|Year : 2014 | Volume
| Issue : 5 | Page : 580-585
|In-silico analysis of heat shock protein 47 for identifying the novel therapeutic agents in the management of oral submucous fibrosis
Jayasankar P Pillai1, Girish J Parmar2, Rakesh Rawal3, Faraz Shaikh4, Girish R Chauhan5, Rajarajeswari J Pillai6
1 Department of Oral Pathology, Government Dental College and Hospital, Ahmedabad, Gujarat, India
2 Department of Conservative Dentistry and Endodontics, Government Dental College and Hospital, Ahmedabad, Gujarat, India
3 Department of Medicinal Chemistry, Division of Medicinal Chemistry and Pharmacogenomics, Gujarat Cancer and Research Institute, Ahmedabad, Gujarat, India
4 Department of Chemistry, Saurastra University, Rajkot, Gujarat, India
5 Department of Oral Pathology, Government Dental College and Hospital, Jamnagar, Gujarat, India
6 Consulting Dental Surgeon, Ahmedabad, Gujarat, India
Click here for correspondence address and email
|Date of Submission||11-Jun-2013|
|Date of Decision||18-Apr-2014|
|Date of Acceptance||14-Oct-2014|
|Date of Web Publication||16-Dec-2014|
| Abstract|| |
Background: Heat shock proteins-47 (HSP47) is a collagen specific molecular chaperone, involved in the processing and/or secretion of procollagen. It seems to be regularly upregulated in various fibrotic or collagen disorders. Hence, this protein can be a potential target for the treatment of various fibrotic diseases including oral submucous fibrosis (OSF), which is a collagen metabolic disorder of oral cavity and whose etiopathogeneic mechanism and therapeutic protocols are still not well documented.
Aim: The aim of this study is to identify the novel therapeutic agents using in-silico methods for the management of OSF.
Objectives: The objectives of this study are to identify the binding sites of HSP47 on the collagen molecule and to identify the lead compound with anti-HSP47 activity from the library of natural compounds, using in-silico methodology.
Materials and Methods: The web-based and tool based in-silico analysis of the HSP47 and collagen molecules are used in this study. The crystal structure of collagen and HSP47 were retrieved from Protein Data Bank website. The binding site identification and the docking studies are done using Molegro Virtual Docker offline tool.
Results: Out of the 104 Natural compounds, six ligands are found to possess best binding affinity to the binding amino acid residues. Silymarin binds with the 4AU2A receptor and the energy value are found to be −178.193 with four Hbonds. The other best five natural compounds are hesperidin, ginkgolides, withanolides, resveratrol, and gingerol. Our findings provide the basis for the in-vitro validation of the above specified compounds, which can possibly act as "lead" molecules in designing the drugs for OSF.
Conclusion: HSP47 can be a potential candidate to target, in order to control the production of abundance collagen in OSF. Hence, the binding sites of HSP47 with collagen are identified and some natural compounds with a potential to bind with these binding receptors are also recognized. These natural compounds might act as anti-HSP47 lead molecules in designing novel therapeutic agents for OSF, which are so far unavailable.
Keywords: Docking, heat shock protein 47, in-silico, natural compounds, oral submucous fibrosis
|How to cite this article:|
Pillai JP, Parmar GJ, Rawal R, Shaikh F, Chauhan GR, Pillai RJ. In-silico analysis of heat shock protein 47 for identifying the novel therapeutic agents in the management of oral submucous fibrosis. Indian J Dent Res 2014;25:580-5
Heat shock proteins (HSPs) are a distinctive class of proteins that play a crucial role in the assembly and folding of intracellular polypeptides and help to restore the biological activities of abnormal proteins. They are synthesized within the cells in response to temperature and other stresses including oxidative stress. They help the cells to repair and adapt to such stresses, either by refolding damaged proteins or by degrading them.  In 1962, Ritossa first observed the heat shock response in Drosophila and this response is now widely accepted as a universally conserved cellular defense system.  There are various types of HSPs, which act as Molecular Chaperones in maintaining cellular homeostasis. , Thus they regulate the folding and assembly of nascent and unfolded peptides and help in transporting proteins to particular sub cellular compartments and assist in the degradation of misfolded proteins. 
|How to cite this URL:|
Pillai JP, Parmar GJ, Rawal R, Shaikh F, Chauhan GR, Pillai RJ. In-silico analysis of heat shock protein 47 for identifying the novel therapeutic agents in the management of oral submucous fibrosis. Indian J Dent Res [serial online] 2014 [cited 2019 Jul 23];25:580-5. Available from: http://www.ijdr.in/text.asp?2014/25/5/580/147094
| Heat Shock Protein 47|| |
One such HSP, which acts as a specific chaperone for procollagen molecule is HSP47. It is a stress inducible 47 kD collagen-binding glycoprotein that is present in the endoplasmic reticulum (ER) of collagen-secreting Cells.  It belongs to the serine protease inhibitor (serpin) super family  and it plays an essential role in normal procollagen folding. The defined role of HSP47 in the processing of procollagens has not been recognized. , However, there are reports showing increased expression of HSP47 in fibrotic diseases in human ,,, and in animal models. , Type I collagen, a typical fibril-forming collagen and a major component of extra cellular matrix, is synthesized as a precursor molecule, termed procollagen, which undergoes extensive post-translational processing before the formation of triple helix.  The HSP47 binds and thereby stabilizes the triple helix of procollagen in the pH-neutral ER, subsequently dissociates under low pH conditions of the cis-Golgi or ER-Golgi intermediate compartment and recycles back to the ER. , In the extracellular environment, the procollagen molecule undergoes further modification, such as cleavage of the C- and N-terminal propeptides and deamination of lysines before incorporation into stable, cross-linked fibrils. , New insights into the role of HSP47 in collagen biosynthesis were revealed in the earlier works of Nagai et al.  The binding of HSP47 to the triple helix, protects it from intracellular degradation and helps the procollagen to retain its form in the ER, thus giving it additional time to achieve the correct structure. This event slows down the transport of procollagen peptides from the ER and subsequently increasing the concentration of propeptides in the subcellular compartment. In the absence of HSP47, the procollagen chain would be transported normally by vesicular transport. It might also be possible that HSP47 binds to regions of the helix to prevent micro-unfolding, particularly during heat stress. According to Koide et al., the triple helix formation is a prerequisite for the interaction of collagen-like model peptides with HSP47 and that Gly-Xaa-Arg sequence, which is harbored in the triple helix structure serves as a binding motif for HSP47. , Nearly, 25 HSP47 molecules bind to a single folded type I collagen triple helix  with no significant binding to unfolded chains. 
Role of heat shock protein 47 in oral submucous fibrosis
Oral submucous fibrosis (OSF) is a potentially malignant disorder of oral mucous membrane due to the consumption of areca nut and commercially available areca products. This condition is characterized by the stiffness of oral mucosa, leading to an inability to open mouth and intolerance to spicy food.  The fibro-elastic changes in OSF are due to the excess deposition and/or decreased degradation of collagen in the connective tissue of oral mucosa. Kaur et al. (2001) demonstrated that increased levels of HSP47 in OSF and suggested a possible role of HSP47 in its pathogenesis.  An earlier in-vitro study by Chang et al. (1999) clearly indicated that arecoline in areca nut enhanced collagen synthesis in human gingival fibroblasts.  Furthermore in 2008, Yang et al. evaluated the role of HSP47 expression in buccal mucosal fibroblasts (BMFs), stimulated with arecoline. Their study proved that arecoline is capable of stimulating HSP47 messenger ribonucleic acid expression in human BMFs. Thus, they proposed that the accumulation of collagen in oral mucosal connective tissue may be caused by a simultaneous effect on HSP47 by arecoline during areca chewing, which is considered as an important etiological factor in the OSF. The expression of HSP47 is found to be upregulated in the human buccal fibroblasts cells stimulated with arecoline  and it suggests that increased collagen deposition in the oral mucosa of OSF patients might be secondary to arecoline induced over expression of HSP47. It was also proposed by Taguchi et al. that HSP47 could be a biomarker of phenotypically altered collagen-producing cells during wound healing and fibrosis.  Furthermore, it is documented that as a part of heat shock/stress response HSP47 expression is upregulated under stress conditions, like elevated temperature, heavy metals and oxidative stress.  The role of Oxidative stress in OSF was studied by Gupta et al.  also studies have revealed the role of copper in the pathogenesis of OSF. , Thus based on the above studies ,, it is rational to hypothesize the role HSP47 in excess collagen deposition in OSF, secondary to stimuli like arecoline, oxidative stress and copper. Hence, we propose that HSP47 could be a rationale therapeutic target for anti-fibrotic therapy in OSF. Accordingly an in-silico research was designed first for identifying the actual binding sites or the amino acids involved in the binding of HSP47 with collagen molecule and secondly for identifying those scaffolds from natural compounds, which have the potential to block these binding sites.
| Materials and methods|| |
A comprehensive study on the OSF was done and protein structural data were retrieved from Protein Data Bank (PDB) (Available from: www.rcsb.org/pdb/explore.do?structureid = 4au2).
Binding site identification
The binding site study was done on the 4AU2 (Crystal Structure of HSP47-collagen complex), which contain the six collagen chain and the four HSP47 chain [Figure 1]. Each collagen chain was converted to the ligand and its interaction with the HSP47 four chain proteins was checked. This was done for the finding of specific residue interaction between the HSP47 and collagen. Each chain of collagen treated as the ligand by option of Molegro Virtual Docker 4.2 (MVD). Each ligand was used as an active ligand and its hydrogen bond (H-bond) interaction was checked with the HSP47 four chain proteins. All possible interactive residues were noted down.
|Figure 1: The binding of Heat Shock Protein 47 molecule with collagen chains|
Click here to view
The natural compound was docked against 4AU2 (A) (Crystal structure of HSP47 chain A) using MVD.  The structure represents a crystal structures of HSP47 in its free form and in complex with homotrimeric synthetic collagen model peptides, each peptide composed of one HSP47-binding site represented by an arginine at the Yaa-position of a Xaa-Yaa-Gly triplet.  So we have selected one HSP47 chain to target its particular interaction with the collagen peptides, for the inhibition of its extensive contacts with the leading and trailing strands, which is occupied by the HSP47. The target protein structures were prepared after careful removal of hetero atoms and water molecules. The binding site identified the residue selected in the workspace and 16 Aº constrain were generated at the region of selected amino acid residue. The docking study was subjected to that constrain with 104 ligands to target the specific interaction of collagen and the HSP47 in MVD. The scoring system of MVD is based on the new hybrid search algorithm called 'guided differential evolution'. This algorithm combines the differential evolution optimization technique with a cavity prediction algorithm. The Moldock simplex evolution was taken as search algorithm with an energy threshold of 100 and the one giving lowest energy was chosen. After the docking simulation, over generated pose were sorted by the Moldock score. 
| Results|| |
0Interaction site prediction
The binding of HSP47 and collagen molecule is well characterized by numerous studies. It becomes quite imperative that their site of interaction be targeted in order inhibition of the HSP47-collagen complex. The protein-protein interaction between HSP47 and collagen peptide was analyzed and the precise site of interaction was identified by the MVD [Figure 2]. The list of amino acid residues of HSP47 which are involved in the interaction with the collagen molecule is listed in [Table 1].
|Figure 2: The amino acid residue involved in the interaction between the heat shock protein 47 and each collagen peptides analyzed by Molegro Virtual Docker|
Click here to view
|Table 1: List of amino acid residue involved in the interaction between the HSP47 and collagen peptides analyzed by MVD |
Click here to view
The docking results were tabulated for all compounds against the 4AU2 (A) chain. The structure contains four chains which are similar at their structural forms. The best docking pose which possess the highest moldock score and relatively good H-bond interaction toward the binding site were chosen. The best six compounds, which exhibited very good affinity and targeting site of our interest were listed in the [Table 2]. That molecule interaction site toward receptors studied and the best ligand binding pose was picked out on the basis of aforementioned criteria. Out of the 104 compounds, six ligands were found to possess best binding affinity towards the receptors and were found to form H-bond interaction. [Figure 3].
|Figure 3: Illustrations of the docking pose of 6 best ligands binding with the 4AU2A carried out on Molegro Virtual Docker 4.2. silymarin, hesperidin, ginkgolides B, withanolides with withaferin A, resveratrol, gingerol all ligand with the specific interaction site at heat shock protein 47 depicted in figure. Images depict best six ligands and their interaction with the 4AU2 site dotted Green lines represent hydrogen bonds interaction. The protein is depicted in ball and stick model and the ligand in Wireframe. Corey, Pauling, Koltun coloring has been employed|
Click here to view
|Table 2: The best six ligands which possessed the lowest MolDock score and with the number of hydrogen bond and interactive amino acid residue were chosen and listed in the table |
Click here to view
| Discussion|| |
Heat Shock Proteins 47 is an ATP-independent HSP. Its expression pattern indicates that it is collagen-specific rather than general ER chaperone.  Contrary to general chaperones, HSP47 is not released upon procollagen folding. Instead, it travels together with procollagen from the ER to the cis-Golgi network, where it dissociates (due to lower pH) and returns to the ER.  Findings from various human and experimental fibrotic diseases clearly suggests that HSP47 expression is upregulated and since HSP47 has a single substrate protein, collagen, the de novo expression of HSP47 acts not only as a novel histological biomarker but also a unique therapeutic target for selective inactivation in fibrotic diseases like OSF. An animal study by Hagiwara et al. in 2007 showed that the introduction of antisense oligonucleotides against HSP47 effectively decreased HSP47 levels and reduced the bleomycin induced pulmonary fibrosis.  A similar experimental study revealed that antisense oligonucleotides against HSP47 suppress collagen accumulation in glomerulonephritis.  Till date many research on OSF were designed mainly either to explore the possible etiopathogeneic mechanism or as epidemiological surveys. Very few papers report on the treatment protocols of this particular disease. We have made an attempt to identify those ligands from the library of natural compounds which can block the binding sites of HSP47 with collagen. Here we suggest a novel strategy for the management of fibrotic conditions like OSF, based on the earlier findings that down-regulation of HSP47 could lead to reduction in collagen deposition. Our study was to target the site of interaction between the HSP47 and collagen. The key step was to itemize the amino acid residues involved at the site of interaction. The extensive study of interaction between the HSP47 and collagen gave complete list of all the residues. Our interaction study results showed that amino acids residues of HSP47 were involved in its binding with possible collagen residues Arg222, Arg228, Asp385 with H-bond and other residue like Met218, Met225, His238, His274, Tyr303 with electrostatic and Van der waals bond. This result of interaction coherent with the earlier published report (Widmer et al.  ), which depicts the recognition of conserved Asp with Arginine residue within the Xaa-Arg-Gly triplet. Silymarin which bind with the highest affinity and inhibit specific site of the interested makes it viable drug candidate for the OSMF. Other compounds, which possess relatively lesser affinity and H-bond can also be subjected to the in vitro validation. Earlier, when the crystal structure of HSP47 was not available, we predicted the structure using homologous modeling and the docking study using the predicted structure with the library of natural compounds revealed Curcumin, Theaflavin, Gingerol, silymarin, Resveratrol and Ellagic acid as some of the potential anti-HSP47 compounds.
But in August, 2012, after the X-ray diffraction study by Widmer et al.  the crystal structure of HSP47 was made available in the protein structure databank (PDB id 4AU2, 4AU3, 4AU4). Hence, we utilized this specific crystal structure of HSP47 for the present docking study, identified and listed some seven best ligands [Table 2], which possessed the best Moldock score. Also we found that compounds like Silymarin, Gingerol, resveratrol and curcumin possessed best affinity toward the binding amino acids of the HSP47-collagen complex both in the recent crystal structure study and also in our previous predicted structure study.
| Conclusion|| |
Heat Shock Protein 47 is the specific molecular chaperone in the collagen synthesis; hence, it can be a potential candidate to target, in order to control the production of abundance collagen in disease like OSF. In this report, using in-silico methodology, we revealed the interacting sites of HSP47 with collagen and also recognized some natural compounds with a potential to bind with these binding receptors. These natural compounds can act as anti-HSP47 lead molecules in designing novel therapeutic agents for OSF, which so far are unavailable.
| Acknowledgment|| |
We extend our sincere gratitude to the Directorate of Medical Education and Research, Department of Health and Family Welfare Government of Gujarat for funding this project.
| References|| |
Georgopoulos C, Welch WJ. Role of the major heat shock proteins as molecular chaperones. Annu Rev Cell Biol 1993;9:601-34.
Ritossa FM. A new puffing pattern induced by temperature shock and DNP in Drosophila
. Experientia 1962;18:571-3.
Becker J, Craig EA. Heat-shock proteins as molecular chaperones. Eur J Biochem 1994;219:11-23.
Hendrick JP, Hartl FU. Molecular chaperone functions of heat-shock proteins. Annu Rev Biochem 1993;62:349-84.
Hartl FU, Hayer-Hartl M. Converging concepts of protein folding in vitro
and in vivo
. Nat Struct Mol Biol 2009;16:574-81.
Nagata K. Hsp47: A collagen-specific molecular chaperone. Trends Biochem Sci 1996;21:22-6.
Whisstock J, Skinner R, Lesk AM. An atlas of serpin conformations. Trends Biochem Sci 1998;23:63-7.
Tasab M, Batten MR, Bulleid NJ. Hsp47: A molecular chaperone that interacts with and stabilizes correctly-folded procollagen. EMBO J 2000;19:2204-11.
Dafforn TR, Della M, Miller AD. The molecular interactions of heat shock protein 47 (Hsp47) and their implications for collagen biosynthesis. J Biol Chem 2001;276:49310-9.
Razzaque MS, Nazneen A, Taguchi T. Immunolocalization of collagen and collagen-binding heat shock protein 47 in fibrotic lung diseases. Mod Pathol 1998;11:1183-8.
Brown KE, Broadhurst KA, Mathahs MM, Brunt EM, Schmidt WN. Expression of HSP47, a collagen-specific chaperone, in normal and diseased human liver. Lab Invest 2005;85:789-97.
Kuroda K, Tsukifuji R, Shinkai H. Increased expression of heat-shock protein 47 is associated with overproduction of type I procollagen in systemic sclerosis skin fibroblasts. J Invest Dermatol 1998;111:1023-8.
Naitoh M, Hosokawa N, Kubota H, Tanaka T, Shirane H, Sawada M, et al.
Upregulation of HSP47 and collagen type III in the dermal fibrotic disease, keloid. Biochem Biophys Res Commun 2001;280:1316-22.
Liu D, Razzaque MS, Cheng M, Taguchi T. The renal expression of heat shock protein 47 and collagens in acute and chronic experimental diabetes in rats. Histochem J 2001;33:621-8.
Kawada N, Kuroki T, Kobayashi K, Inoue M, Nakatani K, Kaneda K, et al.
Expression of heat-shock protein 47 in mouse liver. Cell Tissue Res 1996;284:341-6.
Prockop DJ, Kivirikko KI. Collagens: Molecular biology, diseases, and potentials for therapy. Annu Rev Biochem 1995;64:403-34.
Lamandé SR, Bateman JF. Procollagen folding and assembly: The role of endoplasmic reticulum enzymes and molecular chaperones. Semin Cell Dev Biol 1999;10:455-64.
Mala JG, Rose C. Interactions of heat shock protein 47 with collagen and the stress response: An unconventional chaperone model? Life Sci 2010;87:579-86.
Prockop DJ, Sieron AL, Li SW. Procollagen N-proteinase and procollagen C-proteinase. Two unusual metalloproteinases that are essential for procollagen processing probably have important roles in development and cell signaling. Matrix Biol 1998;16:399-408.
Siegel RC. Collagen cross-linking. Synthesis of collagen cross-links in vitro
with highly purified lysyl oxidase. J Biol Chem 1976;251:5786-92.
Nagai N, Hosokawa M, Itohara S, Adachi E, Matsushita T, Hosokawa N, et al.
Embryonic lethality of molecular chaperone hsp47 knockout mice is associated with defects in collagen biosynthesis. J Cell Biol 2000;150:1499-506.
Koide T, Takahara Y, Asada S, Nagata K. Xaa-Arg-Gly triplets in the collagen triple helix are dominant binding sites for the molecular chaperone HSP47. J Biol Chem 2002;277:6178-82.
Koide T, Nishikawa Y, Asada S, Yamazaki CM, Takahara Y, Homma DL, et al.
Specific recognition of the collagen triple helix by chaperone HSP47. II. The HSP47-binding structural motif in collagens and related proteins. J Biol Chem 2006;281:11177-85.
Koide T, Asada S, Takahara Y, Nishikawa Y, Nagata K, Kitagawa K. Specific recognition of the collagen triple helix by chaperone HSP47: Minimal structural requirement and spatial molecular orientation. J Biol Chem 2006;281:3432-8.
Pindborg JJ, Sirsat SM. Oral submucous fibrosis. Oral Surg Oral Med Oral Pathol 1966;22:764-79.
Kaur J, Rao M, Chakravarti N, Mathur M, Shukla NK, Sanwal BD, et al.
Co-expression of colligin and collagen in oral submucous fibrosis: Plausible role in pathogenesis. Oral Oncol 2001;37:282-7.
Chang YC, Tai KW, Lii CK, Chou LS, Chou MY. Cytopathologic effects of arecoline on human gingival fibroblasts in vitro
. Clin Oral Investig 1999;3:25-9.
Yang SF, Tsai CH, Chang YC. The upregulation of heat shock protein 47 expression in human buccal fibroblasts stimulated with arecoline. J Oral Pathol Med 2008;37:206-10.
Taguchi T, Nazneen A, Al-Shihri AA, Turkistani KA, Razzaque MS. Heat shock protein 47: A novel biomarker of phenotypically altered collagen-producing cells. Acta Histochem Cytochem 2011;44:35-41.
Morimoto RI, Kline MP, Bimston DN, Cotto JJ. The heat-shock response: Regulation and function of heat-shock proteins and molecular chaperones. Essays Biochem 1997;32:17-29.
Gupta S, Reddy MV, Harinath BC. Role of oxidative stress and antioxidants in aetiopathogenesis and management of oral submucous fibrosis. Indian J Clin Biochem 2004;19:138-41.
Trivedy C, Meghji S, Warnakulasuriya KA, Johnson NW, Harris M. Copper stimulates human oral fibroblasts in vitro
: A role in the pathogenesis of oral submucous fibrosis. J Oral Pathol Med 2001;30:465-70.
Trivedy CR, Warnakulasuriya KA, Peters TJ, Senkus R, Hazarey VK, Johnson NW. Raised tissue copper levels in oral submucous fibrosis. J Oral Pathol Med 2000;29:241-8.
Available from: http://www.rcsb.org/pdb/explore/explore.do?structureId=4au2. [Last accessed on 2013 Jan 10].
Widmer C, Gebauer JM, Brunstein E, Rosenbaum S, Zaucke F, Drögemüller C, et al
. Molecular basis for the action of the collagen-specific chaperone Hsp47/SERPINH1 and its structure-specific client recognition. Proc Natl Acad Sci U S A 2012;109:13243-7.
Thomsen R, Christensen MH. MolDock: A new technique for high-accuracy molecular docking. J Med Chem 2006;49:3315-21.
Koide T, Nagata K. Collagen biosynthesis. In: Collagen. New York: Springer-Verlag; 2005. p. 85-114.
Nagata K. HSP47 as a collagen-specific molecular chaperone: Function and expression in normal mouse development. Semin Cell Dev Biol 2003;14:275-82.
Hagiwara S, Iwasaka H, Matsumoto S, Noguchi T. Antisense oligonucleotide inhibition of heat shock protein (HSP) 47 improves bleomycin-induced pulmonary fibrosis in rats. Respir Res 2007;8:37.
Sunamoto M, Kuze K, Tsuji H, Ohishi N, Yagi K, Nagata K, et al.
Antisense oligonucleotides against collagen-binding stress protein HSP47 suppress collagen accumulation in experimental glomerulonephritis. Lab Invest 1998;78:967-72.
Jayasankar P Pillai
Department of Oral Pathology, Government Dental College and Hospital, Ahmedabad, Gujarat
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2]
| Article Access Statistics|
| Viewed||1755 |
| Printed||31 |
| Emailed||1 |
| PDF Downloaded||147 |
| Comments ||[Add] |