Mechanism of the Rpn13-induced activation of Uch37

Abstract

Uch37 is a de-ubiquitinating enzyme that is activated by Rpn13 and involved in the proteasomal degradation of proteins. The full-length Uch37 was shown to exhibit low iso-peptidase activity and is thought to be auto-inhibited. Structural comparisons revealed that within a homodimer of Uch37, each of the catalytic domains was blocking the other’s ubiquitin (Ub)-binding site. This blockage likely prevented Ub from entering the active site of Uch37 and might form the basis of auto-inhibition. To understand the mode of auto-inhibition clearly and shed light on the activation mechanism of Uch37 by Rpn13, we investigated the Uch37-Rpn13 complex using a combination of mutagenesis, biochemical, NMR, and smallangle X-ray scattering (SAXS) techniques. Our results also proved that Uch37 oligomerized in solution and had very low activity against the fluorogenic substrate ubiquitin-7-amino-4-methylcoumarin (Ub-AMC) of de-ubiquitinating enzymes. Uch37Δ^Hb,Hc,KEKE, a truncation removal of the C-terminal extension region (residues 256–329) converted oligomeric Uch37 into a monomeric form that exhibited iso-peptidase activity comparable to that of a truncation-containing the Uch37 catalytic domain only. Wealso demonstrated that Rpn13C (Rpn13 residues 270–407) could disrupt the oligomerization of Uch37 by sequestering Uch37 and forming a Uch37-Rpn13 complex. Uch37 was activated in such a complex, exhibiting 12-fold-higher activity than Uch37 alone. Time-resolved SAXS (TR-SAXS) and FRET experiments supported the proposed mode of auto-inhibition and the activation mechanism of Uch37 by Rpn13. Rpn13 activated Uch37 by forming a 1:1 stoichiometric complex in which the active site of Uch37 was accessible to Ub.

Keywords

Uch37-Rpn13 complex / de-ubiquitination / SAXS analysis / oligomerization / iso-peptidase

References

[1] Armstrong CT, Vincent TL, Green PJ, Woolfson DN(2011) SCORER 2.0: an algorithm for distinguishing parallel dimeric and trimeric coiled-coil sequences. Bioinformatics27: 1908-1914

[2] Brunger AT, Adams PD, Clore GM, DeLano WL, Gros P, Grosse-Kunstleve RW, Jiang J-S, Kuszewski J, Nilges M, Pannu NSet al (1998) Crystallography & NMR System: a new software suite for macromolecular structure determination. Acta Crystallogr Sect D54: 905-921

[3] Burgie SE, Bingman CA, Soni AB, Phillips GN Jr (2011) Structural characterization of human Uch37. Proteins80: 649-654

[4] Chen X, Lee BH, Finley D, Walters KJ(2010) Structure of proteasome ubiquitin receptor hRpn13 and its activation by the scaffolding protein hRpn2. Mol Cell38: 404-415

[5] wDas C, Hoang QQ, Kreinbring CA, Luchansky SJ, Meray RK, Ray SS, Lansbury PT, Ringe D, Petsko GA(2006) Structural basis for conformational plasticity of the Parkinson’s disease-associated ubiquitin hydrolase UCH-L1. Proc Natl Acad Sci USA103: 4675-4680

[6] Duggan B, Legge G, Dyson HJ, Wright P(2001) SANE (Structure Assisted NOE Evaluation): an automated model-based approach for NOE assignment. J Biomol NMR19: 321-329

[7] Fischer H, de Oliveira Neto M, Napolitano HB, Polikarpov I, Craievich AF(2010) Determination of the molecular weight of proteins in solution from a single small-angle X-ray scattering measurement on a relative scale. J Appl Crystallogr43: 101-109

[8] Glickman MH, Zhang DN, Chen T, Ziv I, Rosenzweig R, Matiuhin Y, Bronner V, Fushman D(2009) Together, Rpn10 and Dsk2 can serve as a polyubiquitin chain-length sensor. Mol Cell36: 1018-1033

[9] Groll M, Schreiner P, Chen X, Husnjak K, Randles L, Zhang NX, Elsasser S, Finley D, Dikic I, Walters KJ(2008) Ubiquitin docking at the proteasome through a novel pleckstrin-homology domain interaction. Nature453: 548-552

[10] Güntert P, Mumenthaler C, Wüthrich K (1997) Torsion angle dynamics for NMR structure calculation with the new program Dyana. J Mol Biol273: 283-298

[11] Hamazaki J, Iemura S, Natsume T, Yashiroda H, Tanaka K, Murata S(2006) A novel proteasome interacting protein recruits the deubiquitinating enzyme UCH37 to 26S proteasomes. EMBO J25: 4524-4536

[12] Husnjak K, Elsasser S, Zhang N, Chen X, Randles L, Shi Y, Hofmann K, Walters KJ, Finley D, Dikic I(2008) Proteasome subunit Rpn13 is a novel ubiquitin receptor. Nature453: 481-488

[13] Johnson B, Blevins R(1994) NMR view: acomputer program for the visualization and analysis of NMR data. J Biomol NMR4: 603-614

[14] Johnston SC, Larsen CN, Cook WJ, Wilkinson KD, Hill CP(1997) Crystal structure of a deubiquitinating enzyme (human UCH-L3) at 1.8 angstrom resolution. EMBO J16: 3787-3796

[15] Johnston SC, Riddle SM, Cohen RE, Hill CP(1999) Structural basis for the speciflcity of ubiquitin C-terminal hydrolases. EMBO J18: 3877-3887

[16] Konarev PV, Petoukhov MV, Svergun DI(2001) MASSHA—a graphics system for rigid-body modelling of macromolecular complexes against solution scattering data. J Appl Crystallogr34: 527-532

[17] Konarev PV, Volkov VV, Sokolova AV, Koch MHJ, Svergun DI(2003) PRIMUS: a Windows PC-based system for small-angle scattering data analysis. J Appl Crystallogr36: 1277-1282

[18] Koradi R, Billeter M, Wüthrich K (1996) MOLMOL: a program for display and analysis of macromolecular structures. J Mol Graph14: 51-55

[19] Koulich E, Li X, DeMartino GN(2008) Relative structural and functional roles of multiple deubiquitylating proteins associated with mammalian 26S proteasome. Mol Biol Cell19: 1072-1082

[20] Kozin MB, Svergun DI(2001) Automated matching of high- and lowresolution structural models. J Appl Crystallogr34: 33-41

[21] Krissinel E, Henrick K(2007) Inference of macromolecular assemblies from crystalline state. J Mol Biol372: 774-797

[22] Lam YA, Xu W, DeMartino GN, Cohen RE(1997) Editing of ubiquitin conjugates by an isopeptidase in the 26S proteasome. Nature385: 737-740

[23] Larzabal M, Mercado EC, Vilte DA, Salazar-Gonzalez H, Cataldi A, Navarro-Garcia F (2010) Designed coiled-coil peptides inhibit the type three secretion system of enteropathogenic Escherichia coli. PLoS One5: e9046

[24] Laskowski R, Rullmann JA, MacArthur M, Kaptein R, Thornton J(1996) AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR. J Biomol NMR8: 477-486

[25] Liu CW, Li X, Thompson D, Wooding K, Chang TL, Tang Z, Yu H, Thomas PJ, DeMartino GN(2006) ATP binding and ATP hydrolysis play distinct roles in the function of 26S proteasome. Mol Cell24: 39-50

[26] Maiti TK, Permaul M, Boudreaux DA, Mahanic C, Mauney S, Das C(2011) Crystal structure of the catalytic domain of UCHL5, a proteasome-associated human deubiquitinating enzyme, reveals an unproductive form of the enzyme. FEBS J278: 4917-4926

[27] Markley JL, Bax A, Arata Y, Hilbers CW, Kaptein R, Sykes BD, Wright PE, Wuthrich K(1998) Recommendations for the presentation of NMR structures of proteins and nucleic acids—(IUPAC Recommendations 1998). Pure Appl Chem70: 117-142

[28] Misaghi S, Galardy PJ, Meester WJ, Ovaa H, Ploegh HL, Gaudet R(2005) Structure of the ubiquitin hydrolase UCH-L3 complexed with a suicide substrate. J Biol Chem280: 1512-1520

[29] Nederveen AJ, Doreleijers JF, Vranken W, Miller Z, Spronk CAEM, Nabuurs SB, Güntert P, Livny M, Markley JL, Nilges M (2005) RECOORD: a recalculated coordinate database of 500+proteins from the PDB using restraints from the BioMagResBank. Proteins59: 662-672

[30] Nishio K, Kim SW, Kawai K, Mizushima T, Yamane T, Hamazaki J, Murata S, Tanaka K, Morimoto Y(2009) Crystal structure of the de-ubiquitinating enzyme UCH37 (human UCH-L5) catalytic domain. Biochem Biophys Res Commun390: 855-860

[31] Niu F, Shaw N, Wang YE, Jiao L, Ding W, Li X, Zhu P, Upur H, Ouyang S, Cheng G (2013) Structure of the Leanyer orthobunyavirus nucleoprotein-RNA complex reveals unique architecture for RNA encapsidation. Proc Natl Acad Sci USA110: 9054-9059

[32] Popp MW, Artavanis-Tsakonas K, Ploegh HL(2009) Substrate flltering by the active site crossover loop in UCHL3 revealed by sortagging and gain-of-function mutations. J Biol Chem284: 3593-3602

[33] Qiu XB, Ouyang SY, Li CJ, Miao S, Wang L, Goldberg AL(2006) hRpn13/ADRM1/GP110 is a novel proteasome subunit that binds the deubiquitinating enzyme, UCH37. EMBO J25: 5742-5753

[34] Reyes-Turcu FE, Ventii KH, Wilkinson KD(2009) Regulation and cellular roles of ubiquitin-speciflc deubiquitinating enzymes. Annu Rev Biochem78: 363-397

[35] Schuck P(2000) Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and lamm equation modeling. Biophys J78: 1606-1619

[36] Shen Y, Delaglio F, Cornilescu G, Bax A(2009) TALOS+: a hybrid method for predicting protein backbone torsion angles from NMR chemical shifts. J Biomol NMR44: 213-223

[37] Svergun D(1992) Determination of the regularization parameter in indirect-transform methods using perceptual criteria. J Appl Crystallogr25: 495-503

[38] Svergun DI, Petoukhov MV, Koch MH(2001) Determination of domain structure of proteins from X-ray solution scattering. Biophys J80: 2946-2953

[39] Ventii KH, Wilkinson KD(2008) Protein partners of deubiquitinating enzymes. Biochem J414: 161-175

[40] Voges D, Zwickl P, Baumeister W(1999) The 26S proteasome: a molecular machine designed for controlled proteolysis. Annu Rev Biochem68: 1015-1068

[41] Wicks SJ, Haros K, Maillard M, Song L, Cohen RE, Dijke PT, Chantry A(2005) The deubiquitinating enzyme UCH37 interacts with Smads and regulates TGF-beta signalling. Oncogene24: 8080-8084

[42] Yao T, Song L, Xu W, DeMartino GN, Florens L, Swanson SK, Washburn MP, Conaway RC, Conaway JW, Cohen RE(2006) Proteasome recruitment and activation of the Uch37 deubiquitinating enzyme by Adrm1. Nat Cell Biol8: 994-1002

[43] Yao T, Song L, Jin J, Cai Y, Takahashi H, Swanson SK, Washburn MP, Florens L, Conaway RC, Cohen RE (2008) Distinct modes of regulation of the Uch37 deubiquitinating enzyme in the proteasome and in the Ino80 chromatin-remodeling complex. Mol Cell31: 909-917

[44] Yin ST, Huang H, Zhang YH, Zhou ZR, Song AX, Hong FS, Hu HY(2011) A fluorescence assay for elucidating the substrate speciflcities of deubiquitinating enzymes. Biochem Biophys Res Commun416: 76-79

[45] Young P, Deveraux Q, Beal RE, Pickart CM, Rechsteiner M(1998) Characterization of two polyubiquitin binding sites in the 26 S protease subunit 5a. J Biol Chem273: 5461-5467

[46] Zhang H, Chen J, Wang Y, Peng L, Dong X, Lu Y, Keating AE, Jiang T(2009) A computationally guided protein-interaction screen uncovers coiled-coil interactions involved in vesicular trafflcking. J Mol Biol392: 228-241

[47] Zhou ZR, Zhang YH, Liu S, Song AX, Hu HY(2012) Length of the active-site crossover loop deflnes the substrate speciflcity of ubiquitin C-terminal hydrolases for ubiquitin chains. Biochem J441: 143-149

[48] Zhu QZ, Wani G, Wang QE, El-mahdy M, Snapka RM, Wani AA(2005) Deubiquitination by proteasome is coordinated with substrate translocation for proteolysis in vivo. Exp Cell Res307: 436-451

RIGHTS & PERMISSIONS

2014 This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.