## 3 Discussion
The high-throughput proteomics analysis suggests that the most probable interaction partners of FAHD1 are carnitine palmitosyltransferase 2 (CPT2), clustered mitochondria homolog (CLUH), NADH dependent ubiquinone oxidoreductase subunit S6 (NDUFS6), polyribonucleotide nucleotidyltransferase 1 (PNPT1) and putative ubiquitin protein ligase E3 component n-recognin 3 (UBR3). Putative UBR3 is an E3 ubiquitin-protein ligase, which is a component of the \(N\)-end rule pathway, leading to ubiquitination and the subsequent degradation of its target proteins [33; 34]. Thus, we were curious to know about the specific E3 ligases that regulate the ubiquitination and degradation of FAH at the physiological and pathological levels. APC/C\({}^{\text{Cdh1}}\) catalyzes the direct attachment of ubiquitin monomers to the protein target at multiple lysine sites in a random pattern. It has been reported that the multiple monoubiquitination of cyclin B1 is triggered by APC, which provides a signal for proteolysis [35]. Previous work has revealed that APC/C\({}^{\text{Cdh1}}\) regulates craniofacial development through the monoubiquitination of Gsc [36]. Besides monoubiquitination, APC associates with two E2 conjugating enzymes, Ube2S and Ube2C, to induce K11-linked ubiquitin chains on the substrates, thus orchestrating the progression of cells through mitosis [37]. In [PERSON]'s disease and stroke pathogenesis, APC/C\({}^{\text{Cdh1}}\) but not Cdc20, is expressed in post-mitotic mammalian neurons and the inactivation of Cdh1 by phosphorylation results in the accumulation of cyclin B1 [38]. Thus, we predicted that finding an E3 ligase governing FAH ubiquitination might serve as a therapeutic target in HT1. Hepatocellular carcinoma is one of the most prevalent causes of cancer-related deaths worldwide and is commonly associated with viral hepatitis, chronic exposure to toxins and hereditary liver diseases [39]. FAA is a thiol-reacting and organelle/mitotic spindle-disturbing agent with mutagenic activities. Accumulation of the metabolite FAA is the hallmark of HT1. Hence, liver injury in HT1-deficient organisms has been mainly attributed to the immediate toxicity of the metabolite [40]. Thus, these studies prompted us to investigate the expression of FAH in HCC patient tissues. By using in silico analysis, we found that FAH protein expression is restricted to the liver and adipose tissues (Figure S1). Additionally, _FAH_ was upregulated in LIHC (Figure S2). Furthermore, Immunohistochemistry distinctive (IHC) staining for FAH in liver cancer tissues showed very high expression levels in most of the cancer patients when compared with normal tissues (Figure 1). Thus, we initiated our research on whether FAH protein is regulated by 26S proteasome and on screening the E3 ligases responsible for FAH protein turnover. Here, we delineate the role of APC/C\({}^{\text{Cdh1}}\) in maintaining FAH enzyme levels in cells. For the first time, we demonstrated that FAH protein indeed undergoes 26S proteasomal degradation (Figure 2). Furthermore, the overexpression of Cdh1 promotes FAH protein ubiquitination and destabilizes it in a dose-dependent manner (Figure 3). To support our data for identifying Cdh1 as a specific E3 ligase, _CRISPR/Cas9_-mediated knockout of the Cdh1 gene showed a stabilizing effect on FAH protein levels by preventing FAH protein degradation (Figure 4). We further demonstrated that Cdh1 and FAH protein co-localize in both the cytoplasm and the nucleus. Additionally, we showed that both proteins interact at exogenous and endogenous levels (Figure 5). As a functional consequence of interaction, Cdh1 promotes FAH protein polyubiquitin and subsequently decreases the half-life of FAH protein (Figure 6). Thus, our data signifies that the depletion of Cdh1 results in the stabilization of FAH protein by preventing its ubiquitination and signifies the importance of E3 ligase in FAH-mediated metabolism.