# Computational Simulations to Predict Creatine Kinase-Associated Factors: Protein-Protein Interaction Studies of Brain and Muscle Types of Creatine Kinases

Authors: Wei-Jiang Hu; Sheng-Mei Zhou; Joshua SungWoo Yang; Fan-Guo Meng
Journal: Enzyme Research
 (2011)

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## Abstract

Creatine kinase (CK; EC 2.7.3.2) is related to several skin diseases such as psoriasis and dermatomyositis. CK is important in skin energy homeostasis because it catalyzes the reversible transfer of a phosphoryl group from MgATP to creatine. In this study, we predicted CK binding proteins via the use of bioinformatic tools such as protein-protein interaction (PPI) mappings and suggest the putative hub proteins for CK interactions. We obtained 123 proteins for brain type CK and 85 proteins for muscle type CK in the interaction networks. Among them, several hub proteins such as NFKB1, FHL2, MYOC, and ASB9 were predicted. Determination of the binding factors of CK can further promote our understanding of the roles of CK in physiological conditions.


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## Body

## 1. Introduction

Creatine kinase (CK) (ATP: creatine kinase N-phosphotransferase, EC 2.7.3.2) is thought to be crucial for intracellular transport and the storage of high energy phosphate because it catalyzes the reversible transfer of a phosphoryl group from MgATP to creatine, which leads to the creation of phosphocreatine and MgADP [1]. CK plays an important role in the cellular energy metabolism of vertebrates, and it is widely distributed in tissues that require a lot of energy [2]. Several types of CK are expressed in various tissues: the muscle and brain types of CK are the most common, and three different isoenzymes that include CK-MM (the muscle type homodimer), CK-BB (the brain type homodimer), and CK-MB (the muscle plus brain type heterodimer) originate from these two common types. CK is an important serum marker for myocardial infarction. Various types of CKs (the muscle, brain, and mitochondrial types) are thought to be important not only in the diagnosis of myocardial infarction, cardiac hypertrophy, and muscular dystrophy but also for studies of some other serious diseases, including Alzheimer’s disease, Parkinson's disease, and psoriasis [3–8].CK-BB is associated with several pathologies, including neurodegenerative and age-related diseases. Recently, Chang et al. [9] reported an important role for CK-BB in osteoclast-mediated bone resorption, which was found using a proteomics approach. They found that CK-BB is greatly increased during osteoclastogenesis and suggested that it represents a potential target for antiresorptive drug development. CK-BB interacts with the potassium-chloride cotransporter 3, which is involved in the pathophysiology of hereditary motor and sensory neuropathy with agenesis of the corpus callosum [10]. Previous studies [11, 12] have reported that CK-BB is involved in Alzheimer's disease (AD) as an oxidatively modified protein. This suggests that oxidatively damaged CK-BB may be associated with aging and age-related neurodegenerative disorders such as AD.CK-MM is a good model to use for studying folding pathways because of several characteristics: (i) it is a dimer that consists of two identical subunits, each with an N-terminal domain with about 100 residues and a C-terminal domain with about 250 residues connected by a long linker [13]; (ii) extensively denatured CK can be renatured spontaneously with restoration of its enzymatic activity in the absence of any external assistance [14]; (iii) its folding pathway is complicated and involves several intermediates [15, 16]; (iv) conformational changes of the secondary and tertiary structures can be easily measured by monitoring activity changes [14, 15]; (v) protein-protein interactions, including molecular chaperones, are observed during refolding [17, 18].In this study, we obtained computational predictions of the binding proteins by using two types of CK (CK-BB and CK-MM) as hub proteins in bioinformatic algorithms. As a result, we obtained 208 protein lists in the interaction networks via application of both muscle and brain types of CK. Determination of the binding factors and functions of CK can further promote our understanding of the physiological roles of CK.


## 2. Materials and Methods

### 2.1. PPI Mappings: PEIMAP and PSIMAP Algorithms

We present the functionally classified protein-protein interactions on the basis of the cell cycle, cell transport, oxidoreductase, and apoptosis. PPI resources were assembled from a combination of several experimental protein interaction databases. The protein interaction resources included six databases: DIP [19], BIND [20], IntAct [21], MINT [22], HPRD [23], and BioGrid [24]. We performed a redundancy test to remove identical protein sequences from the interaction databases. The databases contain 116,773 proteins and 229,799 interactions.PPI prediction uses most of the major types of PPI algorithms. They are (1) Protein Structural Interactome MAP (PSIMAP), a method that uses the structural domain of the SCOP (Structural Classification of Proteins) database [25] and (2) Protein Experimental Interactome MAP (PEIMAP), a common method that uses public resources of experimental protein interaction information such as HPRD, BIND, DIP, MINT, IntAct; and BioGrid. The basic procedure of PSIMAP is to infer interactions between proteins by using their homologs. Interactions among domains or proteins for known PDB (Protein Data Bank) structures are the basis for the prediction. If an unknown protein has a homolog to a domain, then PSIMAP assumes that the query has the probability to interact with its homolog’s partners. This concept is called “homologous interaction.” The original interaction between two proteins or domains is based on the Euclidean distance. Therefore, PSIMAP gives a structure-based interaction prediction [26]. PEIMAP was constructed by combining several experimental protein-protein interaction databases. We carried out a redundancy check to remove identical protein sequences from the source interaction databases. The image was made by the Pajak2.00 program (http://vlado.fmf.uni-lj.si/pub/networks/pajek/).


## 2.1. PPI Mappings: PEIMAP and PSIMAP Algorithms

We present the functionally classified protein-protein interactions on the basis of the cell cycle, cell transport, oxidoreductase, and apoptosis. PPI resources were assembled from a combination of several experimental protein interaction databases. The protein interaction resources included six databases: DIP [19], BIND [20], IntAct [21], MINT [22], HPRD [23], and BioGrid [24]. We performed a redundancy test to remove identical protein sequences from the interaction databases. The databases contain 116,773 proteins and 229,799 interactions.PPI prediction uses most of the major types of PPI algorithms. They are (1) Protein Structural Interactome MAP (PSIMAP), a method that uses the structural domain of the SCOP (Structural Classification of Proteins) database [25] and (2) Protein Experimental Interactome MAP (PEIMAP), a common method that uses public resources of experimental protein interaction information such as HPRD, BIND, DIP, MINT, IntAct; and BioGrid. The basic procedure of PSIMAP is to infer interactions between proteins by using their homologs. Interactions among domains or proteins for known PDB (Protein Data Bank) structures are the basis for the prediction. If an unknown protein has a homolog to a domain, then PSIMAP assumes that the query has the probability to interact with its homolog’s partners. This concept is called “homologous interaction.” The original interaction between two proteins or domains is based on the Euclidean distance. Therefore, PSIMAP gives a structure-based interaction prediction [26]. PEIMAP was constructed by combining several experimental protein-protein interaction databases. We carried out a redundancy check to remove identical protein sequences from the source interaction databases. The image was made by the Pajak2.00 program (http://vlado.fmf.uni-lj.si/pub/networks/pajek/).


## 3. Results and Discussion

We identified potential candidates through protein-protein interaction predictions made using various protein interaction resources. By analyzing the hub protein of the networks with metrics such as degree and centrality, we detected 123 potential candidates for CKB interacting (direct or indirect) factors and 85 candidates for CKM.In Figure1, interacting factors such as NFKB1 (NP_003989, nuclear factor of kappa light polypeptide gene enhancer in B-cells 1), MYOC (NP_000252; myocilin, trabecular meshwork inducible glucocorticoid response), MYOM2 (NP_003961; myomesin (M-protein) 2, 165 kDa), FHL2 (NP_001034581, four-and-a-half LIM domains 2), HIF1AN (NP_060372, hypoxia-inducible factor 1, alpha subunit inhibitor), ASB9 (NP_076992, ankyrin repeat and SOCS box-containing 9), and CKM (NP_001815, creatine kinase, muscle) were elucidated. Interestingly, NFKB1 was detected as a hub protein interacting with CK-BB in our results. In Figure 2, we obtained results similar to those from Figure 1, where NFKB1, MYOC, MYOM2, FHL2, HIF1AN, ASB9, and CKM were detected as interacting factors that were directly or indirectly associated with CKB. NFKB1, CKM, and ASB9 interacted with CKB directly.Figure 1
PPI map for CKB as a target hub protein with the 80% identity. Labels with red color indicate the hub protein of targeting. The image was made by the aiSee program (http://www.aisee.com/).Figure 2
PPI map for CKB as a target hub protein with the 100% identity. The methodological conditions were the same as for Figure1 except the identity.In the same way, we detected the CKM-associated proteins as shown in Figure3 with 80% sequence identity. As a result, we found that CKB, FHL2, MYOC, ASB9, HIF1AN, NFKB1, TTN (NP_596870, titin), MYH9 (NP_002464, myosin, heavy chain 9, non-muscle), and ITGA7 (NP_002197, integrin, alpha 7) mainly interacted with CKM at 80% sequence identity. At the level of 100% identity, we found that MYOM2, CKB, FHL2, and MYOC directly interacted with CKM as shown in Figure 4. In addition to these factors, complete lists of factors that interacted with CKB and CKM in a direct or indirect manner are shown in Tables 1 and 2. After overlapping the results from Figures 1 to 4, we found that NFKB1, FHL2, and MYOC were still detected as hub proteins in Figure 5.Table 1
Gene lists for the analyses of the PEIMAP and PSIMAP using CK-BB as a hub protein with 100% identity.
Gene IDGene symbolFull name6256RXRARetinoid X receptor, alpha3309HSPA5Heat shock 70 kDa protein 5 (glucose-regulated protein, 78 kDa)3320HSP90AA1Heat shock protein 90 kDa alpha (cytosolic), class A member 12908NR3C1Nuclear receptor subfamily 3, group C, member 1 (glucocorticoid receptor)6778STAT6Signal transducer and activator of transcription 6, interleukin-4 induced3146HMGB1High-mobility group box 13301DNAJA1DnaJ (Hsp40) homolog, subfamily A, member 14221MEN1Multiple endocrine neoplasia I3312HSPA8Heat shock 70 kDa protein 83840KPNA4Karyopherin alpha 4 (importin alpha 3)2274FHL2Four-and-a-half LIM domains 24792NFKBIANuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha57805KIAA1967KIAA19673185HNRNPFHeterogeneous nuclear ribonucleoprotein F203068TUBBTubulin, beta6774STAT3Signal transducer and activator of transcription 3 (acute-phase response factor)4670HNRNPMHeterogeneous nuclear ribonucleoprotein M1997ELF1E74-like factor 1 (ets domain transcription factor)113457TUBA3DTubulin, alpha 3D1999ELF3E74-like factor 3 (ets domain transcription factor, epithelial-specific)5591PRKDCProtein kinase, DNA-activated, catalytic polypeptide708C1QBPComplement component 1, q subcomponent binding protein2274FHL2Four-and-a-half LIM domains 23313HSPA9Heat shock 70 kDa protein 9 (mortalin)8600TNFSF11Tumor necrosis factor (ligand) superfamily, member 113659IRF1Interferon regulatory factor 184617TUBB6Tubulin, beta 67280TUBB2ATubulin, beta 2A2908NR3C1Nuclear receptor subfamily 3, group C, member 1 (glucocorticoid receptor)2908NR3C1Nuclear receptor subfamily 3, group C, member 1 (glucocorticoid receptor)8517IKBKGInhibitor of kappa light polypeptide gene enhancer in B-cells, kinase gamma7295TXNThioredoxin10318TNIP1TNFAIP3 interacting protein 14793NFKBIBNuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, beta3065HDAC1Histone deacetylase 13551IKBKBinhibitor of kappa light polypeptide gene enhancer in B-cells, kinase beta1152CKBCreatine kinase, brain4069LYZLysozyme (renal amyloidosis)140462ASB9Ankyrin repeat and SOCS box-containing 94653MYOCMyocilin, trabecular meshwork inducible glucocorticoid response6774STAT3Signal transducer and activator of transcription 3 (acute-phase response factor)3660IRF2Interferon regulatory factor 27278TUBA3CTubulin, alpha 3c4221MEN1Multiple endocrine neoplasia I5966RELv-rel reticuloendotheliosis viral oncogene homolog (avian)1147CHUKConserved helix-loop-helix ubiquitous kinase55922NKRFNFKB repressing factor2113ETS1v-ets erythroblastosis virus E26 oncogene homolog 1 (avian)64332NFKBIZNuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, zeta51773RSF1Remodeling and spacing factor 15971RELBv-rel reticuloendotheliosis viral oncogene homolog B1832DSPDesmoplakin347733TUBB2BTubulin, beta 2B2353FOSv-fos FBJ murine osteosarcoma viral oncogene homolog9325TRIP4Thyroid hormone receptor interactor 44435CITED1Cbp/p300-interacting transactivator, with Glu/Asp-rich carboxy-terminal domain, 122984PDCD11Programmed cell death 11790CADCarbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase1326MAP3K8Mitogen-activated protein kinase kinase kinase 81917EEF1A2Eukaryotic translation elongation factor 1 alpha 29172MYOM2Myomesin (M-protein) 2, 165 kDa10856RUVBL2RuvB-like 2 (E. coli)1158CKMCreatine kinase, muscle808CALM3Calmodulin 3 (phosphorylase kinase, delta)672BRCA1Breast cancer 1, early onset801CALM1Calmodulin 1 (phosphorylase kinase, delta)293SLC25A6Solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator), member 63310HSPA6Heat shock 70 kDa protein 6 (HSP70B′)2908NR3C1Nuclear receptor subfamily 3, group C, member 1 (glucocorticoid receptor)136319MTPNMyotrophin2274FHL2Four-and-a-half LIM domains 29093DNAJA3DnaJ (Hsp40) homolog, subfamily A, member 34628MYH10Myosin, heavy chain 10, non-muscle4221MEN1Multiple endocrine neoplasia I6774STAT3Signal transducer and activator of transcription 3 (acute-phase response factor)3839KPNA3Karyopherin alpha 3 (importin alpha 4)57805KIAA1967KIAA19672908NR3C1Nuclear receptor subfamily 3, group C, member 1 (glucocorticoid receptor)1869E2F1E2F transcription factor 155662HIF1ANHypoxia-inducible factor 1, alpha subunit inhibitor79155TNIP2TNFAIP3 interacting protein 29532BAG2BCL2-associated athanogene 26421SFPQSplicing factor proline/glutamine-rich (polypyrimidine tract binding protein associated)2908NR3C1Nuclear receptor subfamily 3, group C, member 1 (glucocorticoid receptor)10627MRCL3Myosin regulatory light chain MRCL37431VIMVimentin672BRCA1Breast cancer 1, early onset2274FHL2Four-and-a-half LIM domains 24221MEN1Multiple endocrine neoplasia I672BRCA1Breast cancer 1, early onset4221MEN1Multiple endocrine neoplasia I3836KPNA1Karyopherin alpha 1 (importin alpha 5)3093UBE2KUbiquitin-conjugating enzyme E2K (UBC1 homolog, yeast)805CALM2Calmodulin 2 (phosphorylase kinase, delta)5970RELAv-rel reticuloendotheliosis viral oncogene homolog A (avian)9782MATR3Matrin 38600TNFSF11Tumor necrosis factor (ligand) superfamily, member 118607RUVBL1RuvB-like 1 (E. coli)4627MYH9Myosin, heavy chain 9, nonmuscle23421ITGB3BPIntegrin beta 3 binding protein (beta3-endonexin)140462ASB9Ankyrin repeat and SOCS box-containing 94841NONONon-POU domain containing, octamer-binding9276COPB2Coatomer protein complex, subunit beta 2 (beta prime)4221MEN1Multiple endocrine neoplasia I1213CLTCClathrin, heavy chain (Hc)292SLC25A5Solute carrier family 25 (Mitochondrial carrier; adenine nucleotide translocator), member 54066LYL1Lymphoblastic leukemia-derived sequence 164332NFKBIZNuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, zeta5531PPP4CProtein phosphatase 4 (formerly X), catalytic subunit8091HMGA2High-mobility group AT-hook 26202RPS8Ribosomal protein S81051CEBPBCCAAT/enhancer binding protein (C/EBP), beta222643UNC5CLUnc-5 homolog C (C. elegans)-like4790NFKB1Nuclear factor of kappa light polypeptide gene enhancer in B-cells 171ACTG1Actin, gamma 13312HSPA8Heat shock 70 kDa protein 89782MATR3Matrin 33320HSP90AA1Heat shock protein 90 kDa alpha (cytosolic), class A member 14637MYL6Myosin, light chain 6, alkali, smooth muscle and nonmuscle2908NR3C1Nuclear receptor subfamily 3, group C, member 1 (glucocorticoid receptor)4793NFKBIBNuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, beta688KLF5Kruppel-like factor 5 (intestinal)672BRCA1Breast cancer 1, early onsetTable 2
Gene lists for the analyses of the PEIMAP and PSIMAP using CK-MM as a hub protein with 100% identity.
Gene IDGene symbolFull name1889ECE1Endothelin-converting enzyme 15981RFC1Replication factor C (activator 1) 1, 145 kDa226ALDOAAldolase A, fructose-bisphosphate2335FN1Fibronectin 19372ZFYVE9Zinc finger, FYVE domain containing 960ACTBActin, beta3688ITGB1Integrin, beta 1 (fibronectin receptor, beta polypeptide, antigen CD29 includes MDF2, MSK12)7273TTNTitin2274FHL2Four-and-a-half LIM domains 24853NOTCH2Notch homolog 2 (Drosophila)2512FTLFerritin, light polypeptide1192CLIC1Chloride intracellular channel 12274FHL2Four-and-a-half LIM domains 25313PKLRPyruvate kinase, liver and RBC302ANXA2Annexin A27704ZBTB16Zinc finger and BTB domain containing 162200FBN1Fibrillin 127332ZNF638Zinc finger protein 63892086GGTLC1Gamma-glutamyltransferase light chain 1713C1QBComplement component 1, q subcomponent, B chain3029HAGHHydroxyacylglutathione hydrolase5664PSEN2Presenilin 2 (Alzheimer disease 4)7086TKTTransketolase (Wernicke-Korsakoff syndrome)4176MCM7Minichromosome maintenance complex component 71152CKBCreatine kinase, brain1499CTNNB1Catenin (cadherin-associated protein), beta 1, 88 kDa140462ASB9Ankyrin repeat and SOCS box-containing 99457FHL5Four-and-a-half LIM domains 54653MYOCMyocilin, trabecular meshwork inducible glucocorticoid response3029HAGHHydroxyacylglutathione hydrolase7704ZBTB16Zinc finger and BTB domain containing 163675ITGA3Integrin, alpha 3 (antigen CD49C, alpha 3 subunit of VLA-3 receptor)226ALDOAAldolase A, fructose-bisphosphate3689ITGB2Integrin, beta 2 (complement component 3 receptor 3 and 4 subunit)3688ITGB1Integrin, beta 1 (fibronectin receptor, beta polypeptide, antigen CD29 includes MDF2, MSK12)302ANXA2Annexin A292086GGTLC1Gamma-glutamyltransferase light chain 13679ITGA7Integrin, alpha 72023ENO1Enolase 1, (alpha)9172MYOM2Myomesin (M-protein) 2, 165 kDa1158CKMCreatine kinase, muscle4790NFKB1Nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (p105)2A2MAlpha-2-macroglobulin3688ITGB1Integrin, beta 1 (fibronectin receptor, beta polypeptide, antigen CD29 includes MDF2, MSK12)56944OLFML3Olfactomedin-like 31281COL3A1Collagen, type III, alpha 1 (Ehlers-Danlos syndrome type IV, autosomal dominant)2274FHL2Four-and-a-half LIM domains 23688ITGB1Integrin, beta 1 (fibronectin receptor, beta polypeptide, antigen CD29 includes MDF2, MSK12)118427OLFM3Olfactomedin 322900CARD8Caspase recruitment domain family, member 83488IGFBP5Insulin-like growth factor binding protein 57132TNFRSF1ATumor necrosis factor receptor superfamily, member 1A226ALDOAAldolase A, fructose-bisphosphate3675ITGA3Integrin, alpha 3 (antigen CD49C, alpha 3 subunit of VLA-3 receptor)51455REV1REV1 homolog (S. cerevisiae)6421SFPQSplicing factor proline/glutamine-rich (polypyrimidine tract binding protein associated)302ANXA2Annexin A24633MYL2Myosin, light chain 2, regulatory, cardiac, slow8880FUBP1Far upstream element (FUSE) binding protein 12274FHL2Four-and-a-half LIM domains 23688ITGB1Integrin, beta 1 (fibronectin receptor, beta polypeptide, antigen CD29 includes MDF2, MSK12)27332ZNF638Zinc finger protein 6383673ITGA2Integrin, alpha 2 (CD49B, alpha 2 subunit of VLA-2 receptor)203AK1Adenylate kinase 14627MYH9Myosin, heavy chain 9, non-muscle5213PFKMPhosphofructokinase, muscle140462ASB9Ankyrin repeat and SOCS box-containing 97076TIMP1TIMP metallopeptidase inhibitor 15176SERPINF1Serpin peptidase inhibitor, clade F (alpha-2 antiplasmin, pigment epithelium derived factor), member 13694ITGB6Integrin, beta 659ACTA2Actin, alpha 2, smooth muscle, aorta4176MCM7Minichromosome maintenance complex component 710487CAP1CAP, adenylate cyclase-associated protein 1 (yeast)7046TGFBR1Transforming growth factor, beta receptor I (activin A receptor type II-like kinase, 53 kDa)3543IGLL1Immunoglobulin lambda-like polypeptide 15313PKLRPyruvate kinase, liver and RBC51455REV1REV1 homolog (S. cerevisiae)10296MAEAMacrophage erythroblast attacher3911LAMA5Laminin, alpha 52597GAPDHGlyceraldehyde-3-phosphate dehydrogenase975CD81CD81 molecule92086GGTLC1Gamma-glutamyltransferase light chain 11915EEF1A1Eukaryotic translation elongation factor 1 alpha 15664PSEN2Presenilin 2 (Alzheimer disease 4)1278COL1A2Collagen, type I, alpha 2Figure 3
PPI map for CKM as a target hub protein with the 80% identity. Labels with red color indicate the hub protein of targeting. The image was made by the aiSee program (http://www.aisee.com/).Figure 4
PPI map for CKM as a target hub protein with the 100% identity. The methodological conditions were the same as for Figure3 except the identity.Figure 5
Overlapping map between CKB and CKM PPI maps. Data were input by using the results from Figures1 to 4.NFKB1 (also known as p50 or NF-kappaB) is a well-known transcription regulator that is responsible for the expression and regulation of many genes for immune response, cell adhesion, differentiation, proliferation, angiogenesis, and apoptosis [27–31]. It translocates into the nucleus and stimulates the expression of many genes involved in various biological functions. NFKB1 is also associated with a number of inflammatory diseases such as lymphoma [32], Alzheimer disease [33], psoriatic arthritis [34], breast cancer [35, 36], and rheumatoid arthritis [37]. Activation of NFKB1 requires binding of NF-kappaB essential modulator (NEMO) to ubiquitinated substrates [38]. With respect to an association with CK, it has been reported that NFKB1 is mostly associated with myocardial ischemia/reperfusion. During reperfusion, the absence of poly(ADP-ribose) polymerase-1 (PARP-1) leads to a reduction of myocardial apoptosis, which is associated with reduced NFKB1 activation [39, 40], and proteasome inhibition ablates activation of NFKB1 in myocardial reperfusion and reduces reperfusion injury [41]. Myocardial injury was assessed by measuring the serum levels of CK, and CK was reduced in serum along with reduction of NFKB1 activation.FHL2 is a member of the human four-and-a-half-LIM-only protein family, which consists of the members FHL1, FHL2, FHL3, FHL4, and ACT. These proteins function in various cellular processes, including regulation of cell survival, transcription, and signal transduction [42]. FHL2 contains an LIM domain, one of the protein-protein interaction motifs, which allows specific proteins to combine with certain partners. The specificity of a protein-protein interaction can be obtained by an interaction code predicted by conserved amino acid sequences. The interaction of FHL2 with transcription factors and other proteins involved in cancer development was examined. Since transcription factors control all fundamental developmental and homeostatic processes, transcriptional cofactors such as FHL2 are likely to contribute to human carcinogenesis and are of clinical importance in various forms of cancer [43], including leukemia [44]. With respect to an association with CK, Chung et al. [45] reported that FHL2 (developmentally enhanced phosphotransfer enzyme-anchoring protein) amalgamated the myofibrillar CK metabolic signaling circuit, providing an energetic continuum between mitochondria and the nascent contractile machinery in a murine embryonic stem cell cardiac differentiation model. They reported that CK-M clustered around developing myofibrils, sarcolemma, and the perinuclear compartment, whereas CK-B was tightly associated with myofibrillar alpha-actinin, forming wire-like structures extending from the nuclear compartment to the sarcolemma. FHL2 was also increased in myocardial ischemia-reperfusion injury, where IL-6 and IL-8 mRNA are upregulated in human cardiac myocytes [46].Recently, ASB9 was found to interact with ubiquitous mitochondrial CK [47]. The ankyrin repeat domains of ASB9 can associate with the substrate binding site of CK in a SOCS box-independent manner. The overexpression of ASB9 induces ubiquitination of CK. ASB9 reduces CK activities and cell growth and negatively regulates cell growth. ASB9 is a member of the ankyrin repeat and is a suppressor of the cytokine signaling (SOCS) box protein family. It can interact with the SOCS box domain of the elongin B-C adapter complex and can further complex with the cullin and ring box proteins to form E3 ubiquitin ligase complexes [48]. These complexes may be involved in specific substrate-recognition for ubiquitination and degradation and mediate the substrate-recognition of the E3 ubiquitin ligases.The interaction between CK and MYOC has not been elucidated. However, MYOC has a cytoskeletal function, and this implies that it may interact with CK somehow. MYOC is expressed in many ocular tissues including the trabecular meshwork [49], which is a specialized eye tissue that is essential in regulating intraocular pressure. MYOC mutations have been identified as the cause of hereditary juvenile-onset open-angle glaucoma [50].Researchers could apply computational prediction by PPI mapping to help determine target proteins. Since the next step in the functional study of interesting proteins/genes is a time- and cost-consuming process, the number of target proteins is limited; hence, for the right choice, computational prediction on the basis of database information could be critical at this step. Functional studies can be further conducted using a mouse model and a large number of clinical samples. Final confirmation and CK mechanisms could then be more clearly evaluated for developing drugs to effectively treat CK-related diseases.The functions of most of the candidate proteins predicted in this study have not been well reported in skin diseases or in the pathogenesis of other diseases. We provide new information regarding these candidate proteins’ interaction with CK, as well as the involvement of several hub proteins such as NFKB1, FHL2, ASB9, and MYOC. Although we do not suggest a direct role of any candidate protein in skin diseases, we provide candidate proteins to be targeted in further studies of CK-associated diagnostic markers and/or treatment of corresponding skin conditions. Furthermore, we also provide some insights into understanding the responses of CK in skin.



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*Source: 328249-2011-08-03.xml*