RA Mediated T-reg Differentiation

Introduction

T-regulatory cells (T-regs) are a small subset of CD4+ T-cells that exhibit strong down regulation of immune system activity in their local environment. They are distinguished from other CD4+ T-cells by the expression of FOXP3, a gene regulator. ^[1] The exact mechanisms used by T-regs to down regulate the immune system has not yet been clearly elucidated. These cells have been shown to differentiate from CD4+ T-helper cells upon activation and exposure to the following cytokines: tumor growth factor β (TGF-β), Interleukin-2 (IL-2) and retinoic acid (RA). ^[2] Both TGF-β and IL-2 are used in other immune system differentiation, however, RA has been shown to bias T-cells to the T-reg phenotype. ^[3] When acting upon T-reg cells, RA acts as the ligand for the Retinoic Acid Receptor-α (RARα) / Retinoid X Receptor-α (RXRα) heterodimer. This heterodimer is of the nuclear receptor family, and each chain consists of the same three part structure: a Ligand binding domain (LBD), a DNA binding domain (DBD), and a hinge region connecting the two binding domains. ^[4] (PDB entry 1dkf).

Ligand Binding Domain

The Ligand binding domain for each piece of the dimer has a nearly identical structure of an . These alpha helices form a total of 12 domains per protein (referred to as H1-12), with an additional 2 beta sheets as well. Additionally, the α-helical sandwich formed has been shown to bind All-Trans Retinoic Acid (ATRA), the isomer of RA used by the body. Both monomers contain two regions of activity, the and the .^[5]

RARα-RXRα Heterodimer

When these two proteins form a heterodimer, the overall structure of the larger dimer is comparable to that of an RXRα homodimer, likely due to the many similarities these two molecules share. RARα and RXRα rely on residues from the H7, H8, H9, H10, L8-9, and L9-10 domains of both molecules to form the . The sequence identity between the two molecules on the dimer interface is 0.33, demonstrating that 33% of the interacting residues are homologous between the different proteins.

The residues of α that are interacting in the heterodimer are as follows: Hydrophobic residues: L356, F374, P375, L378, M379, I381 and A389 (yellow); Negatively charged residues: D338, D349, E353, E357, D383, and E393 (red); Positively charged residues: K360, R364, H372, K376, K380, and R385 (blue); Hydrophilic residues: Q315, Q352, T382, and S386 (green).

The residues of α that are interacting in the heterodimer are as follows: Hydrophobic residues: Y402, P417, F420, A421, L424, L425, L427, P428, A429, and L435 (yellow); Negatively charged residues: E357, D384, E395, E399, E406, and E439 (red); Positively charged residues: R353, K361, R398, K410, K422, R426, R431, and K436 (blue); Hydrophilic residues: S432 (green). ^[6]

RARα-RXRα Ligand Interactions

Upon binding of the ligand ATRA in the cytoplasm, RARα and RXRα form a heterodimer and alter the C-terminals on domain H12 of both subunits in a manner that allows them to change the conformation of their DNA binding domains. The two proteins have 29% identity in their . For the ligand used in RARα crystallization, BMS614, 21 primarily hydrophobic residues form the . BMS614 is not the natural ligand for this molecule, but acts as an more stable agonist for crystallization. The largest difference between BMS614 and ATRA upon binding to the pocket are at Ile 412, where BMS614 pushes much closer to the amino acid than ATRA does. Residues that form the binding pocket are found on H1, H3, H5, H11, L6-7, and L11-12 on RARα. The between RARα, RARβ and RARγ are present in this area: Residue 270: α:Ile β:Ile γ:Met; Residue 232: α:Ser β:Ala γ:Ala; Residue 395: α:Val β:Val γ:Ala

The is comprised of 16 primarily hydrophobic residues, found on the H3, H5, H7, H11, and L11-12 domains. The ligand used in the crystal, Oleic Acid, is similar to RA, and RA is capable of binding to the RXRα pocket.^[7]

DNA Binding Domain

(PDB entry 1by4). When RXRα homodimers assemble on DNA, they form a four poplypeptide complex assembled via head to tail interactions along DR-1 repeated sequences. The structures of the polypeptides sit in the major grooves of the DNA chain, allowing for interaction with specific bases, giving a sequence specificity for the protein. The two do not alter their configuration upon DNA binding, but are used to guide the DNA into the correct position. Upon binding to DNA, the C-terminal end of the protein, referred to as the alters its conformation from alpha helical to an extended conformation. This extended conformation allows Glu74 to move away from the DNA binding pocket and moves it so it interacts with the Zn(II) domain of the next polypeptide. ^[8]

RXRα-DNA Interactions

RXRα homodimers preferrentially assemble on DR-1 repeat sequences. DR-1 sequences are composed of an AGGTCA tandem repeat, with a single nucleotide spacer in between the repeats. Only Lys22, Lys26, Glu19 and Arg27 interact with the DNA bases directly. interact with the phosphate backbone of the DNA molecule, making sure it is in position for base recognition. RXRα homodimers have also been shown to assemble on DR-2 tandem repeats, sequences with the same organization as DR-1, but with two nucleotides as a spacer. The DNA interaction is similar with DR-2 repeats, just spaced further apart. ^[9]

DR-5 Specificity

RXRα/RARα homodimers have not yet been crystallized on DNA, but have been shown to associate with extended DR-5 tandem repeats. The sequence of this DR-5 repeat is AGGTCA-nnnnn-AGGTCA. Additonally, when incubated with retinoic acid, cells expressing RARα showed high expression of genes located downstream of DR-5 tandem repeats. ^[10]

Biological Significance

Since T-regulatory cells are so highly regulated in the body, elucidating the exact mechanism of activation can show how these immune processes work, and use them in the treatment of disease. Two clear mechanisms of regulation arise from the studies, both which are related to the heterodimer itself. First, the ligand specificity for RA in both molecules allows for specific signaling of these molecules. RA is not normally expressed in cells, and therefore will limit when this heterodimer is activated. Likewise, the propensity for the heterodimer to associate with DR-5 repeats limits the number of genes it will activate to a select few. All of this in addition to the other cytokines necessary, TGF-β and IL-2, show the complex mechanisms regulating the differentiation of T-helper cells into T-regulatory cells.