RESUMEN
Direct transduction of the homeobox (HOX) protein HOXB4 promotes the proliferation of hematopoietic stem cells (HSCs) without induction of leukemogenesis, but requires frequent administration to overcome its short protein half-life (â¼1 hour). We demonstrate here that HOXB4 protein levels are post-translationally regulated by the CUL4 ubiquitin ligase, and define the degradation signal sequence (degron) of HOXB4 required for CUL4-mediated destruction. Additional HOX paralogs share the conserved degron in the homeodomain and are also subject to CUL4-mediated degradation, indicating that CUL4 likely controls the stability of all HOX proteins. Moreover, we engineered a degradation-resistant HOXB4 that conferred a growth advantage over wild-type HOXB4 in myeloid progenitor cells. Direct transduction of recombinant degradation-resistant HOXB4 protein to human adult HSCs significantly enhanced their maintenance in a more primitive state both in vitro and in transplanted NOD/SCID/IL2R-γ(null) mice compared with transduction with wild-type HOXB4 protein. Our studies demonstrate the feasibility of engineering a stable HOXB4 variant to overcome a major technical hurdle in the ex vivo expansion of adult HSCs and early progenitors for human therapeutic use.
Asunto(s)
Células Madre Adultas/fisiología , Proliferación Celular , Proteínas Cullin/fisiología , Células Madre Hematopoyéticas/fisiología , Proteínas de Homeodominio/metabolismo , Factores de Transcripción/metabolismo , Adulto , Células Madre Adultas/metabolismo , Animales , Técnicas de Cultivo de Célula/métodos , Células Cultivadas , Proteínas Cullin/genética , Proteínas Cullin/metabolismo , Estudios de Factibilidad , Células HeLa , Células Madre Hematopoyéticas/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/fisiología , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Ratones Transgénicos , Cultivo Primario de Células/métodos , Ingeniería de Proteínas , Proteolisis , Factores de Transcripción/genética , Factores de Transcripción/fisiologíaRESUMEN
It is believed that memory reactivation transiently renders consolidated memory labile and that this labile or deconsolidated memory is reconsolidated in a protein synthesis-dependent manner. The synaptic correlate of memory deconsolidation upon reactivation, however, has not been fully characterized. Here, we show that 3,5-dihydroxyphenylglycine (DHPG), an agonist for group I metabotropic glutamate receptors (mGluRI), induces synaptic depotentiation only at thalamic input synapses onto the lateral amygdala (T-LA synapses) where synaptic potentiation is consolidated, but not at synapses where synaptic potentiation is not consolidated. Using this mGluRI-induced synaptic depotentiation (mGluRI-depotentiation) as a marker of consolidated synapses, we found that mGluRI-depotentiation correlated well with the state of memory deconsolidation and reconsolidation in a predictable manner. DHPG failed to induce mGluRI-depotentiation in slices prepared immediately after reactivation when the reactivated memory was deconsolidated. DHPG induced mGluRI-depotentiation 1 h after reactivation when the reactivated memory was reconsolidated, but it failed to do so when reconsolidation was blocked by a protein synthesis inhibitor. To test the memory-specificity of mGluRI-depotentiation, conditioned fear was acquired twice using two discriminative tones (2.8 and 20 kHz). Under this condition, mGluRI-depotentiation was fully impaired in slices prepared immediately after reactivation with both tones, whereas mGluRI-depotentiation was partially impaired immediately after reactivation with the 20 kHz tone. Consistently, microinjection of DHPG into the LA 1 h after reactivation reduced fear memory retention, whereas DHPG injection immediately after reactivation failed to do so. Our findings suggest that, upon memory reactivation, consolidated T-LA synapses enter a temporary labile state, displaying insensitivity to mGluRI-depotentiation.