GRAIL (gene related to anergy in lymphocytes), as its name indicates, is involved in the induction of anergy in CD4+ T cells. However, until now, the factors that regulate the activity of this RING-type E3 ligase (which attaches ubiquitin to proteins that are destined for degradation) were unknown. Soares et al. now report in Nature Immunology that two isoforms of the ubiquitin-specific protease otubain-1 control the expression and function of GRAIL in the induction of anergy.

Full activation of CD4+ T cells requires two signals, and anergy results when a signal is received through the T-cell receptor (TCR) in the absence of a co-stimulatory signal. It is thought that GRAIL might induce anergy by ubiquitylation of membrane-associated targets that are required for T-cell activation. To investigate this system further, the authors used a yeast two-hybrid system to identify GRAIL-binding partners. Two isoforms of a gene containing a OTU domain, otubain-1 and alternative reading frame otubain-1 (otubain-1 ARF1), were shown to bind with high affinity to GRAIL and to co-precipitate with GRAIL in further assays.

What effect do these otubain isoforms have on GRAIL function in T cells? Co-expression of otubain-1 and GRAIL by mouse T-cell hybridomas resulted in the degradation of GRAIL and decreased GRAIL-mediated inhibition of interleukin-2 (IL-2) production, whereas co-expression of otubain-1 ARF1 caused an increase in GRAIL expression and enhanced inhibition of IL-2 production. Further experiments showed that the otubain-1 protein, rather than directly affecting the ubiquitylation of GRAIL, binds a deubiquitylating enzyme USP8 in a trimolecular complex with GRAIL, which regulates GRAIL ubiquitylation and degradation. Otubain-1 ARF1 binds GRAIL, not USP8, allowing USP8 to deubiquitylate polyubiquitylated GRAIL and so stabilize GRAIL function.

Next, the authors investigated the effects of overexpression of otubain-1/otubain-1 ARF1 on GRAIL function and the induction of anergy. Lethally irradiated mice were reconstituted with bone marrow from TCR-transgenic mice that had been retrovirally transduced to express one of these otubain isoforms. CD4+ T cells that constitutively expressed otubain-1 showed increased IL-2 production and enhanced proliferation in response to antigen in comparison to control cells. By contrast, cells expressing otubain-1 ARF1 were functionally anergic and they responded poorly to antigen in terms of IL-2 production and proliferation.

This study shows that two isoforms of otubain-1, in conjunction with the deubiquitylating enzyme USP8, have opposing effects on the expression and function of GRAIL in the induction of anergy.