Mass-spectrometric mapping of VDAC (voltage dependent anion channel) from mouse brain membranes with high sequence coverage

Year 2018, Volume: 3 Issue: 4, 225 - 248, 29.12.2018

Natalia Akentıeva

https://doi.org/10.26453/otjhs.458683

Abstract

VDACs (voltage
dependent anion channels) are integral membrane proteins serving as nonspecific
diffusion pores or as specific systems for the transport of substrates through
mitochondrial membranes. The functional role of VDAC has been investigated in
many studies, and different functions of VDAC have been shown.
Posttranslational modifications of VDAC are significant for its regulation. The
aim of our research was to develop approach for characterization of primary
structure and posttranslational modifications of VDACs and other membrane
proteins. Mouse brain membranes were isolated from mouse brains by differential
centrifugation. Primary structure of mitochondrial isoform VDAC1 from mouse
brain membranes has been identified almost completely (95 %, 258 of 283 amino
acids) by combination of SDS-PAGE and LTQ-FTMS mapping of peptide mixtures
after proteolytic degradation with trypsin. Sequence of each found peptide of
VDAC has been analysed and confirmed according to accurate mass, isotopic
distribution and MS/MS tandem analysis. Posttranslational modifications of
VDAC’speptides have been shown. High sequence coverage of VDAC has been
obtained, including 11 transmembranes domains. Extensive sequence coverage has
been also detected for some other proteins at 30-34 kDa. A repressor of
estrogen receptor activity has been identified with 76 % coverage, malate
dehydrogenase with 55 % sequence coverage, syntaxin 1A and syntaxin 1B2 have
been sequenced with 60 % and 65 % coverage, respectively. These results demonstrated
that mass spectrometric mapping is reliable and sensitive approach for
characterization of primary structure membrane proteins and identification of
their posttranslational modifications. 

Keywords

VDAC, primary structure

Yüksek dizi kapsama alanı ile fare beyin zarlarından VDAC (voltaj bağımlı anyon kanalı) kütle spektrometrik haritalama.

Abstract

VDAK'lar
(voltaj bağımlı anyon kanalları) spesifik olmayan difüzyon gözenekleri veya
mitokondriyal membran aracılı substratların taşınması için özel sistemler
olarak hizmet veren entegre membran proteinleridir. VDAK'ın fonksiyonel rolü
birçok çalışmada araştırılmış ve VDAK'ın farklı işlevleri gösterilmiştir.
VDAK'ın post-translasyon modifikasyonları regülasyonu için önemlidir. VDAK'ın
post-translasyon modifikasyonları regülasyonu için önemlidir. Araştırmamızın
amacı, VDAK'ların post-translasyonel ve diğer zar proteinlerinin
modifikasyonları ve primer yapının karakterizasyonu için yaklaşım
geliştirmektir. Fare beyin
zarları diferansiyel santrifüjleme ile fare beyninden izole edildi. Fare beyin
zarlarından gelen mitokondriyal izoform VDAK'ın primer yapısı tripsin ile
proteolitik yıkımdan sonra peptit karışımlarının LTQ-FTMS haritalama ve
SDS-PAGE 'nin kombinasyonu ile  neredeyse
tamamı (% 95, 283 amino asitin 258'i) tanımlanmıştır. VDAK’ın bulunan her bir
peptid dizisi doğru kütle, izotopik dağılım ve MS / MS tandem analizine göre
analiz edilmiş ve doğrulanmıştır. VDAK’ın peptitlerinin post-translasyon
modifikasyonları gösterilmiştir. VDAK'ın yüksek dizi içeriği 11 transmembran
alanı dahil olmak üzere elde edilmiştir. 
30-34 kDa'da diğer bazı proteinler için geniş çaplı sekans içeriği de
tespit edilmiştir. Sırasıyla; östrojen reseptorü baskılayıcısı aktivitesinin
içeriğinin %76’sı, malat dehidrogenazın %55’i, sintaksin 1A ve sintaksin
1B2’nin %60 ve %65’i belirlenmiştir. Bu sonuçlar, kütle spektrometrik haritalamanın
ana yapı membran proteinlerinin karakterizasyonu ve bunların post-translasyonel
modifikasyonlarının tanımlanması için güvenilir ve duyarlı bir yaklaşım
olduğunu göstermiştir.

Keywords

VDAC, birincil yapı

References

• Colombini M. A candidate for the permeability pathway of the outer mitochondrial membrane.Nature.1979;279(5714):643-645.PMID:450112.
• De Pinto V, Messina A, AccardiR, et al.New functions of an old protein: the eukaryotic porin or voltage dependent anion selective channel (VDAC). Ital. J. Biochem.2003; 52 (1): 17-24. PMID: 12833633.
• Tsujimoto Y, Shimizu S. Biochimie. The voltage-dependent anion channel: an essential player in apoptosis. 2002; 84 (2-3): 187-193. PMID: 12022949.
• Crompton M, BarksbyE, Johnson N, et al. Mitochondrial intermembranejunctional complexes and their involvement in cell death. Biochimie. 2002; 84 (2-3): 143-152. PMID: 12022945.
• Crompton M. The mitochondrial permeability transition pore and its role in cell death.Biochem J. 1999; 341 (Pt2): 233-249. PMID: 10393078.
• Lawen A, Ly JD, Lane DJ, et al. Voltage-dependent anion-selective channel 1 (VDAC1)-a mitochondrial protein, rediscovered as a novel enzyme in the plasma membrane. Internat. J.Biochemistry and Cell Biology. 2005; 37 (2): 277-282. DOI: 10.1016/j.biocel.2004.05.013.
• Shimizu S, Narita M, Tsujimoto Y. Bcl-2 family proteins regulate the release of apoptogenic cytochrome c by the mitochondrial channel VDAC. Nature. 1999;399 (6735): 483–487.DOI: 10.1038/20959.
• Rostovtseva T, Colombini M. VDAC channels mediate and gate the flow of ATP: implications for the regulation of mitochondrial function. Biophys. J. 1997; 72 (5):1954-1962. DOI: 10.1016/S0006-3495(97)78841-6.
• Hodge T, Colombini M. Regulation of metabolite flux through voltage-gating of VDAC channels. J.Membr. Biol. 1997; 157 (3): 271-279. PMID: 9178614.
• Schiltz E, Kreusch A, Nestel U. et al. Primary structure of porin from Rhodobactercapsulatus. Eur. J. Biochem. 1991, 199 (3), 587-594. PMID: 1651239.
• Przybylski M, Glocker MO, Nestel U, et al.X-ray crystallographic and mass spectrometric structure determination and functional characterization of succinylatedporin from Rhodobactercapsulatus: implications for ion selectivity and single-channel conductance. Protein Sci. 1996; 5 (8): 1477-1489. DOI: 10.1002/pro.5560050804.
• Buhler S, Michels J, Wendt S, et al. Mass spectrometric mapping of ion channel proteins (porins) and identification of their supramolecular membrane assembly.Proteins. 1998; Suppl. 2:63-73. PMID: 9849911.
• Bureau MH, Khrestchatisky M, Heeren MA, et al. Isolation and cloning of a voltage-dependent anion channel-like Mr 36, 000 polypeptide from mammalian brain. J. Biol.Chem. 1992; 267 (12): 8679-8684. PMID: 1373732.
• Blachly-Dyson E, Baldini A, Litt M, et al. Human genes encoding the voltage-dependent anion channel (VDAC) of the outer mitochondrial membrane: mapping and identification of two new isoforms. Genomics.1994; 20 (1): 62-67. DOI: 10.1006/geno.1994.1127. Ha H, Hjajek P, Bedwell DM, et al. A mitochondrial porincDNA predicts the existence of multiple human porins. J. Biol.Chem.1993; 268 (16):12143-12149. PMID: 7685033.
• Zhang H, Li XJ, Martin DB, et al. Identification and quantification of N-linked glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry. Natl.Biotechnol. 2003; 21(6): 660-666. DOI: 10.1038/nbt827.
• Chalmers MJ, Hakansson K, Johnson R, et al. Protein kinase A phosphorylation characterized by tandem Fourier transform ion cyclotron resonance mass spectrometry. Proteomics. 2004; 4(4): 970-981. DOI: 10.1002/pmic.200300650.
• Sapp DW, Witte U, Turner DM, et al. Regional variation in steroid anesthetic modulation of [35S]TBPS binding to gamma-aminobutyricacidA receptors in rat brain. J.Pharmacol.Exp.Ther. 1992; 262 (2): 801-808. PMID: 1323664.
• Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature. 1970; 227(5259): 680-685. PMID: 5432063.
• Shevchenko A, Jensen ON, Podtelejnikov AV, et al. A strategy for identifying gel-separated proteins in sequence databases by MS alone.Proc. Natl. Acad. Sci. USA. 1996; 93(25): 14440-14445. PMID: 8962070.
• Clauser KR, Baker P, Burlingame AL.Role of accurate mass measurement (+/- 10 ppm) in protein identification strategies employing MS or MS/MS and database searching. Anal. Chem. 1999; 71 (14): 2871-2882. PMID: 10424174.
• Dermietzel R, Hwang TK, Buettner R, et al. Cloning and in situ localization of a brain-derived porin that constitutes a large-conductance anion channel in astrocytic plasma membranes. Proc. Natl. Acad. Sci. U S A. 1994; 91(2): 499-503. PMID: 7507248
• Sabirov RZ,Sheiko T, Liu H, et al. Genetic demonstration that the plasma membrane maxianion channel and voltage-dependent anion channels are unrelated proteins. J.Biol.Chem. 2006; 281(4): 1897-904. DOI: 10.1074/jbc.M509482200.
• Yu WH, Forte M. Is there VDAC in cell compartments other than the mitochondria? J. Bioenerg. Biomembr. 1996; 28 (2):93-100. PMID: 9132422. Rostovtseva TK, Tan W, Colombini M. On the role of VDAC in apoptosis: fact and fiction. J.BioenergBiomembr. 2005; 37 (3):129-142. DOI: 10.1007/s10863-005-6566-8.
• Bera AK, Ghosh S. Dual mode of gating of voltage-dependent anion channel as revealed by phosphorylation. J. Struct. Biol. 2001; 135 (1): 67-72. DOI: 10.1006/jsbi.2001.4399.
• Bera AK, Ghosh S, Das S. Mitochondrial VDAC can be phosphorylated by cyclic AMP-dependent protein kinase. Biochem.Biophys. Res. Commun.1995; 209 (1):213-217. DOI: 10.1006/bbrc.1995.1491.
• Baines ChP, Song ChXu, ZhengYuT, et al. Protein kinase C interacts with and inhibits the permeability transition pore in cardiac mitochondria. Circ. Res. 2003; 92 (8): 873-880. DOI: 10.1161/01.RES.0000069215.36389.8D.
• Liberatori S, Canas B, TaniCh, et al. Proteomic approach to the identification of voltage-dependent anion channel isoforms in guinea pig brain synaptosomes. Proteomics. 2004; 4 (5):1335-1340. DOI: 10.1002/pmic.200300734.
• Shafir I, Feng W, Shoshan_Barmataz VV. Voltage-dependent anion channel proteins in synaptosomes of the Torpedo electric organ: immunolocalization, purification, and characterization. J. Bioenergetics and Bomembranes. 1998; 30 (5): 499-510. PMID: 9932652.
• Blumental A, Kahn K, Beja O, et al.Purification and characterization of the voltage-dependent anion-selective channel protein from wheat mitochondrial membranes. Plant Physiol. 1993; 101 (2): 579-587. PMID: 12231713.
• Brugiere S, Kowalski S, Ferro M, et al. The hydrophobic proteome of mitochondrial membranes from Arabidopsis cell suspensions.Phytochemistry.2004; 65 (12): 1693-1707. DOI: 10.1016/j.phytochem.2004.03.028.
• Prinz T, Muller J, Kuhn K, et al.Characterization of low abundant membrane proteins using the protein sequence tag technology. J. Proteome Res. 2004; 3 (5):1073-1081. DOI: 10.1021/pr049925u.
• Florke H, Thinnes FP, Winkelbach H, et al. Channel active mammalian porin, purified from crude membrane fractions of human B lymphocytes and bovine skeletal muscle, reversibly binds adenosine triphosphate(ATP). Biol. Chem. Hoppe Seyler. 1994; 375 (8):513-520. PMID: 7529026.
• Taylor S, Warnock D, Glenn G, et al.An alternative strategy to determine the mitochondrial proteome using sucrose gradient fractionation and 1D PAGE on highly purified human heart mitochondria. J. Proteome Res. 2002; 1(5): 451-458. PMID: 12645917.
• Da Cruz S, Xenarios I, Langbridge J. et al. Proteomic analysis of the mouse liver mitochondrial inner membrane. J. Biol. Chem. 2003;278 (42):41566-41571. DOI: 10.1074/jbc.M304940200.
• Bogdanov B, Smith RD. Proteomics by FTICR mass spectrometry: top down and bottom up. Mass Spectrum. Rev. 2005; 24 (2): 168-200. DOI: 10.1002/mas.20015.
• Aebersold R, Mann, M. Mass spectrometry-based proteomics. Nature. 2003; 422 (1): 198-207. DOI: 10.1021/bc034150l
• Distler A, Kerner J, Peterman S, et al. A targeted proteomic approach for the analysis of rat liver mitochondrial outer membrane proteins with extensive sequence coverage.Analytical Biochemistry. 2006;356 (1):18-29. DOI: 10.1016/j.ab.2006.03.053.
• Song J, Midson C, Blachy-Dyson E, et al.The sensor regions of VDAC are translocated from within the membrane to the surface during the gating processes. Biophys. J. 1998;74 (6):2926-2944. DOI: 10.1016/S0006-3495(98)78000-2.
• Weiss M, Kreusch A, Schiltz E, et al.The structure of porin from Rhodobactercapsulatus at 1.8 A resolution.FEBS Lett. 1991; 280 (2):379-382. PMID: 1707373.
• Cowan S, Schirmer T, Rummel G, et al. Crystal structures explain functional properties of two E. coliporins. Nature. 1992; 358 (6389): 727-733. DOI: 10.1038/358727a0.
• Kreusch A, Neubuser A, Schiltz E, et al. Structure of the membrane channel porin from Rhodopseudomonasblastica at 2.0 A resolution. Protein Sci. 1994; 3 (1):58-63. DOI: 10.1002/pro.5560030108.
• Schirmer T, Keller T, Wang Y, et al. Structural basis for sugar translocation through maltoporin channels at 3.1 A resolution.Science. 1995; 267 (5197): 512-512. PMID: 7824948.
• Forte M, Guy HR, Mannella CA. Molecular genetics of the VDAC ion channel: structural model and sequence analysis. J.Bioenerg. Biomembr. 1987; 19 (4):341-350. PMID: 2442148.
• Rauch G, Moran O. On the structure of mitochondrial porins and its homologies with bacterial porins.Biochem.Biophys. Res. Commun. 1996; 200 (2): 908-915. DOI: 10.1006/bbrc.1994.1536.
• Blachly-Dyson E, Peng SZ, Colombini M, et al. Selectivity changes in site-directed mutants of the VDAC ion channel: structural implications. Science. 1990; 247 (4947): 1233-1236. PMID: 1690454.
• Song J, Midson C, Blachy-Dyson E, et al.The sensor regions of VDAC are translocated from within the membrane to the surface during the gating processes. Biophys. J.1998; 74 (6):2926-2944. DOI: 10.1016/S0006-3495(98)78000-2.