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57th Annual Meeting of the German Society for Neuropathology and Neuroanatomy (DGNN)

German Society for Neuropathology and Neuroanatomy

12. - 15.09.2012, Erlangen

57th Annual Meeting of the German Society for Neuropathology and Neuroanatomy (DGNN)

Heteromeric p97/p97R155C complexes induce dominant negative changes in wild-type and autophagy 9-deficient Dictyostelium cells

Meeting Abstract

  • presenting/speaker Khalid Arhzaouy - Institut for Biochemistry I, Cologne, Germany
  • Karl-Heinz Strucksberg - Institute of Neuropathology, Erlangen, Germany
  • Sze Man Tung - Institut for Biochemistry I, Cologne, Germany
  • Karthikeyan Tangavelou - Institut for Biochemistry I, Cologne, Germany
  • Maria Stumpf - Institut for Biochemistry I, Cologne, Germany
  • Jan Faix - Institute for Biophysical Chemistry, Hannover, Germany
  • Rolf Schröder - Institute of Neuropathology, Erlangen, Germany
  • Christoph Clemen - Institut for Biochemistry I, Cologne, Germany
  • Ludwig Eichinger - Institut for Biochemistry I, Cologne, Germany

Deutsche Gesellschaft für Neuropathologie und Neuroanatomie. 57th Annual Meeting of the German Society for Neuropathology and Neuroanatomy (DGNN). Erlangen, 12.-15.09.2012. Düsseldorf: German Medical Science GMS Publishing House; 2012. Doc12dgnnOP05

DOI: 10.3205/12dgnn005, URN: urn:nbn:de:0183-12dgnn0057

Published: September 11, 2012

© 2012 Arhzaouy et al.
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Outline

Text

p97 (VCP or valosin containing protein in mammals and Cdc48p in yeast) is a ubiquitously expressed and evolutionarily highly conserved hexameric member of the magnesium-dependent Walker P-loop AAA-ATPases. p97 has been associated with various essential cellular processes including ubiquitin-proteasome protein degradation and autophagy. Point mutations in the human p97 gene cause autosomal-dominant IBMPFD (Inclusion Body Myopathy with early-onset Paget disease and Frontotemporal Dementia) or ALS14 (Amyotrophic Lateral Sclerosis). Most prevalent is the R155C mutation. We studied the function of p97 in Dictyostelium discoideum, a social amoeba and well-established model organism for the study of basic biological processes, and have generated strains that ectopically express p97-RFP or p97R155C-RFP in AX2 wild-type and autophagy 9 (ATG9) knock-out cells. Co-immunoprecipitation studies using an anti-RFP antibody showed that endogenous p97 and p97R155C-RFP form heteromers. The mutant strains displayed changes in cell growth, phagocytosis and phototaxis indicating misregulation of multiple essential cellular processes. Immunofluorescence analysis revealed an increase of protein aggregates in ATG9KO/p97R155C-RFP and ATG9KO cells. They were positive for ubiquitin in both strains, however, immunoreactive for p97 only in the ATG9KO mutant. Immunoblotting showed an increase of ubiquitinated proteins and of the autophagy marker ATG8(LC3). Proteasomal activity was slightly reduced in AX2/p97R155C-RFP cells, but nearly completely inhibited in the ATG9KO mutant and partially rescued in the ATG9KO/p97R155C-RFP double mutant. The observed R155C specific cellular alterations in the single versus the double mutants suggest a novel mode of p97 regulation based on the interaction and mutual inhibition of wild-type p97 and core autophagy proteins.