Structural and functional studies of human ADAM 12 in myoblast fusion and Ebola virus VP40 in assembly

Timmins, Joanna Ruth (2003). Structural and functional studies of human ADAM 12 in myoblast fusion and Ebola virus VP40 in assembly. PhD thesis The Open University.



Part I:
ADAM 12 is a member of the growing ADAM protein family that displays a conserved domain organisation: a signal sequence, a pro-, a metalloprotease, adisintegrin, a cysteine-rich, a transmembrane and a cytoplasmic domain. ADAM proteins are found in higher eukaryotes and in most mammalian tissues, and appear to be involved in very diverse developmental processes. In this work, a number of biochemical and biophysical techniques were used to obtain structural information on the disintegrin and cysteine-rich domains of ADAM 12 for which no structural data is available. We also investigated the potential role of ADAM 12in myogenesis and found that ADAM 12 could specifically be retained, by an unknown mechanism, in a perinuclear compartment when expressed in muscle cells, and was unable to provoke myogenesis by itself. A dimerisation partner of ADAM 12 was isolated, namely ADAM 19, which could specifically bind to ADAM 12 in vitro.

Part II:
The Ebola virus matrix protein, VP40, is targeted to the plasma membrane where it is thought to induce assembly and budding of virions. Ebola virus VP40 is a monomer in solution consisting of two domains. Cellular localisation studies ofdifferent VP40 mutants showed that the C-terminal domain is required for plasma membrane association. Moreover, we showed that wild type VP40 and an N-terminally truncated form VP40 (31-326) are both released into the cell culture supernatant, when expressed in 293T cells, in vesicular structures that resemblevirus-like particles as detected by electron microscopy. These results suggest thatVP40 is sufficient for virus assembly and budding. Moreover, VP40 contains two motifs at its N-terminus, which are required for binding to an ubiquitin ligase(Nedd4) and to an inactive E2 enzyme (Tsg101),both of which are here shown to interact with VP40 in vitro. These interactions are believed to improve the budding efficiency.

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