although bilirubin has long been considered as a cytotoxic waste product , the beneficial properties of bilirubin have been identified during the last few decades . one possible physiologic role of bilirubin is as an antioxidant , and most current studies on the physiological functions of bilirubin focus on this effect . although other functions of bilirubin besides the antioxidant effects are not well documented , increasing evidence suggests that bilirubin possesses potential immunomodulatory properties . liu and colleagues have demonstrated that bilirubin possesses powerful immunomodulatory activity and suppresses experimental autoimmune encephalomyelitis ( eae ) . fischman and colleagues have shown that higher serum total bilirubin levels are protective against rheumatoid arthritis ( ra ) based on the secondary analysis of national health and nutrition examination survey data collected between 2003 and 2006 . peng and colleagues have observed that serum bilirubin concentrations in patients with multiple sclerosis ( ms ) are significantly reduced . vtek and colleagues have found that serum bilirubin levels in systemic lupus erythematosus ( sle ) patients are significantly lower compared to the control population . one of the plausible mechanisms of the immunomodulatory activity of bilirubin would be derived from the antioxidant activity . however , the results by liu and colleagues have clearly demonstrated that the potent immunosuppressive effects of bilirubin can not be attributed to its antioxidant activity . they also have shown that bilirubin has a broad suppressive effect on t cell reactivity . considering the background above and the well - known facts that certain autoimmune diseases are closely associated with specific types of human leukocyte antigens ( hla ) , we have hypothesized that bilirubin might significantly interact with the relevant hla molecules and hinder the binding of antigenic peptides that trigger the immune responses . the crystal structures of various types of hla molecules have revealed that their three - dimensional structures are highly conserved . the suppression of autoimmune diseases by bilirubin could be due to the common interactions between the disease - associated hla molecules and bilirubin . however , no experimental data that indicate the direct interactions between bilirubin and the hla molecules have been found in the literature so far . as the possibility of such interactions can be explored by theoretical docking simulations with reasonable accuracy now , we have undertaken the docking simulations between bilirubin and the relevant hla molecules . a software system moe ( molecular operating environment )   was used throughout this study . judging from the immunosuppressive effects of bilirubin , it is highly possible that bilirubin binds to the antigenic - peptide binding groove located between two  helices as illustrated in figure 1 . as the groove is relatively wide , we have predicted the possible binding sites of bilirubin by the use of the alpha site finder function   implemented in moe . small spheres named alpha spheres shown in figure 1 correspond to locations of tight atomic packing at the antigenic - peptide binding groove in this case . a site where the alpha spheres are clustered is designated alpha site which is considered to be the potential binding site of small molecules . one is an ase model , which is defined by the combination of alpha spheres generated at a concave in a protein and the excluded volumes around the concavity . the other is an ase score , which evaluates the shape similarity between the ligand and the ase model . the ase score selects and refines the initial pose by maximizing the overlap between the alpha spheres and the ligand , and minimizing the overlap between the excluded volume and the ligand . because the ase score makes good use of the gaussian - type function for evaluating and optimizing the overlap between the ligand and the site model , it can pose a ligand onto the docking site relatively faster and more effectively than using potential energy functions . because the posing algorithm of asedock is free from any bias except for shape a validation study using 59 high - quality x - ray structures of the complexes between drug - like molecules and the target proteins has demonstrated that asedock can faithfully reproduce experimentally determined docking modes of various drug like molecules in their target proteins . the binding affinity of bilirubin to the hla molecule was judged by a scoring function of gbvi / wsa   which is considered to express protein - ligand binding free energy .   for docking validation , redocking was performed using the recently published x - ray structure of the complex between an anti - hiv drug abacavir and the hla - b  57:01 molecule . this is the only x - ray structure of the complex between a hla molecule and a small organic molecule reported so far . the three - dimensional coordinates ( 3vri ) were obtained from the protein data bank ( pdb ) . the root - mean square deviation(rmsd ) between nonhydrogen atoms of abacavir in the crystal and docked structures is 0.99   . since prediction within rmsd of 2.0   is held as the passing standard , the results indicate that asedock is suitable for the docking simulations of the hla - bilirubin system . since the 4z , 15z - bilirubin ix form with negatively charged carboxylic groups as illustrated in figure 2 is the predominant form of bilirubin in vivo , this isomer was adopted in this study . the pdb structures of 3o6f and 2wbj were used for the hla dr4 and dr2 molecules associated with ra and sle , respectively . the x - ray structure of the hla dp5 molecule associated with ms was not available when we started the present study . therefore the 3d structure was built by homology modeling using the corresponding most - homologous hla molecule deposited in pdb . the pdb structure of 3lqz was selected as a template because of its high sequence identity with hla dp5 and the high - quality structure . the method is based on a combination of the segment - matching procedure   and an approach to the modeling of indels . ten models were generated by making a series of boltzmann - weighted choices of side chain rotamers and loop conformations from a set of protein fragments selected from a library of high - resolution protein structures . the simulations have demonstrated that bilirubin should bind to the peptide - binding grooves with relatively high affinity , albeit the binding positions are somewhat different depending on the hla molecule . the binding modes between bilirubin and the three hla molecules are illustrated in figure 3 . bilirubin is a relatively bulky molecule and it occludes over a large area of the peptide - binding groove . in addition , a part of the bound bilirubinprotrudes towards the space where the t cell receptor is supposed to approach to recognize the antigenic peptide . the gbvi / wsa values for the docked structures between bilirubin and the dr2 , dr4 , and dp5 molecules are 8.83 , 8.72 , and 7.02  kcal / mol , respectively . the corresponding pic50 values are estimated to be 6.49 , 6.41 , and 5.16 , respectively , at t = 298  k. the values suggest that the bound bilirubin molecules would significantly block the binding of antigenic peptides to the peptide - binding grooves leading to suppression of immunological responses . predicted high affinities of the bilirubin molecules against the relevant hla molecules support the above hypothesis . this hypothesis suggests that the immunoregulatory activity of bilirubin could be derived from its binding affinity to the relevant hla molecules . the hypothesis would foster new suspicions that different antigenic peptides might bind to the bilirubin - hla complexes . recently , illing and colleagues   have clearly shown that binding of an anti - hiv drug abacavir at the antigen - binding groove guides the selection of new peptides . abacavir is a relatively small molecule which binds to the bottom of the antigen - binding groove . therefore , the new endogenous peptide can bind to top of the drug - modified hla causing adverse effect of abacavir . however , since bilirubin is much bulkier molecule than abacavir , it can not bind to the bottom of the groove . instead , bilirubin binds near the surface of antigen - binding groove as illustrated in figure 3 . moreover , the protruding moiety of the bound bilirubin would prevent any peptides from further binding . this particular binding mode of bilirubin might be the reason for nonspecific suppression of several autoimmune diseases by bilirubin . the evaluation results of our hypothesis strongly suggest that much more studies about the relationships between bilirubin and autoimmune diseases should be undertaken . the hypothesis also implies the clinical possibility of bilirubin for treatment or alleviation of particular autoimmune diseases . however , recently khan and poduval have demonstrated that clinically relevant concentrations of unconjugated bilirubin induce apoptosis and necrosis in immune cells by depleting cellular glutathione . in addition , since there are no rich natural sources of bilirubin and it is rather expensive to synthesize it , the clinical applications of bilirubin itself do not seem to be practical . from the point of clinical applications , we must find compounds which can be effectively substituted for bilirubin . it is very interesting to start a drug discovery project to search for safer compounds which can mimic bilirubin . it is proposed that specific interactions between bilirubin and the hla molecules associated with autoimmune diseases might contribute to the immunomodulatory activities . if this hypothesis turns out to be correct , it would certainly lead to the development of new drugs for treatment of autoimmune diseases for which adequate pharmacotherapy is still lacking .
bilirubin is an endogenous product of heme degradation in mammals . 
 bilirubin has long been considered as a cytotoxic waste product that needs to be excreted . however 
 , increasing evidence suggests that bilirubin possesses multiple biological activities . 
 in particular , recent studies have shown that bilirubin should be a protective factor for several autoimmune diseases such as rheumatoid arthritis , multiple sclerosis , and systemic lupus erythematosus . 
 since these autoimmune diseases are closely associated with specific types of human leukocyte antigens ( hlas ) , we have hypothesized that bilirubin might bind to the antigenic peptide - binding groove of the hla molecules and exert its immunosuppressive actions . in order to evaluate the hypothesis , 
 theoretical docking studies between bilirubin and the relevant hla molecules have been undertaken . 
 the in silico studies have clearly shown that bilirubin may bind to the antigenic peptide - binding groove of the hla molecules relevant to the autoimmune diseases with significant affinity . 
 the bound bilirubin may block the binding of antigenic peptides to be presented to t cell receptors and lead to suppression of the autoimmune responses . 
 based on this hypothesis new drug discovery research for autoimmune diseases will be conducted .