Identification of VCP/p97, carboxyl terminus of Hsp70-interacting protein (CHIP), and amphiphysin II interaction partners using membrane-based human proteome arrays.
Proteins mediate their biological function through interactions with other proteins. Therefore, the systematic identification and characterization of protein-protein interactions have become a powerful proteomic strategy to understand protein function and comprehensive cellular regulatory networks. For the screening of valosin-containing protein, carboxyl terminus of Hsp70-interacting protein (CHIP), and amphiphysin II interaction partners, we utilized a membrane-based array technology that allows the identification of human protein-protein interactions with crude bacterial cell extracts. Many novel interaction pairs such as valosin-containing protein/autocrine motility factor receptor, CHIP/caytaxin, or amphiphysin II/DLP4 were identified and subsequently confirmed by pull-down, two-hybrid and co-immunoprecipitation experiments. In addition, assays were performed to validate the interactions functionally. CHIP e.g. was found to efficiently polyubiquitinate caytaxin in vitro, suggesting that it might influence caytaxin degradation in vivo. Using peptide arrays, we also identified the binding motifs in the proteins DLP4, XRCC4, and fructose-1,6-bisphosphatase, which are crucial for the association with the Src homology 3 domain of amphiphysin II. Together these studies indicate that our human proteome array technology permits the identification of protein-protein interactions that are functionally involved in neurodegenerative disease processes, the degradation of protein substrates, and the transport of membrane vesicles.
Grelle
Gerlinde
G
Max-Delbrück-Centrum für Molekulare Medizin, Robert-Rössle-Strasse 10, D-13125 Berlin-Buch, Germany.
Kostka
Susanne
S
Otto
Albrecht
A
Kersten
Birgit
B
Genser
Klaus F
KF
Müller
Eva-Christina
EC
Wälter
Stephanie
S
Böddrich
Annett
A
Stelzl
Ulrich
U
Hänig
Christian
C
Volkmer-Engert
Rudolf
R
Landgraf
Christiane
C
Alberti
Simon
S
Höhfeld
Jörg
J
Strödicke
Martin
M
Wanker
Erich E
EE
D000251:Adenosine Triphosphatases; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000378:metabolism*; D002522:Chlorocebus aethiops; D050883:HSC70 Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D008567:Membranes, Artificial; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D040081:Protein Array Analysis; D011485:Protein Binding; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D020543:Proteome; D000074405:Valosin Containing Protein
D018797:Cell Cycle Proteins; D050883:HSC70 Heat-Shock Proteins; D008567:Membranes, Artificial; D009419:Nerve Tissue Proteins; D020543:Proteome; C075637:amphiphysin; D000251:Adenosine Triphosphatases; C000614178:VCP protein, human; D000074405:Valosin Containing Protein