Crystal structure of suboptimal viral fragments of Epstein Barr Virus Rta peptide-HLA complex that stimulate CD8 T cell response.

Peptides presented by Human leukocyte antigen (HLA) class-I molecules are generally 8-10 amino acids in length. However, the predominant pool of peptide fragments generated by proteasomes is less than 8 amino acids in length. Using the Epstein - Barr virus (EBV) Rta-epitope (ATIGTAMYK, residues 134-142) restricted by HLA-A*11:01 which generates a strong immunodominant response, we investigated the minimum length of a viral peptide that can constitute a viral epitope recognition by CD8 T cells. The results showed that Peripheral blood mononuclear cells (PBMCs) from healthy donors can be stimulated by a viral peptide fragment as short as 4-mer (AMYK), together with a 5-mer (ATIGT) to recapitulate the full length EBV Rta epitope. This was confirmed by generating crystals of the tetra-complex (2 peptides, HLA and ß2-microglobulin). The solved crystal structure of HLA-A*11:01 in complex with these two short peptides revealed that they can bind in the same orientation similar to parental peptide (9-mer) and the free ends of two short peptides acquires a bulged conformation that is directed towards the T cell receptor. Our data shows that suboptimal length of 4-mer and 5-mer peptides can complement each other to form a stable peptide-MHC (pMHC) complex.

HNF4α Combinatorial Isoform Heterodimers Activate Distinct Gene Targets that Differ from Their Corresponding Homodimers.

Hepatocyte nuclear factor 4α (HNF4α) is a dimeric transcription factor that controls as much as 60% of all liver genes. However, how it achieves such broad functional diversity is unknown. Here, we show that inflammation and immune pathway genes are differentially regulated in an isoform-dependent manner, confirming that each isoform homodimer preferentially regulates a subset of HNF4α targets. With all 12 human HNF4α isoform clones, we tested combinatorial pairings to determine whether isoform heterodimers are functional. Indeed, synergistic and potent pairing combinations of isoform heterodimers were noted for HNF4α3-8, HNF4α6-12, and HNF4α5-8 that activated CYP7A1, IL6, and IL17A genes, respectively. Surprisingly, these genes are not at all activated by their corresponding isoform homodimers, suggesting that a particular heterodimer pair can regulate its own subset of target genes. Given the combinatorial possibility of 66 isoform heterodimers, our data provide the basis for a more detailed understanding of the diverse influence of HNF4α.