Identification of VCP/p97, Carboxyl Terminus of Hsp70-interacting Protein (CHIP), and Amphiphysin II Interaction Partners Using Membrane-based Human Proteome Arrays

Abstract

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. Molecular & Cellular Proteomics 5:234 -244, 2006.As protein-protein interactions (PPIs) 1 are central to most biological processes, their systematic identification is considered a key strategy for uncovering the complex organization principles of functional cellular networks (1-3). A number of experimental and computational techniques have been developed to determine all the potential and actual PPIs in selected model organisms (4 -8) and humans (9 -11).Because protein arrays allow the parallel detection of addressable elements in a single experiment (12-18), they have been recognized as a promising technology for the identification of PPIs (19,20) or other specific biochemical activities (20 -23), and much effort has been devoted to their development in recent years. Zhu et al. (20) e.g. printed 5,800 purified yeast proteins onto coated glass slides and used them successfully in proof-of-principle experiments for the detection of novel calmodulin-and phospholipid-interacting proteins. Protein arrays were also used efficiently for identifying novel targets of protein kinases (24 -26). In principle, the production of protein arrays and their utilization for systematic large scale interaction and activity screens has proven viable. One major drawback, however, has been the necessity to use purified proteins. Protein purification is usually difficult, time-consuming, and expensive. For array-based studies, especially high throughput systematic screening (19), technologies are needed that permit...

Deletion of the Ubiquitin Ligase CHIP Leads to the Accumulation, But Not the Aggregation, of Both Endogenous Phospho- and Caspase-3-Cleaved Tau Species

“…In addition to interacting with HSP70 and HSP90 (Grelle et al, 2006), it was also demonstrated that CHIP was capable of facilitating HSF-1 activity, demonstrating the regulatory role that this protein may have in the inducible chaperone response. Thus, because reductions in HSP70 have been shown to facilitate caspase-3 activation (Li et al, 2000), one could certainly argue that the deletion of CHIP could result in a number of consequences because of the number of HSP70/ HSP90 client proteins that are also potential CHIP substrates.…”

“…These findings correlate well with the recent finding that CHIP levels were elevated in AD samples relative to control tissue (Sahara et al, 2005), further suggesting that CHIP plays an important role in mature tangle formation. Additionally, a recent proteomic study identified HSP70, HSP90, and tau as CHIP binding partners (Bomar et al, 2003;Grelle et al, 2006). Here, we have characterized the effect of CHIP deletion on endogenous mouse tau as well as overexpressed mutant (P301L) human tau (Lewis et al, 2000;Dai et al, 2003).…”

CHIP Overexpression Reduces Mutant Androgen Receptor Protein and Ameliorates Phenotypes of the Spinal and Bulbar Muscular Atrophy Transgenic Mouse Model

“…A wide range of different proteins have been identified as CHIP substrates, including members of the steroid hormone receptor family (Connell et al, 2001;Tateishi et al, 2004;Wang and DeFranco, 2005), the cystic-fibrosis transmembraneconductance regulator (Meacham et al, 2001;Younger et al, 2006), E2A transcription factors (Huang et al, 2004), raf-1 protein kinase (Demand et al, 2001), ErbB2 (Zhou et al, 2003), nucleophosminanaplastic lymphoma kinase (Bonvini et al, 2004), dual leucine zipper-bearing kinase (Daviau et al, 2006), caytaxin (Grelle et al, 2006), ␣B-crystallin (Chavez Zobel et al, 2003), tau (Hatakeyama et al, 2004;Petrucelli et al, 2004;Sahara et al, 2005;Dickey et al, 2006), ␣-synuclein (Shin et al, 2005), the p53 tumor suppressor (Esser et al, 2005), apoptosis signal-regulating kinase 1 (Hwang et al, 2005), and polyQ-disease causative proteins (Jana et al, 2005;Miller et al, 2005;Al-Ramahi et al, 2006). CHIP can directly interact with and degrade the wildtype AR in a phosphorylation-dependent or -independent manner (Cardozo et al, 2003;Rees et al, 2006) and can repress AR transcriptional activity, suggesting that CHIP may play a role in regulating AR function in the cell (He et al, 2004).…”

Induction of cell proliferation, clonogenicity and cell accumulation in S phase as a consequence of human UBE2Q1 overexpression

“…These results open a landscape towards exploring the interacting partners of this novel protein and identifying its upstream and downstream molecules involved in carcinogenesis. UBE2Q1 has been also observed to be a potential binding partner of carboxyl terminus of heat shock protein 70-interacting partner (34), which participates in protein quality control and stress response (35), and functions as an E3 ligase for p53 (36). Notably, UBE2Q2, a close related homolog of UBE2Q1 that belongs to the class II E2 family of enzymes (30), has been demonstrated to be differentially expressed in several human malignancies, including HNSCC (14), breast cancer (3), pediatric ALL (15) and CRC (16).…”