**5. Deubiquitinating Enzymes Regulate Molecular Chaperones**

Approximately 100 putative DUBs have been identified in humans. These large ubiquitin-cleaving proteases are classified into seven families: ubiquitin-specific proteases, ubiquitin C-terminal hydrolases, ovarian tumor proteases, Machado-Joseph disease domain proteases, Jab1/Mpn/Mov34 metalloenzymes, monocyte chemotactic protein-induced proteases, and zinc finger with UFM1-85 specific peptidase domain proteins, but most of their functions and substrates have not ye<sup>t</sup> been characterized [84]. Some of the important functions of DUBs in the ubiquitin pathway include generating free ubiquitin monomers by processing inactive ubiquitin precursors, acting as an E3 ligase antagonist by cleaving the ubiquitin molecule from the substrate proteins, and maintaining a ubiquitin pool by recycling cleaved ubiquitin molecules. DUBs are known to be involved in physiological processes and thus are predicted to be involved in cancers [85], neurodegeneration [84], and infectious diseases [86]. Interestingly DUBs also regulate the members of another major degradation pathway in PQC called autophagy. Misfolded proteins are recognized by molecular chaperones in the HSP family, which coordinates with the UPS for protein refolding and the removal of misfolded proteins [87].

Ubiquitination and deubiquitination both play crucial roles in the dynamic regulation of di fferent stages of the autophagic process. To induce proper autophagy, post-translational modification of its initiators is essential. It is a well-organized game of "on" and "o ff" between the E3 ligase and DUBs in controlling autophagy signals [87]. Numerous E3-chaperone complexes work in parallel to target misfolded proteins. For example, the E3 ligase carboxy-terminus of Hsc70 interacting protein (CHIP) tightly regulates the function of Hsp70/Hsp90 to orchestrate cellular protein folding and degradation. Ubiquitination of substrate proteins is antagonized by DUBs, allowing misfolded proteins to escape from degradation [88]. A growing body of evidence suggests crosstalk between the DUBs and the HSPs as well. For instance, proteasome-bound USP14 protein was found to interact with molecular chaperone Hsc70 to modulate autophagy in neuroblastoma cells. Striatal neuronal cells expressing mutant huntingtin protein had a defect in autophagosome maturation that was influenced by Hsc70 and proteasome free-USP14, indicating a link between the proteasome-independent function of USP14 and Hsc70 in mediating crosstalk among autophagy, ER stress signaling, and the proteasome [89]. Similarly, the DUB USP19 has two major isoforms. One isoform contains a transmembrane domain at its C-terminus and is associated with ERAD for an unfolded protein response; the other isoform contains an EEVD extension at the C-terminus that interacts with CHIP. The N-terminus of both isoforms interacts with the Hsp90 chaperone. The regulatory function of USP19 was recently confirmed in a study demonstrating that USP19 interacts directly with chaperone Hsp90 and upregulates the aggregation of poly-Q containing the proteins Ataxin-3 and Huntingtin, which causes spinocerebellar ataxia type-3 and Huntington's disease, respectively [90]. Direct evidence indicates that chaperone Hsp90 enhances USP19 DUB activity by promoting its substrate recognition [91]. Hsp90 recruits misfolded proteins for refolding, and should the protein fail to refold, the co-chaperone CHIP ubiquitinates the misfolded protein for degradation with the help of Hsp90, or the misfolded protein is deubiquitinated

by USP19, allowing it to avoid degradation and promoting aggregation [90]. This process is perfectly synchronized as a defense mechanism against proteins whose aggregation is cytotoxic to the cells. However, to enhance the rescue of functional mutant proteins, understanding the regulatory mechanism of DUBs and molecular chaperones is beneficial.

### **6. Rapid Degradation of Misfolded PAH and FAH Proteins**

More than 1000 variants in the human *PAH* gene are recorded in the locus-specific database *PAH*vdb (http://www.biopku.org/home/pah.asp), and certain missense mutations in the regulatory and catalytic domains cause protein instability and folding defects of the PAH protein, resulting in its rapid degradation and loss of function [31,32,92]. Thus, PKU was generally considered to be the paradigm of misfolded metabolic diseases [93]. The destabilized mutants of PAH are precisely degraded by the cellular PQC system. Mutation-dependent destabilization and accelerated proteolytic degradation are the main pathogenic mechanisms in PKU [94]. PAH is reported to be a substrate for Ub-conjugating enzyme and is likely degraded by the UPS. Døskeland et al. demonstrated that PAH isolated from rat liver is conjugated with mono- and multi-/poly-ubiquitination at its catalytic domain [29]. More recently, in an ENU1/<sup>2</sup> heteroallelic mouse model of HPA, mutant PAH was highly ubiquitinated, which corresponded with an increased rate of degradation [24]. The mutant proteins were degraded more rapidly than the wild type enzyme [62]. The wild type is reported to have a half-life of 2 days in rat liver and 7–8 h in hepatoma cells; in contrast, mutants are degraded rapidly, due to the destabilization of their protein structure [29]. Molecular chaperones such as DNAJC12/HSP70 play a role in processing mutant PAH for UPS-mediated degradation or ubiquitin-mediated autophagy [28,30].

Likewise, more than 100 mutations of the *FAH* gene cause HT1. Like PAH, most of the mutations produce FAH destabilization, causing the enzyme to be rushed to the aggregation pathway. When cells expressing the FAH protein were subjected to the proteasomal inhibitor MG132, FAH protein levels were restored. Therefore, the FAH protein undergoes proteasomal degradation [7]. FAH is also conjugated with Ub at multiple lysine residues according to the PhosphoSitePlus (www.phosphosite.org) database. However, no evidence indicates the type of ubiquitin linkage and whether it targets FAH for degradation or tags it for further cellular processes. The reduced activity and deficiency of FAH found in HT1 could result from the rapid degradation of destabilized mutant proteins [95], similar to PAH in PKU.

### **7. Residual Catalytic Activity of PAH and FAH Can Be Rescued by Deubiquitination or Molecular Chaperones**

Certain cases of PKU result from genetic mutations that impede the normal folding of the wild type PAH protein, leading to reduced or no enzyme activity. Genotype-based prediction of metabolic phenotypes, including patients with homozygosity and those with functional hemizygosity, has been studied for several years [16]. Two alleles, both with severe mutations in the *PAH* gene, produce an enzyme with little or no enzyme activity, whereas the presence of two mild mutations or one severe and one mild mutation produces high residual enzyme activity, producing HPA or mild PKU (>30% activity compared with wild type PAH) [93]. Certain combinations of mutations in the genotype and their predicted residual enzyme activity have already been reported [96,97]. Some mutations characterized by high residual activity were found to be responsive to natural co-factor BH4 [98]. BH4 responsiveness has a multifactorial basis, including intragenic polymorphisms and non-genetic factors. The main molecular mechanism underlying BH4 responsiveness is its chaperone-like e ffect on PAH, whereby it protects PAH protein integrity and rescues it from Ub-dependent degradation [99].

It is increasingly apparent that molecular chaperones could help mutant PAH proteins that are partially functional serve their purpose and help to prevent the pathogenic mechanisms that underlie genetic diseases.

Given the importance of chaperones, mutations in the chaperones themselves can be lethal. Multiple diseases are associated with mutations in the regulating chaperones. For example, a missense mutation in the equatorial domain of HSP60 causes spastic paraplegia, and mutation in tubulin-specific

chaperone E causes hypoparathyroidism, mental retardation, and facial dysmorphism [100]. Similarly, in PKU an autosomal recessive mutation in DNAJC12, a PAH co-chaperone, reduced the activity of wild type PAH, leading to HPA. DNAJC12 is involved in PAH folding and interacts with the monoubiquitinated *PAH* variant, marking it for the Ub-dependent proteasomal/autophagy degradation system. Further studies are ongoing to elucidate the role of DNAJC12 in regulating PAH and PAH mutants [25,101]. Gene therapy and the ectopic expression of wild type chaperones might help to restore the partially functional mutant proteins [102,103].

Some patients with HT1 who are treated with NTBC (2-[2-nitro-4-(trifluoromethyl) benzoyl] cyclohexane-1,3-dione) also suffer from chronic hepatopathy and the development of hepatocellular carcinoma [104]. In a murine model of HT1, chaperones such as HSPB and HSPA were found to be associated with the anti-apoptotic proteins BCL-2 and BAG in the hepatocarcinogenetic process [105]. However, the role of molecular chaperones in FAH protein stability and degradation needs to be investigated.

Another system that can be targeted to rescue defective proteins is the UPS. As discussed in the previous section, the UPS is mainly driven by E1-E2-E3 enzymes that tag substrate proteins with ubiquitin molecules to mark them for degradation via the 26S proteasome, and DUBs can reverse that process. The role of DUBs in disease regulation has been imagined ever since their discovery, because they are involved in almost all cellular processes [106]. In PQC, the ubiquitin-mediated proteolytic pathway is a dynamic system responsible for regulating the fate of many proteins. In loss-of-function diseases, saving functional misfolded proteins from degradation can be a better alternative than dealing with a deficiency of proteins caused by rapid degradation. DUBs can rescue proteins from degradation by cleaving their degradative signals. Thus, DUBs act as proofreaders for mis-tagged substrate proteins and prevent them from degradation. In that way, DUBs could be used to curb protein misfolding diseases. It is unsurprising that direct evidence on this point is sparse. Most studies dealing with diseases related to protein folding problems aim to clear the misfolded proteins from the cells rapidly, and thus they target DUBs or the proteasome via specific inhibitors to prevent the pathogenesis of defective protein accumulation [107,108]. However, in diseases such as PKU and HT1, artificial manipulation of those systems could prove advantageous and pave the way for new therapeutic approaches. Nonetheless, the regulation of the proteostasis is not possible for those missense mutations which are present at the active site of the enzyme and other mutations causing truncation and splice variant. Therefore, controlling the proteostasis might be favorable only to the missense mutations that are located outside the active site.

PAH and FAH enzyme proteins are ubiquitinated and degraded by the Ub-dependent system, and therefore PAH and FAH mutants with high residual enzyme activity could be deubiquitinated by DUBs, which might suffice to create an adequate supply of functional protein. However, the mutations in certain genotypes can show dramatically different disease severities. Thus, in targeting DUBs as therapeutics for diseases with misfolded protein, it is important to understand the genotype-phenotype correlation and the allelic combination of mutations present in the genotype. A grea<sup>t</sup> deal of work remains to be done to improve understanding of how DUBs, molecular chaperones, and their combination can help to regulate enzyme deficiencies.

### **8. Current Treatments and Currently Ongoing Research**
