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.

Pleiotropic tumor suppressor functions of WWOX antagonize metastasis

“…In fact, another study has shown via MS that WWOX and SCAMP3 co-immunoprecipitate, further emphasizing a possible functional role for this interaction. 56 CDC5L, cell division cycle 5-like, is a pre-mRNA splicing factor that is known to regulate mitotic progression. One study reported that depletion of CDC5L causes dramatic mitotic arrest, chromosome misalignment and sustained activation of the spindle assembly checkpoint, which eventually leads to mitotic catastrophe.…”

The WWOX gene in brain development and pathology

“…Pathway analysis of identified WWOX binding proteins also confirmed its important role in metabolic pathways, setting it as one of the main regulators of glycolysis, fatty acid degradation, TCA cycle, de novo synthesis of lipids and amino acids. 26 …”

“… 16 , 20 , 21 , 41 , 42 Noteworthy, Dvl1 and Dvl2, core components of WNT pathway, were amongst the highest confidence protein interactors identified in TAP-MS WWOX protein interactome study. 26 …”

Strategies by which WWOX-deficient metastatic cancer cells utilize to survive via dodging, compromising, and causing damage to WWOX-positive normal microenvironment

“…Importantly, WWOX gene is one of the 5 recently discovered risk factors in Alzheimer’s disease 8 . WWOX interacts with specific cytosolic proteins, mainly functioning in normal cell physiology and death 1–5 and metabolism such as glycolysis, fatty acid degradation and acetyl-CoA generation 9 .…”