GRAIL: a unique mediator of CD4 T-lymphocyte unresponsiveness

Abstract

GRAIL (gene related to anergy in lymphocytes, also known as RNF128), an ubiquitin-protein ligase (E3), utilizes a unique single transmembrane protein with a split-function motif, and is an important gatekeeper of T-cell unresponsiveness. Although it may play a role in other CD4 T-cell functions including activation, survival and differentiation, GRAIL is most well characterized as a negative regulator of T-cell receptor responsiveness and cytokine production. Here, we review the recent literature on this remarkable E3 in the regulation of human and mouse CD4 T-cell unresponsiveness.

Figure 1

Schematic representation of the structural domains of GRAIL (Gene Related to Anergy In Lymphocytes). GRAIL is a 428 amino acids type I transmembrane single subunit ubiquitin E3 ligase protein with a cytosolic zinc-binding RING finger domain and a luminal or extracellular protease-associated (PA) domain. The RING finger domain is C2H2C3 type and functions as an ubiquitin E3 ligase, the PA domain captures transmembrane protein targets for ubiquitination. This split function motif is unique for a single protein E3 ligase. Unlike other E3 ligases, GRAIL is uniquely localized to the transferrin-recycling endocytic pathway.

Figure 2

Molecular basis of GRAIL regulation. GRAIL is associated with and regulated by two isoforms of the ubiquitin-specific protease otubain 1 (Otub1). Otub1, a deubiquitinating enzyme or DUB, binds to ub-GRAIL but does not deubiquitinate it. USP8, is a DUB that binds to GRAIL and to Otub1 in a tri-molecular complex. USP8 can function as a DUB for auto-ubiquitinated GRAIL, however USP8's DUB function is blocked by Otub1, but not catalytic mutants of Otub1 or it's alternatively spliced isoform, Otub1-ARF1. USP8 must be ubiquitinated to function as a DUB for auto-ubiquitinated GRAIL. Otub1, (but not catalytic mutants), de-ubiquitinates ubiquitinated USP8, inactivating it and allowing auto-ubiquitinated. GRAIL to be degraded by the 26S proteosome.

Figure 3

GRAIL and Otub1 regulation by the mTOR pathway controls naïve CD4 T cell proliferation. (left panel) Productive activation of naive CD4 T cells leading to proliferation comes about through TCR engagement (signal 1) and CD28 costimulation (signal 2); IL-2 production, signaling through the IL-2R leading to phosphorylation of Akt and activation of mTOR, expression of Otub1 protein, and subsequent GRAIL degradation, allowing proliferation to occur. (Right panel) Three independent mechanisms that block mTOR activation result in inhibition of naïve T cell proliferation. CTLA4-Ig blocks CD28 costimulation, does not allow IL-2 production, thus prevents Akt phosphorylation, mTOR is inactive, and Otub1 protein is absent, leading to the maintenance of GRAIL, inhibiting proliferation. Anti-IL-2 blocks IL-2R engagement, thus preventing Akt phosphorylation, mTOR is inactive, and Otub1 protein is absent, leading to the maintenance of GRAIL, inhibiting proliferation. Rapamycin blocks the activity of mTOR, prevents protein expression of Otub1, leading to the maintenance of GRAIL, inhibiting proliferation.