Identification of proteins that interact with alpha A-crystallin using a human proteome microarray

Purpose To identify proteins interacting with alpha A-crystallin (CRYAA) and to investigate the potential role that these protein interactions play in the function of CRYAA using a human proteome (HuProt) microarray. Methods The active full-length CRYAA protein corresponding to amino acids 1–173 of CRYAA was recombined. A HuProt microarray composed of 17,225 human full-length proteins with N-terminal glutathione S-transferase (GST) tags was used to identify protein–protein interactions. The probes were considered detectable when the signal to noise ratio (SNR) was over 1.2. The identified proteins were subjected to subsequent bioinformatics analysis using the DAVID database. Results The HuProt microarray results showed that the signals of 343 proteins were higher in the recombinant CRYAA group than in the control group. The SNR of 127 proteins was ≥ 1.2. The SNR of the following eight proteins was > 3.0: hematopoietic cell-specific Lyn substrate 1 (HCLS1), Kelch domain-containing 6 (KLHDC6), sarcoglycan delta (SGCD), KIAA1706 protein (KIAA1706), RNA guanylyltransferase and 5′-phosphatase (RNGTT), chromosome 10 open reading frame 57 (C10orf57), chromosome 9 open reading frame 52 (C9orf52), and plasminogen activator, urokinase receptor (PLAUR). The bioinformatics analysis revealed 127 proteins associated with phosphoproteins, alternative splicing, acetylation, DNA binding, the nuclear lumen, ribonucleotide binding, the cell cycle, WD40 repeats, protein transport, transcription factor activity, GTP binding, and cellular response to stress. Functional annotation clustering showed that they belong to cell cycle, organelle or nuclear lumen, protein transport, and DNA binding and repair clusters. CRYAA interacted with these proteins to maintain their solubility and decrease the accumulation of denatured target proteins. The protein–protein interactions may help CRYAA carry out multifaceted functions. Conclusions One-hundred and twenty-seven of 17,225 human full-length proteins were identified that interact with CRYAA. The advent of microarray analysis enables a better understanding of the functions of CRYAA as a molecular chaperone.

[1]  M. Kantorow,et al.  Autophagy and mitophagy participate in ocular lens organelle degradation. , 2013, Experimental eye research.

[2]  C. Lieber.  S-adenosyl-L-methionine: its role in the treatment of liver disorders. , 2002, The American journal of clinical nutrition.

[3]  H. Xiang,et al.  Human αA- and αB-crystallins bind to Bax and Bcl-XS to sequester their translocation during staurosporine-induced apoptosis , 2004, Cell Death and Differentiation.

[4]  F. Bai,et al.  Cell kinetic status of mouse lens epithelial cells lacking alphaA- and alphaB-crystallin. , 2004, Molecular and cellular biochemistry.

[5]  Li Wang,et al.  Identification of New Autoantigens for Primary Biliary Cirrhosis Using Human Proteome Microarrays* , 2012, Molecular & Cellular Proteomics.

[6]  M. Gerstein,et al.  Global Analysis of Protein Activities Using Proteome Chips , 2001, Science.

[7]  S. Cole,et al.  Mechanism of RPE Cell Death in α-Crystallin Deficient Mice: A Novel and Critical Role for MRP1-Mediated GSH Efflux , 2012, PloS one.

[8]  L. Hancock,et al.  Differential Binding of Mutant (R116C) and Wildtype AlphaA Crystallin to Actin , 2007, Current eye research.

[9]  M. Kantorow,et al.  Methionine sulfoxide reductase A (MsrA) restores alpha-crystallin chaperone activity lost upon methionine oxidation. , 2009, Biochimica et biophysica acta.

[10]  R. Maddala,et al.  α-Crystallin localizes to the leading edges of migrating lens epithelial cells , 2005 .

[11]  H. Xiang,et al.  Human alphaA- and alphaB-crystallins bind to Bax and Bcl-X(S) to sequester their translocation during staurosporine-induced apoptosis. , 2004, Cell death and differentiation.

[12]  Alexander K. Buell,et al.  Binding of the molecular chaperone αB-crystallin to Aβ amyloid fibrils inhibits fibril elongation. , 2011, Biophysical journal.

[13]  U. Andley.  Effects of alpha-crystallin on lens cell function and cataract pathology. , 2009, Current molecular medicine.

[14]  J. Clark,et al.  Lens cytoskeleton and transparency: A model , 1999, Eye.

[15]  M. Takeichi,et al.  EPLIN mediates linkage of the cadherin–catenin complex to F-actin and stabilizes the circumferential actin belt , 2008, Proceedings of the National Academy of Sciences.

[16]  Keyang Wang,et al.  alpha-crystallin stabilizes actin filaments and prevents cytochalasin-induced depolymerization in a phosphorylation-dependent manner. , 1996, European journal of biochemistry.

[17]  S. Bhat,et al.  alpha A-crystallin is expressed in non-ocular tissues. , 1992, The Journal of biological chemistry.

[18]  L. Fan,et al.  Down‐regulation and CpG island hypermethylation of CRYAA in age‐related nuclear cataract , 2012, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[19]  E. Wawrousek,et al.  The Molecular Chaperone αA-Crystallin Enhances Lens Epithelial Cell Growth and Resistance to UVA Stress* , 1998, The Journal of Biological Chemistry.

[20]  R. Maddala,et al.  alpha-Crystallin localizes to the leading edges of migrating lens epithelial cells. , 2005, Experimental cell research.

[21]  F. Bai,et al.  Alpha‐crystallin expression affects microtubule assembly and prevents their aggregation , 2006, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[22]  Xiangjia Zhu,et al.  αA Crystallin May Protect against Geographic Atrophy—Meta-Analysis of Cataract vs. Cataract Surgery for Geographic Atrophy and Experimental Studies , 2012, PloS one.

[23]  M. Delaye,et al.  Short-range order of crystallin proteins accounts for eye lens transparency , 1983, Nature.

[24]  Brad T. Sherman,et al.  Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources , 2008, Nature Protocols.

[25]  F. Bai,et al.  Cell kinetic status of mouse lens epithelial cells lacking αA- and αB-crystallin , 2004, Molecular and Cellular Biochemistry.

[26]  Jef D. Boeke,et al.  Rapid Identification of Monospecific Monoclonal Antibodies Using a Human Proteome Microarray* , 2012, Molecular & Cellular Proteomics.

[27]  F. Bai,et al.  Reduced survival of lens epithelial cells in theα A-crystallin-knockout mouse , 2003, Journal of Cell Science.

[28]  D. Hinton,et al.  Novel roles for α-crystallins in retinal function and disease , 2012, Progress in Retinal and Eye Research.

[29]  E. Li,et al.  ECM-related gene expression profile in vascular smooth muscle cells from human saphenous vein and internal thoracic artery , 2013, Journal of Cardiothoracic Surgery.

[30]  A. Doseff,et al.  The anti-apoptotic function of human αA-crystallin is directly related to its chaperone activity , 2010, Cell Death and Disease.

[31]  U. Andley.  Effects of α-Crystallin on Lens Cell Function and Cataract Pathology , 2009 .

[32]  R. Quinlan.  Cytoskeletal competence requires protein chaperones. , 2002, Progress in molecular and subcellular biology.

[33]  K. Kato,et al.  Immunoreactive alpha A crystallin in rat non-lenticular tissues detected with a sensitive immunoassay method. , 1991, Biochimica et biophysica acta.