Bioinformatics analysis of HCN1 gene and protein in human_Journal of Epilepsy

Authors：

LI Yinchao ¹ , LIN Wanrong ¹ , CHEN Shuda ¹ , ZHAO Yiran ¹ , CHEN Aohan ¹ ,  ZHOU Liemin ^1,2

1. Department of Neurology, the Seven Affiliated Hospital, Sun Yat-Sen University, Shenzhen 518107, China;
2. Department of Neurology, the First Affiliated Hospital, Sun Yat-Sen University, Guangzhou 510030, China;

Corresponding author：

ZHOU Liemin, Email: lmzhou56@163.com

Keywords：

Hyperpolarization activated cyclic nucleotide gated channel 1; Bioinformatics; Promoter; Transcription factor; Gene; Protein

DOI：

10.7507/2096-0247.20200048

Video：

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Objective To lay a theoretical foundation for the research of regulation of Hyperpolarization activated cyclic nucleotide gated channel 1 (HCN1) gene expression and its involvement in the pathogenesis of Mesio-temporal lobe epilepsy (MTLE) and other related diseases, the bioinformatics methods were used to analyze sequence characteristic, transcription factors and their binding sites in the promoter region of human HCN1 gene, and the physicochemical properties, signal peptides, hydrophobicity, transmembrane regions, protein structure, interacting proteins and functions of HCN1 proteins.Method Biological software and website, such as Protparam, Protscale, MHMM, SignalP 5.0, NetPhos 3.1, Swiss-Model, Promoter 2.0, AliBaba2.1 and EMBOSS were used to analyze and predict physicochemical properties, structural functions, localized expression, phylogenetic relationships and protein interactions with human HCN1 protein, and promoter, CpG island and transcription factor characteristics of HCN1 gene.Results The evolutionary analysis of HCN1 protein showed that the genetic distance between human and Pongo abelii was the smallest, indicating the closest genetic relationship between human and Pongo abelii. Human HCN1 protein was an unstable hydrophilic protein located on the plasma membrane, which contained two transmembrane structure. However, the predicted results showed that there was no signal peptide and nuclear localization sequence in this protein. The secondary structure of HCN1 protein was mostly random coil and alpha helix, and it contained multiple potential phosphorylation sites. The ontology analysis results of HCN1 protein were showed as follows. The cellular component of HCN1 protein was located in the plasma membrane (GO:0005886); the molecular functionof HCN1 protein were cyclic adenosine monophosphate binding (GO:0030552) and voltage-gated ion channel activity (GO:0005244); the biological process of this protein were reacting to cAMP (GO:0071320) and transmembrane transport of potassium (GO:0071805). The analysis results of String database showed that the proteins that had close interaction with human HCN1 protein mainly included the ten proteins (HCN2, HCN4, PEX5L, MARCH7, KCTD3, GNAT3, SHKBP1, KCNQ2, FLNA and NEDD4L). These proteins were mainly involved in regulation of ion transport and transmembrane transport of potassium (GO:0071805). The HCN1 gene was located at 5p12 and contained 8 exons and 7 introns.There were at least three promoter regions in the nucleotide sequence of 2 000 bp from the upstream of the HCN1 gene to the 5 'flanks, and contained a 158 bp CpG island in the promoter region and one TATA boxes and one CAAT boxes in the 5' regulation region ofHCN1 gene; niceteen transcription factors, including NF-κB, NF-1, AP-1, TBP, IRF-1, c-Ets-1, Elf-1, HNF-3, HNF-1, YY1, GATA-1, RXR-α, GR, AP-2αA, ENKTF-1, C/EBPβ, C/EBPα, c-Fos and c-Jun, binding in the promoter region of the HCN1 gene were predicted by both softwares (AliBaba2.1 and PROMO2).Conclusion The analysis results provide important information for further studies on the role of HCN1. Bioinformatics analysis of the promoter region can improve the research efficiency of gene promoters, and provide theoretical basis for subsequent experiments to construct expression vectors of HCN1 gene promoters and identify their functions.

Citation： LI Yinchao, LIN Wanrong, CHEN Shuda, ZHAO Yiran, CHEN Aohan, ZHOU Liemin. Bioinformatics analysis of HCN1 gene and protein in human. Journal of Epilepsy, 2020, 6(4): 296-306. doi: 10.7507/2096-0247.20200048 Copy

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1. He C, Chen F, Li B, et al. Neurophysiology of HCN channels: from cellular functions to multiple regulations. Prog Neurobiol, 2014, 112: 1-23.
2. Notomi T, Shigemoto R. Immunohistochemical localization of Ih channel subunits, HCN1-4, in the rat brain. J Comp Neurol, 2004, 471(3): 241-276.
3. Powell KL, Ng C, O'Brien TJ, et al. Decreases in HCN mRNA expression in the hippocampus after kindling and status epilepticus in adult rats. Epilepsia, 2008, 49(10): 1686-1695.
4. Kumar S, Stecher G, Tamura K. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol, 2016, 33(7): 1870-1874.
5. Wilkins MR, Gasteiger E, Bairoch A, et al. Protein identification and analysis tools in the ExPASy server. Methods Mol Biol, 1999, 112: 531-552.
6. Reddy BV. Structural distribution of dipeptides that are identified to be determinants of intracellular protein stability. J Biomol Struct Dyn, 1996, 14(2): 201-210.
7. Almagro Armenteros JJ, Tsirigos KD, Sonderby CK, et al. SignalP 5.0 improves signal peptide predictions using deep neural networks. Nat Biotechnol, 2019, 37(4): 420-423.
8. Kosugi S, Hasebe M, Tomita M, et al. Systematic identification of cell cycle-dependent yeast nucleocytoplasmic shuttling proteins by prediction of composite motifs. Proc Natl Acad Sci USA, 2009, 106(25): 10171-10176.
9. Kosugi S, Hasebe M, Matsumura N, et al. Six classes of nuclear localization signals specific to different binding grooves of importin alpha. J Biol Chem, 2009, 284(1): 478-485.
10. Chen Y, Yu P, Luo J, et al. Secreted protein prediction system combining CJ-SPHMM, TMHMM, and PSORT. Mamm Genome, 2003, 14(12): 859-865.
11. Blom N, Sicheritz-Ponten T, Gupta R, et al. Prediction of post-translational glycosylation and phosphorylation of proteins from the amino acid sequence. Proteomics, 2004, 4(6): 1633-1649.
12. Letunic I, Bork P. 20 years of the SMART protein domain annotation resource. Nucleic Acids Res, 2018, 46(D1): D493-D496.
13. Sapay N, Guermeur Y, Deleage G. Prediction of amphipathic in-plane membrane anchors in monotopic proteins using a SVM classifier. BMC Bioinformatics, 2006, 7: 255.
14. Waterhouse A, Bertoni M, Bienert S, et al. SWISS-MODEL: homology modelling of protein structures and complexes. Nucleic Acids Res, 2018, 46(W1): W296-W303.
15. Szklarczyk D, Morris JH, Cook H, et al. The STRING database in 2017: quality-controlled protein-protein association networks, made broadly accessible. Nucleic Acids Res, 2017, 45(D1): D362-D368.
16. Reese MG. Application of a time-delay neural network to promoter annotation in the Drosophila melanogaster genome. Comput Chem, 2001, 26(1): 51-56.
17. Knudsen S. Promoter2.0: for the recognition of PolII promoter sequences. Bioinformatics, 1999, 15(5): 356-361.
18. Solovyev VV, Shahmuradov IA, Salamov AA. Identification of promoter regions and regulatory sites. Methods Mol Biol, 2010, 674: 57-83.
19. Plake C, Schiemann T, Pankalla M, et al. AliBaba: PubMed as a graph. Bioinformatics, 2006, 22(19): 2444-2445.
20. Farre D, Roset R, Huerta M, et al. Identification of patterns in biological sequences at the ALGGEN server: PROMO and MALGEN. Nucleic Acids Res, 2003, 31(13): 3651-3653.
21. Carver T, Bleasby A. The design of Jemboss: a graphical user interface to EMBOSS. Bioinformatics, 2003, 19(14): 1837-1843.
22. Li LC, Dahiya R. MethPrimer: designing primers for methylation PCRs. Bioinformatics, 2002, 18(11): 1427-1431.
23. Brown HF, DiFrancesco D, Noble SJ. How does adrenaline accelerate the heart? Nature, 1979, 280(5719): 235-236.
24. Tibbs GR, Posson DJ, Goldstein PA. Voltage-gated ion channels in the PNS: novel therapies for neuropathic pain? Trends Pharmacol Sci, 2016, 37(7): 522-542.
25. Poller WC, Bernard R, Derst C, et al. Lateral habenular neurons projecting to reward-processing monoaminergic nuclei express hyperpolarization-activated cyclic nucleotid-gated cation channels. Neuroscience, 2011, 193: 205-216.
26. Noam Y, Ehrengruber MU, Koh A, et al. Filamin A promotes dynamin-dependent internalization of hyperpolarization-activated cyclic nucleotide-gated type 1 (HCN1) channels and restricts Ih in hippocampal neurons. J Biol Chem, 2014, 289(9): 5889-5903.
27. Weng XC, Liu SJ. [Role of HCN channels in the nervous system: membrane excitability and various modulations]. Zhongguo Ying Yong Sheng Li Xue Za Zhi, 2014, 30(6): 506-510.
28. Sarkisian MR, Bartley CM, Rakic P. Trouble making the first move: interpreting arrested neuronal migration in the cerebral cortex. Trends Neurosci, 2008, 31(2): 54-61.
29. Cao-Ehlker X, Zong X, Hammelmann V, et al. Up-regulation of hyperpolarization-activated cyclic nucleotide-gated channel 3 (HCN3) by specific interaction with K+ channel tetramerization domain-containing protein 3 (KCTD3). J Biol Chem, 2013, 288(11): 7580-7589.
30. Zagotta WN, Olivier NB, Black KD, et al. Structural basis for modulation and agonist specificity of HCN pacemaker channels. Nature, 2003, 425(6954): 200-205.
31. DeBerg HA, Brzovic PS, Flynn GE, et al. Structure and Energetics of Allosteric Regulation of HCN2 Ion Channels by Cyclic Nucleotides. J Biol Chem, 2016, 291(1): 371-381.
32. Jones PA. Functions of DNA methylation: islands, start sites, gene bodies and beyond. Nat Rev Genet, 2012, 13(7): 484-492.
33. Sulewska A, Niklinska W, Kozlowski M, et al. DNA methylation in states of cell physiology and pathology. Folia Histochem Cytobiol, 2007, 45(3): 149-158.

Journal of Epilepsy

Bioinformatics analysis of HCN1 gene and protein in human

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