A Split-Luciferase-Based Trimer Formation Assay as a High-throughput Screening Platform for Therapeutics in Alport Syndrome

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A Split-Luciferase-Based Trimer Formation Assay as a High-throughput Screening Platform for Therapeutics in Alport Syndrome Graphical Abstract

Authors Kohei Omachi, Misato Kamura, Keisuke Teramoto, ..., Tsuyoshi Shuto, Mary Ann Suico, Hirofumi Kai

Correspondence [email protected] (M.A.S.), [email protected] (H.K.)

In Brief Omachi et al. showed that the type IV collagen a345(IV) trimer is a therapeutic target for the hereditary kidney disease, Alport syndrome. They established an HTS-applicable a345(IV) trimer assay and showed that chemical chaperones rescued trimer formation of mutant a5(IV).

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Mutant a5(IV) monomers are stable intracellularly despite harboring a mutation a345 (IV) trimer is a preferred therapeutic target for Alport syndrome Developing HTS-applicable a345 (IV) trimer assay Several chemical chaperones have potential to promote a345(IV) trimer formation

Omachi et al., 2018, Cell Chemical Biology 25, 1–10 May 17, 2018 ª 2018 Elsevier Ltd. https://doi.org/10.1016/j.chembiol.2018.02.003

Please cite this article in press as: Omachi et al., A Split-Luciferase-Based Trimer Formation Assay as a High-throughput Screening Platform for Therapeutics in Alport Syndrome, Cell Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.02.003

Cell Chemical Biology

Resource A Split-Luciferase-Based Trimer Formation Assay as a High-throughput Screening Platform for Therapeutics in Alport Syndrome Kohei Omachi,1,2 Misato Kamura,1,2 Keisuke Teramoto,1,2 Haruka Kojima,1 Tsubasa Yokota,1 Shota Kaseda,1,2 Jun Kuwazuru,1 Ryosuke Fukuda,1 Kosuke Koyama,1 Shingo Matsuyama,1 Keishi Motomura,1 Tsuyoshi Shuto,1 Mary Ann Suico,1,* and Hirofumi Kai1,2,3,* 1Department of Molecular Medicine, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto City 862-0973, Kumamoto, Japan 2Program for Leading Graduate School ‘‘HIGO (Health Life Science: Interdisciplinary and Glocal Oriented) Program’’, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto City 862-0973, Kumamoto, Japan 3Lead Contact *Correspondence: [email protected] (M.A.S.), [email protected] (H.K.) https://doi.org/10.1016/j.chembiol.2018.02.003

SUMMARY

Alport syndrome is a hereditary glomerular disease caused by mutation in type IV collagen a3–a5 chains (a3–a5(IV)), which disrupts trimerization, leading to glomerular basement membrane degeneration. Correcting the trimerization of a3/a4/a5 chain is a feasible therapeutic approach, but is hindered by lack of information on the regulation of intracellular a(IV) chain and the absence of high-throughput screening (HTS) platforms to assess a345(IV) trimer formation. Here, we developed sets of split NanoLuc-fusion a345(IV) proteins to monitor a345(IV) trimerization of wild-type and clinically associated mutant a5(IV). The a345(IV) trimer assay, which satisfied the acceptance criteria for HTS, enabled the characterization of intracellular- and secretiondependent defects of mutant a5(IV). Small interfering RNA-based and chemical screening targeting the ER identified several chemical chaperones that have potential to promote a345(IV) trimer formation. This split luciferase-based trimer formation assay is a functional HTS platform that realizes the feasibility of targeting a345(IV) trimers to treat Alport syndrome. INTRODUCTION Alport syndrome (AS) is a hereditary glomerular disease caused by mutation in the COL4A3 (Lemmink et al., 1994), COL4A4 (Jefferson et al., 1997), or COL4A5 (Barker et al., 1990) gene encoding type IV collagen a3, a4, and a5 chains (a3–a5(IV)), respectively, which are components of the glomerular basement membrane (GBM) (Harvey et al., 2003; Lennon et al., 2014). The most common mutations are those in COL4A5, which comprise more than 80% of AS-associated mutations (Crockett et al., 2010). Mutant a chains cannot form a345(IV) trimer, which leads to abnormal GBM (Randles et al., 2017; Suleiman et al., 2013). In

current therapeutic approaches for the management of AS, inhibitors of the renin-angiotensin-aldosterone system (RAAS) are typically prescribed (Gross et al., 2003). Although early intervention by RAAS blockade suppresses the progression of nephritis (Gross et al., 2012; Stock et al., 2017), patients with AS taking RAAS inhibitors eventually develop end-stage renal disease (Savva et al., 2016). Numerous basic studies have revealed the molecules that are associated with the progression of AS (Chen et al., 2003; Kruegel et al., 2013). However, candidate drug targets have not been assessed in clinical applications, and a novel therapeutic strategy is urgently needed (Miner et al., 2014). Protein misfolding is a promising target in various diseases, including cystic fibrosis (CF) (Hutt et al., 2010; Okiyoneda et al., 2013), familial epilepsy (Yokoi et al., 2015), and primary biliary cirrhosis (Cortez and Sim, 2014). In fact, the US Food and Drug Administration has approved corrector compounds, such as ivacaftor and lumacaftor, for CF (Accurso et al., 2010; Ramsey et al., 2011; Van Goor et al., 2011), that can normalize the causal protein. Notably, AS is also classified as a misfolded protein disease. In an AS mouse model carrying a missense mutation of a3(IV), mutant a3/4/5 chains lost the ability to form heterotrimers, causing an unfolded protein response (Pieri et al., 2014). Several reports have described basic and clinical studies on the targeting of misfolded proteins in AS. For example, normalizing the a345(IV) network may be a promising therapy (Lin et al., 2014a). In addition, patients with AS who express even low amounts of the a5(IV) chain in GBM exhibit milder clinical manifestations (Hashimura et al., 2014), particularly with respect to end-stage renal disease and hearing loss (Hashimura et al., 2014). Thus, the presence of a345(IV) in GBM determines prognosis in AS, suggesting that normalizing a345(IV) in GBM may constitute a promising therapeutic strategy. However, to date no effective molecules or compounds have been developed that directly target or specifically correct the a345(IV) trimer. Several factors may contribute to the lack of agents targeting the a345(IV) trimer, one of which is that it is unclear which molecules affect the intracellular behavior of a345(IV). Moreover, there is no available assay for assessing a345(IV) trimer Cell Chemical Biology 25, 1–10, May 17, 2018 ª 2018 Elsevier Ltd. 1

Please cite this article in press as: Omachi et al., A Split-Luciferase-Based Trimer Formation Assay as a High-throughput Screening Platform for Therapeutics in Alport Syndrome, Cell Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.02.003

Figure 1. Intracellular a5(IV) Monomers Are Stable Despite Harboring a Mutation (A) Immunoblots of Myc-tagged WT and mutant a5(IV) monomer in HEK293T cells treated with cycloheximide for chase experiments. (B) Quantification of intracellular a5(IV) expression normalized to vinculin in (A). Data are presented as percentage of the amount detected at 0 hr. Graphs indicate mean ± SE (n = 3). (C) Immunoblots of Myc-tagged WT and mutant a5(IV) monomer in HEK293T cells treated with the indicated reagents for 24 hr. (D) Quantification of intracellular a5(IV) expression normalized to vinculin in (C). Data are presented as percentage of the amount detected in the Mock group. Error bars indicate mean ± SE (n = 3). *p < 0.05, **p < 0.01 versus Mock (Tukey-Kramer test). (E) Intracellular expression of Myc-tagged WT and C1567R a5(IV) monomers in HEK293T cells cotransfected with SAR1 GTP. (F) Quantification of intracellular a5(IV) expression normalized to vinculin in (E). Data are presented as percentage of the amount detected in the Mock group. Error bars indicate the mean ± SE (n = 3). *p < 0.05, **p < 0.01 versus Mock (Student’s t test). Blots in (A), (C), and (E) were probed with anti-Myc or anti-vinculin antibodies. See also Figure S1.

tion ability of mutant a5(IV). These results may have a marked impact on therapeutic drug discovery in AS. RESULTS AND DISCUSSION

formation that is applicable to high-throughput screening (HTS). Previously, an immunoprecipitation (IP)-based approach was used to evaluate the a345(IV) trimer (Kobayashi et al., 2008; Kobayashi and Uchiyama, 2003, 2010). However, this costprohibitive and labor-intensive assay is not ideal for screening compounds that promote a345(IV) trimer formation. Here, we took advantage of the Split Nanoluciferase binary technology (Nanoluc BiT) system in which a subunit (Large BiT [LgBiT] or Small BiT [SmBiT]) is fused to an a(IV) monomer. When the split NanoLuc-tagged proteins interact, the LgBiT and SmBiT complementation system produces luminescence. We established this split NanoLuc-based quantitative and HTS-applicable method as an a345(IV) trimer assay system and demonstrated that some chemical chaperones could rescue the trimer forma2 Cell Chemical Biology 25, 1–10, May 17, 2018

Intracellular a5(IV) Monomers Are Stable Despite Harboring a Mutation Several mutant proteins have reduced intracellular stability and defects in proper trafficking (Gregersen et al., 2006). Thus, we determined the intracellular stability of several mutant a5(IV) monomers (G869R, G1107R, P1517T, C1567R, and L1649R). Before we assessed the stability of a5(IV) monomers, we checked the validity of each a chain and the duration of its transient expression by using antibodies against protein tags (hemagglutinin [HA], FLAG, and Myc) and specific a(IV) chain. The expression of a3–a5(IV) proteins were detected at 48 and 72 hr after plasmid transfection (Figures S1A–S1F), indicating the robustness of this transient expression system. Because a5 mutations accounted for more than 80% of AS-associated mutations, the a5(IV) chain was considered the most important chain for evaluation of defects in a5(IV). Although the rate of degradation of some mutant a5(IV) chains was greater than that of wildtype (WT) a5(IV) monomer, the difference was not significant (Figures 1A and 1B), suggesting that the mutation itself is not a critical factor of the intracellular stability of a5(IV) monomer. Brefeldin A (BFA), an inhibitor of ER-Golgi transport, increased a5(IV) monomer protein expression, but no significant changes

Please cite this article in press as: Omachi et al., A Split-Luciferase-Based Trimer Formation Assay as a High-throughput Screening Platform for Therapeutics in Alport Syndrome, Cell Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.02.003

Figure 2. WT and Mutant a5(IV) Chains Are Similarly Regulated by Typical ER Chaperones (A and B) Blots of intracellular and extracellular Myc-tagged a5(IV) monomer expression in HEK293T cells co-transfected with the indicated ER chaperones (A) or siRNA (B). O.E., overexpression. Blots were probed with antibodies indicated at the right side of the panels. See also Figure S2.

were observed between WT and mutant a5(IV) monomer even in the non-secretory mutant C1567R a5(IV) (Figures 1C and 1D). To verify this result, we inhibited ER-Golgi transport by expressing SAR1 guanosine triphosphate (GTP), which regulates COPIIdependent membrane trafficking in a dominant-negative manner. ER-Golgi transport inhibition by SAR1 GTP increased WT and mutant a5(IV) monomer expression (Figures 1E and 1F), indicating that intracellular collagen degradation occurs after proteins pass through the ER (Ripley et al., 1993) and that intracellular mutant a5(IV) monomer is trafficked similarly as WT.

Mutant a5(IV) Chains Are Regulated by Typical ER Chaperones and Exhibit Similar ER Localization as WT a5(IV) Secretory proteins are folded in the ER, and this process is mediated by several ER chaperones (Araki and Nagata, 2012). Collagen folding is also mediated by ER chaperones such as binding immunoglobulin protein (BiP), glucoseregulated protein (GRP) 94, protein disulfide isomerase (PDI), and heat shock protein 47 (HSP47) (Makareeva et al., 2011). Therefore, we examined the difference in intracellular regulation Cell Chemical Biology 25, 1–10, May 17, 2018 3

Please cite this article in press as: Omachi et al., A Split-Luciferase-Based Trimer Formation Assay as a High-throughput Screening Platform for Therapeutics in Alport Syndrome, Cell Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.02.003

Table 1. Intracellular Regulation of a5(IV) Monomer BiP

GRP94

PDI

HSP47

CRT

+

+

+




NC

Overexpression Intracellular a5 (IV)

Extracellular a5 (IV)

WT G869R

+

+

+




NC

C1567R

+

+

+




NC

WT




+










G869R




+










C1567R

ND

ND

ND

ND

ND

siRNA Intracellular a5 (IV)

Extracellular a5 (IV)

WT







NC




NC

G869R







NC




NC

C1567R







NC




NC

WT













NC

G869R













NC

C1567R

ND

ND

ND

ND

ND

+, increased;
, decreased; siRNA, small interfering RNA; NC, not changed; ND, not detected.

by ER chaperones between WT and mutant a5(IV) (G869R, C1567R). BiP and PDI overexpression increased the intracellular expression of WT and mutant a5(IV) but decreased the extracellular expression of these molecules (Figure 2A and Table 1). CRT decreased only the extracellular expression of a5(IV). Both WT and C1567R a5(IV) were predominantly localized in the ER as detected by immunofluorescence (Figure S2). The similarity in ER localization of WT and mutant a5(IV) monomers is consistent with the results of BFA and Sar1 GTP experiments (Figures 1C–1F), which suggested that intracellular a(IV) regulation or degradation occurs after a(IV) passes through the ER. In addition to BiP, PDI, and CRT enhanced the immunofluorescence of a5(IV), consistent with the increase in intracellular a5(IV). Because exogenous BiP, PDI, and CRT decreased the extracellular a5(IV), these data collectively indicated that BiP, PDI, and CRT induced the ER retention of a5(IV). GRP94 increased both intracellular and extracellular a5(IV) expression, and slightly increased the immunofluorescence of ER-localized a5(IV) (Figures 2A and S2; Table 1), suggesting a positive effect of GRP94 on the expression and secretion of a5(IV). Conversely, HSP47 decreased a5(IV) expression in the cell lysate and culture medium (Figure 2A and Table 1). Knockdown of BiP, GRP94, or HSP47 decreased WT and mutant a5(IV) expression in both the cell lysate and culture medium. Knockdown of PDI decreased a5(IV) expression only in the culture medium. Knockdown of CRT did not change a5(IV) expression in both the cell lysate and the culture medium (Figure 2B and Table 1). Although the detailed ER quality control mechanism is yet to be fully elucidated, we found that both WT and mutant a5(IV) monomers were regulated by these ER chaperones in a similar manner. Establishment of a Method for Assessing a345(IV) Trimer Formation that Reflected Established Features of Collagen Regulation The similar intracellular regulation between WT and mutant a5(IV) monomers makes the intracellular trafficking pathway of a5(IV) a 4 Cell Chemical Biology 25, 1–10, May 17, 2018

complicated target for therapy. These data also suggested that trimer formation may be a critical defect in mutant a5(IV). Therefore, we focused on trimer formation, which necessitated the development of a method to assess trimer formation and to screen for compounds targeting a345(IV) trimers. To establish the a345(IV) trimer assay, we used the split NanoLuc complementation system (Dixon et al., 2016) because split NanoLuc has several advantageous features, including small size and high sensitivity. We applied this technology for assessing a345(IV) assembly because proper heterotrimerization of a(IV) chains facilitates LgBiT-SmBiT complementation that generates quantifiable luminescence (Figure 3A). First, we constructed plasmids of COL4A3, COL4A4, and COL4A5 containing C-terminal-tagged SmBiT or LgBiT (Figure 3B), and identified the best combination for detecting the a345(IV) trimer. Following transfection into HEK293T cells, luminescence was detected with high sensitivity in the medium and cell lysates from heterotrimer (a3/a4/a5)-expressing cells but not from homodimer (a3/a3, a4/ a4, a5/a5)- and heterodimer (a3/a4, a3/a5, a4/a5)-expressing cells (Figure 3B, secreted; Figure S3A, intracellular). The combination of a3-SmBiT and a5-LgBiT in the presence of a4 had the highest luminescence. Using the combination of a3-SmBiT and a5-LgBiT, we next generated N-terminal-tagged plasmids and checked whether it was possible to detect luminescence using N-terminal-tagged a3-SmBiT and a5-LgBiT. Luminescence of C- or N-terminal split NanoLuc was detected with high sensitivity in the media and cell lysates from a345(IV)-transfected cells but not from cells transfected with a3 or a5 alone or combined (Figures 3C, 3D, S3B, and S3C). These results indicate that a3-SmBiT and a5-LgBiT are able to form stable heterotrimer complex only in the presence of a4(IV). To confirm the validity of the NanoLuc-based a345(IV) trimer assay, we co-transfected cells with a3, a5, and serial dilutions of a4(IV) plasmid. There was an a4(IV) concentrationdependent increase in luminescence in media and cell lysates (Figures 3E and S3D). Luminescence was competitively inhibited by a3(IV)-HA and a5(IV)-Myc (Figures 3F, 3G, S3E, and S3F). Type IV collagens consist of a COL domain (Gly-X-Y repeats) and an NC1 domain, which are essential for the assembly of a chains (Boutaud et al., 2000; Kalluri, 2003). To investigate the importance of these domains in the NanoLuc-based a345(IV) assay, we generated COL and NC1 deletion mutants of a5(IV). Luminescence was significantly decreased in culture media from both DCOL- and DNC1 a5(IV)-expressing cells (Figure 3H). However, the luminescence of intracellular lysates from DCOL-a5(IV)-expressing cells was higher than that for WT although the increase was not significant (Figure S3G). NC1 domain is responsible for trimer formation. The absence of COL domain may enhance intracellular interaction, but the COL domain may be important for trimer maturation; thus DCOL had decreased extracellular luminescence. Because ascorbic acid (AsA) is important for the folding and secretion of almost all collagens (Murad et al., 1981), we next treated cells with AsA. As expected, luminescence in the culture media but not in intracellular lysates was increased in the presence of AsA (Figures 3I and S3H), revealing that AsA enhanced the secretion of properly assembled a345(IV) trimer. a345(IV) has been shown to form heterotrimers intracellularly but not extracellularly (Kobayashi and Uchiyama, 2003), so we checked

Please cite this article in press as: Omachi et al., A Split-Luciferase-Based Trimer Formation Assay as a High-throughput Screening Platform for Therapeutics in Alport Syndrome, Cell Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.02.003

Figure 3. Establishment of a Method for Assessing a345(IV) Trimer Formation that Reflected Established Features of Collagen Regulation (A) The scheme of split NanoLuc-based a345 (IV) trimer assay. (B–J) Luminescence was measured in the media (extracellular) from HEK293T cells transfected with: (B) C-terminal LgBiT- or SmBiT-a3/a4/a5(IV) in the indicated combination. (C) C-terminal or (D) N-terminal LgBiT-a5(IV), SmBiT-a3(IV), and COL4A4-3FLAG in the indicated combinations. Left panels: scheme of a(IV)-tagged (legend continued on next page)

Cell Chemical Biology 25, 1–10, May 17, 2018 5

Please cite this article in press as: Omachi et al., A Split-Luciferase-Based Trimer Formation Assay as a High-throughput Screening Platform for Therapeutics in Alport Syndrome, Cell Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.02.003

Figure 4. The Split NanoLuc a345(IV) Trimer Assay Enabled the Assessment of Clinically Reported a5(IV) Mutants (A and B) Luminescence was measured in the media from cells expressing a3, a4, and C-terminal-tagged (A) or N-terminal-tagged (B) WT or the indicated mutant a5(IV). Error bars indicate the mean ± SE (n = 4). **p < 0.01 versus WT (TukeyKramer test). See also Figure S4. (C and D) Scatterplot of the intracellular/secreted RLU ratio from cells expressing the WT or mutant a5 chain. Solid line: y = x; dotted line: y = x + 30, y = x
30. White square, WT; black circles, intracellular formation-dependent defect; gray circles, secretion-dependent defect.

whether the split NanoLuc-based trimer assay reflected this assembly characteristic. We compared the luciferase signal among single a3, a4, and a5(IV)-expressing cells, co-cultures of single a-chain-expressing cells, and a345(IV) (triple)-expressing cells. Luminescence was detected only in a345(IV)-expressing cells (Figures 3J and S3I), thus confirming that this assay reflects known a345(IV) assembly characteristic. The Split NanoLuc a345(IV) Trimer Assay Enabled the Assessment of Clinically Reported a5(IV) Mutants For application of this system to the development of a therapeutic strategy targeting a345(IV) trimer formation, it is important to correlate the split NanoLuc-based luminescence with clinically

reported a5(IV) mutants (Bekheirnia et al., 2010; Crockett et al., 2010; Savige et al., 2016). To address this issue, we generated several mutant a5(IV) C- or N-terminal LgBiT fusion constructs by site-directed mutagenesis. Among the 32 mutants investigated, 12 mutant constructs (37.5%) of a5(IV) N- and C-terminal fusions exhibited >50% reduction in luminescence compared with WT. Specifically, 9 mutant a5(IV) N-terminal fusions (28% of the total) had >50% reduction, whereas 4 mutant a5(IV) C-terminal fusions (12.5% of the total) had >50% reduction. In comparison, 7 mutant a5(IV) N- and C-terminal fusion constructs (21.9% of the total) had <50% reduction in luminescence compared with WT a5(IV) (Figures 4A, 4B, S4A, and S4B; Table 2). In addition to each result from the C- or N-terminal split NanoLuc assay, we considered the type of trimerization defect in terms of intracellular/secreted relative light units (RLU) ratio. The comprehensive parameter showed that trimer defects in 15 N-terminal fusion mutants and 14 C-terminal fusion mutants depended on intracellular trimer formation, whereas the other defects were related to secretion defects (Figures 4C and 4D; Table S1). Almost all a5(IV) mutants showed different patterns of folding states of N and C termini, even in similar Gly-substituted mutants. By analyzing the intracellular/extracellular trimer ratio, we characterized the type of defect for each missense mutant. More than half of the a5 mutants assessed in the present study have a secretion-dependent defect, indicating that correcting the protein folding and promoting secretion is a better

constructs. S.S., signal sequence. (E) C-terminal LgBiT-a5(IV), SmBiT-a3(IV), and empty vector or serial amounts of expression vectors encoding COL4A43FLAG. (F and G) C-terminal LgBiT-a5(IV), SmBiT-a3(IV), COL4A4-3FLAG, and empty vector or expression vectors encoding COL4A3-HA (F) or COL4A5-Myc (G). (H) C-terminal LgBiT-WT, -DCOL, or -DNC1 a5(IV), SmBiT-a3(IV), and COL4A4-3FLAG. Left panel: scheme of a(IV)-tagged constructs. (I) C-terminal LgBiTa5(IV), SmBiT-a3(IV), and COL4A4-3FLAG treated with 200 mM ascorbic acid for 24 hr. (J) Single a-chain-expressing cells (a3, a4, a5), co-cultured single a-chainexpressing cells (a3+a4+a5), or triple a-chain-expressing cells (a345). In (B) to (J), error bars indicate mean ± SE (n = 4). **p < 0.01 versus a3, a5, and a35 (C and D); **p < 0.01 versus WT (H); **p < 0.01 versus a3, a4, a5 and a3+a4+a5 (J) (B–E, J, Tukey-Kramer; F–I, Student’s t test). See also Figure S3.

6 Cell Chemical Biology 25, 1–10, May 17, 2018

Please cite this article in press as: Omachi et al., A Split-Luciferase-Based Trimer Formation Assay as a High-throughput Screening Platform for Therapeutics in Alport Syndrome, Cell Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.02.003

Table 2. Trimer Formation of Mutant a5(IV) Chain MT a5(IV) Luminescence -LgBit versus WT Fusion a5(IV) Mutations

Ratio

N term

<50%

G129E, G227S,G325R, 28% (9/32) G521D, G573D, L664S, G675S, G911E, G1030S

C term

<50%

G624D, R1569Q, L1649R, R1683Q

N,C term

<50%

G426R, G475S, G650D, 37.5% (12/32) G869R, G1107R, G1143D, G1220D, G1241C, G1241V, G1244D, G1448R, C1567R

N,C term

>50%

G594D, G594S, G796R, 21.9% (7/32) S916G, S953V, P1517T, M1607I

12.5% (4/32)

therapeutic focus than intracellular stabilization. DCOL and DNC1 a5(IV) mutants were not secreted even though they partially formed a complex intracellularly. This result could be explained by the controversial relationships among mutation locations, substitution residues, and clinical manifestations in AS. Single Targeting of ER Chaperones Did Not Rescue the Trimer Formation Ability of Mutant a5(IV) We investigated whether typical ER chaperones corrected the trimer formation of mutant a5(IV). Overexpression of BiP, PDI, and CRT reduced the amount of secreted mutant a345(IV) trimer. Conversely, overexpression of GRP94 slightly increased the amount of secreted a345(IV) trimer, whereas overexpression of HSP47 did not induce a change (Figure 5A). Intracellularly, CRT did not change trimer formation while HSP47 suppressed the amount of a345(IV) trimer (Figure S5A). Knockdown of BiP, GRP94, and PDI slightly reduced the secreted a345(IV) trimer, while HSP47 and CRT knockdown had no effect (Figure 5B). Knockdown of these molecular chaperones decreased the amount of intracellular a345(IV) trimer (Figure S5B). These data indicated that although BiP, GRP94, and PDI are important for trimer formation, increasing their expression could not improve the secretion of mutant a345(IV) trimer. To establish a molecular chaperone-based therapy, it is necessary to screen for molecules that could improve mutant a5(IV) trimer formation using split NanoLuc-based a345(IV) trimer assay as platform together with large-scale knockdown or knockout library of molecules involved in ER quality control. Chemical Chaperones Rescued Mutant a5(IV) Trimer Formation The lack of suitable HTS methods for targeting the a345(IV) trimer has been a major barrier in developing a causal proteinbased therapy for AS. To determine whether the split NanoLuc-based a345(IV) trimer assay was applicable to HTS, we determined its validity in a 96-well format. The split NanoLuc-based a345(IV) trimer assay satisfied the acceptance criteria for HTS (Iversen et al., 2004) (CVG869R = 6.74 % 20, S/B = 2.38 > 2, Z0 factor = 0.57 R 0.4; Figure 5C). Using this condition,

we investigated whether it is feasible to correct the trimerization of mutant a5(IV). Because G869R a5(IV) is one of the most frequent missense mutations and because luminescence of G869R a5(IV) was decreased in both the N-terminal and C-terminal trimer assays, we chose G869R as the model mutant to provide proof of concept for correcting the a345(IV) trimer. Many reports have described the effects of chemical chaperones on misfolded proteins consequent to gene mutation (Matsuda et al., 2003; Winter et al., 2014; Zode et al., 2011). Chemical chaperones are low-molecular-weight compounds that enhance protein folding, decrease aggregation, correct mislocalization, and/or stabilize misfolded proteins (reviewed in Perlmutter, 2002). So here we examined the effects of chemical chaperones (Okiyoneda et al., 2013) on the trimer formation of G869R a5(IV). The secreted trimer of G869R a5(IV) C-terminal LgBiT was decreased compared with that of WT protein. Treatment with taurine, glucose, sucrose, mannitol, trehalose, and trimethylamine N-oxide (TMAO) rescued the trimerization of G869R a5(IV) (Figure 5D). These compounds are classified as osmolytes, which affect protein folding and aggregation. Both mannitol and TMAO increased the intracellular and secreted trimer of G869R a5(IV) in a concentration-dependent manner, with TMAO showing the greatest effect in correcting trimer formation (Figures 5E, 5F, S5C, and S5D). Moreover, mannitol and TMAO also rescued the trimerization of other glycine missense mutants G1107R, G1244D, and G1143D a5(IV) (Figures 5G–5I and S5E–S5G) but not of C1567R mutant a5(IV), which exhibits complete loss of heterotrimerization intracellularly and extracellularly (Figures 5J and S5H). Thus, our findings demonstrated a potential trimer-based therapeutic strategy for AS. Previously, Wang et al. (2017) reported that the chemical chaperone 4-phenylbutyric acid (4-PBA) reduces ER stress and increases a5(IV) expression in cultured fibroblasts from patients with X-linked AS with G908R and G624D mutations. Although 4-PBA showed an effect on ER stress and a5(IV) expression, the authors did not describe its effect on collagen trimer formation. In our trimer assay system, however, 4-PBA did not rescue the trimerization of G869R a5(IV). This finding reveals that different glycine mutations have different foldingstate characteristics. Therefore, the responsiveness to chemical chaperones may differ depending on the mutation. The NanoLuc-based a345(IV) assay could help to elucidate the responsiveness of various a5(IV) mutants to chemical chaperones. The IP-based a345(IV) trimer assay previously used to measure a345(IV) trimer formation (Kobayashi et al., 2008; Kobayashi and Uchiyama, 2003, 2010) was crucial for understanding the molecular mechanisms of type IV collagen in AS. However, IP-based assay is not suitable for quantitative and highthroughput applications because IP is a non-homogeneous assay and requires a relatively large-scale cell culture. The split NanoLuc-based a345(IV) trimer assay overcomes these limitations and reflects known collagen regulatory characteristics. The split NanoLuc-based a345(IV) trimer assay is also useful for evaluating the trimer formation of various clinically relevant a5(IV) mutants. In addition, this assay enables the assessment of partial misfolding of a5(IV) mutants by using N- and C-terminal split NanoLuc assay variants. Cell Chemical Biology 25, 1–10, May 17, 2018 7

Please cite this article in press as: Omachi et al., A Split-Luciferase-Based Trimer Formation Assay as a High-throughput Screening Platform for Therapeutics in Alport Syndrome, Cell Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.02.003

Figure 5. Some Chemical Chaperones but Not Typical ER Chaperones Were Able to Correct the Trimer Formation of Mutant a5(IV) (A and B) Luminescence was measured in media from cells expressing C-terminal-tagged a34 and WT or G869R a5 co-transfected with the indicated ER chaperone expression vectors (A) or siRNA (B). Error bars indicate mean ± SE (n = 4). ##p < 0.01 versus Mock WT a5; *p < 0.05, **p < 0.01 versus Mock G869R a5 (Tukey-Kramer test). (C) Validity of the 96-well format of the C-terminal split NanoLuc-based a345 trimer assay. (D–F) Luminescence was measured in media from cells expressing C-terminal-tagged a34 and G869R a5 treated with the indicated chemical chaperones (D), or serial concentrations of mannitol (E) and TMAO (F). Data are presented as mean ± SE (n = 4). *p < 0.05, **p < 0.01 versus Mock G869R a5 (Tukey-Kramer test). (G–J) Luminescence was measured in media from cells expressing C-terminal-tagged a34 and G1107R (G), G1244D (H), G1143D (I), and C1567R (J) a5(IV) treated with the indicated chemical chaperones. Data are presented as mean ± SE (n = 4). ##p < 0.01 versus Mock WT a5; **p < 0.01 versus Mock mutant a5 (Tukey-Kramer test). See also Figure S5.

misfolding diseases, particularly those whose causal proteins have complex structures. SIGNIFICANCE

Despite the advantages of the split NanoLuc-based trimer assay, the assay is limited to assessing physiological and pathological intracellular responses. In vivo, the a345(IV) trimer is produced by podocytes, which are glomerular epithelial cells (Abrahamson et al., 2009). Podocytes are highly differentiated cells that have specialized characteristics, such as foot processes. The foot processes are essential for the glomerular filtration barrier and are damaged in various nephrotic syndromes, including AS (Fukuda et al., 2016). Given the cell type difference, we cannot investigate the physiological and pathological responses by expressing mutant a5(IV) chains in split NanoLuc-based a345(IV) assay. Similarly, whether the conditions in the split NanoLuc-based a345(IV) assay mimic that of in vivo GBM is still unknown. However, as a tool for screening of drugs that induce trimer formation of mutant a5(IV), the split NanoLuc-based a345(IV) trimer assay could be a powerful tool. To the best of our knowledge, this is the first HTS platform targeting misfolding diseases with multimer formation abnormalities, providing a useful basis for establishing screening systems for other genetic 8 Cell Chemical Biology 25, 1–10, May 17, 2018

While the causal protein in Alport syndrome (AS) has long been identified, therapies for this genetic kidney disease are symptomatic, and to date no curative approach targeting the mutant collagen has been developed. This is in part due to our poor understanding of the regulation of WT and mutant a345(IV) in the cells. Targeting the causal protein is a sound therapeutic approach for misfolding diseases, but in AS the disrupted trimerization of a345(IV) further presents a challenge in that a trimerization assay needs to be established. Moreover, the assay system requires that it is amenable to HTS for evaluation of possible drugs or compounds that can promote or correct trimer formation. Here, we firstly assessed the intracellular regulation of WT and clinically relevant mutant a5(IV) monomers. WT and mutant a5(IV) monomers are relatively stable intracellularly. Mutant a5(IV) has characteristics similar to those of the WT monomer in terms of the degradation mechanism and the regulation of intracellular localization by ER chaperones such as BiP, GRP94, PDI, HSP47, and CRT. This argues for a strategy of focusing on trimer formation rather than the intracellular regulation of mutant a5(IV) monomer. With this aim, we developed a NanoLucbased assay that serves as a useful quantitative and HTSapplicable method to detect a345(IV) trimer formation and

Please cite this article in press as: Omachi et al., A Split-Luciferase-Based Trimer Formation Assay as a High-throughput Screening Platform for Therapeutics in Alport Syndrome, Cell Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.02.003

evaluate the trimerization ability of mutant a5(IV). By using the validated NanoLuc-based a345(IV) trimer assay, we ascertained that chemical chaperones classified as osmolytes were able to correct the trimer formation of mutant a5(IV). The findings presented here are highly relevant to causal protein-based therapy for AS and may be applicable to other diseases caused by dysfunctional heterocomplex formation. STAR+METHODS Detailed methods are provided in the online version of this paper and include the following: d d d d

d

KEY RESOURCES TABLE CONTACT FOR REAGENT AND RESOURCE SHARING EXPERIMENTAL MODEL AND SUBJECT DETAILS B Cell Culture METHOD DETAILS B Plasmids B Transfection and Treatment B Cell Lysis, Gel Electrophoresis, and Immunoblotting B Immunofluorescence Staining B Luciferase Assay QUANTIFICATION AND STATISTICAL ANALYSIS

SUPPLEMENTAL INFORMATION Supplemental Information includes five figures and two tables and can be found with this article online at https://doi.org/10.1016/j.chembiol.2018. 02.003. ACKNOWLEDGMENTS We thank Editage (http://www.editage.com) for editing and reviewing this manuscript for English language and Y. Sado for providing the a3–5(IV) specific antibodies (H31, H43, H53). This work was supported by the Japan Society for the Promotion Science (JSPS) KAKENHI (grant nos. JP26460098 and JP17K08309 [to M.A.S.]), the Alport Syndrome Research Funding Program of the Alport Syndrome Foundation (ASF) and the Pedersen family, Kidney Foundation of Canada (KFOC [to H.K.]), Kumamoto University HIGO Program Research Funding Project (Fiscal Year 2013–2016 [to K.O.]), Grant-in-Aid for JSPS Fellows for Young Researchers (grant no. 17J11628 [to K.O.]), and JSPS Program for Advancing Strategic International Networks to Accelerate the Circulation of Talented Researchers (grant no. S2803 [to H.K.]). AUTHOR CONTRIBUTIONS K.O. designed the research, conducted experiments, and wrote the manuscript. M.A.S. designed the research and wrote the manuscript. T.S. and H.K. designed the research. M.K., K.T., H.K., T.Y., S.K., J.K., R.F., K.K., S.M., and K.M. conducted experiments. All authors discussed the results and provided input on the manuscript. DECLARATION OF INTERESTS

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H.K. holds a patent related to this work, Japanese Patent Application No. 2017-99497.

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Received: October 11, 2017 Revised: December 11, 2017 Accepted: February 5, 2018 Published: March 8, 2018

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Please cite this article in press as: Omachi et al., A Split-Luciferase-Based Trimer Formation Assay as a High-throughput Screening Platform for Therapeutics in Alport Syndrome, Cell Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.02.003

STAR+METHODS KEY RESOURCES TABLE

REAGENT or RESOURCE

SOURCE

IDENTIFIER

Antibodies anti-Myc monoclonal antibody (9E10)

Santa Cruz Biotechnology

Catalog # sc-40; RRID: AB_627268

anti-Vinculin polyclonal antibody (H-300)

Santa Cruz Biotechnology

Catalog # sc-5573; RRID: AB_2214507

anti-HSP47 monoclonal antibody (M16.10A1)

Enzo Life Sciences

Catalog # ADI-SPA-470-F; RRID: AB_10618557

anti-PDI polyclonal antibody

Novus

Catalog # NB100-1921; RRID: AB_10001061

anti-calreticulin polyclonal antibody (SPA-600)

Enzo Life Sciences

Catalog # ADI-SPA-600; RRID: AB_10618853

anti-KDEL monoclonal antibody (10C3)

Enzo Life Sciences

Catalog # ADI-SPA-827; RRID: AB_10618036

anti-COL4A3 monoclonal antibody (H31)

Shigei Medical Research Institute

Clone # H31

anti-COL4A4 monoclonal antibody (H43)

Shigei Medical Research Institute

Clone # H43

anti-COL4A5 monoclonal antibody (H53)

Shigei Medical Research Institute

Clone # H53

MG-132

Calbiochem

Catalog # 474790

Bafilomycin A1 (BafA1)

Calbiochem

Catalog # 196000

Brefeldin A (BFA)

Calbiochem

Catalog # 500583

Cycloheximide (CHX)

Sigma

Catalog # C104450

L-ascorbic acid 2-phosphate trisodium salt

Wako Pure Chemical Industries

Catalog # 323-44822

4-PBA

Sigma

Catalog # P21005

TUDCA

Tokyo Chemical Industry

Catalog # T1567

betaine

Tokyo Chemical Industry

Catalog # B0455

trehalose

Tokyo Chemical Industry

Catalog # T0832

glycine

Nacalai Tesque

Catalog # 17109-35

Chemicals, Peptides, and Recombinant Proteins

dimethylsulfoxide

Nacalai Tesque

Catalog # 13409-12

glycerol

Nacalai Tesque

Catalog # 12404-92

glucose

Nacalai Tesque

Catalog # 16806-25

sucrose

Nacalai Tesque

Catalog # 30403-55

sorbitol

Nacalai Tesque

Catalog # 32021-82

mannitol

Nacalai Tesque

Catalog # 21302-55

taurine

Nacalai Tesque

Catalog # 32708-15

beta-alanine

Sigma

Catalog # 146064

TMAO

Sigma

Catalog # 317594

Promega

Catalog # N2012

Human reference nucleotide NCBI Human COL4A3

NCBI Reference Sequence: NM_000091.4

https://www.ncbi.nlm.nih.gov/ nuccore/NM_000091.4

Human reference nucleotide NCBI Human COL4A4

NCBI Reference Sequence: XM_005246281.3

https://www.ncbi.nlm.nih.gov/ nuccore/XM_005246281.3

Human reference nucleotide NCBI Human COL4A5

NCBI Reference Sequence: NM_033380.2

https://www.ncbi.nlm.nih.gov/ nuccore/NM_033380.2

Critical Commercial Assays Nano-Glo Live Cell Assay System Deposited Data

Experimental Models: Cell Lines HEK293T cells

RIKEN BRC

Cell # RCB2202

HeLa cells

RIKEN BRC

Cell # RCB0007 (Continued on next page)

Cell Chemical Biology 25, 1–10.e1–e4, May 17, 2018 e1

Please cite this article in press as: Omachi et al., A Split-Luciferase-Based Trimer Formation Assay as a High-throughput Screening Platform for Therapeutics in Alport Syndrome, Cell Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.02.003

Continued REAGENT or RESOURCE

SOURCE

IDENTIFIER

Oligonucleotides GL2-LUC siRNA (control siRNA)

N/A

BiP siRNA

N/A

GRP94 siRNA

N/A

ON-TARGET plus Human P4HB (PDI) SMART pool

GE Dharmacon

Catalog # L-003690-00-0005

ON-TARGET plus Human CALR (CRT) SMART pool

GE Dharmacon

Catalog # L-0008197-00-0005

Hs_SERPINH1 (HSP47). 6 FlexiTube siRNA

QIAGEN

Catalog # SI02777138

pEB multi-Neo vector

Wako

Catalog # 057-08131

pEB multi-Hyg vector

Wako

Catalog # 050-08121

pEF6 Myc vector

Thermo Fisher Scientific

Catalog # V96220

pEF Myc ER vector

Thermo Fisher Scientific

Catalog # V89120

pEB multi-Neo Human COL4A3-HA

This paper

N/A

pEB multi-Hyg Human COL4A4-3FLAG

This paper

N/A

pEF6 Human COL4A5-Myc

This paper

N/A

Recombinant DNA

pFN35K SmBiT TK-neo Flexi vector

Promega

Catalog # N195A

pFN33K LgBiT TK-Neo Flexi vector

Promega

Catalog # N194A

pFC36K SmBiT TK-neo Flexi vector

Promega

Catalog # N193A

pFC34K LgBiT TK-Neo Flexi vector

Promega

Catalog # N192A

pFN35K SmBiT TK-neo Flexi Human COL4A3 vector

This paper

N/A

pFN33K LgBiT TK-Neo Flexi Human COL4A5 vector

This paper

N/A

pFC36K SmBiT TK-neo Flexi Human COL4A3 vector

This paper

N/A

pFC36K SmBiT TK-neo Flexi Human COL4A4 vector

This paper

N/A

pFC36K SmBiT TK-neo Flexi Human COL4A5 vector

This paper

N/A

pFC34K LgBiT TK-Neo Flexi Human COL4A3 vector

This paper

N/A

pFC34K LgBiT TK-Neo Flexi Human COL4A4 vector

This paper

N/A

pFC34K LgBiT TK-Neo Flexi Human COL4A5 vector

This paper

N/A

pEF6 Myc Human HSP47

This paper

N/A

pEF Myc ER Human BiP

This paper

N/A

pEF Myc ER Human GRP94

This paper

N/A

pEF Myc ER Human PDI

This paper

N/A

pEF Myc ER Human CRT

This paper

N/A

pIRES2-DsRed Human SAR1 GTP(H79G)

Okiyoneda et al., 2004

N/A

SAS

N/A

Thermo Fisher Scientific

Catalog # 34580

Software and Algorithms JMP 13 Statistical DiscoveryTM Other SuperSignal West Pico Chemiluminescent Substrate LumiNunc

TM

96-well white plate format

GloMax Navigator system

Thermo Fisher Scientific

Catalog # 136102

Promega

Catalog # GM2000

CONTACT FOR REAGENT AND RESOURCE SHARING Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead contact, Hirofumi Kai ([email protected]) EXPERIMENTAL MODEL AND SUBJECT DETAILS Cell Culture Human embryonic kidney HEK293T cells, derived from female embryo (Lin et al., 2014b), were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100-U penicillin and streptomycin in tissue culture e2 Cell Chemical Biology 25, 1–10.e1–e4, May 17, 2018

Please cite this article in press as: Omachi et al., A Split-Luciferase-Based Trimer Formation Assay as a High-throughput Screening Platform for Therapeutics in Alport Syndrome, Cell Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.02.003

plates coated with Cellmatrix type I-C. HeLa cells, derived from a female patient with cervical cancer, were maintained in MEM supplemented with 10% FBS and 100-U penicillin and streptomycin in tissue culture plates. For the luciferase assay, HEK293T cells were maintained in DMEM low glucose supplemented with 10% FBS, 100-U penicillin and streptomycin, 2 mM glutamine, and 200 mM L-ascorbic acid 2-phosphate trisodium salt. METHOD DETAILS Plasmids The human COL4A3 coding sequence was inserted into the pEB multi-Neo vector, and human COL4A4 and COL4A5 coding sequences were each inserted into the pEB multi-Hyg vector. HA, 3FLAG, and Myc sequences were added, followed by COL4A3, COL4A4, and COL4A5 coding sequences, respectively. Human BiP, GRP94, PDI, and CRT coding sequences were inserted into the pEF Myc ER vector. The human HSP47 coding sequence was inserted into the pEF6 Myc vector. Human Sar1-GTP (H79G) in the pIRES2-DsRed2 vector was described previously (Okiyoneda et al., 2004). For the C-terminal fusion split luciferase complementation assay, human COL4A3, COL4A4, and COL4A5 coding sequences were inserted into the pFC36K SmBiT TK-neo Flexi vector and pFC34K LgBiT TK-Neo Flexi vector. For the N-terminal fusion split luciferase complementation assay, human COL4A3 and COL4A5 coding sequences were subcloned into the pFN35K SmBiT TK-neo Flexi vector and pFN33K LgBiT TK-Neo Flexi vector, respectively. The signal sequence was added proximal to the SmBiT and LgBiT tag sequences in the pFN35K SmBiT TK-neo Flexi and pFN33K LgBiT TK-Neo Flexi vectors, respectively. COL4A5 mutants (pFC34K LgBiT and pFN33K LgBiT) were generated by site-directed mutagenesis as previously described (Suzuki et al., 2014). Primer sequences are shown in Table S2. The COL domain deletion mutant was generated by inverse PCR, and the NC1 domain deletion mutant was subcloned from the WT COL4A5 coding sequence. For the untagged competition assay, COL4A5-Myc (pEB-multi Hyg) and COL4A3-HA (pEB-multi Neo) expression vectors were used. Transfection and Treatment Transient transfection of plasmid DNA was performed using TransIT-LT1 as described previously (Sato et al., 2012). Most experiments were performed at 48 h after transfection. siRNA (siBiP, siGRP94, siPDI, siCRT, and siHSP47) or control siRNA was transiently transfected into HEK293T cells at a concentration of 100 nM using Lipofectamine RNAiMAX according to the manufacturer’s instructions. Cells were typically assayed 48-72 h after transfection. In the case of co-transfection with siRNA and complementary DNA plasmids, plasmid transfection was performed at 24 h after siRNA transfection, and cells were then cultured for an additional 48 h. The knockdown efficiency was confirmed by immunoblotting. For evaluation of protein stability in the cells, cycloheximide (CHX) chase experiment was performed as previously described (Sugiyama et al., 2011). Briefly, plasmid-transfected cells were treated with CHX (100 mM) for the time periods indicated, lysed, and analyzed by immunoblotting. For analysis of intracellular regulation and trafficking, HEK293T cells were treated with 20 mM MG132, 200 nM bafilomycin A1 (BafA1), or 5 mg/mL brefeldin A (BFA) for 24 h. For chemical chaperones treatment, the following reagents were used: 4-PBA (10 mM), TUDCA (200 mM), glycine (150 mM), taurine (150 mM), beta-alanine (150 mM), betaine (150 mM), DMSO (2%), glycerol (5%), glucose (150 mM), sucrose (150 mM), mannitol (150 mM), sorbitol (150 mM), trehalose (150 mM), or TMAO (150 mM). Cells were treated for 24 hr. Cell Lysis, Gel Electrophoresis, and Immunoblotting Cells were washed twice with ice-cold phosphate-buffered saline and lysed in RIPA buffer (0.05 M Tris-HCl [pH 7.5], 0.15 M NaCl, 1% v/v Nonidet P-40, 1% w/v Na deoxycholate, and 1% protease inhibitor cocktail) or 1% Triton buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, and 1% Triton-X-100) supplemented with 1% protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). The lysates were cleared by centrifugation at 13,000 3 g for 15 min. The protein concentration of the lysates was determined using a bicinchoninic acid kit (Sigma-Aldrich), and equal amounts of protein lysates were loaded and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotted with the indicated antibodies, and visualized using SuperSignal West Pico Chemiluminescent Substrate. Immunofluorescence Staining COL4A5-Myc plasmids were transfected into HEK293T cells. After 48 h, cells were washed twice with PBS and fixed in 4% paraformaldehyde for 10 min at room temperature, incubated with 1% bovine serum albumin in PBS for 1 h and washed with PBS. Cells were incubated overnight with anti-Myc antibody (1:100) for COL4A5, anti-PDI antibody (1:100) for ER marker in 0.1% Triton-X in PBS at 4 C, washed three times with 0.1% Triton-X in PBS and incubated with secondary antibodies conjugated with Alexa Flour 488 or 546 (1:2000) for 1 h at room temperature. Cells were washed three times with 0.1% Triton-X in PBS, incubated with DAPI solution for 20 min at room temperature, washed three times with PBS and mounted with Vectashield mounting medium (Vector Laboratories). Fluorescence images were observed by fluorescence microscope BZ-X700 (Keyence).

Cell Chemical Biology 25, 1–10.e1–e4, May 17, 2018 e3

Please cite this article in press as: Omachi et al., A Split-Luciferase-Based Trimer Formation Assay as a High-throughput Screening Platform for Therapeutics in Alport Syndrome, Cell Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.02.003

Luciferase Assay COL4A3-SmBiT, COL4A5-LgBiT, and COL4A4-3FLAG plasmids were transfected into HEK293T cells. After 48 h, culture media were changed. At 72 h after transfection, Nano-Glo Live Cell Assay reagent was added, and the luciferase activity in the medium and cells was measured using a GloMax Navigator system (Promega). All luciferase assays were conducted in LumiNunc 96-well white plates. QUANTIFICATION AND STATISTICAL ANALYSIS Statistical parameters are reported in the Figure Legends. Immunoblot experiments were performed in triplicate starting from 3 cell cultures. Luciferase assays were performed in quadruplicate starting from 4 cell cultures. Immunofluorescence was performed in duplicate from 2 separate cell cultures. All data are presented as means ± standard errors (SEs). The significance of differences between two groups was assessed using Student’s unpaired two-tailed t-tests. For three-group comparisons, we used analysis of variance (ANOVA) with Tukey-Kramer post-hoc tests. Differences with P values of less than 0.05 were considered statistically significant.

e4 Cell Chemical Biology 25, 1–10.e1–e4, May 17, 2018