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. 2015 Sep 25;290(39):23850-62.
doi: 10.1074/jbc.M115.664375. Epub 2015 Aug 10.

NEDD8 Ultimate Buster 1 Long (NUB1L) Protein Suppresses Atypical Neddylation and Promotes the Proteasomal Degradation of Misfolded Proteins

Jie Li  1 , Wenxia Ma  1 , Huizhong Li  1 , Ning Hou  2 , Xuejun Wang  3 , Il-man Kim  1 , Faqian Li  2 , Huabo Su  4
Affiliations

NEDD8 Ultimate Buster 1 Long (NUB1L) Protein Suppresses Atypical Neddylation and Promotes the Proteasomal Degradation of Misfolded Proteins

Jie Li et al. J Biol Chem. .

Abstract

Neddylation is a posttranslational modification that controls diverse biological processes by covalently conjugating the ubiquitin-like protein NEDD8 to specific targets. Neddylation is commonly mediated by NEDD8-specific enzymes (typical neddylation) and, sometimes, by ubiquitin enzymes (atypical neddylation). Although typical neddylation is known to regulate protein function in many ways, the regulatory mechanisms and biological consequence of atypical neddylation remain largely unexplored. Here we report that NEDD8 conjugates were accumulated in the diseased hearts from mouse models and human patients. Proteotoxic stresses induced typical and atypical neddylation in cardiomyocytes. Loss of NUB1L exaggerated atypical neddylation, whereas NUB1L overexpression repressed atypical neddylation through promoting the degradation of NEDD8. Activation of atypical neddylation accumulated a surrogate misfolded protein, GFPu. In contrast, suppression of atypical neddylation by NUB1L overexpression enhanced GFPu degradation. Moreover, NUB1L depletion accumulated a cardiomyopathy-linked misfolded protein, CryAB(R120G), whereas NUB1L overexpression promoted its degradation through suppressing neddylation of ubiquitinated proteins in cardiomyocytes. Consequently, NUB1L protected cells from proteotoxic stress-induced cell injury. In summary, these data indicate that NUB1L suppresses atypical neddylation and promotes the degradation of misfolded proteins by the proteasome. Our findings also suggest that induction of NUB1L could potentially become a novel therapeutic strategy for diseases with increased proteotoxic stress.

Keywords: NEDD8; NUB1L; cardiomyopathy; posttranslational modification (PTM); proteasome; protein degradation; ubiquitin.

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Figures

FIGURE 1.
FIGURE 1.
Increased NEDD8 conjugates in diseased hearts. A and B, representative Western blots (A) of the indicated proteins and densitometric analysis (B) of 2-month-old CryABR120G and D7-Des Tg mouse hearts. n = 4–6 for each group. Conj, conjugate. C, NEDD8 immunohistochemistry of myocardium sections of explanted failed human hearts with ischemic or dilated cardiomyopathy and nonfailing hearts from donors without cardiovascular disorders. Normal rabbit serum was used as the primary antibody for a negative control. Shown are representative images from five sets of staining.
FIGURE 2.
FIGURE 2.
Activation of typical and atypical neddylation by multiple stresses in cardiomyocytes. A, NRVMs were treated with the proteasome inhibitor bortezomib (BZM) for the indicated times. HMW, high molecular weight; conj, conjugate. B, NRVMs were treated with the proteasome inhibitors BZM (100 nm), MG132 (5 μm), and MG262 (1 μm) for 6 h. The NAE inhibitor MLN4924 (MLN) was added 1 h after the administration of proteasome inhibitors. Veh, vehicle. C, NRMMs were treated with vehicle or BZM (100 nm) in the absence or presence of MLN4924 (1 μm) for 6 h. Polyubiquitinated proteins were purified from cell extracts using TUBEs under denaturing or non-denaturing conditions and immunoblotted with the indicated antibodies. D, NRMMs were subjected to sI for 12 h. E, NRMMs were infected with Ad-SF-NEDD8 for 24 h and treated with H2O2 for another 8 h. A–E, Shown are representative Western blots and the densitometric analysis from three independent experiments. The asterisks and # denote high and low molecular weight NEDD8 conjugates respectively.
FIGURE 3.
FIGURE 3.
NUB1L suppresses neddylation of ubiquitinated proteins. A, extracts from NUB1L wild-type (+/+) or deficient (−/−) MEFs treated with BZM (100 nm) for 6 h were precipitated with TUBEs to pull down polyubiquitinated proteins under denaturing conditions and analyzed by Western blots. IP, immunoprecipitation. B and C, NRVMs expressing Strep- and FLAG-tagged NEDD8 (SF-NEDD8) were infected with Ad expressing FLAG-tagged NUB1L (Ad-NUB1L) or β-gal (Ad-Gal) or transfected with siRNA against UBE1 (siUBE1). BZM (100 nm) or MLN4924 (1 μm) was added 6 h before cell harvest. B, representative Western blots of total cell lysates. Conj, conjugate. C, polyubiquitinated or neddylated proteins were pulled down with TUBEs or NEDD8 antibody from cell extracts under denaturing conditions and analyzed by Western blots. D, CHX chase assay. Representative Western blots (top panel) and densitometric analysis (bottom panel) are shown. The NEDD8 densitometries were relative to its level at 0 h.
FIGURE 4.
FIGURE 4.
Activation of atypical neddylation accumulates a proteasome surrogate substrate GFPu. A and B, HEK293 cells stably expressing GFPu and RFP were transfected with the SF-NEDD8 or SF-NEDD8ΔGG expression vectors for 48 h. Conj, conjugate. C and D, NRVMs were infected with Ad-GFPu and Ad-RFP before infection with Ad-SF-NEDD8 for another 48 h. Representative immunoblots of the indicated proteins (A and C) and quantitative analyses (B and D) are shown. **, p < 0.01; ***, p < 0.001.
FIGURE 5.
FIGURE 5.
NUB1L overexpression enhances the degradation of GFPu. NRVMs were first infected with Ad expressing GFPu (Ad-GFPu) and RFP (Ad-RFP) for 24 h and subsequently with Ad-NUB1L or Ad-Gal. A, representative Western blots of total cell extracts are shown. Ad-NUB1L was infected at 5, 10, and 20 MOI and Ad-Gal at 20 MOI. BZM (100 nm) was added 6 h prior to cell harvesting. B, quantitative real-time PCR analysis of GFPu transcripts. N.S., not significant. C and D, CHX chase assay. Representative Western blots (C) and the quantification (D) are shown. GFPu protein levels at 0 h were set as 1. E and F, cells were treated with H2O2 (100 μm) for 8 h (E), sI for 6 h, or sI followed by reperfusion for another 6 h (sI/R) (F) prior to cell harvesting. Ad-NUB1L was infected at 10 and 20 (E) or 10 MOI (F). Representative Western blots of total cell extracts are shown. The fast-migrating band of GFPu is its N-terminal truncated form. The densitometries of GFPu normalized to RFP or NUB1L are denoted in A, C, E, and F.
FIGURE 6.
FIGURE 6.
NUB1L reduces misfolded protein CryABR120G in cardiomyocytes. NRVMs were infected with Ad expressing HA-tagged CryABR120G (Ad-HA-CryABR120G) for 24 h and subsequently infected with Ad-NUB1L (20 MOI) or transfected with siRNA against NUB1L (siNUB1L) for another 72 h. A—D, the Triton X-100 (1%) soluble and insoluble fractions of cell extracts were subjected to Western blot analysis (A and C) and protein dot blot to quantify the abundance of CryABR120G (B and D). Arrows point to the modified forms of CryABR120G. Exp, exposure.
FIGURE 7.
FIGURE 7.
NUB1L promotes the proteasomal degradation of CryABR120G. NRVMs were infected with Ad-HA-CryABR120G or WT CryAB for 24 h and subsequently infected with Ad-NUB1L (20 MOI). A and B, Western blots of total cell extracts (A) and quantification analysis (B). NUB1L overexpression reduced CryABR120G but not WT-CryAB protein levels. Endo, endogenous; N.S., not significant. C, quantitative PCR analysis of CryABR120G transcripts. D and E, proteasome inhibition attenuates the NUB1L-induced reduction of CryABR120G. Representative Western blots (D) of total cell extracts and densitometric analysis (E) are shown. BZM was added 24 h before cell harvesting. F and G, CHX chase assay. Representative Western blots of total cell extracts (F) and pooled densitometry data (G) of CryABR120G are shown. The changes of the native form and modified forms (arrows) of CryABR120G are shown in the short and long exposure (expos) blots, respectively. The abundance of NUB1L is denoted under the blot. A.U., arbitrary unit.
FIGURE 8.
FIGURE 8.
NUB1L overexpression accelerates the turnover of ubiquitinated misfolded proteins. A, the effects of NUB1L on proteasome abundance. Western blots of the indicated proteasome subunits in total cell extracts are shown. B, the effects of NUB1L on proteasome peptidase activities. n = 6 biological repeats. N.S., not significant. C and D, NRVMs were infected with Ad-NUB1L or Ad-Gal for 72 h. Ad-HA-CryABR120G was infected 24 h prior to Ad-NUB1L infection, and BZM was added 24 h before cell harvesting. C, NUB1L overexpression reduced Lys-48-linked ubiquitinated CryABR120G, and proteasome inhibition attenuated the reduction. CryABR120G was immunoprecipitated (IP) with anti-HA antibodies from cell lysate and blotted (IB) with the indicated antibodies. Data are representative of three independent repeats. D, polyubiquitinated proteins were precipitated from cell lysates using TUBEs under denaturing conditions and blotted with the indicated antibodies. Data are representative of three independent repeats.
FIGURE 9.
FIGURE 9.
NUB1L ameliorates proteotoxic stress-induced cytotoxicity. A–C, NRVMs were infected with the indicated adenovirus (A and B) or transfected with the indicated siRNA (C) for 48 h before being subjected to BZM (100 nm) treatment for another 24 h. Representative Western blots of total cell extracts (A) and the densitometric analysis (B) are shown. n = 4–6/group. Veh, vehicle. C, culture medium was collected for the lactate dehydrogenase (LDH) activity assay. D–F, NUB1L wild-type (+/+) or deficient (−/−) MEFs were treated with BZM (100 nm) for 24 h. D, representative Western blots of total cell lysates and the densitometries of cleaved caspase 3 relative to those of GAPDH. E, cell viability assay by measuring cellular ATP levels. F, LDH activity assay. For cell viability and LDH release assays (C, E, and F), data are representative of three independent experiments. n = 6–8 samples/group for each replication. G, model of the function of NUB1L in proteasomal proteolysis.
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