Identification of novel and disease-related VCP/p97 binding partners and effects of disease-causing VCP/p97 mutations on protein interactions and protein quality control systems in Dictyostelium and mouse
Final Report Abstract
In our project we analyzed aspects of the complex molecular pathophysiology of VCP/p97-related myofibrillar myopathies employing a broad spectrum of experimental techniques in vitro and in cell as well as Dictyostelium, zebrafish and mouse models. VCP/p97 is a very abundant and evolutionarily highly conserved member of the triple-A ATPase family involved in a plethora of cellular processes such as membrane dynamics, protein quality control, cell cycle, apoptosis, and DNA damage response. More than 40 different disease-causing missense mutations of VCP/p97 have been described in human, which cause neurodegenerative disorders, e.g. IBMPFD, ALS and HSP. Our search for novel and disease-relevant VCP/p97 binding partners resulted in the identification of strumpellin. In human, missense mutations of strumpellin also cause a form of HSP. Moreover, our genetic analysis of a cohort of 43 ALS-FTD patients for mutations in VCP/p97, strumpellin, WASH1, FAM21C, CCDC53, SWIP, CapZα, and CapZβ uncovered one patient with a putatively pathogenic heterozygous CAPZA1 variant. Thus, mutant forms of VCP/p97 or WASH complex subunits may have a concerted pathogenic role in various human neurodgenerative diseases. Another focus of our work was on the functional consequences of point mutated VCP/p97 in mouse, zebrafish and Dictyostelium with special emphasis on autophagy and proteasome activity. In our VCP/p97 haploinsufficient mouse model the reduced amount of VCP/p97 protein caused a decrease in proteasomal activity in conjunction with an increase in ubiquitinated proteins. Still ongoing work on our R155C VCP/p97 knock-in mouse strain focuses on the analysis of the consequences of the expression of R155C VCP/p97 in heterozygous animals. Thus far, we could not detect any alterations, which mirror the human disease phenotype, however, we will further investigate unexpected alterations which have not been linked to IBMPFD, ALS, or HSP. Furthermore, we identified another novel direct VCP/p97 interaction partner, the endogenous proteasome inhibitor PSMF1/PI31. This led to novel insights into the basic regulation of proteasomal activity and we anticipate that our findings may have substantial implications for the molecular understanding of ageing and human diseases. In Dictyostelium, the induction of dominant-negative changes through expression of mutant VCP/p97 revealed another novel mode of VCP/p97 interaction. The results imply that VCP/p97 and the core autophagy protein ATG9 mutually inhibit each other. As a further protein-protein interaction, we analyzed the interaction of VCP/p97 with UBXD9 and demonstrated that both human and Dictyostelium UBXD9 proteins very efficiently could disassemble wild-type, but to a lesser extent mutant VCP/p97 hexamers into monomers. We hypothesize that the various biochemical differences caused by VCP/p97 point mutations which we uncovered in the course of our project could be responsible for the human VCP-related disease pathology in the long-term.
Publications
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Proteasomal activity in skeletal muscle: a matter of assay design, muscle type and age. Anal Biochem 2010; 399: 225-229
Strucksberg K-H, Tangavelou K, Schröder R, Clemen CS
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Strumpellin is a Novel VCP Binding Protein interlinking Hereditary Spastic Paraplegia with Protein Aggregation Diseases. Brain 2010; 133: 2920-2941
Clemen CS, Tangavelou K, Strucksberg K-H, Just S, Gaertner L, Regus-Leidig H, Stumpf M, Reimann J, Coras R, Morgan RO, Fernandez M-P, Hofmann A, Müller S, Schoser B, Hanisch F-G, Rottbauer W, Blümcke I, von Hörsten S, Eichinger L, Schröder R
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Heteromeric p97/p97R155C complexes induce dominant negative changes in wild-type and autophagy 9-deficient Dictyostelium strains. PLoS ONE 2012; 7:e46879
Arhzaouy K, Strucksberg K-H, Tung SM, Tangavelou K, Stumpf M, Faix J, Schröder R, Clemen CS, Eichinger L
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VCP: A jack of all trades in neuro- and myodegeneration? – Reply to the Letter to the Editor "Hereditary Spastic Paraplegia caused by a mutation in the VCP gene". Brain 2012; 135: 12: e224
Clemen CS, Eichinger L, Schröder R
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In vivo characterization of human myofibrillar myopathy genes in zebrafish. Biochem Biophys Res Commun 2015; 461: 217-223
Bührdel JB, Hirth S, Keßler M, Westphal S, Forster M, Manta L, Wiche G, Schoser B, Schessl J, Schröder R, Clemen CS, Eichinger L, Fürst DO, van der Ven PFM, Rottbauer W, Just S
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VCP and PSMF1: Antagonistic regulators of proteasome activity. Biochem Biophys Res Cmmun 2015; 463:1210-1217
Clemen CS, Marko M, Strucksberg KH, Behrens J, Wittig I, Gärtner L, Winter L, Chevessier F, Matthias J, Türk M, Tangavelou K, Schütz J, Arhzaouy K, Klopffleisch K, Hanisch FG, Rottbauer W, Blümcke I, Just S, Eichinger L, Hofmann A, Schröder R
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Mutant p97 exhibits species-specific changes of its ATPase activity and compromises the UBXD9- mediated monomerisation of p97 hexamers. Eur J Cell Biol 2016; 95: 195-207
Rijal R, Arhzaouy K, Strucksberg K-H, Cross M, Hofmann A, Schröder R, Clemen CS, Eichinger L
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Genetic analysis of VCP and WASH-complex genes in a German cohort of sporadic ALS-FTD patients. Neurobiol Aging 2017; 56: 213.e1-213.e5
Türk M, Schröder R, Khuller K, Hofmann A, Berwanger C, Ludolph AC, Dekomien G, Müller K, Weishaupt JH, Thiel CT, Clemen CS
