USP14 protein, human

Western blotting and IP assays were performed as described previously (25 (link), 26 (link), 28 (link)). Briefly, HEK293T cells were transiently transfected with mock as control vector, Flag-USP14, Myc-Beclin 1 wild type (wt), Myc-Beclin 1 truncated mutants, Flag-TRAF6 wt, Flag-TRAF6 truncated mutants, Myc-USP14 wt, HA-Ub, HA-TAB 2 wt, HA-TAB 2 truncated mutants, or Myc-Ub vector indicated in each experiment by using Lipofectamine 2000 (Invitrogen). At 38 h after transfection, transfected cells were extracted and immunoprecipitated with anti-Flag (Cell Signaling Technology, Beverly, MA, USA), anti-HA (Cell Signaling Technology), or anti-Myc antibody (Cell Signaling Technology). Immunoprecipitated complexes were separated by 6–10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and probed with anti-HA, anti-Myc, or anti-Flag antibody. Ctrl and USP14^KD THP-1 cells treated with or without 3-MA (5 mM) and pepstatin A (10 µM) were stimulated with LPS (200 ng/ml) for 6 h. Whole cell lysates were subjected for immunoblot analysis of LC3A/B (4108, Cell Signaling Technology) and GAPDH (Cell Signaling Technology) as a loading control. Ctrl and USP14^KD THP-1 cells were treated with or without LPS (200 ng/ml) for different times. Cells were extracted, separated by 6–10% SDS-PAGE, and probed with the following antibodies: IκB-α, pho-p38, p38, pho-IKKαβ, IKKβ, and GAPDH purchased from Cell Signaling Technology (Beverley, MA, USA).

USPs were first discovered and cloned in Saccharomyces cerevisiae (Tobias & Varshavsky, 1991 (link)). They are the largest subfamily of DUBs having a total of 58 members. The number of USPs has increased since the evolution of E3 ubiquitin ligases (Semple, 2003 (link)). The size of USPs ranges from 330 amino acids to 3,500 amino acids with an average size of 800–1,000 amino acids for full-length enzymes. The catalytic structural domain of USPs contains 295-850 amino acids; the catalytic structural domain of 27 USPs contains 300–400 amino acids, while that of 29 USPs contains 400–850 amino acids (Ye et al., 2009 (link)). USPs have also other diverse domains in terms of size and structure (Hariri & St-Arnaud, 2021 (link)). However, there is a high degree of homology within the catalytic domain. The catalytic core of USPs contains three motifs, consisting of very conserved catalytic Cys residues, catalytic His residues, and catalytic Asp/Asn residues, which form the catalytic triad (Nijman et al., 2005 (link); Ye et al., 2009 (link)). In addition to the catalytic domain, USPs also have domains for subcellular localization, substrate specificity, zinc binding, and ubiquitin recognition (Hariri & St-Arnaud, 2021 (link); Nijman et al., 2005 (link); Ye et al., 2009 (link)). Figure 2A shows USP4, USP7, USP14, USP19, and USP44 as examples of USPs having major domains.
The ubiquitin-like (UBL) domain of USPs can regulate their catalytic activity; however, the mechanism of action of the UBL domain in each USP varies. For example, the UBL domain of USP14 is important for its localization on proteasome and might enhance its catalytic capability., while that of USP4 binds to the catalytic domain, showing a competitive relationship with ubiquitin. As shown in Fig. 2B, the UBL4 and UBL5 domains of USP7 are located on its C-terminal and can affect its deubiquitinating activity by promoting conformational changes and facilitating the formation of a catalytic center (Faesen, Luna-Vargas & Sixma, 2012 (link)).
Different USPs have specific substrate proteins; therefore, they can regulate different signaling pathways (Ye et al., 2009 (link)). USPs can stabilize various oncoproteins or alter their cellular localization by deubiquitination, which can cause the development and progression of cancer (Chauhan et al., 2021 (link)). Numerous studies have shown that targeting USPs might be a promising therapeutic approach for cancer treatment (Dai et al., 2020 (link); Du et al., 2021 (link); Li et al., 2020 (link); Ma et al., 2019 (link); Nininahazwe et al., 2021 (link); Zhu et al., 2020 (link)).

USP14 promotes the proliferation of PCa cells and is closely associated with its progression (Liao et al., 2017 (link)). It can also promote the development of PCa by stabilizing AR protein and ATF2 (activating the transcription factor 2) (Geng et al., 2020 (link); Liao et al., 2017 (link)). USP14 is a novel regulator of AR (Liao et al., 2017 (link)). AR protein is overexpressed in PCa and plays a key role in its growth and progression (Liao et al., 2017 (link)). ATF2, an oncogene of PCa, facilitates the proliferation of PCa cells (Geng et al., 2020 (link)).

Protein–protein interactions were predicted using the STRING functional protein association network [66 (link)]. Coordinate files for homology models were determined using sequence identity criteria (average identity 37%). The DSS1 model structures were created using the Phyre2 protein folding recognition server by uploading the following sequences: AtDSS1(I) (Uniprot:AT1G64750) and AtDSS1(V) (Uniprot:AT5G45010) [67 (link)]. The best template structure, human 26S proteasome bound to ubiquitin carboxyl-terminal hydrolase 14 (USP14)-ubiquitin-like modifier-activating enzyme 1 (UbAl) (pdb:5gjg) has 66% identity with a query sequence [68 (link)]. After preparing the protein data bank (PDB) model, the docking protocol was applied using the PatchDock algorithm with a cluster root mean square deviation (RMSD) of 4 Å [69 (link)]. The top 20 cluster structures were subjected to FireDock refinement based on transformations [70 (link)]. The scoring function is based on geometric fitting and atomic desolvation energy [71 (link)]. The parameters used to calculate the solution global binding energy (GBE) are the atomic contact energy or desolvation energy for the two proteins at transit from the unbound state to the complex (ACE), hydrogen and disulfide bonds (HB) and aliphatic interactions (ALIPH), attractive and repulsive van der Walls forces, short- and long-range Coulomb forces, cation-π and π-π stacking interactions.