engleski

Induction of apoptosis by transduction of p27

The present study was examined, how transduced proteins influence on the regulation of cell cycle. TAT peptides, derived from the HIV-1 TAT protein, facilitate intracellular delivery of proteins and small colloidal particles into the cells. TAT fusion protein enters into the cells when added to surrounding media, in concentration depended manner. After entering, TAT component is degraded by proteolytic enzymes, protein is refolded with the cell chaperons (Hsp proteins) and ready for its physiological function. Cell plasma membranes are impermeable for proteins and peptides. Because of that, the potential for intracellular therapeutic use of proteins, peptides and oligonucleotides has been limited by the impermeable nature of the cell membrane to these compounds. Thus protein transduction has been overcame that obstacle, and it has been widely used to analyze biochemical processes in living cells. Recombinant proteins (p27, Mp27 and p23) were expressed in BL21 bacteria, purified on Ni-NTA column, and desalted on PD10 column. The proteins (p27, p23, Mp27), at concentration of 100 nM, were transduced into different cell lines (NALM, MOLT, Raji, SuDHL, and K562). Their effects on the proliferation and regulation of cell cycle were measured. Transduced p27 did not remarkable influence on proliferation of examined cell lines. Mutated p27 inhibited the proliferation of examined cell lines up to 30 %. On the other hand, a transduction of p23 protein, truncated form of p27, inhibited the proliferation all of examined cell lines 30-60 %. Also, the effects of transduced proteins on induction of apoptosis, was examined. By Western blot was shown that transduction of p27 and Mp27 induced the activation of Caspase 3, a proteolytic enzyme responsible for apoptosis.

apoptosis; p27; cell cycle; protein transduction

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