Delineating WWOX Protein Interactome by Tandem Affinity Purification-Mass Spectrometry: Identification of Top Interactors and Key Metabolic Pathways Involved

Abstract

It has become clear from multiple studies that WWOX (WW domain-containing oxidoreductase) operates as a "non-classical" tumor suppressor of significant relevance in cancer progression. Additionally, WWOX has been recognized for its role in a much wider array of human pathologies including metabolic conditions and central nervous system related syndromes. A myriad of putative functional roles has been attributed to WWOX mostly through the identification of various binding proteins. However, the reality is that much remains to be learned on the key relevant functions of WWOX in the normal cell. Here we employed a Tandem Affinity Purification-Mass Spectrometry (TAP-MS) approach in order to better define direct WWOX protein interactors and by extension interaction with multiprotein complexes under physiological conditions on a proteomic scale. This work led to the identification of both well-known, but more importantly novel high confidence WWOX interactors, suggesting the involvement of WWOX in specific biological and molecular processes while delineating a comprehensive portrait of WWOX protein interactome. Of particular relevance is WWOX interaction with key proteins from the endoplasmic reticulum (ER), Golgi, late endosomes, protein transport, and lysosomes networks such as SEC23IP, SCAMP3, and VOPP1. These binding partners harbor specific PPXY motifs which directly interact with the amino-terminal WW1 domain of WWOX. Pathway analysis of WWOX interactors identified a significant enrichment of metabolic pathways associated with proteins, carbohydrates, and lipids breakdown. Thus, suggesting that WWOX likely plays relevant roles in glycolysis, fatty acid degradation and other pathways that converge primarily in Acetyl-CoA generation, a fundamental molecule not only as the entry point to the tricarboxylic acid (TCA) cycle for energy production, but also as the key building block for de novo synthesis of lipids and amino acids. Our results provide a significant lead on subsets of protein partners and enzymatic complexes with which full-length WWOX protein interacts with in order to carry out its metabolic and other biological functions while also becoming a valuable resource for further mechanistic studies.

Keywords: TAP-MS; WW domains; WWOX; interactome; metabolic pathways; protein transport.

Figure 1

TAP-MS based Proteomic Profiling of WWOX Interactome. Schematic diagram showing the major steps involved in the TAP-MS procedure and data analysis. HEK293T cells stably expressing WWOX with C-terminal SFB-tag (Streptavidin binding protein (SBP)-tag, S-tag, and Flag-tag) were generated by stable transfection and puromycin selection. Following the described tandem affinity purification steps, purified protein complexes were identified by mass spectrometry analysis (LC-MS/MS), and final interacting proteins were identified after proper annotation and MUSE algorithm-based analysis to define interaction scores. Data annotation was done using Bioconductor. To remove the non-specific bindings or contaminants data was analyzed using CRAPome database to obtain a list of high confidence 216 binding partners. Finally, data were subjected to motif and pathway analysis.

Figure 2

High confidence WWOX protein interactors. Graph showing candidate WWOX interacting partners with interaction score (IS) > 0.2. Bars in green represent the set of 14 proteins with an extremely high likelihood of being direct physical WWOX binding proteins with IS > 0.3. Bars in blue represent good candidate WWOX direct binding proteins with IS < 0.3 and > 0.2. Numbers in parenthesis next to bars represent the number of potential WWOX binding motifs for each respective interacting protein. The orange line graph represents CRAPome frequency for each WWOX binding protein.

Figure 3

(A) Direct physical interaction between WWOX and SEC23IP, SCAMP3, and VOPP1 proteins. Reciprocal co-immunoprecipitation of GFP-WWOX and Myc-SEC23IP, Myc-SCAMP3 or Myc-VOPP1 in HEK293T cells. Cells were co-transfected with either 1 μg of Myc-DDK-VOPP1, Myc-DDK-SCAMP3, or Myc-DDK-SEC23IP along with 1 μg of GFP-WWOX plasmid. After 36 h transfected cells were lysed and were immunoprecipitated with either rabbit IgG (control), anti-Myc, or anti-GFP antibodies. The immunoprecipitates were immunoblotted with anti-Myc or anti-WWOX antibodies as indicated. (B) Western blot and corresponding Coomassie blue staining of GST pull-down assay. HEK293T cells were transfected with either Myc-SEC23IP or Myc-SCAMP3 expression vectors. Cell lysates were subjected to pulldowns with the indicated GST fusion proteins: either wild-type WW domains, i.e., WW1 + WW2 (GST-WW1-2); mutant WW1 domain (GST-Mut-WW1), i.e., WW1 W44F/P47A + WW2 WT; mutant WW2 domain (GST-Mut-WW2), i.e., WW1 WT + WW2 Y85A/P88A; WW1 WT (GST-WW1), WW2 WT (GST-WW2), full length wild-type WWOX (GST-WWOX), or GST alone. Bound protein was detected by anti-Myc immunoblot.

Figure 4

WW domain binding motifs in WWOX interacting proteins. (A) ExPASY PROSITE analysis was performed on the list of 216 WWOX interactors (Supplementary File 2). Candidate WWOX WW1 domain binding motifs were identified in 31% (67 out of 216) proteins. Out of 67 proteins with putative WW1 binding motifs, 12 candidate WWOX binding interactors displayed both PPX(Y/F) and LPX(Y/F) motifs. (B) Amino acid sequence of candidate WWOX WW1 domain binding motifs. The probability of the residue at each position is proportional to the size of the letters. The image was generated using the Weblogo 3.4 program. Thirty-nine proteins displayed PPX(Y/F) motifs at 56 sites and 40 proteins displayed LPX(Y/F) motif at 49 sites. (C) Box and whisker plot showing the comparison of median interaction scores between proteins with- and without-motifs. Proteins with-motifs display higher interaction scores (mean interaction score = 0.1875) vs. proteins with No-motifs (mean interaction score = 0.1287). Statistical analysis was done using two-tailed unpaired Student's t-test, ^**p < 0.001. (D) Comparison of SFB-WWOX-TAP-MS data with GST-WW1-pull-down-MS data from Abu-Odeh (34). Only 16 common WWOX interactors were identified overlapping both datasets. (E) Bar graph showing a comparison of the percentage of proteins with WWOX binding motifs between the aforementioned datasets. Thirty-one percent of proteins from the SFB-WWOX-TAP-MS dataset (this study) displayed candidate WW1 binding motifs vs. 46.8% of proteins in the GST-WW1-pull-down-MS dataset (34).

Figure 5

Metabolic Pathways significantly associated with WWOX protein interactome. (A) Bar graph represents biological pathways that are significantly over-represented within the list of 216 WWOX interactors. See Supplementary File 4 for detail of interactors and p-values of pathway shown (B) Among the most significantly associated pathways we identified key metabolic pathways including: “Valine, Leucine, and Isoleucine degradation” (KEGG pathway hsa00280), “Glycolysis/Gluconeogenesis” (KEGG pathway hsa00010), “Pyruvate metabolism” (KEGG pathway hsa00620), and “Fatty acid degradation” (KEGG pathway hsa00071). All pathways converge to Acetyl-CoA generation the key molecule that delivers its acetyl group for oxidation and energy production to the tricarboxylic acid (TCA) cycle. WWOX interactors identified in our TAP-MS dataset are highlighted in red.