「様式３－B」

課題番号（Project number） 22

Models for normal human aging by focusing on Werner syndrome
and bioenergetics in cells and mice; the presence of a
WRN-interacting protein, Polymerase  in mitochondria［１］組織（Research group）
代表者（Principal Investigator (PI)）：
Vilhelm Bohr（National Institute on Aging, NIH）
対応者（Host researcher at IDAC）：
Akira Yasui, Shin-ichiro Kanno（IDAC, Tohoku University）
研 究 費 (Expenditure report of researchfunds)： consumable goods 300,000 Yen，
［２］研究経過（Research setup）
Werner syndrome (WS) is a progeroid disease caused bymutations in the gene coding for Werner protein (WRN).
WS patients demonstrate many symptoms of normal
human aging when they are adolescent or young adultpatients. Therefore, it is generally accepted that WS is agood model for normal human aging. At the cellular level,
evidence of cellular pathology is also similar in cells from
WS patients and older normal individuals. Importantly,
WS shares features with human mitochondrial disorders.
While WRN contributes directly to nuclear genome
integrity (Bohr, 2008), very little is known about
the role of WRN in mitochondrial metabolism andwhy WS patient shows early aging remains still obscure.

Fig. 1 Werner syndrome patient

Since we showed previously the interaction
between WRN and Pol and the presence of
WRN in mitochondria, we wanted to knowwhether pol is present in mitochondria as well.
Until now DNA polymerase  is believed to be theonly DNA polymerase in mitochondria. We haveseparately determined the presence of Pol inhuman mitochondria by immune-staining andwestern blotting. Here we wanted to characterize
mitochondrial proteins interacting with Pol byaffinity screening of human mitochondrial
extracts. Mitochondrial fraction was preparedfrom human cell in NIA/NIH and transferred toIDAC for affinity screening of interacting proteinsto Pol. We discussed the project and the resultswith Skype and e-mail communication.
Mitochondrial extracts were prepared inNIA/NIH and sent to IDAC/Tohoku University
for affinity screening of the proteins in theextracts to GST-fused Pol (domains). The resultsof this collaboration are now in a paper submitted
for publication, and, therefore, we are not able todisclose the data in details in this report.
［３］成果（Research outcomes）（３－１）研究成果（Results）For the analysis of the function of Pol inmitochondria our approach is to identify thehuman mitochondrial proteins, which interactwith Pol. We prepared recombinant proteins ofGST-fused Pol-N and Pol-C domains andscreened
binding
proteins
inhuman
mitochondrial extracts in vitro (Fig. 2A and 2B).
The bound proteins to each domain wereseparated by SDS gel and 18 proteins weredetermined with mass spectrometry using nano
LC-MS/MS (Fig. 3). Thus determined
mitochondrial proteins were classified as 1. DNA
maintenance. 2. Stress response 3. RNAprocessing. 4. Metabolism. All the data are novelinteractions between Pol and mitochondrial
proteins, which should be further characterized
to understand the function of Pol inmitochondria.
In addition to the determination by mass
spectrometry through the screening, we applied
western analysis of the blotted mitochondrial
proteins by using antibodies against proteins
important for protein transport into mitochondria,
TOM70 and TIM50, for replication andtranscription, TFAM as well as for helicase,
TWINCLE. We found the presence of these
proteins on the blot of affinity screening done with
GST-Pol-N (not shown). The interaction betweenPol and WRN in mitochondria remains to bedetermined.
Fig. 2. Experimental procedure
Fig. 3. Gel electrophoresis of affinity screening
with GST-Pol-N and -C

AdRP Lyase
domain

Polb

1

Polb-N

1

Polb-CB

Polymerase
domain
335
141

141

335

［４］成果資料（List of Papers）
The results shown in this report is a part of paper
submitted for publication.