Abstract
Von Hippel-Lindau tumor suppressor protein (pVHL) functions to induce neuronal differentiation of neural stem/progenitor cells (NSCs) and skin-derived precursors (SKPs). Here we identified a neuronal differentiation domain (NDD) in pVHL. Neuronal differentiation of SKPs was induced by intracellular delivery of a peptide composed of the amino-acid sequences encoded by the NDD. Neuronal differentiation mediated by the NDD was caused by the binding between it and elongin C followed by Janus kinase-2 (JAK2) ubiquitination of JAK2 and inhibition of the JAK2/the signal transducer and activator of transcription-3(STAT)3 pathway. The NDD in pVHL contained the BC-box motif ((A,P,S,T)LXXX (A,C) XXX(A,I,L,V)) corresponding to the binding site of elongin C. Therefore, we proposed that other BC-box proteins might also contain an NDD; and subsequently also identified in them an NDD containing the amino-acid sequence encoded by the BC-box motif in BC-box proteins. Furthermore, we showed that different NDD peptide-delivered cells differentiated into different kinds of neuron-like cells. That is, dopaminergic neuron-like cells, cholinergic neuron-like cells, GABAnergic neuron-like cells or rhodopsin-positive neuron-like cells were induced by different NDD peptides. These novel findings might contribute to the development of a new method for promoting neuronal differentiation and shed further light on the mechanism of neuronal differentiation of somatic stem cells.
Highlights
We demonstrated that von Hippel-Lindau tumor suppressor protein promotes the neuronal differentiation of neural stem/progenitor cells (NSCs) and skin-derived precursors (SKPs) and suggested a relationship between it and neuronal differentiation [1,2,3]. pVHL-derived peptide containing the BC-box motif ((A,P,S,T)LXXX (A,C) XXX(A,I,L,V)) [4] functions to promote neuronal differentiation in those cells: neural stem cells [5,6], bone marrow stromal cells [7] and skin-derived precursors (SKPs) [8,9]
We recognized that the BC-box motif plus five amino acids (VHL(157–171)) played a role as an neuronal differentiation domain (NDD) within pVHL but that the BC-box motif plus two amino acids (VHL(157–168)) scarcely had neuronal differentiation activity in the morphological study, immunocytochemistry, immunohistochemistry, electophysiological study, and Wesntern blotting analysis
The immunoprecipitation study showed a firm binding between elongin C and BC-box motif plus five amino acids (VHL(157–171)) and a very weak binding between elongin C and BC-box motif plus two amino acids (VHL(157–168))
Summary
We demonstrated that von Hippel-Lindau tumor suppressor protein (pVHL) promotes the neuronal differentiation of neural stem/progenitor cells (NSCs) and skin-derived precursors (SKPs) and suggested a relationship between it and neuronal differentiation [1,2,3]. pVHL-derived peptide containing the BC-box motif ((A,P,S,T)LXXX (A,C) XXX(A,I,L,V)) [4] functions to promote neuronal differentiation in those cells: neural stem cells [5,6], bone marrow stromal cells [7] and skin-derived precursors (SKPs) [8,9]. Underline = protein transduction domain; double underline = BC box motif; all species = human To identify this NDD in detail, we first examined the neurite outgrowth activities of TAT(YARAAARQARA), VHL(159–171), VHL(157–166), VHL(157–168), and VHL(157–171). Protein expression levels in various NDD peptide-mediated neuronal differentiation were examined by Western blot analysis, with the following results: Expression of TH was pronounced in SOCS7 peptide-treated cells, and moderate in SOCS 1, 3, 6, WSB2, and VHL(157–171)-treated cells. High rates of positive cells were found for TH-positive cells (dopaminergic neuron-like cells) treated by peptides derived from SOCS 1–3, 5, 6, ASB3, WSB2, LRR1, and VHL, and for rhodopsin-positive cells by peptides derived from SOCS5 and VHL (Figure 6B).
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