Saningong, Akuma Divine:

Functional studies on Par14/Par17 with emphasis on chromatin, the cell cycle, and protein-protein interactions

Duisburg, Essen (2010), XV, 144 Bl.
Dissertation / Fach: Biologie
Fakultät für Biologie
Bayer, Peter (Doktorvater, Betreuerin)
Iliakis, George (GutachterIn)
Duisburg, Essen, Univ., Diss., 2010
Abstract:
Par14 and Par17 are members of the parvulin family of peptidyl-prolyl cis-trans iso-merases. Par14 has been shown to be enriched in the nucleus and Par17 was dem-onstrated to be located in the mitochondrial matrix. It has been suggested that Par14 plays a role in chromatin remodeling on basis of sequence and structural identities to HMGB and HMGN proteins. Both Par14 and Par17 have been shown to bind to double-stranded DNA in vitro. However, the cellular functions of both proteins have not been characterised, and we aimed in this study to give insights to their function. By means of biochemical fractionations, we have shown that Par14 is enriched in chromatin 3-fold higher than in the nuclear matrix. In the same experiments, we also observed that Par14 was released from the chromatin fraction after treatment with DNase I indicating its binding to DNA. In the light of this, we performed another bio-chemical fractionation scheme enriching nucleic acid-binding proteins. Using a phos-pho-cellulose column, we eluted Par14 at 0.35 and 1 M NaCl from the nucleic acid-binding fraction. This gave credit to the fact that Par14 was associated with DNA in vivo. Following Par14’s association with chromatin, pilot experiments were initiated using Chromatin Affinity Purification (ChAP) to search for DNA-binding motifs for Par14. Pin1 is the other member of the parvulin family and it has been characterised as a mitotic regulator. As a result, we were interested to investigate the role of Par14/Par17 in the cell cycle. Using our established cell cycle synchronisation system by serum deprivation, we showed with the aid of qRT-PCR that Par14 and Par17 were 2- and 3-fold up-regulated respectively during the S-phase at the mRNA level. A 3- and 5-fold up-regulation was seen for Par14 and Par17 in the G2/M phase respectively. The transcriptional increase of Par14 in the S and G2/M phases was correlated with a 4-fold translational increase as analysed by western blotting. As concerns Par17, we observed that Par17 was 4- and 3-fold up-regulated when cellular lysates from the individual cell cycle phases were used. Moreover, there was the occurrence of a 28 kDa variant of Par17 in all the phases of the cell cycle before and after the fractionation of the cellular lysates. As a strategy to elucidate the function of Par14/Par17, we used affinity purification coupled with mass spectrometry to find their interaction partners. A Par17 construct fused to Strep-tag was transfected in HCT116 cells and the cellular lysates were passed over a modified streptavidin (Strep-Tactin) column. Elution was done with desthiobiotin and the eluted proteins were sent for tandem mass spectrometry. After filtering spurious and non-specific binders, EZR was among the 17 potential interaction partners of both parvulin proteins. EZR plays a role in the cytoskeletal network; but it remains to be validated by other techniques whether it is a bona fide Par14/Par17 interactor. We were interested to know if some of the potential interaction partners found were up- or down-regulated in the advent of a successful Par14/Par17 knock-down. Our protein-protein interaction data paved the way to perform gene knock-down experiments for Par14/Par17 using siRNA technology. HeLa and HCT116 cells were transfected and knock-down was investigated by performing qRT-PCR and western blot experiments. No appreciable knock-down of Par14/Par17 mRNAs and their gene products was registered. Fluorescence microscopic studies were also done on transfected cells to check for any morphological abnormalities as a result of Par14/Par17 loss. Still, no visible phenotypic changes were observed. Taken together, Par14 can be described as a chromatin and DNA-binding protein. The transcriptional up-regulation of Par14 and Par17 was directly proportional to their translational up-regulation. If EZR is validated as an interaction partner of Par14/Par17, this may suggest a role of Par14/Par17 as anchor proteins that link the cytoskeletal apparatus to a DNA-related process in the nucleus or in the mitochondria.