Recombinant human TAT-OP1 to enhance NGF neurogenic potential: preliminary studies on PC12 cells.
Osteogenic protein 1 (OP1), also known as bone morphogenic protein-7 (BMP7), is a multifunctional cytokine with demonstrated neurogenic potential. As the recombinant OP1 (rhOP1) was shown to provide axonal guidance cues and to prevent the reduction of dendritic growth in the injury-induced cortical cultures, it was suggested that an in vivo efficient rhOP1 delivery could enhance neurite growth and functional reconnectivity in the damaged brain. In the present work, we engineered a chimeric molecule in which rhBMP7 was fused to a protein transduction domain derived from HIV-1 TAT protein to deliver the denatured recombinant BMP7 into cells and obtain its chaperone-mediated folding, circumventing the expensive and not much efficient in vitro refolding procedures. When tested on rat PC12 cells, a widely used in vitro neurogenic differentiation model, the resulting fusion protein (rhTAT-OP1) demonstrated to enter fastly into the cells, lose HIV-TAT sequence and interact with membrane receptors activating BMP pathway by SMAD 1/5/8 phosphorylation. In comparison with nerve growth factor (NGF) and BMP7, it proved itself effective to induce the formation of more organized H and M neurofilaments. Moreover, if used in combination with NGF, it stimulated a significant (P < 0.05) and more precocious dendritic outgrowth with respect to NGF alone. These results indicate that rhTAT-OP1 fused with TAT transduction domain shows neurogenic activity and may be a promising enhancer factor in NGF-based therapies.
Di Liddo
R
R
Department of Pharmaceutical Sciences, University of Padova, Via Marzolo 5, Padua, Italy. rosa.diliddo@unipd.it
Grandi
C
C
Venturini
M
M
Dalzoppo
D
D
Negro
A
A
Conconi
M T
MT
Parnigotto
P P
PP
D000595:Amino Acid Sequence; D000818:Animals; D055419:Bone Morphogenetic Protein 7 / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D049109:Cell Proliferation; D003712:Dendrites / Q000378:metabolism; D015870:Gene Expression; D006678:HIV / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D020932:Nerve Growth Factor / Q000378:metabolism*; D055495:Neurogenesis; D016716:PC12 Cells; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
C525819:BMP7 protein, human; D055419:Bone Morphogenetic Protein 7; D011994:Recombinant Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus; D020932:Nerve Growth Factor