Structural Insights into the Phosphorylation-Enhanced Deubiquitinating Activity of UCHL3 and Ubiquitin Chain Cleavage Preference Analysis

Ubiquitin C-terminal hydrolase-L3 (UCHL3), an important member of the ubiquitin C-terminal hydrolase family, is involved in DNA repair and cancer development. UCHL3 can cleave only complexes of monoubiquitin and its conjugates, such as Ub-AMC, His, or small ubiquitin-like modifier, but not polyubiquitin chains. Phosphorylation of Ser75 promotes the cleavage activity of UCHL3 toward poly-ubiquitin chains in vivo, but biochemical evidence in vitro is still lacking. Here, we first analyzed the structure of simulated phosphorylated UCHL3S75E and the complex of UCHL3S75E with Ub-PA and preliminarily explained the structural mechanism of phosphorylation-enhanced UCHL3 deubiquitinating activity. Additionally, the cleavage activity of UCHL3 toward different types of synthesized poly-ubiquitin chains in vitro was tested. The results showed that purified UCHL3S75E enhanced the cleavage activity toward Ub-AMC compared to UCHL3WT. Meanwhile, UCHL3S75E and UCHL3WT did not show any cleavage activity for different types of di-ubiquitin and tri-ubiquitin chains. However, UCHL3 could hydrolyze the K48 tetra-ubiquitin chain, providing compelling in vitro evidence confirming previous in vivo results. Thus, this study shows that UCHL3 can hydrolyze and has a cleavage preference for polyubiquitin chains, which expands our understanding of the phosphorylation regulation of UCHL3 and lays a foundation for further elucidation of its physiological role.


Introduction
Ubiquitination is a ubiquitous post-translational modification that plays important roles in various life processes, including protein degradation, signal transduction, and DNA damage repair [1][2][3][4][5]. Ubiquitination refers to the covalent attachment of ubiquitin (Ub), a 76-residue polypeptide, to target proteins through a sophisticated three-step enzymatic cascade reaction in which Ub is activated by E1(Ub-activating enzyme) and binds to E2 (Ub-conjugating enzyme), leading to the transfer of Ub from E2 to the target protein through E3 (Ub ligase) [6][7][8]. Ub itself can be ubiquitinated via its seven lysine residues or Met1 to form different types of poly-ubiquitin chains, including the K6-, K11-, K27-, K29-, K33-, K48-, and K63-linked chains and the M1 linear Ub chain [9], thus conferring diverse functions on their linked protein substrates [5,[10][11][12]. Different ubiquitin chains function differently depending on their length, type, and the connected substrates of ubiquitin chains [7,13]. So far, mono-ubiquitination, K48 linkage ubiquitination, and K63 linkage ubiquitination of proteins have been studied extensively [14]. For example, K48 ubiquitin chains usually mediate the ubiquitin-26S proteasome system and are involved in the degradation of proteins, whereas K63 ubiquitin chains are associated with intracellular transport, various types of signal transduction, and other non-degradation pathways, cleavage activity of UCHL3 toward a certain ubiquitin chain remains unclear and requires further study.
In 2016, Luo et al. first found that phosphorylated UCHL3 at Ser75 can cleave single or polyubiquitin chains on the substrate RAD51 and play an important role in DNA damage repair and tumor occurrence. Phosphorylated UCHL3 showed stronger ubiquitin chain cleavage activity against RAD51 in vivo [52]. However, the chain cleavage type and specificity of UCHL3 remains unclear. Furthermore, the mechanism by which phosphorylation confers the ability of UCHL3 to cleave polyubiquitin chains in vitro remains unclear. Ser75 is not conserved in other UCH family members, suggesting that phosphorylation regulation at Ser75 is unique to UCHL3.
In this study, Ser75 on UCHL3 was mutated to Glu to achieve a simulated phosphorylation modification. Wild-type UCHL3 (UCHL3 WT ) and simulated phosphorylated UCHL3 (UCHL3 S75E ) were recombinantly expressed and purified using affinity chromatography, ion exchange chromatography, and gel filtration chromatography. We first analyzed the structure of UCHL3 S75E and the complex of UCHL3 S75E with its substrate, Ubiquitin Propargylamide (Ub-PA), and explained the mechanism of phosphorylation-promoted activity of UCHL3. Finally, the cleavage activity of UCHL3 S75E and UCHL3 WT on different types of synthesized ubiquitin chains was studied in vitro, and it was found that UCHL3 preferentially cleaved activity toward K48 tetramer-Ub (K48 tetra-Ub) chains. These studies are expected to lay a foundation for understanding the molecular mechanism of phosphorylation modification and the ubiquitin chain cleavage preference of UCHL3.

Cloning, Expression, and Purification of UCHL3 S75E from E. coli
The full-length human UCHL3 contained 230 amino acids, which were amplified by polymerase chain reaction (PCR) and ligated into the pET28b vector, yielding an expression construct with an N-terminal His6 tag.
The QuikChange Site-Directed Mutagenesis Kit was used to construct the plasmid UCHL3 S75E , and then UCHL3 S75E was overexpressed and purified. The plasmid and protein purification results for UCHL3 S75E are shown in Supplementary Figure S1.

Crystal Structure of UCHL3 S75E
UCHL3 S75E was constructed by site-directed mutagenesis, overexpressed in Escherichia coli (E. coli) strain BL21 (DE3) using the vector pET-28a (GE Healthcare), and purified to homogeneity. To explore whether phosphorylation changes the deubiquitinating activity of UCHL3 by directly causing its conformational changes, crystallization trials for UCHL3 S75E were carried out. The structure of UCHL3 S75E was successfully determined by molecular replacement using an atomic model of human UCHL3 WT (PDB ID:1UCH [61]). The X-ray structure of UCHL3 S75E was refined to a 2.5 Å resolution ( Figure 1A); the data collection and refinement statistics are given in Table 1. The structure of UCHL3 S75E was similar to that of UCHL3 WT , with an RMSD of 0.273 Å. UCHL3 S75E caused an outward shift of amino acids at positions 74, 75, and 76, resulting in a 1.2 Å outward shift on the α3 helix compared to that in UCHL3 WT ( Figure 1B). In addition, after phosphorylation, residues 91 and 92 from the loop connecting the active site of catalytic residue Cys95 have an outward shift and may contribute to the exposure of the active site and recognition of the substrate ( Figure 1C).

Crystal Structure of UCHL3 S75E in Complex with Ub-PA
The molecular mechanism of phosphorylation-enhanced UCHL3 deubiquitination activity has not been well explained due to the lack of obvious conformational changes. We propose that phosphorylation modification may cause a conformational change in UCHL3 after binding to the ubiquitin substrate, similar to DUBA [38], a member of the OTU family. Therefore, the co-crystal structure of UCHL3 S75E bound to Ub-PA (UCHL3 S75E -Ub-PA) at high resolution (2.5 Å; Figure 2A) was obtained and successfully solved by molecular replacement using the atomic model of human UCHL3 WT bound to Ub-VME(UCHL3 WT -Ub-VME) (PDB ID:1XD3 [62]). Data collection and refinement statistics are presented in Table 1.      We compared the structures of UCHL3 S75E -Ub-PA with UCHL3 WT -Ub-VME and found that the simulated phosphorylation modification still resulted in a 3.5 Å outward shift of the α3 helix in which the amino acids at positions 74, 75, and 76 of UCHL3 S75E -Ub-PA displaced relative to that in UCHL3 WT -Ub-VME ( Figure 2B). In addition, we compared the structure of UCHL3 S75E in the UCHL3 S75E -Ub-PA complex with that of apo UCHLL3 S75E and found that their structures were almost similar to each other with an RMSD of 0.472. Specifically, the amino acids at positions 91 and 92 of UCHL3 S75E -Ub-PA had an outward position change relative to that of apo UCHL3 S75E , which might make it easier to expose and enhance its deubiquitinating activity ( Figure 2C). The amino acids Arg72, Leu73, and Arg 74 are located on the loop at the C-terminus of Ub, which is the region entering the active center of UCHL3. From the structure of UCHL3 WT -Ub-VME, we can see that Pro8 on UCHL3 interacts with Arg74 on Ub via hydrogen bonding. However, the interaction force becomes weaker after phosphorylation because of the larger distance between the two amino acids, which may increase the width of the region where the substrate enters the active center, allowing the entry of larger substrates. Meanwhile, the hydrophobic interaction between Arg 72 on Ub and Gln 12 on UCHL3 and the hydrogen bond between Ala11 on UCHL3 and Leu73 on Ub become stronger after phosphorylation, which may play a role in the recognition of the substrate by UCHL3 ( Figure 2D,E). Therefore, the changes in the interaction force in this region may indicate a change in the recognition and interaction between UCHL3 and the substrate.

Data-Collection and Refinement Statistics of UCHL3 S75E and UCHL3 S75E -Ub-PA
After numerous trials, the crystal structures of UCHL3 S75E and UCHL3 S75E -Ub-PA were successfully solved by molecular replacement using the atomic models of human UCHL3 WT (PDB ID:1UCH [61]) and human UCHL3 WT bound to Ub-VME(UCHL3 WT -Ub-VME) (PDB ID:1XD3 [62]). Data collection and refinement statistics are given in Table 1.

Phosphorylation Enhanced the Deubiquitinating Activity of UCHL3
We observed some changes in the structure of UCHL3 S75E and the complex of UCHL3 S75E -Ub-PA and attempted to confirm whether phosphorylation modification can improve the activity of UCHL3 in vitro. A ubiquitin-7-amino-4-methylcoumarin (Ub-AMC) hydrolysis assay was performed as previously described [63]. Compared with UCHL3 WT , phosphorylation increased Ub-AMC hydrolytic activity by approximately10 times (Figure 3).

Data-Collection and Refinement Statistics of UCHL3 S75E and UCHL3 S75E -Ub-PA
After numerous trials, the crystal structures of UCHL3 S75E and UCHL3 S75E -Ub-PA were successfully solved by molecular replacement using the atomic models of human UCHL3 WT (PDB ID:1UCH [61]) and human UCHL3 WT bound to Ub-VME(UCHL3 WT -Ub-VME) (PDB ID:1XD3 [62]). Data collection and refinement statistics are given in Table 1.

Phosphorylation Enhanced the Deubiquitinating Activity of UCHL3
We observed some changes in the structure of UCHL3 S75E and the complex of UCHL3 S75E -Ub-PA and attempted to confirm whether phosphorylation modification can improve the activity of UCHL3 in vitro. A ubiquitin-7-amino-4-methylcoumarin (Ub-AMC) hydrolysis assay was performed as previously described [63]. Compared with UCHL3 WT , phosphorylation increased Ub-AMC hydrolytic activity by approximately10 times (Figure 3).

Cleavage Activity of UCHL3 to Different Types of Ubiquitin Chains in Vitro
UCHL3 has been considered to cleave only complexes of monoubiquitin and its small conjugates, such as Ub-AMC, Ub-His, and Ub-Sumo [54]. Moreover, the UCH family of DUBs (UCHL1, UCHL3, UCHL5, and BAP1) is reported to be inactive against di-Ub in all linkage types [54]. Luo et al. found that phosphorylated UCHL3 can cleave polyubiquitin chains in vivo, but in vitro evidence is still lacking [52]. To explore the

Cleavage Activity of UCHL3 to Different Types of Ubiquitin Chains In Vitro
UCHL3 has been considered to cleave only complexes of monoubiquitin and its small conjugates, such as Ub-AMC, Ub-His, and Ub-Sumo [54]. Moreover, the UCH family of DUBs (UCHL1, UCHL3, UCHL5, and BAP1) is reported to be inactive against di-Ub in all linkage types [54]. Luo et al. found that phosphorylated UCHL3 can cleave polyubiquitin chains in vivo, but in vitro evidence is still lacking [52]. To explore the cleavage preference of UCHL3 for different types of ubiquitin chains and whether phosphorylation modification gives UCHL3 the ability to cleave ubiquitin chains in vitro, different linkages (K6, K11, K27, K48, and K63) of di-Ub were synthesized and tested for their activity of UCHL3 WT and UCHL3 S75E . However, both UCHL3 WT and UCHL3 S75E were unable to hydrolyze di-Ub ( Figure 4B-F); the complete gel is shown in Figure S3. Previous structural studies have shown that UCHL3 WT can recognize K27 di-Ub and exhibits a more stretched structure of K27 di-Ub, with more exposed isopeptide bonds [55,57,64]. We propose that UCHL3 may recognize K27 di-Ub and cleave longer ubiquitin chains.
We then synthesized K48 tetra-Ub attached to cyclinB1(cyclinB1-K48 tetra-Ub) as a substrate to test whether UCHL3 WT or UCHL3 S75E could hydrolyze longer ubiquitin chains ( Figure 6A). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results showed that both UCHL3 WT and UCHL3 S75E can cleave cyclinB1-K48 tetra-Ub into several bands, which was verified by mass spectrometry. The red arrow presents cyclinB1 and mono-Ub, as the molecular weight of mono-Ub is similar to that of cyclinB1. The blue arrow represents di-Ub, corresponding to the control sample of K48 di-Ub. Additionally, based on the mass spectrometry analysis and molecular weight, the purple and yellow arrows indicate tetra-Ub and tri-Ub, respectively. Therefore, we concluded that UCHL3 WT and UCHL3 S75E can hydrolysis cyclinB1-K48 tetra-Ub into cyclinB1, mono-Ub, di-Ub, tri-Ub, and tertra-Ub ( Figure 6B).

Discussion
PTMs of DUBs play important roles in various cellular activities, and phosphorylation is one of the most common modes. According to existing reports, the molecular mechanism of phosphorylation-regulated deubiquitination activity changes in DUBs is mainly divided into three ways. First, phosphorylation modification directly causes conformational changes in DUBs such as USP14; phosphorylation at Ser432 may open the BL2 loop of USP14 to release self-inhibition and promote hydrolysis of Ub substrates [43]. In the second method, after phosphorylation modification, the conformation of DUBs changes only after binding to the substrate, such as the phosphorylated DUBA bound to Ub, which alters its conformation instead of the phosphorylated DUBA [38]. Another way is that phosphorylation recruits a partner to bind with DUBs, such as USP8, which enables USP8 to interact with 14-3-3 proteins, thereby inhibiting its activity [44]. We found that UCHL3 S75E had a slight structural change in the α3 helix and a loop

Discussion
PTMs of DUBs play important roles in various cellular activities, and phosphorylation is one of the most common modes. According to existing reports, the molecular mechanism of phosphorylation-regulated deubiquitination activity changes in DUBs is mainly divided into three ways. First, phosphorylation modification directly causes conformational changes in DUBs such as USP14; phosphorylation at Ser432 may open the BL2 loop of USP14 to release self-inhibition and promote hydrolysis of Ub substrates [43]. In the second method, after phosphorylation modification, the conformation of DUBs changes only after binding to the substrate, such as the phosphorylated DUBA bound to Ub, which alters its conformation instead of the phosphorylated DUBA [38]. Another way is that phosphorylation recruits a partner to bind with DUBs, such as USP8, which enables USP8 to interact with 14-3-3 proteins, thereby inhibiting its activity [44]. We found that UCHL3 S75E had a slight structural change in the α3 helix and a loop near the active center compared to UCHL3 WT . Therefore, the mechanism of phosphorylation-induced UCHL3 activity changes belong to the conformational changes caused by phosphorylation of the noncatalytic center, and phosphorylated UCHL3 has a greater degree of structural changes when bound to the Ub substrate. However, we did not observe obvious changes in the structures of UCHL3 S75E and UCHL3 S75E -Ub-PA, so as not to provide a clear illustration of the mechanism of phosphorylation-induced activity changes in UCHL3.
We found that UCHL3 had a cleavage preference toward K27/48 tri-Ub and cyclinB1-K48 tetra-Ub chains, but UCHL3 S75E did not show enhanced cleavage activity compared to UCHL3 WT . These results may be due to the difference between the simulated and true phosphorylation by ATM. Similar to DUBA, it is inactive by mimic phosphorylation, whereas it can be activated when it is phosphorylated by CKII kinase; there are structural changes after binding to the Ub substrate instead of apo DUBA [38]. In addition, the polyubiquitin chains on the substrate RAD51 can be cleaved by ATM-phosphorylated UCHL3 instead of simulated phosphorylated UCHL3 in vivo. Nevertheless, these slight structural changes might also provide some clues to explain the phosphorylation-enhanced activity of UCHL3.
Previous studies have analyzed the crystal structure of UCHL3-K27 di-Ub, in which one ubiquitin occupies the S1 site of UCHL3 and the other ubiquitin occupies the S2 site of UCHL3 ( Figure 7A) [57]. It is known that the cleavage of ubiquitin chains by DUBs makes the isopeptide bond as close to the active center as possible. Therefore, if UCHL3 S75E can cleave polyubiquitin chains, then there should also be an S1 site. The Ub moiety that binds to the enzymatic S1 site contributes to Lys, which binds to the carboxyl group contributed by the Ub bound at the S1 site to form an isopeptide bond, penetrating the active center. Therefore, we propose that UCHL3 may use di-Ub as a recognition unit to cleave polyubiquitin chains. That is, ubiquitin occupies the S1 and S2 sites of UCHL3 and then cleaves the isopeptide bond between S1 and S1 ( Figure 7B). The phosphorylation site Ser75 of UCHL3 is located on S1 , and phosphorylation of Ser75 may help S1 to form the correct conformation and enhance the interaction between ubiquitin chains and UCHL3. near the active center compared to UCHL3 WT . Therefore, the mechanism of phosphorylation-induced UCHL3 activity changes belong to the conformational changes caused by phosphorylation of the noncatalytic center, and phosphorylated UCHL3 has a greater degree of structural changes when bound to the Ub substrate. However, we did not observe obvious changes in the structures of UCHL3 S75E and UCHL3 S75E -Ub-PA, so as not to provide a clear illustration of the mechanism of phosphorylation-induced activity changes in UCHL3. We found that UCHL3 had a cleavage preference toward K27/48 tri-Ub and cy-clinB1-K48 tetra-Ub chains, but UCHL3 S75E did not show enhanced cleavage activity compared to UCHL3 WT . These results may be due to the difference between the simulated and true phosphorylation by ATM. Similar to DUBA, it is inactive by mimic phosphorylation, whereas it can be activated when it is phosphorylated by CKII kinase; there are structural changes after binding to the Ub substrate instead of apo DUBA [38]. In addition, the polyubiquitin chains on the substrate RAD51 can be cleaved by ATMphosphorylated UCHL3 instead of simulated phosphorylated UCHL3 in vivo. Nevertheless, these slight structural changes might also provide some clues to explain the phosphorylation-enhanced activity of UCHL3.
Previous studies have analyzed the crystal structure of UCHL3-K27 di-Ub, in which one ubiquitin occupies the S1 site of UCHL3 and the other ubiquitin occupies the S2 site of UCHL3 ( Figure 7A) [57]. It is known that the cleavage of ubiquitin chains by DUBs makes the isopeptide bond as close to the active center as possible. Therefore, if UCHL3 S75E can cleave polyubiquitin chains, then there should also be an S1′ site. The Ub moiety that binds to the enzymatic S1′ site contributes to Lys, which binds to the carboxyl group contributed by the Ub bound at the S1 site to form an isopeptide bond, penetrating the active center. Therefore, we propose that UCHL3 may use di-Ub as a recognition unit to cleave polyubiquitin chains. That is, ubiquitin occupies the S1 and S2 sites of UCHL3 and then cleaves the isopeptide bond between S1 and S1′ ( Figure 7B). The phosphorylation site Ser75 of UCHL3 is located on S1′, and phosphorylation of Ser75 may help S1′ to form the correct conformation and enhance the interaction between ubiquitin chains and UCHL3.  We speculated the mechanism of hydrolysis of polyubiquitin chains by UCHL3 by analyzing the structure of UCHL3 WT complexed with K27 di-Ub, UCHL3 S75E , and UCHL3 S75E -Ub-PA. Meanwhile, we found that UCHL3 can cleave K27/48 tri-Ub into di-Ub and mono-Ub and can cleave cyclinB1-K48 tetra-Ub into tri-Ub, di-Ub, and mono-Ub, but it cannot cleave di-Ub. These biochemical assays first prove that UCHL3 has a preference for the K48 polyubiquitin chain and then prove our idea of how UCHL3 hydrolyzes ubiquitin chains. Therefore, analyzing the high-resolution crystal structure of the UCHL3 complex bound to K48 tri-Ub or K48 tetra-Ub may further clarify the molecular mechanism by which UCHL3 recognizes and specifically cleaves K48 linkage ubiquitin chains.
We know that phosphorylated UCHL3 by ATM can deubiquitinate RAD51 in vivo; however, our studies showed that mimetic phosphorylated UCHL3 can only cleave K48 tri-Ub or longer K48 tetra-Ub with a slower rate in vitro. Just like USP13, it can also catalyze the hydrolysis of K63 and K48 poly-Ub chains, but with a very low hydrolyzing rate [65]. The regulation of UCHL3 may be complex and may require the cooperative participation of multiple DUBs rather than a single UCHL3 in vivo. In addition, phosphorylated UCHL3 by ATM may show excellent hydrolytic properties toward K48 ubiquitin chains compared to wild-type UCHL3. On the other hands, in order to further understand the type of ubiquitin chains hydrolyzed by UCHL3 in vivo, we can obtain the ubiquitinated RAD51 in vivo, which can then be investigated by enzyme digestion and mass spectrometry. From these studies, the functional substrates and mechanisms of UCHL3 may be illustrated clearly.

Protein Preparation
His-tagged full-length UCHL3 WT was cloned into the pET-28a expression vector. The protein was overexpressed and purified, as previously described [57]. The phosphorylated UCHL3 S75E plasmid was generated by PCR using a QuikChange Site-Directed Mutagenesis Kit (TransGen Biotech, Beijing, China), and the protein was overexpressed and purified using the same method.

UCHL3 S75E Bound to Ub-PA
Ub-PA was a gift from the Lei Liu laboratory (Tsinghua University, Beijing, China); UCHL3S75E and Ub-PA were incubated in a molar ratio of 2:1 in a 100 uL reaction system containing 25 mM Tris-HCl, pH 7.5, 150 mM NaCl at 4, and 18 • C, respectively; samples were taken at 1, 2, and 4 h, respectively. The complex was analyzed via SDS-PAGE, and excess UCHL3 S75E and Ub-PA were removed on a Superdex 75 size-exclusion column run with 10 mM Tris pH 7.5 and 150 mM NaCl, and the final complex was concentrated to 20 mg. mL −1 . The results for UCHL3 S75E bound to Ub-PA are shown in Supplementary Figure S2.

Crystallization of UCHL3 S75E -Ub-PA and UCHL3 S75E
Crystals of UCHL3 S75E -Ub-PA were grown for a week at 18 • C by the hanging drop method by mixing the UCHL3 S75E -Ub-PA with an equal volume of reservoir solution containing 25% PEGMME 2000 and 0.1 M Bis-Tris (pH 6.9). Crystals were equilibrated in a cryoprotectant buffer containing reservoir buffer and 20% ethylene glycol (v/v) and were flash frozen in a cold nitrogen stream at -170 • C. Crystals of UCHL3 S75E were grown for 4-7 days by mixing UCHL3 S75E with an equal volume of reservoir solution containing 0.1 M Bis-Tris (pH 8.5), 0.3M MgCl 2 , and 30% PEG 4000 using the hanging drop method; the crystals were equilibrated using the same method as above.

X-ray Data Collection and Structure Determination
Native diffraction datasets for UCHL3 S75E -Ub-PA and UCHL3 S75E were collected on beamline BL17U1 at the Shanghai Synchrotron Radiation Facility and processed using HKL2000. Subsequent processing was performed using programs from the CCP4 suite. The UCHL3 S75E -Ub-PA and UCHL3 S75E structures were solved by molecular replacement using PHASER [66]. Human UCHL3 WT -Ub-VME (PDB ID:1XD3 [62]) and UCHL3 WT (PDB ID:1UCH) were selected as research models for molecular replacement. The structure was refined using PHENIX software [67]. The statistical analysis results of the data are summarized in Table 1.

Ub-AMC Hydrolysis Assay
Ub-AMC was used to measure the deubiquitinating activity of UCHL3 WT and UCHL3 S75E , which were gifts from the Lei Liu laboratory (Tsinghua University, Beijing, China). The reaction mixture contained 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 mg/mL ovalbumin, 5 mM ATP/MgCl2 (freshly prepared), 1 mM DTT (freshly prepared), 0.05% Tween, and 5 nM UCHL3 S75E , UCHL3 WT , or 50nM UCHL3 WT . To start the reaction, 1 µM of Ub-AMC was added to the system. Ub-AMC hydrolysis was measured at Ex355/Em460 on an EnVision plate reader (PerkinElmer). Fluorescence intensity was recorded every 250 s for 50 min or 500 s for 150 min. The experiment was repeated thrice, and the average value was calculated. The result was calculated based on the rate from the start time to the time of starting equilibrium point.

Ubiquitin Chain Cleavage Assays
Different types of ubiquitin chains were obtained from Yiming Li Laboratory [68] (Hefei University of Technology, Hefei, China). These were used to measure the deubiquitinating activity of UCHL3 WT and UCHL3 S75E in vitro. Di-Ub cleavage reactions containing 5 µM substrate and 3 µM enzyme were conducted in a reaction buffer including 25 mM Tris-HCl (pH 8.0) and 150 mM NaCl at room temperature for different time points. Finally, the hydrolysis activity was analyzed using SDS-PAGE gels. For the tri-Ub and tetra-Ub chain hydrolysis assays, the reaction conditions were the same as above.

Conclusions
UCHL3 has been designated as an important DUB in various cancers and for DNA repair. A study also found that phosphorylation of Ser75 promotes the cleavage activity of UCHL3 toward poly-ubiquitin chains in vivo. Therefore, it is necessary to describe the mechanism of phosphorylation modification and the preference of Ub chains in detail. In this study, in vitro assay showed that purified UCHL3 S75E enhanced the cleavage activity toward Ub-AMC compared to UCHL3 WT . We first analyzed the structures of the simulated phosphorylation of UCHL3 S75E and the complex of UCHL3 S75E with Ub-PA. We found that UCHL3 S75E has slight structural changes in the α3 helix and the loop near the active center compared to UCHL3 WT , and it is preliminarily speculated that the exposure of the active center of UCHL3 and the enhanced recognition of substrates are the reasons for the phosphorylation modification to enhance the activity of UCHL3 deubiquitinase. We proved that UCHL3 S75E also cannot cleave any type of di-Ub chains; however, UCHL3 tends to cleave longer Ub chains and has a preference for K48-linked Ub chains. These findings expand our understanding of the phosphorylation regulation of UCHL3 and clarify the multicomplex functions of UCHL3.

Data Availability Statement:
The data that support the findings of this study are available from the corresponding author upon reasonable request.

Acknowledgments:
We would like to thank the staff at the beamline BL17U1 of the Shanghai Synchrotron Radiation Facility for their assistance. We thank L.L. of Tsinghua University for his gift of Ub-AMC. We thank Y.L. from Hefei University of Technology for providing ubiquitin chains. We would like to thank Y.W. from the School of Life Sciences, Tsinghua University, for help in the analysis of the structure of UCHL3S75E and UCHL3S75E-Ub-PA.

Conflicts of Interest:
The authors declare no conflict of interest.