Supplementary information for Identification and characterization of a human mitochondrial NAD kinase Kazuto Ohashi, Shigeyuki Kawai, Kousaku Murata* Laboratory of Basic and Applied Molecular Biotechnology, Division of Food and Biological Science, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan *Corresponding author Kousaku Murata Address: Laboratory of Basic and Applied Molecular Biotechnology, Division of Food and Biological Science, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan Telephone number: +81-774-38-3766 Fax number: +81-774-38-3767 E-mail:
[email protected]
Supplementary Figures S1 to S7 Supplementary Tables S1 to S5 Supplementary Methods Supplementary References
1
Supplementary Figure S1. Sequence information of C5orf33 gene. (a) DNA fragment encoding N-terminal 100 amino acid residues of C5orf33 protein in pMK3635. This DNA fragment was used for pMK3269, pMK3270, pMK3271, and pMK3272 (Supplementary Table S4). This sequence was optimized for human codon usage. Underlined sequence was used for In-fusion cloning. BamHI site is boxed. Start codon is indicated by asterisks. Downstream of the arrow indicates the sequence encoding N-terminally truncated 62C5orf33 protein. (b) mRNA structures of C5orf33 transcript variant 1, NADK, and GAPDH. Gray arrows indicate the genes encoding C5orf33 protein, NADK, and GAPDH. The nucleotide sequences, which are indicated by double-headed arrows and whose sizes are shown, were amplified and inserted into pBluescript II SK (+). The base numbers are indicated by defining the start of each mRNA as 1. Full-length cDNAs corresponding to the mRNAs of human NADK and C5orf33 variant 1 were unable to be amplified.
2
Supplementary Figure S2. Saturation curve and Hanes–Woolf plot. (a) The effect of concentration of ATP on the initial velocity (V, U/mg) of the NADK reaction determined in a mixture (1.0 mL) containing 5.0 mM NAD+, indicated concentrations of ATP, 5.0 mM glucose 6-phosphate, 0.5 U glucose 6-phosphate dehydrogenase, 40 mM MgCl 2 , 100 mM Tris-HCl (pH 8.0), and appropriate concentrations of purified C5orf33 protein. (b) Hanes–Woolf plot of the data in (a).
3
Supplementary Figure S3. In vivo assay of localization of C5orf33 protein in S. cerevisiae. S. cerevisiae NADK triple mutant (utr1 yef1 pos5) carrying YCplac33-UTR1 and the indicated plasmids were spotted onto the solid media and were incubated at 30°C for 4 days. pMK2145 is pRS415 carrying P pos5 driving POS511. POS5 lacking the 16-residue mitochondrial targeting sequence was designated as POS511. ScMTS is the mitochondrial targeting sequence consisting of N-terminal 62 residues of Pos511. SD medium containing Lys and Ura was used as Arg() medium, and SD medium containing Lys, Ura, and Arg was used as Arg(+) medium. Pos5 and ScMTS-fused proteins are targeted to mitochondria; in contrast, 16Pos5 is not11as in Supplementary Fig. S3. The pos5 cell is auxotrophic for Arg, since the NADPH-requiring step in Arg biosynthesis is carried out in mitochondria3,
11, 16
.
Accordingly, we observed obviously slower growth of utr1 yef1 pos5 cell carrying 16POS5 (pMK2145) relative to utr1 yef1 pos5 cell carrying full-length POS5 (pMK2127) on Arg() medium as in Fig. S3. In order to examine the localizations of C5orf33 and 62C5orf33 proteins, we examined the Arg requirements of the utr1 yef1 pos5 cells carrying the plasmids encoding C5orf33, ScMTS-fused 62C5orf33 (ScMTS+62C5orf33), and 62C5orf33 proteins (pMK3269, pMK3601, and pMK3270), respectively. utr1 yef1 pos5 cells carrying the 62C5orf33 gene (pMK3270) displayed slower growth on Arg() medium than utr1 yef1 pos5 cells carrying the full-length C5orf33 gene (pMK3269) or the ScMTS+62C5orf33 gene (pMK3601) (Fig. S3), indicating mitochondrial localization of C5orf33 and ScMTS+62C5orf33 proteins and cytosolic localization of 62C5orf33 protein.
4
Supplementary Figure S4. Specific reactivity of anti-C5orf33. Purified C5orf33 protein (1.4 µg) and purified human NADK (2.4 µg) purified as described29 were analyzed by SDS-PAGE followed by Western blotting. Proteins were detected using anti-C5orf33 (Abnova, rabbit polyclonal, 1:312,500 dilution; middle) and anti-human NADK (Abnova, mouse monoclonal, 1:500 dilutions; right) antibodies. HRP-anti-rabbit IgG and HRP-anti-mouse IgG were used at dilutions of 1:20,000 and 1:10,000, respectively. After electro-blotting, gels were stained with Coomassie brilliant blue R-250 (CBB) to visualize proteins and dried (left). M: Precision Plus Protein Standard.
5
6
Supplementary Figure S5. siRNA transfection. HEK293A cells were transfected with siRNA#2 against C5orf33 (C5orf33 siRNA) or MISSION siRNA Universal Negative Control #1 (control siRNA) and incubated for 1, 2, and 3 days. (a) Transfected cells incubated for 3 days were stained with MitoTracker Red. (b, c) Viability upon menadione treatment. Transfected cells incubated for 1, 2, and 3 days after transfection were treated without (gray bar) or with (black bar) 12.5 µM menadione for 20 h; viability was determined using MTT (b) or calcein AM (c) as described in Supplementary Methods. (d) Intracellular ROS levels. Transfected cells incubated for 1, 2, and 3 days were incubated with 10 µM CM-H2DCFDA for 30 min, and then treated without (gray bar) or with (black bar) 100 µM menadione for 30 min; intracellular ROS levels were measured as described in Supplementary Methods. t-test; * p = 0.0085, ** p = 0.0026, *** p = 0.0022, **** p = 0.0044 × 10-4. Viability and intracellular ROS level are presented as relative values normalized against their respective levels in control siRNA-transfected cells at each incubation time in the absence of menadione treatment; values represent means and SD of 3 independent experiments conducted in duplicate (b, c, d).
7
8
Supplementary Figure S6. Phylogenetic tree. Phylogenetic tree was constructed based on the primary structures of the 107 homologs of the human C5orf33 protein as described in Supplementary Methods. The proteins containing the predicted mitochondrial targeting sequence, detected by Mitoprot28, are indicated by asterisks. Proteins are specified by the names of species (three-letters; e.g. has:human) plus ID in KEGG. Among C5orf33 homologs, species are grouped as either animals or protists, except for tgo (protist) grouped as animals and api (insect; animal) grouped as protists. 1,000 bootstrap trials were conducted; the resulting bootstrap values are shown at the nodes. Human C5orf33 and plant NADK3 are indicated by arrows.
9
Supplementary Fig. S7a
10
Supplementary Fig. S7b
11
Supplementary Figure S7. Multiple alignment of primary structures of C5orf33 protein, NADK3, and NADKs (a), C5orf33 homologs (b), and plant NADK3 homologs (c). The structures were aligned using ClustalW 50. GGDG and NE/D short motifs are shown in green and pale pink, respectively. NADK conserved region, motifs 1 and 2 are boxed by light blue, pale purple, and pink, respectively. The amino acid residue numbers of human C5orf33 protein are specified above (a,b), those of Ppnk are below (a), and those of NADK3 are above (c). The NADK motif (Fig. 1a) consists of 204 amino acid residues (from 118 to 321 residues) of the C5orf33 protein. Asp, Thr, and Tyr residues, corresponding to Asp-189, Thr-200, and Tyr-202 of Ppnk, respectively, are in yellow, while the corresponding Ser, Lys, and Trp residues are in orange (a,b); the Ala ones were in blue (a,c). Identical and similar amino acids are denoted by asterisks and dots, respectively. Proteins are specified, except for C5orf33 protein and NADK3, by the names of species (b,c). The C5orf33 and NADK3 additional regions were boxed in purple (a,b) and pale brown (a,c).
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Supplementary Table S1. Optimization of the condition to express 62C5orf33 proteina.
a
IPTG
Temp.
Period
Sp. act.
IPTG
Temp.
Period
Sp. act.
(µM)
(°C)
(days)
(mU/mg)
(µM)
(°C)
(days)
(mU/mg)
25
16
0.25
1.27
25
25
1
1.82
1,000
16
0.25
1.05
1,000
25
1
1.65
25
16
1
1.59
25
25
3
2.23
1,000
16
1
1.47
1,000
25
3
1.87
25
16
3
1.84
25
25
5
2.02
1,000
16
3
1.97
1,000
25
5
3.00
25
16
5
2.74
25
37
0.25
1.32
1,000
16
5
3.02
1,000
37
0.25
1.11
1,000
16
6
2.82
25
37
1
0.64
1,000
16
7
2.30
1,000
37
1
0.47
1,000
16
8
0.53
25
37
3
0.39
25
25
0.25
1.35
1,000
37
3
0.32
1,000
25
0.25
1.15
As described in Supplementary Methods, we examined the effects on specific NADK activity of
62C5orf33 protein in cell extract. High specific activity resulted from cultivation at 16 or 25°C, but not 37°C; for 5 days, but not more than 6 days; in the presence of either 25 µM or 1.0 mM isopropyl--D-thiogalactopyranoside (IPTG). At 16°C under 25 µM IPTG, cultivations for 4 or 5 days resulted in an approximately the same specific activity. Collectively, we expressed 62C5orf33 protein using the following conditions: cultivation at 16°C, and 4 or 5 days after addition of 25 µM IPTG. We selected 25 µM IPTG at 16°C rather than 1.0 mM IPTG at 25°C, since 25 µM IPTG at 16°C is a milder condition expected to facilitate expression of soluble protein, as observed in the expressions of other NADKs: archaeal MJ0917 (18°C, 25 µM IPTG)51, yeast Pos5 (16°C, 0.2 mM IPTG)11, Escherichia coli YfjB (16°C, 0.1 mM IPTG)39, and yeast Utr1 and Ppnk (18°C, 0.4 mM)14, 52
.
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Supplementary Table S2. siRNA duplexes used to knock down C5orf33 gene in this study. Number
#1 #2 #3 #5 #6 #7
Sequence of sense strand /antisense strand 5’-CAAUAGAGCCCUUAACAUU dTdT-3’ / 5’-AAUGUUAAGGGCUCUAUUG dTdT-3’ 5’-CUAAGCUUGAAUCAGCACA dTdT-3’ / 5’-UGUGCUGAUUCAAGCUUAG dTdT-3’ 5’-CUUCGAUGAUGAUCAAUAA dTdT-3’ / 5’-UUAUUGAUCAUCAUCGAAG dTdT-3’ 5’-CACAAUAGAGCCCUUAACA dTdT-3’ / 5’-UGUUAAGGGCUCUAUUGUG dTdT-3’ 5’-GAAGGAUUUCCUCAUGACA dTdT-3’ / 5’-UGUCAUGAGGAAAUCCUUC dTdT-3’ 5’-CCUACUAUGAGAUUUCAGUTT dTdT-3’ / 5’-ACUGAAAUCUCAUAGUAGG dTdT-3’
14
Supplementary Table S3. Distribution of C5orf33 and NADK homologs in animals and protistsa. Category Species ID b C5orf33 homolog c NADK homolog c d Animals Vertebrates Sus scrofa (pig) ssc ssc 100515177 Equus caballus (horse) ecb ecb 100067696 Xenopus laevis (frog) xla 379705 734906 Nematodes Brugia malayi bmy Bm1 18275 Trichinella spiralis tsp Tsp 08750 Cnidarians Caenorhabditis elegans cel Y77E11A.2 Y17G7B.10 Caenorhabditis briggsae cbr CBG13928 CBG21019 Nematostella vectensis nve NEMVE v1g93522 NEMVE v1g145879 Hydra magnipapillata hmg 100207865 100204951 Placozoans Trichoplax adhaerens tad TRIADDRAFT 26765 Protists e Choanoflagellates Monosiga brevicollis mbr MONBRDRAFT 30952 MONBRDRAFT 12055 Amoeboflagellate Naegleria gruberi ngr Amoebozoa Dictyostelium ddi DDB G0292442 discoideum DDB G0272224 Entamoeba histolytica ehi EHI 151920 Entamoeba dispar edi EDI 113320 Alveolates Plasmodium falciparum pfa PFI 0650c 3D7 Plasmodium falciparum pfd PFDG 01065 Dd2 Plasmodium falciparum pfh PFHG 03353 HB3 Plasmodium yoelii pyo PY04842 Plasmodium chabaudi pcb PC000710.01.0 PC302600.00.0 Plasmodium berghei pbe PB000335.00.0 Plasmodium knowlesi pkn PKH 071060 Plasmodium vivax pvx PVX 099100 Theileria annulata tan TA16300 TA04160 Theileria parva tpv TP01 1095 TP03 0366 Babesia bovis bbo BBOV IV003360 BBOV IV002800
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Supplementary Table S3. Continued. Category Species Toxoplasma gondii Cryptosporidium parvum Cryptosporidium hominis Tetrahymena thermophila
ID b tgo cpv cho tet
NADK homolog c TGME49 044700 cgd8 1970 Chro.80231 TTHERM 00013470 TTHERM 00048990 Paramecium tetraurelia ptm GSPATT 00006279001 GSPATT 00009606001 GSPATT 00015822001 Trypanosoma brucei tbr Tb927.7.5080 Trypanosoma cruzi tcr 509151.4 508175.26 Leishmania major lma LmjF06.0460 Leishmania infantum lif LINJ 06 0480 Leishmania braziliensis lbz LbrM06 V2.0470 Diplomonads Giardia lamblia gla GL50803 17316 Parabasalids Trichomonas vaginalis tva TVAG485920 TVAG056970 TVAG038980 TVAG298380 Oomycetes Phytophthora infestans pif PITG 00257 PITG 02750 a Distribution was judged based on the results of BLASTP search using each of the primary structures of human C5orf33 protein and human NADK. b ID defined in KEGG (Kyoto Encyclopedia of Genes and Genomes). c Gene names deposited in KEGG. d Animals, of which genome sequences were determined, containing either a C5orf33 homolog or NADK homolog. Animals that have both C5orf33 homolog and NADK homolog are not shown. e All protists whose genome sequences have been determined.
16
C5orf33 homolog c TGME49 092300 -
Supplementary Table S4. Plasmids used in this study. Plasmids Description Procedure pRS415 LEU2, CEN, Apr pQE-80L For expression in E. coli, Apr
pFLAG-CM V-5a
For expression in mammalian cells, Apr pBluescript II Cloning vector, SK (+) Apr pCR2.1 Cloning vector, Apr, Kanr YCplac33 5’ -503-bp+UTR1 -UTR1 in SmaI of YCplac33 pMK3635 The gene encoding the N-terminal 100 amino acid residues of C5orf33 protein (Fig. S1a) in pCR2.1 pMK3205 FLJ30596 gene (C5orf33 transcript variant 2) in pME18SFL3 pME18SFL3 Cloning vector, Apr pMK1647 P pos5 +POS5 (NcoI site is introduced in +185) in pRS415 pMK2127 P pos5 +POS5 in SacI/BamHI of pRS415 pMK2145 P pos5 + POS5 in SacI/BamHI of pRS415 P pos5 (BamHI at + pMK3207 b 4) in pRS415 from pMK2127 pMK3269 P pos5 + the gene encoding C5orf33 protein in pRS415
Resource Sikorski et al.53 Qiagen Sigma Agilent Technologies Invitrogen Shi et al.15 Operon Biotechnologies
NBRC a
Toyobo Miyagi et al.11
Miyagi et al.11 Miyagi et al.11 Linearized pMK3207 was amplified by inverse PCR using primers 1 and 2, and pMK2127 as a template.
This study
The gene encoding the N-terminal 99 residues of C5orf33 protein was amplified by PCR using primers 9 and 13, and pMK3635 as a template. This PCR amplicon was inserted into pMK3243, yielding pMK3269 c.
This study
17
Supplementary Table S4. Continued. pMK3270 P pos5 + the gene The gene encoding the N-terminal 37 amino acid encoding residues of62C5orf33 protein was amplified by 62C5orf33 PCR using primers 10 and 13, and pMK3635 as a protein in pRS415 template. This PCR amplicon was inserted into pMK3243, yielding pMK3270 c. pMK3243 P pos5 + the gene The gene encoding 100C5orf33 protein d was encoding100C5o inserted into pMK3207, yielding pMK3243 e. rf33 protein in pRS415 pMK3244 P pos5 + the gene The gene encoding 163C5orf33 protein was encoding163C5o amplified by PCR using primers 4 and 5, and the rf33 protein in cDNA of human C5orf33 variant 2 f as a template. This PCR amplicon was inserted into pMK3207, pRS415 yielding pMK3244 e. pMK3601 P pos5 + ScMTS+ the The gene encoding 62C5orf33 protein was gene encoding amplified by PCR using primers 26 and 27, and 62C5orf33 pMK3270 as a template. This PCR amplicon was protein in pRS415 inserted into pMK1647, yielding pMK3601 g.
pMK3271
The gene encoding C5orf33 protein in BamHI/SmaI of pQE-80L
pMK3272
The gene encoding 62C5orf33 protein in BamHI/SmaI of pQE-80L The gene encoding100C5o rf33 protein in BamHI/SmaI of pQE-80L The gene encoding C-terminally FLAG-tagged C5orf33 protein in pFLAG-CMV-5a The gene encoding C-terminally FLAG-tagged 62C5orf33 protein in pFLAG-CMV-5a PCR amplicon of cDNA encoding GAPDH in pBluescript II SK (+)
pMK3241
pMK3602
pMK3603
pMK3420
This study
This study
This study
This study
This study The gene encoding N-terminal 99 amino acid residues of C5orf33 protein was amplified by PCR using primers 11 and 13, and pMK3635 as a template. This PCR amplicon was inserted into pMK3241, yielding, pMK3271 h. This study The gene encoding the N-terminal 37 amino acid residues of62C5orf33 protein was amplified by PCR using primers 12 and 13, and pMK3635 as a template. This PCR amplicon was inserted into pMK3241, yielding pMK3272 h. This study The gene encoding 100C5orf33 protein d was inserted into BamHI/SmaI site of pQE-80L, yielding pMK3241. The gene encoding C5orf33 protein was amplified This study by PCR using primers 28 and 30, and pMK3270 as a template. This PCR amplicon was inserted into pFLAG-CMV-5a, yielding pMK3602 i. This study The gene encoding 62C5orf33 protein was amplified by PCR using primers 29 and 30, and pMK3270 as a template. This PCR amplicon was inserted into pFLAG-CMV-5a, yielding pMK3603 i. This study cDNA encoding GAPDH was amplified by PCR using primers 14 and 15, and QUICK-Clone cDNA (human) (Clontech) as a template. This PCR amplicon was inserted into pBluescript II SK (+), yielding pMK3420 j.
18
Supplementary Table S4. Continued. pMK3421 PCR amplicon of This study cDNA encoding NADK was amplified by PCR cDNA encoding using primers 16 and 17, and QUICK-Clone cDNA NADK in (human) as template. This PCR amplicon was pBluescript II SK inserted into pBluescript II SK (+), yielding (+) pMK3421 j. pMK3422 PCR amplicon of This study cDNA encoding C5orf33 variant 1 was amplified cDNA encoding by PCR using primers 18 and 19, and C5orf33 variant 1 QUICK-Clone cDNA (human) as template. This in pBluescript II PCR amplicon was inserted into pBluescript II SK SK (+) (+), yielding pMK3422 j. a National Institute of Technology and Evaluation Biological Resource Center. b BamHI was introduced by changing +4 TTTGTC +9 to +4 GGATCC +9. c The plasmids were constructed by In-fusion cloning54 from the PCR product and linearized pMK3243. Linearized pMK3243 was amplified by inverse PCR using primers 6 and 7, and pMK3243 as a template. d The gene encoding 100C5orf33 protein was amplified by PCR using primers 3 and 5, and the cDNA of C5orf33 variant 2 as a template. e This PCR product was digested with BamHI and inserted into the BamHI/SmaI site of pMK3207. f cDNA of C5orf33 transcript variant 2 (NCBI; NM 153013.3 [Fig. 1b]), encoding 100C5orf33 protein, was purchased from NBRC. g The plasmids was constructed by In-fusion cloning from the PCR product and linearized pMK1647. pMK1647 was linearized by NcoI/BamHI. h The plasmids were constructed by In-fusion cloning from the PCR product and linearized pMK3241. Linearized pMK3241 was amplified by inverse PCR using primers 6 and 8, and pMK3241 as a template. i The plasmids was constructed by In-fusion cloning from the PCR product and linearized pFLAG-CMV-5a. pFLAG-CMV-5a was linearized by BamHI. j The plasmids was constructed by In-fusion cloning from the PCR product and linearized pBluescript II SK (+). pBluescript II SK (+) was linearized by KpnI.
19
Supplementary Table S5. Primers used in this study. Number Name Sequence 1 POS5_-18_3_R CATTTTAATCAGTATCCTTAAC 2 POS5BamHI_10_29_F GGATCCAGGGTTAAATTGAATAAACC CGGGATCCCTTGCATTGAAAGGCTCTAG 3 C5orf33_CGBamHI_301_320F 4 C5orf33_CGBamHI_490_511F CGGGATCCATGCTGCTGGCAGCGAGTAAAG 5 C5orf33_1310_1329R TCACTGTTCAAGAAGCACAG 6 C5orf33_301_320F CTTGCATTGAAAGGCTCTAG GGATCCCATTTTAATCAGTATC 7 POS5_-18_3_+BamHI_R 8 PQE80_131-150Rv GGATCCGTGATGGTGATGGT 9 Synth_C5orf33_1_20F ATTAAAATGGGATCCACATG 10 C5orf33_del187_pMK3207F ATTAAAATGGGATCCGATGGCGGGTTTAGGCCG TCA 11 C5orf33_PQE80F CACCATCACGGATCCACATGCTATCGGGGTTTCC TC 12 C5orf33_del187_PQE80F CACCATCACGGATCCGATGGCGGGTTTAGGCCG TCA 13 Synth_C5orf33_308_327R GCCTTTCAATGCAAGCAGCT 14 GAPDHmRNA_infusion_1_20_F CTATAGGGCGAATTGGAAATTGAGCCCGCAGCCT CC 15 GAPDHmRNA_infusion_1310_12 CTCGAGGGGGGGCCCGGGTTGAGCACAGGGTAC TTT 91_R 16 NADKmRNA_infusion_181_200_ CTATAGGGCGAATTGGCGGCATCAGTGTTTTTCT GA F 17 NADKmRNA_infusion_3212_3193 CTCGAGGGGGGGCCCGTTGAAAGGATCTGAAAA GCGTA _R 18 C5orf33mRNA_infusion_5_24_F CTATAGGGCGAATTGGCTTGCTACCGAGGCTTCT TG 19 C5orf33mRNA_infusion_3856_37_ CTCGAGGGGGGGCCCGACAGGGGCCATGAATGT AAA R 20 HA067812-F(GAPDH) GCACCGTCAAGGCTGAGAAC
21 22
HA067812-R(GAPDH) HA092368-F(NADK)
TGGTGAAGACGCCAGTGGA TATGTTCTGCAAGACGCTGTCCTC
23
HA092368-R(NADK)
ATCAGGATCGCACTGAACAGAATG
24
C5orf33mRNAvariant1_227_246_F w C5orf33mRNAvariant1_299_280_ Rv POS5_175_189+C5orf33_187_207 _F p.415_3182_98+C5orf33_1310_29 R pFLAG-CMV-5+C5orf33_4_24_F
AAACCACCCGGTACGAGTTC
25 26
AGCTGCTTCAGGTCCTCCTC
AATCTTTAACCATGGATGGCGGGTTTAGGCCGTC A 27 TGCAGCCCGGGGGATCCTCACTGTTCAAGAAGC ACAG 28 ATCAGTCGACGGATCCACCATGACATGCTATCG GGGTTTCCT 29 pFLAG-CMV-5+C5orf33_187_207 ATCAGTCGACGGATCCACCATGGATGGCGGGTTT AGGCCGTCA _F 30 pFLAG-CMV-5 AATCGGTACCGGATCCTCACTGTTCAAGAAGCAC AG +C5orf33_1310_29R BamHI site (GGATCC) is shown in bold. Sequences for In-fusion cloning are underlined.
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Supplementary Methods Optimization of conditions for expression of 62C5orf33 protein. Plasmid pMK3272, carrying the gene encoding 62C5orf33 protein, was introduced into E. coli Rosetta-gami(DE3)pLysS (Novagen), yielding strain MK3275. To optimize the conditions for expression of 62C5orf33 protein, we examined the effects on NADK activity of varying the following parameters: concentration of IPTG (25 M or 1.0 mM), temperatures after induction (16, 25, or 37°C), and induction periods (up to 8 days). Details follow. MK3275 cells were precultured overnight in 350 mL LB medium (350 mL per 500 mL Sakaguchi flask) supplemented with 100 µg/mL ampicillin and 34 µg/mL chloramphenicol. Cells were collected from these cultures and inoculated into 1.5 L LB media supplemented with the same antibiotics in a 2 L Sakaguchi flask (4 flasks total), or 350 mL of the specified media per 500 mL Sakaguchi flask (2 flasks total), and aerobically cultured at 37°C until A 600 reached 0.80–0.90. IPTG was then added to obtain a final concentration of 25 M or 1.0 mM. Cultivation continued at 16°C (in 1.5 L media, 2 flasks), 25°C (in 1.5 L media, 2 flasks), or 37°C (in 350 mL media, 2 flasks) under aerobic conditions for 8 days, during which 50 mL cultures were sampled at 0.25, 1, 3, 5, 6, 7, and 8 days. The crude extract was prepared, and NADK activity of the extract was measured by the stop method, as described previously29. Estimation of the amounts of soluble and insoluble expressed 62C5orf33 proteins. Centrifugation was conducted at 4°C at 15,000 g for 10 min. MK3275 cells in which expression of 62C5orf33 protein had been induced at 16°C for 4 days after addition of 25 µM IPTG in 13.5 L media (1.5 L media per 2 L flask), described above, were collected, suspended in an approximately equal volume of 10 mM Tris-HCl (pH 8.0), and lysed twice by sonication using an Insonator 201M (Kubota, Tokyo, Japan) at 4°C for 10 min. Before and after sonications, a 1/1,000 volume of 1.0 M phenylmethylsulfonyl fluoride was added to the suspension29. The lysate (10 µL) was centrifugated. The pellet was suspended in 10 µL of 10 mM Tris-HCl (pH 8.0) by briefly vortexing, and subsequently
21
centrifuged. This washing procedure was conducted once more. The resulting pellet was suspended in 30 µL of 10 mM Tris-HCl (pH 8.0). The resultant supernatants were combined and used as cell extract (30 µL). To the suspension and the cell extract, 6 µL of 6 X SDS buffer [12% (w/v) SDS, 36% (v/v) glycerol, 5% (v/v) 2-mercaptoethanol, approximately 0.1 mg/mL bromophenol blue, 250 mM Tris-HCl (pH 6.8)] was added; samples were then boiled for 5 min, yielding 47.5 and 36.0 µL volumes for suspension and cell extract, respectively. The boiled suspension (6.6 µL; 5.0 µL x 47.5 µL/36.0 µL) and boiled cell extract (5 µL) were analyzed by SDS-PAGE. 62C5orf33 proteins in the suspension and cell extract were insoluble and soluble, respectively. The intensities of gel bands corresponding to each protein were estimated using ImageQuant TL (GE Healthcare). Purification of C5orf33 protein. To purify the 62C5orf33 protein, MK3275 cells from 13.5 L culture were treated as in the text, and prepared crude extract was applied to a TALON column (2.5 22.5 cm) (Clontech) equilibrated with 10 mM Tris-HCl (pH 8.0). The column was washed with 2 L of 10 mM Tris-HCl (pH 8.0) containing 0.30 M NaCl and 20 mM imidazole. 62C5orf33 protein was eluted with 10 mM Tris-HCl (pH 8.0) containing 0.30 M NaCl and 150 mM imidazole. Fractions containing 62C5orf33 protein were combined, concentrated to 0.2 mg/mL by ultrafiltration in a Vivaspin (nominal molecular weight limit: 10,000, GE Healthcare). Purified 62C5orf33 protein (11 mg, 0.07 U/mg, total 0.77 U) was obtained from crude extract (1,096 mg, 0.0046 U/mg, total 5.04 U) with 15% yield of total activity; this material was regarded as purified C5orf33 protein.
Assays for sphingosine kinase, diacylglycerol kinase, and 6-phosphofructokinase activities of C5orf33 protein. Stock solutions of D-sphingosine (Sigma, S7049), 1,2-dioleoyl-sn-glycerol (Sigma, D0138), and D-fructose 6-phosphate (Sigma, F3627) were prepared at 50 mM (in ethanol), 32 mM (in dimethylformamide), and 50 mM (in water), respectively. To assay activities of sphingosine kinase, diacylglycerol kinase, and 6-phosphofructokinase, 5.0 μL of a reaction mixture containing 5.0 mM substrates (D-sphingosine, 1,2-dioleoyl-sn-glycerol, or D-fructose 6-phosphate), 5.0 mM MgCl 2 ,
22
100 mM Tris-HCl (pH 8.0), 3.3 g of purified C5orf33 protein, and 5.0 mM ATP was incubated overnight at 37°C. As controls, reaction mixtures containing 5.0 mM NAD+ with or without vehicle [16% (v/v) dimethylformamide or 10% (v/v) ethanol] were also incubated in parallel. Standard compounds and the incubated reaction mixtures were spotted onto a TLC glass plate (silica gel 60 F 254 ; Merck), dried, developed with a solvent system of isobutyrate: 500 mM NH 4 OH (5:3 v/v)44, and detected by exposure to UV light at 254 nm. Kinase activities were assayed by monitoring formation of ADP. Absolute qPCR. Human total RNAs from brain, liver, placenta, and small intestine were purchased from Clontech. Human total RNAs from heart, lung, kidney, skeletal muscle, testes, colon, spleen, and stomach were purchased from Agilent Technologies. cDNAs were prepared from 1.75 µg RNA (RNAs from human tissues and the synthesized mRNAs) in 25 µL, or from 0.2 µg RNA from transfected cells in 15 µL, using the ReverTra Ace qPCR RT kit (Toyobo, Osaka, Japan). Prepared cDNA from samples described above (0.14 µg cDNA from human tissues and the synthesized mRNAs, 0.013 µg cDNA from transfected cells) were quantitated by qPCR using LineGene (BioFlux, Tokyo, Japan) and SYBR Green Real-time PCR Master Mix (Toyobo) in 10 µL reactions under the following conditions: 1 cycle at 95°C for 1 min, and 40 cycles at 95°C for 15 s, 69°C for 15 s, 72°C for 30 s. Primers 20 and 21 (for GAPDH), primers 22 and 23 (for human NADK), primers 24 and 25 (for C5orf33 gene) were used (Supplementary Table S5). For construction of standard curves for absolute quantification of gene expression by qPCR, cDNAs from the synthesized mRNAs (GAPDH, 1.02E+11 copies/µL; human NADK, 4.47E+10 copies/ µL; C5orf33 variant 1, 3.53E+10 copies/µL) were serially diluted in 5-fold steps, and quantified as above. The standard curves were constructed by plotting the Threshold Cycle (Ct) values against the molar concentrations of the synthesized mRNAs. siRNA transfection. The siRNA duplexes (Sigma) for knock down of C5orf33 gene are listed in Supplementary Table S2. HEK293A cells were plated in 0.5 mL Dulbecco’s modification of Eagle’s minimal essential medium (DMEM) containing 10% fetal calf serum (DMEMS) without antibiotics in 12-well plates (3815-012, Iwaki, Asahi Glass,
23
Japan), incubated for 24 h at 37C, transfected with siRNA duplex (siRNA#2 or MISSION siRNA Universal Negative Control #1 [control siRNA, Sigma]) in the presence of Lipofectamine 2000 (Invitrogen) plus Opti-MEM (Invitrogen) to reach a final concentration of 83 nM siRNA in a total 0.6 mL, and further incubated at 37C for 1, 2, and 3 days. This condition was determined as follows: of the six siRNA duplexes we tested (Supplementary Table S2), initial transfections with 33 nM siRNA#1, #2, and #3 siRNA followed by incubation for 1 day resulted in 42, 20, and 0% knockdown, respectively compared to cells transfected with control siRNA. Transfections with 83 nM siRNA #1, #2, #5, #6, #7, and #1 plus #2 (1:1, total 83 nM) followed by incubation for 1 day resulted in 54, 71, 47, 19, 13, and 50% knockdown, respectively; thus, transfection with 83 nM siRNA#2 resulted in maximum C5orf33 knockdown (~70% reduction), and siRNA#2 was selected for subsequent knockdown experiments. Cell viability of siRNA-transfected cells. To siRNA-transfected HEK293A cells incubated after transfection for 1, 2, and 3 days, 1.5 µL of 5.0 mM menadione in dimethyl sulfoxide (DMSO) or 1.5 µL of vehicle (DMSO alone) was added to a final concentration of 12.5 µM (menadione) or 0.25% (v/v) (DMSO). After further incubation at 37°C for 20 h,
cell
viability
of
the
siRNA-transfected
cells
was
measured
using
3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) or calcein AM (Molecular Probes) as described23,
46
with slight modifications. For MTT assay, the
medium was replaced with 0.5 mL DMEM containing MTT at 0.3 mg/mL. After incubation at 37°C for 3 h, the medium was removed, and the generated formazan dissolved in 200 µL DMSO was quantitated by determination of A 600 . In the case of calcein AM assay, cells were washed by PBS three times and incubated in 500 µL PBS plus 3 µM calcein AM at 30˚C for 50 min. Fluorescence was measured at an excitation wavelength of 485 nm and an emission wavelength of 535 nm. Intracellular ROS. Intracellular ROS was measured as described33. siRNA-transfected HEK293A cells that had been incubated after transfection for 1, 2, and 3 days were incubated for 30 min at 37°C with 0.5 mL fresh DMEM plus 2.5 µL of 1.73 mM 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein
24
diacetate,
acetyl
ester
(CM-H2DCFDA, Invitrogen) in DMSO. Next, 10 µL of 5.0 mM menadione in DMSO or 10 µL of vehicle (DMSO alone) was added to reach a final concentration of 100 µM (menadione) or 2 % (v/v) (DMSO). After further incubation for 30 min at 37°C, cells were lysed in 200 µL 10 mM Tris-HCl (pH 7.5) containing 0.2% SDS. Fluorescence was measured at an excitation wavelength of 485 nm and an emission wavelength of 535 nm. Preparation of mitochondria. From transfected HEK293A cells that had been incubated for 3 days, we prepared mitochondria using the Mitochondria Isolation Kit for Cultured Cells (MitoSciences). Frozen and thawed samples of 6 × 106 – 107 cells were suspended in reagent A (250 µL per 106 cells), disrupted using a Dounce homogenizer, and separated as described in the kit instructions except that treatment with reagent B was omitted. The resulting supernatant was used as the cytosolic fraction. The mitochondrial pellet was suspended in 50 µL NP buffer (1% NP-40, 250 mM NaCl, 50 mM Tris-HCl [pH 7.5], 1 mM EDTA, and cOmplete EDTA-free Protease Inhibitor Cocktail [Roche])55, frozen and thawed three times, and centrifuged at 12,000 × g for 15 min. The resulting supernatant was used as the mitochondrial fraction. Protein concentrations of cytosolic and mitochondrial fractions were determined using the BCA Protein Assay Reagent Kit (Pierce) with BSA as a standard. Phylogenetic tree. The 111 homologs of the human C5orf33 protein were initially detected by BLASTP26 using the primary structure of human C5orf33 protein as a query against the eukaryote database in KEGG (Kyoto Encyclopedia of Genes and Genomes website, http://www.genome.jp/kegg/). Four fungal proteins (E-values from 0.61 to 1.5) were included among the 111 proteins. Since the four fungal proteins exhibited higher homology to human NADK (E-values from 1e-47 to 3e-44), the fungal proteins were excluded from the 111 proteins and the resultant 107 proteins were regarded as C5orf33 homologs in this study. The 107 homologs were aligned using ClustalW50. Phylogenetic tree with bootstrap56 was constructed using ClustalX50 with the neighbor-joining method57. The tree was displayed using NJprot 58.
Supplementary References
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Kawai, S. et al. Inorganic polyphosphate/ATP-NAD kinase of Micrococcus flavus and Mycobacterium tuberculosis H37Rv. Biochem. Biophys. Res. Commun. 276, 57-63 (2000).
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Someya, S. et al. Sirt3 mediates reduction of oxidative damage and prevention of age-related hearing loss under caloric restriction. Cell 143, 802-812 (2010).
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Felsenstein, J. Confidence-limits on phylogenies - an approach using the bootstrap. Evolution 39, 783-791 (1985).
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Saitou, N. & Nei, M. The neighbor-joining method - a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406-425 (1987).
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