## 3 Discussion  
FAH is the final enzyme within the tyrosine catabolism pathway that catalyzes the hydrolysis of FAA into fumarate and acetoacetate as the last step in phenylalanine and tyrosine degradation. In 1932, [PERSON] found 4-hydroxyphenylpyruvate within the urine of a 49-year-old man and termed it as \"tyrosinosis\" [23]. Later, in the 1960s, the condition was alluded to as HT1, an outcome of FAH deficiency [24, 25, 26]. Deficiency of FAH leads to the accumulation of upstream metabolites such as FAA and MAA ultimately converting into SUAC, of which FAA and SUAC are known to be both genotoxic  
Figure 6: APC/C\({}^{\text{Cdh1}}\) promotes FAH polyubiquitin and decreases FAH protein half-life. (**A**) Flag-Cdh1, sgRNA1-Cdh1 and HA-ubiquitin were co-transfected into HEK293 cells with Myc-FAH and treated with MG132 (5 \(\upmu\)M) for 8 h. The cells were immunoprecipitated with Myc-antibody followed by immunoblotting with HA and Myc antibodies and the polyubiquitin of FAH was examined. (**B**) Determination of FAH protein half-life. HEK293 cells were transfected with Myc-FAH and control vector and treated with cycloheximide (CHX) (150 \(\upmu\)mL). Cells were harvested at specific time intervals and analyzed by Western blotting with the Myc antibody indicated. (**C**) FAH protein half-life is extended upon the transfection of sgRNA targeting Cdh1. HEK293 cells were transfected with the control vector and sgRNA-Cdh1, treated with CHX and analyzed by Western blotting. (**D**) FAH protein half-life is reduced upon transfection of Flag-Cdh1. HEK293 cells were transfected with control vector and Flag-Cdh1 and treated with CHX and expression levels were analyzed by Western blotting. GAPDH was used as a loading control. The FAH band intensities for (**B–D**) were estimated by ImageJ software and normalized with GAPDH for graphical representation. Data were presented as the mean and standard deviation of three independent experiments. Two way ANOVA followed by [PERSON]’s post hoc test was used for comparison between two groups and the \(p\)-values are indicated. and carcinogenic [27]. HT1 pathogenicity is mostly indefinite but a few missense mutations in the _FAH_ gene may influence catalytic activity, protein stability, protein homeostasis and monomer-dimer equilibrium [11]. For the remedial cure for HT1, dietary restriction in phenylalanine and tyrosine, liver transplantation and an inhibitor of 4-hydroxyphenylpyruvate acid dioxygenase (nitisinone) also known as NTBC (2-[2-nitro-4-(trifluoromethyl)]benzoyl) cyclohexane-1,3-dione) are the known therapies but they have their limitations such as side effects and long-term complications for the patients [28]. Thus, HT1 requires substitute therapeutic intercession lines that would suggest less harmful side impacts. Mutated proteins are susceptible for misfolding which is corrected by protein control systems such as molecular chaperones and UPS. Molecular chaperones are proteins that guide the assembly and conformational folding of macromolecular structures or _vice versa_. These are heat shock proteins (HSPs) produced by cells under stressful conditions that stabilize new proteins to ensure proper folding or refold damaged proteins [29]. Additionally, there are several E3 ligases that intimately coordinate with molecular chaperones for protein refolding or the degrading of misfolded proteins [30]. Quantitative protein interaction analysis suggests that more than 30% of all human E3 ligases interact with Hsp90 [31]. To date, studies related to PTMs and their regulation of FAH protein function have not yet been reported. However, the only described FAH superfamily members in the eukaryotic kingdom are fumarylactoacetate hydrolase domain-containing proteins (FAHDs) 1 and 2, which have been a focus of recent work in aging research [32].