Defining the importance of protein geranylgeranylation in innate immunity

Abstract

RHO family proteins and other intracellular proteins are prenylated with a 20-carbon lipid—a product of the cholesterol synthesis pathway—by protein geranylgeranyltransferase type I (GGTase-I). Prenylation is widely believed to target proteins to membranes where they encounter effector molecules that stimulate GTP-binding and activation. However, my host group found that knockout of GGTase-I in mouse macrophages (Pggt1bΔ/Δ) actually increases GTP-loading of RHO proteins such as RAC1, RHOA, and CDC42, and also increases proinflammatory signaling and cytokine production, and induces severe rheumatoid arthritis. These results suggest that prenylation may inhibit rather than stimulate RHO protein function. The mechanisms underlying increased GTP-loading and exaggerated innate immune responses in the absence of GGTase-I are not known. During my PhD, I have addressed these issues in two independent but interconnected projects. In project 1, we found that there is an imbalance between inflammatory and anti-inflammatory cytokines produced by Pggt1bΔ/Δ macrophages. We also found that knockout of GGTase-I prevents the interaction between KRAS and PI3K catalytic subunit p110δ and that this reduces signalling through the PI3K-AKT-GSK3β pathway. Moreover, Pggt1bΔ/Δ macrophages exhibit increased caspase-1 activity that is directly responsible for the production of active interleukin IL-1β, and that this effect requires the MEFV (pyrin) inflammasome. Thus, we conclude that GGTase-I promotes an association between KRAS and p110δ and thereby controls major inflammatory pathways in macrophages. In project 2, we tested the importance of RHO proteins in the development of arthritis in Pggt1bΔ/Δ mice. We found that knockout of Rac1 (i.e., in Pggt1bΔ/ΔRac1Δ/+ mice), but not Rhoa and Cdc42, markedly reduced inflammatory cytokine production and arthritis in Pggt1bΔ/Δ mice. We also found that non-prenylated RAC1 bound more strongly to the RAS GTPase-activating-like protein 1 (IQGAP1) – which facilitated RAC1 GTP-loading and activation. Knockout of Iqgap1 in Pggt1bΔ/Δ mice abolished cellular phenotypes in vitro and inhibited arthritis in vivo. Thus, we conclude that blocking prenylation stimulates RAC1 effector interactions and activates wide-spread pro-inflammatory signaling. Thus, prenylation normally restrains innate immune responses by inhibiting RAC1 effector interactions.