renales Poliquísticas/genética , Enfermedades Renales Poliquísticas/metabolismo , Reacción en Cadena de la Polimerasa , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/fisiología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Transducción de Señal/fisiologíaRESUMEN
Hematopoietic stem cell transplantation (HSCT) is the treatment of choice for children and certain adults with malignant and nonmalignant hematologic disease. Since viral infections are the major problem, this study examined those that might potentially be transmitted to HSCT recipients via bone marrow (BM) versus umbilical cord blood (UCB). BM progenitor cells, peripheral blood leukocytes, and plasma samples were collected from 30 allogenic BM donors. Umbilical cord blood hematopoietic stem cells and plasma samples were also collected from 34 UCB donors. Viral DNA extracted and purified from collected specimens was processed using nested polymerase chain reactions (PCR) to detect human parvovirus B19 (HPV B19), human herpesvirus-6 (HHV-6), varicella-zoster virus (VZV), human cytomegalovirus (HCMV), and Epstein-Barr virus (EBV). The prevalences of HCMV DNA in collected BM progenitor cells versus UCB hematopoietic stem cells were 73% versus 23%, respectively. Conversely, HHV-6 DNA was not detected in any collected specimen by simple PCR. Distribution of the other investigated virus DNAs except EBV DNA was similar in specimens collected from both groups. EBV DNA was not determined in UCB hematopoietic stem cells. The results indicate that the risk of viral transmission to BM transplant recipients via UCB hematopoietic stem cells is less than that with BM progenitor cells.
