cIAPs promote the proteasomal degradation of mutant SOD1 linked to familial amyotrophic lateral sclerosis

Accepted Manuscript cIAPs promote the proteasomal degradation of mutant SOD1 linked to familial amyotrophic lateral sclerosis Jin Sun Choi, Kidae Kim, Do Hee Lee, Sayeon Cho, Jae Du Ha, Byoung Chul Park, Sunhong Kim, Sung Goo Park, Jeong-Hoon Kim PII:

S0006-291X(16)31743-0

DOI:

10.1016/j.bbrc.2016.10.065

Reference:

YBBRC 36612

To appear in:

Biochemical and Biophysical Research Communications

Received Date: 4 October 2016 Accepted Date: 19 October 2016

Please cite this article as: J.S. Choi, K. Kim, D.H. Lee, S. Cho, J.D. Ha, B.C. Park, S. Kim, S.G. Park, J.-H. Kim, cIAPs promote the proteasomal degradation of mutant SOD1 linked to familial amyotrophic lateral sclerosis, Biochemical and Biophysical Research Communications (2016), doi: 10.1016/ j.bbrc.2016.10.065. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT cIAPs promote the proteasomal degradation of mutant SOD1 linked to familial amyotrophic lateral sclerosis

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Jin Sun Choi1, Kidae Kim1,4, Do Hee Lee7, Sayeon Cho8, Jae Du Ha6, Byoung Chul Park1,4, Sunhong Kim1,5*, Sung Goo Park1,3*, and Jeong-Hoon Kim 2,3*

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Personalized Genomic Medicine

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Disease Target Structure Research Center,

Research Center, Korea Research Institute of Bioscience & Biotechnology (KRIBB),

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Daejeon 305-333, Republic of Korea 3

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Department of Functional Genomics,

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Department of Bio-Analytical Science,

Department of Biomolecular Science, University of Science and Technology (UST),

Daejeon 305-350, Republic of Korea 6

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Bio & Drug Discovery Division, Korea Research Institute of Chemical Technology,

141 Gajeong-ro, Yuseong, Daejeon 305-600, Republic of Korea 7

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Department of Biotechnology, Seoul Women’s University, Seoul, Republic of Korea

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College of Pharmacy, Chung-Ang University, Seoul, Republic of Korea

* Corresponding authors. Sunhong Kim, Disease Target structure Research Center, Korea Research Institute of Bioscience & Biotechnology (KRIBB), Daejeon 34141, Republic of Korea. Tel: +82-42-860-4278; Fax: +82-42-860-4269; E-mail addresses: [email protected]

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ACCEPTED MANUSCRIPT Sung Goo Park, Disease Target structure Research Center, Korea Research Institute of Bioscience & Biotechnology (KRIBB), Daejeon 34141, Republic of Korea. Tel: +82-42-860-4262; Fax: +82-42-860-4269; E-mail addresses: [email protected]

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Jeong-Hoon Kim, Personalized Genomic Medicine Research Center, Korea Research Institute of Bioscience & Biotechnology (KRIBB), Daejeon 34141, Republic of Korea, Tel: +82-42-860-4264; Fax: +82-42-860-4269; E-mail addresses:

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[email protected]

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Contributions

J S Choi and K D Kim: performed experiments; J S Choi, S Cho, S Kim, B C Park, S G Park, J H Kim; analyzed data; J S Choi, K D Kim, S Kim, J D Ha, S Cho, S G Park,

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B C Park, J H Kim; designed experiments; J S Choi, J H Kim wrote the paper.

Competing financial interests

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The authors declare no competing financial interests.

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ACCEPTED MANUSCRIPT ABSTRACT Although the ubiquitin–proteasome system is believed to play an important role in the pathogenesis of familial amyotrophic lateral sclerosis (FALS), caused by

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mutations in Cu/Zn-superoxide dismutase 1 (SOD1), the mechanism of how mutant SOD1 protein is regulated in cells is still poorly understood. Here we have demonstrated that cellular inhibitor of apoptosis proteins (cIAPs) are specifically

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associated with FALS-linked mutant SOD1 (mSOD1) and that this interaction promotes the ubiquitin-dependent proteasomal degradation of mutant SOD1. By

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utilizing cumate inducible SOD1 cells, we also showed that knock-down or pharmacologic depletion of cIAPs leads to H2O2 induced cytotoxicity in mSOD1 expressing cells. Altogether, our results reveal a novel role of cIAPs in FALS-

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associated mutant SOD1 regulation.

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ACCEPTED MANUSCRIPT INTRODUCTION A hallmark of neurodegenerative diseases such as Parkinson’s disease, Huntington’s disease, Alzheimer’s disease (AD), and Amyotrophic lateral sclerosis (ALS) is the

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deposit of ubiquitin-positive protein aggregates in the brains of affected patients (1). ALS is an adult neurodegenerative disease caused by the selective loss of motor neurons in the cerebral cortex, brain stem, and spinal cord. It is characterized by

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progressive paralysis, selective neuronal death, and eventual death of the affected individuals. Although most ALS cases are sporadic with unknown causes, 10 % are

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inherited, in what is known as familial ALS (FALS). The best characterized form of FALS (approximately 20 % of cases) is the dominant mutation in the gene encoding Cu/Zn-superoxide dismutase (SOD1) (2). SOD1 is an abundant antioxidant enzyme, originally identified in the central nervous system, and which catalyzes the

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conversion of superoxide to water and hydrogen peroxide (3). It has been shown that motor neuron death in mice expressing FALS-linked mutant SOD is due to a ‘‘toxic gain-of-function’’ caused by SOD1 mutations (4). At the molecular level, mutant

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SOD1 proteins are rapidly degraded by proteasomes, whose inhibition can induce aggresome formation and subsequent motor neuron death (5). Several E3 ligases

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involved in the proteasomal degradation of mutant SOD1 have been identified. Dorfin, a RING-finger type E3 ubiquitin ligase, protects neuronal cells from the toxic effects of mutant SOD1 proteins by proteasomal degradation (6). Previously, we showed that Hsc70-interacting protein (CHIP), a co-chaperone and quality-control E3 ligase, promotes the degradation of mutant SOD1 (7). Another research group showed that Dorfin-CHIP chimeric proteins are able to degrade mutant SOD1 with high efficiency (8). To define the mechanism underlying the ubiquitin-dependent 4

ACCEPTED MANUSCRIPT degradation of mutant SOD1 and its implications in inclusion body formation, it is therefore crucial to understand how these mutant SOD1 proteins are ubiquitinated and which ubiquitination components are involved in this process.

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The inhibitor of apoptosis protein (IAP) family is an endogenous and negative regulator of apoptosis (9). IAPs are characterized by the presence of the N-terminal baculovirus IAP repeat (BIR) domain (10). Initially, IAP proteins have been shown to

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inhibit apoptosis by directly preventing proteolytic cleavage of caspases (10, 11). Subsequently, some IAP proteins such as cIAP1, cIAP2 and XIAP have been

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demonstrated to possess a RING finger domain which provides them with E3 ubiquitin ligase activity (12). It has been well documented that cIAP1 and cIAP2 regulate the nuclear factor-κB (NF-κB) signaling pathway by means of their E3 ligase activity. In the tumor necrosis factor alpha (TNFα)-mediated canonical NF-kB

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pathways, cIAPs are recruited to TNF receptor 1- and 2-associated complexes through direct interaction with TNF-associated factors 2 (TRAF2) (13). The recruited cIAPs mono-ubiquitinate receptor interacting serine-threonine kinase 1 (RIPK1),

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subsequently leading to the activation of canonical NF-kB pathway (14). On the other hand, under unstimulated conditions, cIAPs together with TRAF1 and TRAF2

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promote the degradation of NF-κB inducing kinase (NIK), suppressing the noncanonical NF-κB signaling target genes, including pro-apoptotic genes (15). Intriguingly, it has been shown that XIAP, a representative of IAPs, is involved in mutant SOD protein induced cell death. The activation of apoptotic caspases such as caspase-12, caspase-9, and caspase-3 have been observed in a transgenic model of ALS (16) and the forced expression of XIAP prolongs survival of ALS transgenic mice by directly inhibiting caspases (16-18). However, in spite of the 5

ACCEPTED MANUSCRIPT structural similarity between XIAP and cIAPs, the relationship between cIAPs and ALS is unknown. In the present study, we demonstrate that cIAPs selectively bind to mutant

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SOD1 proteins, and that this interaction facilitates the ubiquitination of mutant SOD1, leading to the degradation of mutant SOD1. Furthermore, we show that knock-down or pharmacologic depletion of cIAPs induces H2O2 induced cytotoxicity in mutant

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SOD1 expressing cells. Our findings implicate cIAPs as regulators of FALSassociated mutant SOD1 and provide an evidence that cIAPs could be therapeutic

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target for FALS.

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ACCEPTED MANUSCRIPT MATERIALS AND METHODS

Cell culture, transfections, and reagents HEK293T cells (#CRL-11268, ATCC) and HeLa cells (#CCL-2, ATCC) were

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cultured in Dulbecco’s Modified Eagle Medium (DMEM) (GIBCO) supplemented with 10% fetal bovine serum, and penicillin/streptomycin (100 U/mL) in humidified incubators at 37 °C with 5 % CO2. Transfection of HEK293T cells and HeLa cells

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with various expression vectors was carried out using Lipofectamine (Invitrogen) and X-tremeGENE (Roche). Cumate solution (QM100A-1) was obtained from System

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Biosciences (SBI). BV6 (S7463) was obtained from Selleckchem. CellTiter-Glo® Luminescent Cell Viability Assay (G7571) and DeadEnd™ Fluorometric TUNEL System (G3250) were purchased from Promega Corporation. Complete protease

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inhibitor cocktail (13760700) was purchased from Roche.

Immunoprecipitation and western blot analysis For immunoprecipitation experiments, cells were lysed in lysis buffer (20 mM

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Tris–HCl [pH 7.5] containing 150 mM NaCl, 1% (v/v) IGEPAL, 10 % (w/v) glycerol, and 1 mM complete protease inhibitor cocktail). Prepared cell lysates were mixed

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with anti-FLAG M2 affinity gel or anti-HA-agarose at 4 °C for 4 h with gentle agitation or were incubated for 2 h at 4 °C with appropriate antibodies with gentle rotation. Subsequently, protein G plus/protein A agarose was added to each sample and incubated at 4 °C for 2 h with rocking. The beads were collected by centrifugation, washed three times with lysis buffer, and then boiled in SDS-PAGE sample buffer for 5 min to elute the antigen complex from the affinity gel. Samples were resolved by

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ACCEPTED MANUSCRIPT SDS-PAGE, transferred to nitrocellulose membranes (10401116, GE Healthcare Life Sciences), and analyzed by immunoblotting using appropriate antibodies. .

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Establishment of inducible cell lines PiggyBac transposon Mammalian expression vector constructs were obtained as follows. Wild type SOD1 (WT) and mutant SOD1 (G93A) were subcloned into the

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FLAG-tagged PB Cumate switch Transposon vector (System Biosciences). HeLa cells were transfected with PB Transposon plasmid DNA and PiggyBac Transposase

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plasmid (System Biosciences) with X-tremeGENE (Roche). To establish stable inducible cell lines, cells were split after three days at various confluences (1:60 or 1:600 dilution) and placed in media containing 1 µg/ml puromycin. After two weeks of puromycin selection, cumate solution was added (System Biosciences) to induce

siRNA knockdown

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FLAG-SOD1 (WT and G93A) expression.

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RNA interference was carried out using specific siRNA duplexes to silence cIAP1 and cIAP2 in HEK293T or HeLa cells. Chemically synthesized siRNA

used

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duplexes were purchased from Bioneer Corporation. The following sequences were for

all

experiments: cIAP1; 5′-GUCACUGCUUGUUAUGCAU-3′, cIAP2; 5′ CAGUUA CCCUCAUCUACUU-3′. Delivery of sicIAPs or scrambled control into the cells was carried out using the Neon® transfection system (Invitrogen) according to the manufacturer’s instructions; cells were then incubated for 24–48 h before analysis.

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ACCEPTED MANUSCRIPT Cell viability assay and TUNEL assay To perform the cell viability assay, SOD1-G93A cells pretreated with siRNA or with BV6, were incubated in 1mM H2O2 for 0, 6, and 9 h. Cell viability was assessed

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using Cell TiterGlo (CTG) mixture as recommended by the supplier. The fragmented DNA of apoptotic cells was measured by TUNEL, using the DeadEnd Fluorometric TUNEL System (Promega) in accordance with the

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manufacturer’s instructions. In brief, SOD1-G93A cells were seeded in 35 mm glassbottom dishes (MatTek Corp., Ashland, MA, USA) with addition of cumate. The cell

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plate without cumate addition was assayed as the experimental control. After 24 h, the cells were fixed with 4 % formaldehyde in PBS for 25 min at 4 °C and then permeabilized with 0.2 % Triton X-100 for 5 min. The cells were incubated with TdT Reaction Mix for 1 h at 37 °C, washed with distilled water, and incubated for 30 min

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with 2× SSC at room temperature. The stained, mounted cells were counterstained with DAPI. Fluorescence was detected using a Zeiss LSM 510 META confocal

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microscope (Zeiss, Thornwood, NY, USA).

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ACCEPTED MANUSCRIPT RESULTS

cIAPs interact with mutant SOD1 As the survival of mutant SOD1 expressing mice was affected by the expression

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of XIAP, an IAP family protein, we hypothesized that cIAPs could play a role in regulation of mutant SOD1. Therefore, we tested the interaction between cIAP1 or cIAP2 and wildtype SOD1 or FALS associated mutant SOD1 (G93A) using co-

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immunoprecipitation experiments. To this end, either FLAG tagged wildtype SOD1 or G93A SOD1 was expressed with Myc tagged cIAP1 or cIAP2 in HEK293 cells.

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SOD1 proteins were immunoprecipitated using anti-FLAG M2 agarose and the resulting immune complexes were analyzed by immunoblotting with anti-Myc antibody. Of interest, mutant SOD1 selectively interacted with cIAP1 and cIAP2, whereas wildtype SOD1 did not interact with cIAP1, nor cIAP2 (Figure 1A and

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Supplementary Figure S1A). In the reciprocal co-immunoprecipitation experiment, specific association of mutant SOD1 and cIAP1 was also observed (Figure 1B),

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implying that the cIAPs interact with FALS associated mutant SOD1.

cIAPs regulate the stability of mutant SOD1

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cIAPs function as an E3 ubiquitin ligase, regulating the stability of numerous cellular proteins through their own RING domain (19). Direct interaction between cIAPs and mutant SOD1 prompted us to examine whether cIAPs might regulate the stability of mutant SOD1. To avoid the co-transfection artefect, we established HeLa cell lines inducibly expressing either wildtype SOD1 or G93A SOD1 using the PiggyBac cumate switch system. Upon cumate treatment, wildtype SOD1 or G93A SOD1 was expressed in the established cell lines (Figure 2A). Using these cell lines, 10

ACCEPTED MANUSCRIPT we examined SOD1 levels when sicIAP1, sicIAP2 or both was treated in the presence of cumate. Wildtype SOD1 level was not changed while G93A SOD1 level increased by treatment of sicIAPs (Figure 2B). It is of note that sicIAP2 showed

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prominent effects. Similarly, pharmacologic depletion of cIAPs by Smac mimetic BV6, a bivalent IAP antagonist compound which causes the degradation of cIAPs, also induced increased G93A SOD1 level (Figure 2C). To examine whether this effect

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was due to proteasomal degradation, cells were treated with the proteasome inhibitor, MG132 and sicIAPs. Upon MG132 treatment, reduced level of G93A SOD1 by

degradation of mutant SOD1.

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sicIAP1/2 was rescued (Figure 2D), suggesting that cIAPs promote the proteasomal

cIAPs facilitates the ubiquitination of mutant SOD1

In general, proteasomal degradation requires the ubiquitination of target substrates

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(20). Thus, we performed in vivo ubiquitination assay to examine whether cIAPs could promote the ubiquitination of mutant SOD1. FLAG tagged wildtype SOD1 or

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G93A SOD1 was expressed with Myc tagged cIAP1 (Myc-cIAP1) and HA tagged ubiquitin (HA-Ub) in HEK293T cells. SOD1 proteins were immunoprecipitated using

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anti-FLAG M2 agarose and the resulting immune complexes were analyzed by immunoblotting with anti-HA and anti-FLAG antibodies. In the absence of exogenous cIAP1, the ubiquitination of G93A SOD1 was marginal, whilst in the presence of exogenous cIAP1, the ubiquitination of G93A SOD1 strongly increased (Figure 3A). However, wildtype SOD1 was not ubiquitinated regardless of cIAP1 (Figure 3A). Similar results were obtained for cIAP2 (Supplementary Figure S1B). Subsequently, endogenous cIAPs were knocked down using siRNA to investigate the level of 11

ACCEPTED MANUSCRIPT mutant SOD1 ubiquitination. Poly-ubiquitinated G93A SOD1 reduced upon treatment with sicIAP1/2, compared to the scrambled control (Figure 3B), implying that cIAPs directly facilitate the ubiquitination of mutant SOD1. As the RING domain of cIAPs is

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crucial for E3 ligase function, we wondered whether the RING finger domain of cIAP1 is a prerequisite for mutant SOD1 ubiquitination. SOD1-G93A and HAubiquitin were expressed along with wildtype cIAP1 or mutant cIAP1 (H588A) which

resulting

immunocomplex

was

analyzed

by

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lacks E3 ligase activity (19). The SOD1 proteins were immunoprecipitated and the immunoblotting.

Although

the

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overexpression of wildtype cIAP1 augmented the ubiquitination of G93A SOD1, the overexpression of H588A mutant cIAP1 had little effect on the formation of polyubiquitin chains (Figure 3C). This result strongly indicates that RING domain of

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cIAP1 is required for the ubiquitination of mutant SOD1.

Depletion of cIAPs exacerbates H2O2 induced cytotoxicity in mutant SOD1 expressing cells

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Previous reports have shown that overexpression of mutant SOD1 leads to cellular toxicity in cultured neuronal cells and transgenic animal models (21) and that

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mutant SOD1 expressing cells exhibit a dose dependent reduction in cell viability when exposed to H2O2 induced oxidative stress (4, 22, 23). We wondered if cIAPs might contribute to mutant SOD1-mediated cytotoxicity upon H2O2 treatment. To investigate, the cell viability assay was carried out using SOD1-G93A cells, with cIAPs depleted by siRNAs or Smac mimetic. Cumate treatment or cIAP1/2 knockdown induced slight cytotoxicity (20 %~40 %), regardless of H2O2 exposure (Figure 4A). When cells were treated with both sicIAP1/2 and cumate, only 20 % of 12

ACCEPTED MANUSCRIPT the population survived following 6 h and 9 h of H2O2 exposure, indicating that cIAPs are necessary for cell survival when mutant SOD1 is overexpressed (Figure 4A). Similar results were observed in experiments with treatment of BV6. Co-treatment of

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BV6 and cumate induced significant cell death upon H2O2 exposure (Figure 4B). It is of note that treatment of sicIAP1/2 or BV6 did not affect cell viability in cumate induced wildtype SOD1 cells (Figure S2). In addition, we observed that

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downregulation of cIAP1/2 induced greater DNA fragmentation in mutant SOD1 expression using TUNEL assay. (Figure 4C and 4D), suggesting that cIAPs modulate

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mutant SOD1-mediated cell death.

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ACCEPTED MANUSCRIPT DISCUSSION Here we demonstrated that cIAPs selectively bind to FALS-linked mutant SOD1, resulting in its proteasomal degradation. However, this result raises the question of

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how cIAPs can discriminate between mutant and wildtype SOD1. Of interest, cIAPs possess an ubiquitin-associated (UBA) domain in their center. Therefore, we hypothesized that the UBA domain of cIAPs may be essential for this interaction. To

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test this, a UBA domain mutant of cIAP1 (MF/AA) was generated and was used to test the interaction with mutant SOD1. However, UBA mutant cIAP1 also bound to

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mutant SOD1, suggesting that the UBA domain was not essential for this interaction (Supplementary Figure S3). Previously, the carboxyl terminus of CHIP, a cochaperone and a quality-control E3 ligase, was found to interact with mutant SOD1 and promote mutant SOD1 degradation through direct ubiquitination of its interacting

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partner, heat-shock protein 70 or heat-shock cognate 70 (Hsp/Hsc70) (7). Thus we reasoned that CHIP could mediate the interaction between cIAPs and mutant SOD1, facilitating the degradation of mutant SOD1. However, we failed to observe the

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specific interaction between mutant SOD1 and CHIP when mutant SOD1 and CHIP were overexpressed (data not shown). Alternatively, we speculated that the

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association of cIAPs and mutant SOD1 could be mediated by heat-shock proteins, which have been known to be involved in the ubiquitination of abnormal or misfolded proteins (7). This idea is also supported by the fact that cIAP1 regulates cell differentiation through the interaction with Hsp90 (24). SOD1 is an antioxidant enzyme in the cellular antioxidant defense system, scavenging oxygen radicals. Previous studies have shown that FALS-linked mutant SOD1 malfunction, which promotes the production of oxygen radicals, leads to their 14

ACCEPTED MANUSCRIPT intracellular inclusion, which is toxic to motor neurons (25). In our study, we observed that the downregulation of cIAPs by siRNA increases both mutant SOD1 levels and H2O2 induced cell death. Likewise, the smac mimetic compounds (BV6) have been

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shown to lead to mutant SOD1 accumulation. A recent study has demonstrated that NF-κB signaling pathway is constitutively activated in ALS patients and the SOD1G93A mouse model of ALS (26). Given the fact that cIAP2 is not only a modulator in

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the NF-κB signaling pathway, but also a target gene of the NF-κB pathway (27), it is plausible that activated NF-κB signaling pathway induces cIAP2 transcription and

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subsequent expression, helping to remove misfolded and/or malfunctioning mutant

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SOD1 proteins, and preventing cell death in ALS patients or SOD1-G93A mice.

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ACCEPTED MANUSCRIPT Acknowledgments This work was supported by the Korea Research Institute of Bioscience and Biotechnology (KRIBB), a research grant from the National Research Foundation of

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of Science and Technology (CAP-15-11-KRICT).

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Korea (NRF- 2011-0028172), and a research grant from National Research Council

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ACCEPTED MANUSCRIPT FIGURE LEGENDS

Figure 1. cIAPs interact with FALS-linked mutant SOD1. (A) FLAG tagged wildtype SOD1 (F-SOD1 (WT)) or FLAG tagged G93A SOD1 (F-SOD1 (G93A)) was with

Myc

immunoprecipitated

tagged

cIAP1

(Myc-cIAP1)

in

HEK293T cells.

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expressed

The

proteins using FLAG beads were analyzed by immunoblotting

with anti-Myc antibody and anti-FLAG antibody. (B) FLAG tagged wildtype SOD1 (F-

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SOD1 (WT)) or FLAG tagged G93A SOD1 (F-SOD1 (G93A)) was expressed with HA tagged cIAP1 (HA-cIAP1) in HEK293T cells. The immunoprecipitated proteins using

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HA beads were analyzed by immunoblotting with anti-FLAG antibody and anti-HA antibody.

Figure 2. cIAPs promote the degradation of FALS-linked mutant SOD1. (A)

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Establishment of HeLa cell lines inducibly expressing wildtype SOD1 (SOD1-WT) or G93A SOD1 (SOD1-G93A). Expression of SOD1 proteins was detected by immunoblotting following cumate treatment. (B) SOD1-WT or SOD1-G93A cells were

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transfected with siRNA specific for cIAP1, cIAP2 or scrambled control and the SOD1 proteins were induced by cumate. After 24 h, the cell lysates were analyzed by

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immunoblotting using anti-FLAG, anti-cIAP1, anti-cIAP2, and anti-GAPDH antibodies. (C) SOD1-G93A cells were pretreated with cumate for 24 h and then cells were treated with or without Smac mimetic BV6 (1 µΜ) for an additional 7 h. The cell lysates were then analyzed by immunoblotting with anti-FLAG, anti-cIAP1, and antiGAPDH antibodies. (D) SOD1-G93A cells transfected with sicIAP1, sicIAP2 or scrambled control were treated with cumate. After 24 h, the cells were then treated with or without a proteasome inhibitor, MG132 (10 µΜ), for an additional 8 h. The cell 17

ACCEPTED MANUSCRIPT lysates were analyzed by immunoblotting using anti-FLAG, anti-cIAP1, anti-cIAP2, and anti-GAPDH antibodies.

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Figure 3. cIAPs facilitate the ubiquitination of FALS-linked mutant SOD1. (A) FLAG tagged wildtype SOD1 (F-SOD1 (WT)) or G93A mutant SOD1 (F-SOD1 (G93A)) was expressed with Myc tagged cIAP1 (Myc-cIAP1) and HA tagged

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ubiquitin (HA-Ub) in HEK293T cells. After 24 h, the cells were treated with a proteasome inhibitor, MG132 (10 µΜ), for an additional 8 h. The immunoprecipitated

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proteins using FLAG beads were analyzed by immunoblotting using anti-HA antibody and anti-FLAG antibody (top two panels). The expression level was determined using anti-Myc and anti-FLAG antibodies (bottom two panels). (B) HeLa cells were transfected with siRNA specific for cIAP1, cIAP2 or scrambled control for 24 h. The

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next day, cells were transfected with FLAG tagged wildtype SOD1 (F-SOD1 (WT)) or mutant SOD1 (F-SOD1 (G93A)) and HA tagged ubiquitin (HA-Ub). After 24 h, the cells were treated with a proteasome inhibitor, MG132 (10 µΜ), for an additional 8 h. immunoprecipitated

proteins

using

FLAG

beads

were

analyzed

by

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immunoblotting with anti-HA antibody and anti-FLAG antibody (top two panels). The

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expression level was determined using anti-cIAP1, anti-cIAP2, and anti-FLAG antibodies (bottom three panels). (C) FLAG tagged mutant SOD1 (F-SOD1 (G93A)) was expressed with Myc tagged cIAP1 (Myc-cIAP1) or Myc tagged H588A (MycH588A) and HA tagged ubiquitin (HA-Ub) in HEK293T cells. After 24 h, the cells were treated with a proteasome inhibitor, MG132 (10 µΜ), for an additional 8 h. The immunoprecipitated using FLAG beads were analyzed by immunoblotting with antiHA antibody and anti-FLAG antibody (top two panels). The expression level was 18

ACCEPTED MANUSCRIPT determined using anti-Myc and anti-FLAG antibodies (bottom two panels)

Figure 4: Depletion of cIAPs exacerbates H2O2 induced cytotoxicity in mutant

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SOD1 expressing cells. (A) SOD-G93A cells electroporated with siRNA specific for cIAP1, cIAP2 or scrambled control were pretreated with cumate for 24 h and the cells were then treated with H2O2 (1 mM) for 0, 6, and 9 h and cell viabilities were

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measured by the CellTiter-Glo assay. Data are presented as mean ± SEM (error bars) of three independent experiments, as indicated (*P < 0.05, **P < 0.01). (B) SOD-

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G93A cells pretreated with or without cumate for 24 h were treated with or without Smac mimetic BV6 (1 µΜ) for an additional 7 h, and cells were treated with H2O2 (1 mM) for 0, 6 and 9 h before the CellTiter-Glo assay was performed. Data are presented as mean ± SEM (error bars) of three independent experiments, as

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indicated (*P < 0.05, **P < 0.01). (C) SOD-G93A cells electroporated with siRNA specific for cIAP1, cIAP2 or scrambled control were pretreated with cumate for 24 h and the cells were then treated with H2O2 (1 mM) for 3 h, and the TUNEL assay was

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carried out. The images of TUNEL positive cells were captured by a fluorescence microscope (400×). (D) Quantitation of the results from (C). Data are presented as

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mean ± SEM (error bars) of three independent experiments, as indicated (**P < 0.01).

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apoptosis protein XIAP results in fragments with distinct specificities for caspases. The

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Yang, Y. L., and Li, X. M. (2000) The IAP family: endogenous caspase inhibitors with multiple biological activities. Cell research 10, 169-177

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Bertrand, M. J., Milutinovic, S., Dickson, K. M., et al., (2008) cIAP1 and cIAP2 facilitate cancer cell survival by functioning as E3 ligases that promote RIP1 ubiquitination.

Molecular cell 30, 689-700 15.

Zarnegar, B. J., Wang, Y., Mahoney, D. J., et al., (2008) Noncanonical NF-kappaB activation requires coordinated assembly of a regulatory complex of the adaptors cIAP1, cIAP2, TRAF2 and TRAF3 and the kinase NIK. Nature immunology 9, 1371-1378 Inoue, H., Tsukita, K., Iwasato, T., et al., (2003) The crucial role of caspase-9 in the disease

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Wootz, H., Hansson, I., Korhonen, L., et al., (2006) XIAP decreases caspase-12 cleavage and

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calpain activity in spinal cord of ALS transgenic mice. Experimental cell research 312, 19.

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Yang, Y., Fang, S., Jensen, J. P., et al., (2000) Ubiquitin protein ligase activity of IAPs and their degradation in proteasomes in response to apoptotic stimuli. Science 288, 874-877

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Neurology, Research Group on Motor Neuron Diseases 1, 143-161 Richardson, K., Allen, S. P., Mortiboys, H., et al., (2013) The effect of SOD1 mutation on

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cellular bioenergetic profile and viability in response to oxidative stress and influence of mutation-type. PloS one 8, e68256

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ACCEPTED MANUSCRIPT Stehlik, C., de Martin, R., Binder, B. R., et al., (1998) Cytokine induced expression of porcine inhibitor of apoptosis protein (iap) family member is regulated by NF-kappa B.

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Biochemical and biophysical research communications 243, 827-832

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27.

22

ACCEPTED MANUSCRIPT

A F-SOD1 (G93A) Myc-cIAP1

IP: FLAG

- + - - + + + +

- + - - + + + +

70 kDa

α-Myc

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F-SOD1 (WT)

Lysate

25 kDa

Lysate HA-cIAP1 F-SOD1 (G93A) F-SOD1 (WT)

+ +

+ -

IP: HA

+ + -

+ +

25 kDa

+ -

+ + -

M AN U

B

SC

α-FLAG

α-FLAG

70 kDa

AC C

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α-HA

Figure 1

ACCEPTED MANUSCRIPT

A

B F-SOD1 (WT)

cumate

-

+

-

control sicIAP1 sicIAP2

+

25 kDa

α-FLAG

+ - - - + - + - - + +

25 kDa

α-GAPDH

35 kDa

F-SOD1 (G93A)

+ - - - + - + - - + +

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SOD1-WT SOD1-G93A

SC

70 kDa

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70 kDa

α-FLAG α-cIAP1 α-cIAP2 α-GAPDH

35 kDa

D

25 kDa

+ +

AC C

70 kDa

+

EP

1uM BV6 FLAG-G93A

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C

35 kDa

MG132 control

sicIAP1/2 FLAG-G93A 25 kDa

+ +

+ +

+ + +

+ + + α-FLAG

α-FLAG 70 kDa

α-cIAP1

70 kDa

α-cIAP2

35 kDa

α-GAPDH

α-cIAP1 α-GAPDH

Figure 2

ACCEPTED MANUSCRIPT

A

B control

F-SOD1 (G93A) F-SOD1 (WT) HA-Ub

+ +

+ +

+ - + + + +

+ + +

sicIAP1/2 FLAG-WT FLAG-G93A HA-ub

(kDa)

(kDa)

+ + +

+ + +

170 130

170 130

α-HA

100

100

IP: FLAG

70

25

70

SC

IP: FLAG

+ + +

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+ -

Myc-cIAP1

+ + +

α-FLAG

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25

(kDa)

α-HA

α-FLAG

(kDa)

α-Myc

70

Lysate 25

α-FLAG

C

Myc-cIAP1 HA-ub

- - - - + - + + + + +

70

α-cIAP2

25

α-FLAG

+ + +

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FLAG-G93A

+ + -

α-cIAP1

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Myc-H588A

Lysate

70

170 kDa 130 kDa

AC C

100 kDa

IP: FLAG

α-HA

70 kDa

25 kDa

α-FLAG 25 kDa

Lysate 70 kDa

α-FLAG α-MYC

Figure 3

ACCEPTED MANUSCRIPT

A

B G93A (1mM H2O2)

G93A (1mM H2O2)

140 **

** *

**

80

control

60

sicIAP1/2 cumate+control

40

cumate+sicIAP1/2

0

100

* **

80 60 40

DMSO BV6 cumate+DMSO cumate+BV6

0

6 Time(Hrs)

9

0

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0

6 Time(Hrs)

9

D

cumate+control cumate+sicIAP1/2

AC C

G93A (1mM H2O2) Apoptosis rate (%)

sicIAP1/2

EP

control

TE D

C

DAPI

**

20

20

TUNEL

*

ns

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100

*

ns

ns

ns

SC

*

*

*

*

Cell viability (%)

Cell viability (%)

120

*

120

120 100

**

80 60 40 20 0

Figure 4

ACCEPTED MANUSCRIPT  cIAPs selectively interact with FALS-linked mutant SOD1.  cIAPs-mutant SOD1 interaction promotes ubiquitination and degradation of mutant SOD1.  Knock-down or pharmacologic depletion of cIAPs leads to H2O2 induced cytotoxicity in mutant SOD1 expressing cells.

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 Our results reveal a novel role of cIAPs in FALS-associated mutant SOD1 regulation.