In silico identification of epitopes from house cat and dog proteins as peptide immunotherapy candidates based on human leukocyte antigen binding affinity.

The objective of this descriptive study was to determine Felis domesticus (cat) and Canis familiaris (dog) protein epitopes that bind strongly to selected HLA class II alleles to identify synthetic vaccine candidate epitopes and to identify individuals/populations who are likely to respond to vaccines. FASTA amino acid sequences of experimentally validated allergenic proteins of house cat and dog were identified using International Union of Immunological Societies (IUIS) allergen nomenclature database. NetMHCII 2.2 server was used to determine binding affinities in the form of 1-log 50 k and in nM with commonly found HLA II alleles. Screening of house cat and dog allergenic proteins identified 4 (with 2 isoforms for chain 1 and 3 isoforms for chain 2 for fel d 1) and 6 proteins, respectively. Number of strong binders from each protein against each HLA type was determined as potential candidate for allergen immunotherapy. HLA-DRB1*0101 bound maximum number of epitopes (207 and 275 from house cat and dog, respectively) while HLA-DRB1*0802 bound none. We conclude that HLA specific epitope prediction can help identify synthetic peptide vaccine candidates and predict response as well.

A rationale for treatment of hereditary vitamin D-resistant rickets with analogs of 1 alpha,25-dihydroxyvitamin D(3).

Hereditary vitamin D-resistant rickets (HVDRR) is caused by heterogeneous inactivating mutations in the vitamin D receptor (VDR). Treatment of HVDRR patients with high doses of oral calcium and supraphysiologic doses of 1 alpha,25-dihydroxyvitamin D(3) (1,25D(3)) has had limited success. In this study we explored the use of vitamin D analogs as a potential therapy for this disorder. The rationale for the use of vitamin D analogs is that they bind the VDR at different amino acid residues than 1,25D(3), and their ability to modulate VDR functions differs from that of the natural hormone. In this report, we examined the VDR from three HVDRR patients with mutations in the ligand-binding domain of the VDR (histidine 305 to glutamine, arginine 274 to leucine, and phenylalanine 251 to cysteine) for their responses to two vitamin D analogs, 20-epi-1,25D(3) and 1 beta-hydroxymethyl-3-epi-16-ene-26a,27a-bishomo-25D(3) (JK-1626-2). Our results reveal that vitamin D analogs partially or completely restore the responsiveness of the mutated VDR. Analog treatment seemed to be more successful when the mutation affects the amino acids directly involved in ligand binding rather than amino acids that contribute to a functional VDR interface with dimerization partners or coactivators of transcription.

Differential regulation of heterodimerization by 1alpha,25-dihydroxyvitamin D(3) and its 20-epi analog.

Twenty-epi analogs of 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) are 100-1000 times more potent transcriptionally than the natural hormone. To determine to what extent this enhanced activity is mediated through modulation of the dimerization process, we performed quantitative dimerization assays with in vitro translated vitamin D receptor (ivtVDR) and fusion proteins containing glutathione-S-transferase (GST) and either the ligand-binding domain of VDR (GST-VDR) or retinoid X receptor (RXR)alpha (GST-RXR). We found that VDR did not form homodimers in either the presence or absence of ligand, but heterodimerization of the ligand-binding domains of RXRalpha and VDR was primarily deltanoid-dependent. The ED(50) for induction of heterodimerization was 1-2 x 10(-)(9) M for 1alpha,25(OH)(2)D(3) and 0.5 x 10(-)(11) M for 20-epi 1alpha,25(OH)(2)D(3). Mutations in VDR's activation function 2 domain (AF-2) diminished the abilities of 1alpha,25(OH)(2)D(3) to induce a protease-resistant conformation and heterodimerization. These mutations changed neither the potency of 20-epi-1alpha,25(OH)(2)D(3) to induce protease-resistant conformation nor its potency to induce dimerization. Mutations in heptad 9/helix 10 abolished the ability of both 1alpha,25(OH)(2)D(3) and the 20-epi analog to induce dimerization, but not their potency to fold VDR into a protease-resistant conformation. We hypothesize that both the hormone and the analog stabilize receptor conformations that expose VDR's dimerization interface, and that interfaces exposed by these ligands are probably not significantly different. However, the mechanisms by which the two ligands expose the dimerization interface are different with respect to participation of the AF-2 domain.