山羊HSPA6蛋白的特性分析及互作蛋白网络构建

doi:10.16431/j.cnki.1671-7236.2020.03.007

摘要/Abstract

摘要： 为了研究山羊热休克蛋白70（HSP70）家族成员HSPA6基因编码蛋白的结构与功能特性，本研究利用生物信息学方法，分析了山羊HSPA6基因编码蛋白的氨基酸组成、基本理化性质、亲疏水性、跨膜区结构、信号肽、可能的磷酸化修饰位点、亚细胞定位，以及蛋白的二级结构和三级结构；选取多种生物的同源HSP70蛋白序列构建系统进化树，另外还对HSPA6进行了蛋白互作网络分析。结果显示，山羊HSPA6蛋白序列与哺乳动物（牛、猪）的序列同源性较高；山羊HSPA6蛋白分子质量为70.9 ku，理论等电点为5.78，属于酸性蛋白和亲水性蛋白，无跨膜结构和信号肽；蛋白质功能预测结果显示，HSPA6包含10个分值很高的可能磷酸化位点，这些位点分布在N-端的核苷酸结合结构域（NBD）和C-端的多肽结合结构域（PBD）。经序列分析发现，HSPA6 C-端含有EEVD基序，是HSP70家族蛋白定位于细胞质的保守基序，表明HSPA6主要分布在细胞质。蛋白质二级结构主要以α-螺旋和无规则卷曲为主，分别占41.52%和33.75%，为混合型蛋白；蛋白互作网络构建结果显示，和HSPA6相互作用的蛋白包括HSP70家族成员，另外也有HSP40、HSP90家族成员，预示着山羊HSPA6可能在细胞内与HSP40和HSP90形成复合体发挥作用。本试验结果为进一步深入探讨山羊HSPA6响应环境应激的功能机制提供了理论依据。

关键词: 山羊; 热休克蛋白70; HSPA6; 生物信息学

Abstract: In order to investigate the structure and functional characteristics of HSPA6 belongs to heat shock protein 70 family in goat,the amino acid composition,physical and chemical properties,hydrophobicity,transmembrane structure,signal peptide,possible phosphorylation modification sites,subcellular localization,secondary structure and tertiary structure of HSPA6 were analyzed using bioinformatic methods.HSP70 protein sequences of various organisms were selected to construct phylogenetic tree,at the same time,the protein interaction network was analyzed.The results showed that HSPA6 in goat had higher homology sequences with mammalian (cattle,pig);the molecular weight of HSPA6 protein in goat was 70.9 ku;The isoelectric point was about 5.78,which indicated it belonged to acidic protein and hydrophilic protein;Without transmembrane structure and signal peptide.There were 10 phosphorylation sites with high scores,which located in N-terminal(nucleotide binding domain,NBD) and C-terminal (peptide binding domain,PBD),and C-terminal contains EEVD motif,which was the conserved motif of HSP70 family proteins located in the cytoplasm,indicating that it was mainly distributed in cytoplasm.The secondary structure of protein was mainly composed of α-helix (41.52%) and random coil (33.75%).The construction of protein interaction network indicated that HSPA6 might interact with HSP70 family members,and might play a role in the formation of complex with HSP40 and HSP90 in cells.Based on the above,this study provided a theoretical basis for further exploring the functional mechanism of HSPA6 in goat in response to environmental stress.

Key words: goat; heat shock protein 70; HSPA6; bioinformatics

中图分类号:

S811.3

杨佳栋, 刘月琴, 张英杰. 山羊HSPA6蛋白的特性分析及互作蛋白网络构建[J]. 中国畜牧兽医, 2020, 47(3): 706-713.

YANG Jiadong, LIU Yueqin, ZHANG Yingjie. Characterization Analysis of HSPA6 Protein in Goat and Construction of Interactive Protein Network[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(3): 706-713.

参考文献

[1] HUNT C,MORIMOTO R I.Conserved features of eukaryotic HSP70 genes revealed by comparison with the nucleotide sequence of human HSP70[J].Proceedings of the National Academy of Sciences of the United States of America,1985,82(19):6455-6459.
[2] KAMPINGA H H,HAGEMAN J,VOS M J,et al.Guidelines for the nomenclature of the human heat shock proteins[J].Cell Stress Chaperones.2009,14(1):105-111.
[3] WALSH P,BURSAC D,LAW Y C,et al.The J-protein family:Modulating protein assembly,disassembly and translocation[J].EMBO Reports.2004,5(6):567-571.
[4] BENBROOK D M,LONG A.Integration of autophagy,proteasomal degradation,unfolded protein response and apoptosis[J].Experimental Oncology,2012,34(3):286-297.
[5] EDWARDS J L,KING W A,KAWARSKY S J,et al.Responsiveness of early embryos to environmental insults:Potential protective roles of HSP70 and glutathione[J].Theriogenology,2001,55(1):209-223.
[6] MAYS C E,ARMIJO E,MORALES R,et al.Prion disease is accelerated in mice lacking stress-induced heat shock protein 70(HSP70)[J].The Journal of Biological Chemistry,2019,294(37):13619-13628.
[7] LUO L L,LI Y F,SHAN H M,et al.L-glutamine protects mouse brain from ischemic injury via up-regulating heat shock protein 70[J].CNS Neuroscience and Therapeutics,2019,25(9):1030-1041.
[8] 李芳芳,龙开旭,俸祥仁,等.河流型水牛热休克蛋白70的原核表达及其对精液抗冻性的影响[J].中国畜牧兽医,2018,45(9):2417-2424. LI F F,LONG K X,FENG X R,et al.Prokaryotic expression of HSP70 and it's effect on the freezing resistance of Semen in River buffale[J].China Animal Husbandry & Veterinary Medicine,2018,45(9):2417-2424.(in Chinese)
[9] NASOUTI R,KHAKSARI M,MIRZAEE M,et al.Trehalose protects against spinal cord injury through regulating heat shock proteins 27 and 70 and caspase-3 genes expression[J].Journal of Basic and Clinical Physiology and Pharmacology,2019,29(10):1-8.
[10] DONG Y,XIE M,JIANG Y,et al.Sequencing and automated whole-genome optical mapping of the genome of a domestic goat (Capra hircus)[J].Nature biotechnology,2013,31(2):135-141.
[11] BICKHART D M,ROSEN B D,KOREN S et al.Single-molecule sequencing and chromatin conformation capture enable de novo reference assembly of the domestic goat genome[J].Nature Genetics.2017,49(4):643-650.
[12] BANERJEE D,UPADHYAY R C,CHAUDHARY U B,et al.Seasonal variation in expression pattern of genes under HSP70:Seasonal variation in expression pattern of genes under HSP70 family in heat- and cold-adapted goats (Capra hircus)[J].Cell Stress Chaperones,2014,19(3):401-408.
[13] ZHONG C,LI J,MAO L,et al.Proteomics analysis reveals heat shock proteins involved in caprine parainfluenza virus type 3 infection[J].BMC Veterinary Research,2019,15(1):151-160.
[14] HAN Y,KANG Y,YU J,et al.Increase of HSPA1a and HSPA1b genes in the resting B cells of Sirt1 knockout mice[J].Molecular Biology Reports,2019,46(4):4225-4234.
[15] 杨莉,张莉,齐亚银,等.冷应激对湖羊血清因子及热休克蛋白70 mRNA表达的影响[J].中国畜牧兽医,2015,42(4):890-895. YANG L,ZHANG L,QI Y Y,et al.Effect of cold stress on serum factor and HSP70 mRNA in Hu sheep[J].China Animal Husbandry & Veterinary Medicine,2015,42(4):890-895.(in Chinese)
[16] KUMAR A,ASHRAF S,GOUD T S,et al.Expression profiling of major heat shock protein genes during different seasons in cattle (Bos indicus) and buffalo (Bubalus bubalis) under tropical climatic condition[J].Journal of Thermal Biology,2015,51:55-64.
[17] FERNANDEZ-FERNANDEZ M R,VALPUESTA J M.HSP70 chaperone:A master player in protein homeostasis[J].F1000Research,2018,7:1497-1505.
[18] CLOUTIER P,COULOMBE B.Regulation of molecular chaperones through post-translational modifications:Decrypting the chaperone code[J].Biochimica Et Biophysica Acta,2013,1829(5):443-454.
[19] VELASCO L,DUBLANG L,MORO F,et al.The complex phosphorylation patterns that regulate the activity of HSP70 and its cochaperones[J].International Journal of Molecular Sciences,2019,20(17):4122-4131.
[20] NITIKA N,TRUMAN A W.Cracking the chaperone code:Cellular roles for HSP70 phosphorylation[J].Trends in Biochemical Sciences,2017,42(12):932-935.
[21] BELTRAO P,ALBANESE V,KENNER L R,et al.Systematic functional prioritization of protein posttranslational modifications[J].Cell,2012,150(2):413-425.
[22] MORGNER N,SCHMIDT C,BEILSTEN-EDMANDS V,et al.HSP70 forms antiparallel dimers stabilized by post-translational modifications to position clients for transfer to HSP90[J].Cell Reports,2015,11(5):759-769.
[23] ROSENZWEIG R,NILLEGODA N B,MAYER M P,et al.The HSP70 chaperone network[J].Nature Reviews Molecular Cell Biology,2019,20(11):665-680.
[24] WEGELE H,MULLER L,BUCHNER J.HSP70 and HSP90-A relay team for protein folding[J].Reviews of Physiology,Biochemistry and Pharmacology,2004,151:1-44.
[25] ALVIRA S,CUELLAR J,ROHL A,et al.Structural characterization of the substrate transfer mechanism in HSP70/HSP90 folding machinery mediated by Hop[J].Nature Communications,2014,5:5484-5493.
[26] SEYFFER F,KUMMER E,OGUCHI Y,et al.HSP70 proteins bind HSP100 regulatory M domains to activate AAA⁺ disaggregase at aggregate surfaces[J].Nature structural and Molecular Biology,2012,19(12):1347-1355.