*Review* **Protein Degradation and the Pathologic Basis of Phenylketonuria and Hereditary Tyrosinemia**

### **Neha Sarodaya 1, Bharathi Suresh 1, Kye-Seong Kim 1,2,\* and Suresh Ramakrishna 1,2,\***


Received: 27 May 2020; Accepted: 13 July 2020; Published: 15 July 2020

**Abstract:** A delicate intracellular balance among protein synthesis, folding, and degradation is essential to maintaining protein homeostasis or proteostasis, and it is challenged by genetic and environmental factors. Molecular chaperones and the ubiquitin proteasome system (UPS) play a vital role in proteostasis for normal cellular function. As part of protein quality control, molecular chaperones recognize misfolded proteins and assist in their refolding. Proteins that are beyond repair or refolding undergo degradation, which is largely mediated by the UPS. The importance of protein quality control is becoming ever clearer, but it can also be a disease-causing mechanism. Diseases such as phenylketonuria (PKU) and hereditary tyrosinemia-I (HT1) are caused due to mutations in *PAH* and *FAH* gene, resulting in reduced protein stability, misfolding, accelerated degradation, and deficiency in functional proteins. Misfolded or partially unfolded proteins do not necessarily lose their functional activity completely. Thus, partially functional proteins can be rescued from degradation by molecular chaperones and deubiquitinating enzymes (DUBs). Deubiquitination is an important mechanism of the UPS that can reverse the degradation of a substrate protein by covalently removing its attached ubiquitin molecule. In this review, we discuss the importance of molecular chaperones and DUBs in reducing the severity of PKU and HT1 by stabilizing and rescuing mutant proteins.

**Keywords:** deubiquitination; inhibitors; protein quality control; proteolysis; protein stabilization

### **1. Introduction: Overview of Phenylketonuria and Hereditary Tyrosinemia**

Phenylalanine hydroxylase (PAH) and fumarylacetoacetate hydroxylase (FAH) are two highly regulated liver enzymes that catalyze the rate-limiting step in phenylalanine and tyrosine metabolism [1,2]. Mammalian PAH (phenylalanine 4-monooxygenase, E.C. 1.14.16.1) catalyzes the stereospecific hydroxylation of L-phenylalanine into L-tyrosine using tetrahydrobiopterin (BH4), non-heme iron, and dioxygen as co-substrates in the cytosol of the liver and kidney [3]. PAH facilitates oxidation of excess L-phenylalanine into carbon dioxide and water, and is the major enzyme degrading 75% of L-phenylalanine from the diet [2]. PAH assembles as a homotetrameric protein, each subunit composed of N-terminal regulatory domain for allosteric activation by Phe, a central catalytic domain, and C-terminal helix responsible for tetramer formation [4,5].

Likewise, FAH is the last enzyme in the tyrosine catabolism pathway, and it catalyzes the hydrolysis of fumarylacetoacetate into fumarate and acetoacetate as the final step in phenylalanine and tyrosine degradation. FAH is a cytosolic dimer that consists of two <sup>α</sup>–β domains; 300 residues of the C-terminal domain form the active site that binds to Ca2+ and participates in intermolecular interactions at the dimer interface; 120 residues of the N-terminal domain play the regulatory role [6,7]. The FAH dimer is solely considered to be catalytically active [7]. The human *FAH* gene occupies chromosome 15q23–q25,

spans 30–35 kb, and contains 14 exons [8], whereas the *PAH* gene is located on chromosome 12q at position 23.2, spans 90 kb, and contains 13 exons [9].

In 1932, Grace Medes discovered 4-hydroxyphenylpyruvate in the urine of a 49-year-old man and described it as "tyrosinosis" [10]. In the 1960s, the condition was referred to as hereditary tyrosinemia type-I (HT1), and it was later understood to result from FAH deficiency [11–14]. Deficiency of this enzyme leads to the accumulation of upstream metabolites such as fumarylacetoacetate (FAA) and maleylacetoacetate, which are subsequently converted into succinylacetone. FAA and succinylacetone are both genotoxic and carcinogenic [15]. Similarly, in 1934, Dr. Asbjørn Følling recognized elevated levels of phenylketonuric acid in the urine of two mentally retarded siblings and named the condition "phenylpyruvic oligophrenia" or phenylketonuria (PKU) [3]. Elevated levels of blood phenylalanine and its metabolites, such as keto acid and phenylpyruvate, along with reduced blood tyrosine levels, are the characteristics of PKU and its milder variant hyperphenylalaninemia (HPA). PKU is classified as classical PKU (plasma Phe levels > 1200 μM), mild or atypical or variant PKU (600–1200 μM), and non-PKU mild HPA (120–600 μM) [16,17]. PKU is associated with mental retardation, epilepsy, brain damage, and neurological and behavioral problems due to the accumulation of phenylalanine byproducts. Tyrosine is the precursor for multiple molecules; therefore, tyrosine deficiency leads to deficiency of catecholamine neurotransmitters, melanin, and L-thyroxine [3,18].

HT1 pathogenicity is largely unknown; however, missense mutations in the *FAH* gene may influence catalytic activity, protein stability, and/or protein homeostasis and monomer-dimer equilibrium [7]. Despite being studied extensively since years, the pathophysiology of PKU is not fully elucidated. Mutation-driven PAH protein instability, misfolding, and aggregation are the hallmark associated with the disease resulting in subsequent protein turnover [19–21]. The regulation of L-Phe by PAH is a complex mechanism associated with transition between oligomeric state, changes in conformation, phosphorylation and substrate activation, and cofactor inhibition [4,5]. The newly discovered crystal structure supports the notion that PAH exists in two native states: resting state-PAH (RS-PAH) and activated-PAH (A-PAH). The RS-PAH and A-PAH was determined by X-ray crystallography and small-angle X-ray scattering respectively [4]. The RS-PAH has low affinity for Phe and helps maintain the basal level of Phe essentially available for cellular functions. Also, BH4 is complexed with RS-PAH, thus acting as a negative regulator for L-Phe activation [4,22]. BH4 serves as a pharmacological chaperone stabilizing PAH and increasing the steady state level of enzyme [20]. As the concentration of Phe increases, excess Phe acts as an activator and binds to A-PAH allosterically, shifting the equilibrium from RS-PAH to A-PAH. Binding of Phe induces large conformational change and dimerization of regulatory domain of the enzyme, thus exposing the active site for the conversion of Phe to Tyr [4,5,22]. BH4 and Phe binding drives the newly synthesized, partially folded, PAH into equilibrium of native structure [22]. PKU disease-associated alleles affect several different operations (like allosteric activation by Phe, stabilization by BH4) that join forces for efficient degradation of excess Phe. Therefore, it is important to maintain the PAH structure equilibrium which is hampered due to disease-associated mutations.

The crystal structure of the PAH tetramer providing information about PAH allostery and BH4 associated stability was recently discovered [22,23]. The allosterically activated form of PAH is majorly responsible for the conversion of phenylalanine to tyrosine; however, stability calculations are not possible for this form as its high resolution structure is not ye<sup>t</sup> available. Nonetheless, certain experimental reports suggested increased aggregation, high instability, and accelerated degradation of the PAH mutant expressed in Enu1/<sup>1</sup> and Enu1/<sup>2</sup> heteroallelic mouse model, primary hepatocytes and COS-7 cells [24–26]. The mutant PAH proteins (e.g., p.V106A) expressed in Enu1/<sup>1</sup> mouse model, are also known to be highly ubiquitinated in vitro and in vivo, targeting it for proteasome-mediated degradation and selective autophagy [24].

To combat the pathogenic accumulation of defective proteins, the cells are equipped with the protein quality control (PQC) system, mainly including molecular chaperones and the ubiquitin proteasomal system (UPS). The supplementation with cofactor BH4, also acting as a pharmacological chaperone,

stabilizes the PAH tetramer structure, providing a rationale for the BH4-responsive *PAH*-variants [20]. When *PAH* variants are co-expressed with GroEL/ES bacterial chaperone in *Escherichia coli*, decrease in dimer portions, increase in tetramer formation, and increase in residual activity were observed. These results sugges<sup>t</sup> that co-expression with GroEL/ES bacterial chaperone might affect the PAH folding in *Escherichia coli* [19]. These results indicate that molecular chaperones have the potential to prevent protein misfolding and help to stabilize a range of mutant proteins. The proteins that cannot be stabilized by chaperones undergo degradation to avoid its interaction with other native and non-native proteins [27]. UPS is the major cellular degradation pathway, responsible for degrading more than 80% of intracellular proteins [28]. The proteins have to be tagged with ubiquitin moiety, in order to be degraded by the UPS. The PAH and FAH protein is reported to be ubiquitinated [7,24,28–30] and the variants are prone to aggregation and/or degradation [22]. However, the *PAH* and *FAH* variants exert some amount of residual activity depending upon the severity of mutation. Therefore, certain *PAH* and *FAH* mutants with folding defects are still functional, but they nonetheless suffer rapid degradation [21,31–33]. The degradative system therefore needs a way to differentiate between lethal defects and negligible defects. In this review, we discuss different strategies for stabilizing and increasing the concentrations of those functional mutant proteins, that display instability and folding defects and which are conjugated with ubiquitin molecule for degradation. We propose recruiting members of the ubiquitin proteasomal system (UPS) and protein quality control (PQC) chaperones into therapeutic endeavors to rescue functional misfolded proteins from accelerated degradation.
