Cloning and functional characterization of the guinea pig apoptosis inhibitor protein Survivin.
BACKGROUND
The guinea pig is widely used as a model to study (patho)physiological processes, such as neurodegenerative disorders. Survivin's dual function as an apoptosis inhibitor and a mitotic regulator is crucial not only for ordered development but its modulation seems crucial also under disease conditions. However, data on the expression and function of the guinea pig Survivin protein (Survivin(Gp)) are currently lacking.


RESULTS
Here, we here report the cloning and functional characterization of Survivin(Gp). The respective cDNA was cloned from spleen mRNA, containing a 426 bp open reading frame encoding for a protein of 142aa. Survivin(Gp) displays a high homology to the human and murine orthologue, especially in domains critical for function, such as binding sites for chromosomal passenger complex (CPC) proteins and the nuclear export signal (NES). Notably, phylogenetic analyses revealed that Survivin(Gp) is more related to humans than to rodents. Ectopic expression studies of a Survivin(Gp)-GFP fusion confirmed its dynamic intracellular localization, analogous to the human and murine counterparts. In interphase cells, Survivin(Gp)-GFP was predominantly cytoplasmic and accumulated in the nucleus following export inhibition with leptomycin B (LMB). A typical CPC protein localization during mitosis was observed for Survivin(Gp)-GFP. Microinjection experiments together with genetic knockout demonstrated that the NES is essential for the anti-apoptotic and regulatory role of Survivin(Gp) during cell division. In vivo protein interaction assays further demonstrated its dimerization with human Survivin and its interaction with human CPC proteins. Importantly, RNAi-depletion studies show that Survivin(Gp) can fully substitute for human Survivin as an apoptosis inhibitor and a mitotic effector. Immunohistochemistry, immunofluorescence, and western blotting were employed to detect Survivin expression in guinea pig tissues. Besides its expression in proliferating tissues, such as spleen and liver, we also found Survivin in terminally differentiated cell types. Importantly, Survivin was detectable also in the cochlea, suggesting a potential role for Survivin in the auditory system.


CONCLUSIONS
We provide the first experimental evidence for the expression of Survivin in the guinea pig. As Survivin(Gp) can substitute for known functions of human Survivin, the guinea pig model will now also allow investigating Survivin's (patho)physiological role and to test Survivin-directed potential therapeutic strategies.
Habtemichael
Negusse
N
Institute for Molecular and Cellular Oncology, University Hospital of Mainz, Langenbeckstrasse 1, 55101 Mainz, Germany.
Wünsch
Desiree
D
Bier
Carolin
C
Tillmann
Sarah
S
Unruhe
Britta
B
Frauenknecht
Katrin
K
Heinrich
Ulf-Rüdiger
UR
Mann
Wolf J
WJ
Stauber
Roland H
RH
Knauer
Shirley K
SK
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D007758:Ear, Inner / Q000378:metabolism; D006168:Guinea Pigs / Q000235:genetics*; D051034:Inhibitor of Apoptosis Proteins / Q000235:genetics*; D008869:Microtubule-Associated Proteins; D008938:Mitosis; D023421:Models, Animal; D008969:Molecular Sequence Data; D010802:Phylogeny; D017386:Sequence Homology, Amino Acid; D000077022:Survivin; D014162:Transfection
C107489:BIRC5 protein, human; D051034:Inhibitor of Apoptosis Proteins; D008869:Microtubule-Associated Proteins; D000077022:Survivin