Purification and Identification of the 20S Proteasome Complex from Zebrafish

Abstract

The proteasome is a large polymeric protease complex responsible for degradation of intracellular proteins and generation of peptides. In this study, we purified a native 20S proteasome protein complex from zebrafish (Danio rerio) from the whole body. The cytosolic fraction of zebrafish hydrolyzed Suc-Leu-Leu-Val-Tyr-MCA (Suc-LLVY-MCA), a well-known substrate for the proteasome, in the presence of sodium dodecyl sulfate. From the cytosolic fraction, the 20S proteasome was purified using five column chromatography steps: DEAE cellulose, Q-Sepharose, Sephacryl S-300 gel, hydroxylapatite, and phenyl Sepharose. Electrophoresis and Western blot analyses showed that zebrafish 20S proteasome subunits have molecular masses ranging from 22 to 33 kDa. The subunit composition of the purified 20S proteasome was identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis after two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) separation. Fourteen kinds of 20S subunits were found. As a special characteristic of zebrafish, two proteins of the α1 subunit were identified. In addition, the results suggested that the α8 subunit is in the 20S complex instead of the α4 subunit. In this study, we demonstrated the subunit composition of the 20S proteasome complex present in zebrafish cells.

Introduction

Eukaryotic cells contain a large nonlysosomal protease known as a proteasome (or multicatalytic protease), which is found in all eukaryotes, from yeast to human.¹ The ubiquitin–proteasome degrading system plays a major role in the nonlysosomal proteolytic pathway in eukaryotes.^2,3 Proteasomes are separated into two types, 20S and 26S. The 20S proteasome forms the catalytic core of the 26S proteasome.⁴ The 20S particles are built up by two types of subunits, α and β. This barrel-shaped complex has three cavities, whereby the two outer cavities are jointly formed by one α and one β subunit ring and the inner cavity is formed by the two β rings.

Based on this intriguing structure and because the proteasome degrades target proteins with high specificity, researchers proposed that after unfolding, degradation of the substrate proteins occurred in the inner cavity of the proteasome barrel.^5–7 Of these 14 subunits, β1, β2, and β5 have catalytic activities referred to as caspase-like, trypsin-like, and chymotrypsin-like activity, respectively.⁸ Proteasome inhibitors have effective antitumor activity against cultured cells, inducing apoptosis by disrupting the regulated degradation of progrowth cell cycle proteins.⁹

This approach of selectively inducing apoptosis in tumor cells has proven effective in animal models and human trials. Proteasome inhibitors can kill some types of cultured leukemia cells that are resistant to glucocorticoids.¹⁰ The molecule ritonavir, marketed as Norvir, was developed as a protease inhibitor and used to target HIV infection.¹¹ Bortezomib was the first proteasome inhibitor approved for clinical use as a chemotherapy agent.^12,13 More recently, major efforts have been made to assess the proteasome for the development of novel diagnostic markers and strategies. An improved and comprehensive understanding of the pathophysiology of the proteasome should lead to clinical applications in the future. Currently, zebrafish have been used as a model worldwide in genetic, pharmacological, and behavioral research.

Zebrafish have a similar genetic structure and 74% of genes are similar to humans, and 84% of genes known to be associated with human diseases have a zebrafish counterpart. Some reports about syndromes caused by mutations in proteasome subunits or related genes have used zebrafish.^14,15 Although proteasomes are important, purification and characterization of proteasomes have not been reported in zebrafish. Thus, in this study, we tried to purify the natural 20S proteasome complex from zebrafish.

Materials and Methods

Animals

The zebrafish were bred and maintained at 28.5°C under a 14-h light/10-h dark cycle.¹⁶ All zebrafish experiments were carried out with approval from the institutional ethics committee of Shizuoka University, Japan (approval No. 2021F-2); the guidelines set by this committee for the use of animals were strictly followed.

Reagents

The fluorogenic peptide substrate Suc-Leu-Leu-Val-Tyr-MCA (Suc-LLVY-MCA) was purchased from Peptide, Inc., Corp. (Osaka, Japan). DEAE cellulose (DE52) was purchased from Whatman. Q-Sepharose, phenyl Sepharose Fast Flow, and Sephacryl S-300 gel were obtained from Pharmacia. Hydroxylapatite was obtained from Tosoh Corp. (Tokyo, Japan). Other chemicals were purchased from Wako Pure Chemical Industries, Ltd. (Osaka Japan).

Purification of the 20S proteasome

The procedure of purification of the zebrafish 20S proteasome was based on the method established for the purification of Xenopus and goldfish 20S proteasomes.^17,18 Three hundred zebrafish were dissected, and the digestive system (stomach and intestine) and fins were removed. Dissected fishes were stored at −30°C until use. Whole bodies of zebrafish (64 g of 300 fish) were homogenized with six volumes of homogenizing buffer (25 mM Tri-HCl, 0.25 M sucrose, and 10 mM 2-mercaptoethanol, pH 7.5) using a food blender (NAKASA Corp., Japan). The homogenate was centrifuged at 3800 g, 30 min. After collection of the supernatant, it was centrifuged at 100,000 g for 60 min.

The supernatant was collected and filtered with filter paper (Whatman). The clear sample was applied on a DEAE cellulose column (2.6 × 12.0 cm) equilibrated with 50 mM Tri-HCl pH 7.5, which contained 20% glycerol and 10 mM 2-mercaptoethanol (TGM buffer). The column was also washed with TGM buffer, and bound materials were eluted using a linear gradient (total volume of 400 mL) of 0–0.4 M NaCl in TGM buffer at a flow rate of 60 mL/h (fraction volume: 10 mL). Active fractions were pooled and loaded into a 100 mL Q-Sepharose column (2.6 × 19.0 cm) equilibrated with TGM buffer, and after application, the column was washed with the same buffer and eluted using a linear gradient (total volume is 100 mL) of 0–0.4 M NaCl in TGM buffer at a flow rate of 60 mL/h (fraction volume: 5 mL).

Active fractions were pooled and concentrated to <5 mL by using a Q-Sepharose column (0.9 × 4.0 cm). The concentrated fraction was further purified by gel chromatography on a Sephacryl S-300 gel column equilibrated with TGM buffer at a flow rate of 60 mL/h (fraction volume: 10 mL). The active fractions were pooled and applied on a hydroxylapatite column (0.9 × 4.0 cm) equilibrated with 25 mM PGM buffer, and after application, the column was washed with the same buffer and eluted with a linear gradient of phosphate from 25 mL to 300 mM in PGM buffer at a flow rate of 60 mL/h (fraction volume: 5 mL).

Active fractions were pooled and bound to a DEAE cellulose column (0.9 × 4.0 cm) after dilution with 20 mM Tris-HCI, pH 8.0 (TN buffer). The column was washed extensively with TN buffer to remove glycerol. Bound proteins were eluted in high salt buffer (TN buffer containing 2 M NaCl), and the active fraction concentration of NaCl in the active fractions was adjusted to 2 M by adding solid NaCl. Samples were passed through a phenyl Sepharose Fast Flow column (1.0 × 6.4 cm) equilibrated with TN buffer containing 2 M NaCl (fraction volume: 2 mL).

Active fractions were diluted for 20 times with milli-Q water and applied on Q-Sepharose mini column (1.0 × 0.5 cm) equilibrated with TGM buffer. By eluting with TGM buffer containing 0.5 M NaCl (fraction volume: 0.5 mL), concentrated active fractions were obtained. Purified samples were pooled and stored at −80°C. All column chromatography was conducted by using AKTA Prime Plus chromatography system (GE Healthcare, USA) set in chromatography chamber at 4°C.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot analysis

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed on a 12% polyacrylamide gel under denaturing conditions according to the method of Laemmli. For Western blotting, the separated proteins were transferred to Immobilon membranes (Millipore, Billerica, MA, USA). Then, the membranes were blocked in 5% nonfat powdered milk in 20 mM Tris buffer saline, pH 7.6 (TBS) containing 0.1% Tween 20 (TTBS) for 1–2 h at room temperature. After blocking, the membranes were washed with TTBS buffer three times for 5 min each. Subsequently, the membranes were incubated for 1 h with primary antibodies (monoclonal antibody GC3α raised against 20S proteasome from goldfish ovary) that were diluted 10-fold in TBS buffer.¹⁸

After that, the blot was washed with TTBS buffer three times for 5 min each and then incubated for 1 h with secondary antibodies (HRP-antimouse) that were diluted 2000-fold in TBS buffer. Visualization of the target protein was performed by enhanced chemiluminescence (ECL) using an ECL detection kit (Perkin Elmer, Whatman, MA, USA), a method based on chemiluminescence mediated by peroxidase conjugated to a secondary antibody. The signals were digitized using a CCD camera system (luminescent image analyzer LAS-4000 mini; Fujifilm, Tokyo, Japan). The image was analyzed by densitometry to determine the relative amount of protein, and the results were quantified.

Assay of proteasome activity

In proteasome assays, 100 μL of the reaction mixture containing 50 μL of deionized distilled water, 20 μL of 0.5 M Tris-HCl, 10 μL of 0.4% SDS (fluorogenic substrate), and 10 μL of the chromatography fractions was incubated for 10 min at 37°C. The reaction was terminated by the addition of 50 μL of 10% SDS and 50 μL of 1 mM Tris-HCl pH 9.0. The fluorescence was determined on a fluorescence spectrophotometer (Varioskan LUX, Skanlt-5.0) with an excitation wavelength of 360 nm and emission wavelength of 460 nm for the MCA substrate.

Peptide mass fingerprint analysis by matrix-assisted laser desorption/ionization-time of flight mass spectrometry

Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis was used to identify the proteins of the purified 20S proteasome as previously described.^19,20 The two-dimensional polyacrylamide gel electrophoresis of the purified 20S proteasome was stained with Coomassie Brilliant Blue R-250 (CBBR). The spots containing the 20S proteasome subunits were excised, and the proteins in gel spots were digested by trypsin (Promega, Madison, WI, USA) at 37°C for 16 h.

Then, the peptide fragments were collected on a ZipTip (Merck Millipore, Darmstadt Germany) and eluted by 2 μL of a solution of 60% acetonitrile, 0.1% trifluoroacetic acid, and 5 mg/mL α-cyano-4-hydroxycinnamic acid (CHCA) (Sigma Aldrich, St. Louis, MO, USA). The plate was used for loading the samples, which were layered with CHCA. Autoflex MALDI-TOF/MS equipment (Bruker Daltonics, Billerica USA) was used to detect the peptide mass spectrum in positive ion mode. The spectra that were obtained from MALDI-TOF/MS were calibrated by a mixture of molecular weight standards. At least 300 laser shots were used to take the averaged spectrum result.

The peptide fingerprint was analyzed using MASCOT software (Matrix Science, London, United Kingdom). It was searched against peptides from zebrafish taxonomy using the NCBInr data set, and the following parameters were used: cysteine modification by carbamidomethyl, trypsin digestion, zero missed cleavages, and peptide mass tolerance ±0.4. Probability-based MOWSE (for MOlecular Weight SEarch) was used to identify the zebrafish proteins.

Results

Purification of the 20S proteasome

The cytosolic fraction from zebrafish whole bodies showed peptide hydrolyzing activity against Suc-LLVY-MCA, a well-known substrate for the 20S proteasome. To detect the activity of the 20S proteasome, we determined the best concentration of SDS to activate the Suc-LLVY-MCA hydrolyzing activity of the 20S proteasome. The highest activity was shown at 0.04% SDS, similar to the goldfish 20S proteasome (Supplementary Fig. S1). During the following purification steps, the proteasome activity was determined using this assay condition.

The zebrafish 20S proteasome was purified by five chromatography steps: DEAE cellulose, Q-Sepharose, Sephacryl S300, hydroxylapatite, and phenyl Sepharose (Supplementary Fig. S2). The protein contents of the eluted fractions were also assessed by SDS-PAGE and immunoblotting using a monoclonal antibody (antigoldfish 20S proteasome component GC3α). The antibody cross-reacts universally with eukaryotic α subunits from yeast to humans.^18,21 All characteristics of the zebrafish 20S proteasome during the purification steps were the same as those of the goldfish 20S proteasome.

The complex bound on anion exchange columns (DEAE cellulose and Q-Sepharose) and the hydroxylapatite column. In gel column chromatography (Sephacryl S300), the zebrafish 20S proteasome eluted in the fractions corresponding to a high molecular mass of ∼700 kDa. The zebrafish 20S proteasome did not bind to the hydrophobic column (phenyl Sepharose). Column chromatography was effective in removing other proteins from the zebrafish 20S proteasome fraction. In this way, 0.67 mg of purified zebrafish 20S proteasome complex was obtained from the cytosol of 300 fish (total soluble protein of 438 mg).

Protein bands of the zebrafish 20S proteasome appeared with molecular masses ranging from 21 to 33 kDa in one-dimensional PAGE (Fig. 1). Strong signals were detected by Western blotting using three kinds of monoclonal antibodies against the goldfish 20S proteasome (Fig. 1). It is thought that single bands detected by anti-α2 subunit (GC4/5) and anti-α4 (GC3β) subunits correspond to zebrafish α2 subunit and α4 subunits. In two-dimensional (2D) gel electrophoresis, 20 spots were detected by CBBR staining (Fig. 2). Proteins in these spots were identified by TOF-MS analysis (Table 1). All 15 gene products estimated from annotated genes in the zebrafish genome were detected.

FIG. 1.

SDS-PAGE and Western blot analysis of purified zebrafish 20S proteasomes. Purified 20S proteasomes were electrophoresed under denaturing conditions (12.5% gel), stained with CBBR-250, and immunostained with three kinds of monoclonal antibodies (GC3α, GC3β, and GC4/5). Molecular weights of standard proteins are indicated on the left. SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis.

FIG. 2.

2D-PAGE of zebrafish 20S proteasome. Purified 20S proteasomes were separated using a pH 3–10 nonlinear IPG strip in the first dimension followed by separation using 12% SDS-PAGE in the second dimension. The 2D gel was stained with CBBR-250. All labeled spots were identified by MALDI-TOF-MS and database searching. The major spots for each subunit are indicated as α1–α8 or β1–β7. Minor spots are indicated by names for corresponding subunits. The asterisk indicate the protein spot of α-tropomyosin that copurified in some fractions with the 20S proteasome. 2D, two-dimensional; 2D-PAGE, two-dimensional polyacrylamide gel electrophoresis; MALDI-TOF-MS, matrix-assisted laser desorption/ionization-time of flight mass spectrometry.

Table 1.

Summary of Zebrafish 20S Proteasome Subunits Identified by Peptide Mass Fingerprinting

Systematic nomenclature	Gene name	Gene ID	Theoretical mass (M_r)	Theoretical pl	Matched peptides	Sequence coverage (%)
α1a	psma6a	171585	27,868	6.02	12	50
α1b	psma6b	436862	27,808	6.02	10	45
α2	psma2	554193	25,945	5.99	9	47
α2′	psma2	554193	25,945	5.99	7	34
α3	psma4	326687	29,599	7.57	9	49
α3′	psma4	326687	29,599	7.57	7	28
α5	psma5	403011	26,448	4.76	9	49
α6	psma1	445033	29,515	6.20	13	50
α6′	psma1	445033	29,515	6.20	9	48
α6″	psma1	445033	29,515	6.20	10	46
α7	psma3	564370	28,619	4.94	9	37
α8	psma8	406445	28,254	8.94	4	14
β1	psmb6	30388	25,297	5.08	7	41
β2	psmb7	64278	23,134	6.58	9	37
β3	psmb3	573095	23,882	5.36	10	40
β4	psmb2	436882	18,953	7.71	10	43
β4′	psmb2	436882	18,953	7.71	7	34
β5	psmb5	30387	29,510	6.00	4	17
β6	psmb1	445413	26,312	6.32	9	49
β7	psmb4	550499	28,514	5.70	9	27

Among the 7 α subunits, the psma7 gene is missing in zebrafish, but a paralog of psma7, psma8, is present. The protein spot of the α8 subunit encoded by the psma8 gene was identified. Thus, the α8 subunit instead of the α4 subunit forms the α subunit ring of the zebrafish 20S proteasome complex. Although the amount of protein was significantly higher in α1a than in α1b, both protein spots of the α1 subunit encoded by two paralogs of psma6a and psma6b were found. In this article, we named the gene products from psma6a and psma6b subunits α1a and α1b. In contrast to the results from the α subunits, 7 β subunits encoded by psma1–7 genes were identified. Some spots were identified as the same subunit (α2, α3, α6, and β4). These spots are thought to be products of protein modifications.

Discussion

In this study, we purified the natural protein complex of the zebrafish 20S proteasome and identified its subunit composition. The purified zebrafish 20S proteasome showed 14 subunits with molecular masses ranging from 21 to 33 kDa, as expected. All three monoclonal antibodies prepared using the goldfish 20S proteasome as an antigen showed extremely high reactivity. The GC3α antibody, which is presumed to recognize the common sequence of α subunits, showed cross-reactivity with many bands.

As a special characteristic of the zebrafish 20S proteasome, the α4 subunit-encoding gene psma7 is missing in the genome. Thus, psma8 was identified in TOF-MS analysis of 2D gel protein spots. psma8 has been shown to be a testis-specific subunit (α4s) in mice and other organisms. Since a closely related species of zebrafish, goldfish, has both psma7 and psma8, we believe that psma7 was deleted in the zebrafish. It has been confirmed that instead of psma7, a duplicated gene of psma7, psma8, acts as a component of the general 20S proteasome in zebrafish. Another specific characteristic of the zebrafish 20S proteasome is that there are two types of α1 subunits.

For the α1 subunit, zebrafish has two duplicated genes, psma6a and psma6b. However, this is quite normal in zebrafish, and there are duplicated genes for each gene by whole genome duplication, with only one that contains duplicated genes among 14 kinds of subunit genes. Although Psma6a (α1a) was more abundant than Psmab (α1b), a spot of Psma6b was identified. In the case of the Xenopus 20S proteasome, we showed that duplicated genes for α4 subunits were expressed at the same levels.²²

In the case of Xenopus α4 subunits, amino acid sequences from two duplicated genes are the same (just one amino acid deletion in the C-terminus of one of them), but some amino acids differ between α1a and α1b (92.7% identity). Thus, the proteasomes composed of α1a and α1b may share different functions. The different tissue distribution patterns of α1a and α1b should be analyzed.

The importance of regulating proteasome activity by modifying the subunits of proteasomes is beginning to be recognized.²³ Various types of post-translational modifications, especially phosphorylation, on proteasome subunits have been identified.²³ Recent discoveries have provided compelling evidence to support the exact opposite insomuch as the 26S proteasome undergoes dynamic and reversible phosphorylation under a variety of physiopathological conditions.²³ Several subunits (α1, α2, α3, and β4) of the zebrafish 20S proteasome showed modified minor spots in this study.

In goldfish, we identified meiotic cell cycle-dependent phosphorylation of the α4 subunit.²⁴ Furthermore, we identified a protein kinase for the α4 subunit as casein kinase II.²⁵ Phosphorylation of the α4 subunit is suggested to be involved in the regulation of cyclin B digestion activity of the 26S proteasome.^26,27 Modifications of the zebrafish 20S proteasome should be involved in the regulation of proteasome activity. Further analysis of the type of modifications and regulatory mechanisms by these modifications should be conducted.

Owing to its importance as a disease-related target, the proteasome and its relationship with disease have been investigated in zebrafish.^14,15,28 For example, mis-sense mutation in the b6 subunit (Tyr103His in human PSMB1 gene) was related to neurodevelopmental disorder.²⁹ Genetically identified human disorder was linked by using the zebrafish model. Purification and identification of the zebrafish 20S proteasome in this study provide the basis for further detailed analysis.

Footnotes

Acknowledgments

We thank T. Fukuyo for zebrafish maintenance. We also acknowledge the scholarship for foreign students from Honda Benjiro to Md. Maisum Sarwar Jyoti and Md. Hasan Ali.

Disclosure Statement

No competing financial interests exist.

Funding Information

This study was supported by grants-in-aid for scientific research in priority areas from the Ministry of Education, Culture, Sports, Science and Technology of Japan. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the article.

Supplementary Material

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Supplementary Material

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