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Molecular and Cellular Biology, August 2009, p. 4484-4494, Vol. 29, No. 16
0270-7306/09/$08.00+0     doi:10.1128/MCB.00252-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Hereditary Inclusion Body Myopathy-Linked p97/VCP Mutations in the NH2 Domain and the D1 Ring Modulate p97/VCP ATPase Activity and D2 Ring Conformation{triangledown}

Dalia Halawani,1 Andréa C. LeBlanc,2 Isabelle Rouiller,1 Stephen W. Michnick,3 Marc J. Servant,4 and Martin Latterich1,4*

Department of Anatomy and Cell Biology, McGill University, 3640 University Street, Montréal, Québec H3T 1E2,1 Department of Neurology and Neurosurgery, McGill University, 3801 University Street, Montréal, Québec H3A 2B4,2 Département de Biochimie, Université de Montréal Casier Postal 6128, Succursale Centre-Ville, Montréal, Québec H3C 3J7,3 Faculty of Pharmacy, Université de Montréal, Montréal, Québec H3T 1J4, Canada4

Received 25 February 2009/ Returned for modification 6 April 2009/ Accepted 29 May 2009

Hereditary inclusion body myopathy associated with early-onset Paget disease of bone and frontotemporal dementia (hIBMPFTD) is a degenerative disorder caused by single substitutions in highly conserved residues of p97/VCP. All mutations identified thus far cluster within the NH2 domain or the D1 ring, which are both required for communicating conformational changes to adaptor protein complexes. In this study, biochemical approaches were used to identify the consequences of the mutations R155P and A232E on p97/VCP structure. Assessment of p97/VCP oligomerization revealed that p97R155P and p97A232E formed hexameric ring-shaped structures of ~600 kDa. p97R155P and p97A232E exhibited an ~3-fold increase in ATPase activity compared to wild-type p97 (p97WT) and displayed increased sensitivity to heat-induced upregulation of ATPase activity. Protein fluorescence analysis provided evidence for conformational differences in the D2 rings of both hIBMPFTD mutants. Furthermore, both mutations increased the proteolytic susceptibility of the D2 ring. The solution structures of all p97/VCP proteins revealed a didispersed distribution of a predominant hexameric population and a minor population of large-diameter complexes. ATP binding significantly increased the abundance of large-diameter complexes for p97R155P and p97A232E, but not p97WT or the ATP-binding mutant p97K524A. Therefore, we propose that hIBMPFTD p97/VCP mutants p97R155P and p97A232E possess structural defects that may compromise the mechanism of p97/VCP activity within large multiprotein complexes.

* Corresponding author. Present address: The Nicholas Conor Institute for Pediatric Cancer Research, 9710 Scranton Road, Suite 170, San Diego, CA 92121. Phone: (858) 353-9667. Fax: (858) 450-9834. E-mail: mlatterich{at}gmail.com

{triangledown} Published ahead of print on 8 June 2009.

Molecular and Cellular Biology, August 2009, p. 4484-4494, Vol. 29, No. 16
0270-7306/09/$08.00+0     doi:10.1128/MCB.00252-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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