[The apoptosis-inducing activity of human selenoprotein P shorter isoform].
OBJECTIVE
Human selenoprotein P (HSelP) is unique protein that contains 10 selenocysteines encoded by 10 inframe UGA, which typically function as stop codon. The function of HSelP remains unclear, in part due to the inability to express it by gene recombinant technique. This study is to investigate expression and purification of recombinant HSelP in prokaryotic expression system, and its activity to induce apoptosis in vitro.


METHODS
The shorter HSelP isoform was cloned. After the selenocysteine (SeCys) at 40th position from N terminus of the HSelP shorter isoform was mutated into cysteine by PCR, it was expressed in E. coli. The expressed product was purified with DEAE column and identified by Western blot. Subsequently, its function on induction of mitochondrial apoptotic activity was studied.


RESULTS
The mutant HSelP shorter isoform expressed in prokaryotic system was purified by DEAE column to 90% homogeneity. The purified product, HSelP280m, induced the opening of mitochondrial permeability transition pore (PTP) and decreased the transmembrane potential in a dose-dependent manner. These events could be abolished by PTP specific inhibitors.


CONCLUSIONS
HSelP280m can induce the opening of mitochondrial PTP, which provides a basis for investigating the structure and function of recombinant HSelP.
Fang
Qing
Q
Department of Molecular Oncology, Cancer Institute (Hospital), Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing 100021, China.
Yi
Yao
Y
Zheng
Yan-hua
YH
Chen
Quan
Q
Ning
Li
L
Zha
Yuan-yuan
YY
Bi
Sheng-li
SL
Yang
Jian-guo
JG
Lin
Chen
C
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D003001:Cloning, Molecular; D003545:Cysteine / Q000235:genetics; D004926:Escherichia coli / Q000378:metabolism*; D006801:Humans; D007473:Ion Channels / Q000187:drug effects*; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects; D051379:Mice; D008807:Mice, Inbred BALB C; D008930:Mitochondria, Liver / Q000502:physiology*; D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; D009154:Mutation; D020033:Protein Isoforms; D011506:Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D012643:Selenium; D017279:Selenocysteine / Q000235:genetics; D051149:Selenoprotein P; D051140:Selenoproteins
D007473:Ion Channels; D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; D020033:Protein Isoforms; D011506:Proteins; D051149:Selenoprotein P; D051140:Selenoproteins; D017279:Selenocysteine; D012643:Selenium; D003545:Cysteine