Jaw1/LRMPの細胞内機能と生理的機能の解明

概要

Jawl/LRMP is a Type Π integral membrane protein that is localized at the endoplasmic reticulum (ER) and outer nuclear membrane (ONM). Howevei; its physiological fonctions have been poorly xmderstood. In this study the functional analysis of Jawl was performed from the intracellular and physiological perspectives.

Chapter 2 describes the role of Jawl to maintain nuclear shape as a KASH protein. In the nuclear envelope (NE), the linker of nucleus and cytoskeletons (LJNC) complex, a hetero hexamer consisting of KASH proteins and SUN proteins, fimctions to maintain the nuclear shape, nuclear position etc. KASH proteins are localized at the ONM while SUN proteins are localized at the inner nuclear membrane. The KASH domain, a carboxyl terminal region of KASH proteins, interacts with SUN domain of SUN proteins in the perinuclear space, resulting in the LINC complex. KASH proteins and SUN proteins are connected with cytoskeletons and nuclear lamina, respectively This physical linkage across the NE maintains the nuclear integrity Intriguingly an alignment of amino acid sequence of Jawl with KASH proteins reveals that Jawl has a partial homology to the KASH domain. Therefore, the fimctional analysis of Jawl at NE was performed. Here, the siRNA mediated Jawl knockdown in mouse melanoma cell line B16F10 caused an aberrant nuclear shape. Fxirthermore, co-immunoprecipitation indicates that Jawl oligomerizes and interacts with SUN proteins and microtubules. These data suggest that Jawl has a role in maintaining nuclear shape as a component of the LINC complex in the form of an oligomer.

In chapter 3, the role of Jawl cytosolic regions for oligomerization is described. Although some KASH proteins form an oligomei; it has not been uncovered yet which regions of Jawl is essential for this interaction. Furthermore, the fimction of Jawl cytosolic regions is still unknown. Therefore, this study focused on the role of three cytosolic regions for oligomerization· N-terminal region, coiled_coil domain and stem region. Co-immunoprecipitation showed that the coiled-coil domain is the oligomerization site of Jawl. Furthermore, the N-terminal region of Jawl is essential for the ordered oligomerization because the expression of the mutant lacking N-terminal region in B16F10 cells caused an organized smooth ER (OSER) which formed in the cells due to the overexpression or disordered oligomerization of ER resident proteins. This OSER was not significantly observed in the cells expressing mutant lacking both N'terminal region and coiled-coil domain. These results suggest that N-terminal region prevents disordered oligomerization of Jawl, inhibiting the structural exposure of coiled-coil domain.

In chapter 4, the physiological fimction of Jawl in the small intestinal tuft cell is descried. Although Jawl has been reported to be expressed in lymphoid organs and taste cells on the tongue, this is the first to identify the expression of Jawl in the tuft cell using Jawl IRES EGFP reporter mice. Tuft cell is a chemosensoiy cell that exists in a small population in intestinal epitheEum, senses the helminth infection and induces type2 immunity to expulse worms. With these in consideration, the role of Jawl in tuft cell-mediated type2 immunity was investigated using Jawl knockout (Jawl KO) mice.

Here, the helminth infection was mimicked with the administration of 200 mM succinate to the mice which can induce type2 immunity in the same pathway In the downstream of type 2 immunity the number of tuft cells is expanded to enhance the signal of helminth infection as well as the number of goblet cell to produce the mucosal barrier on the epithelium. As ejected, the administration of succinate to Jawl wildtype mice increased the number of tuft cells and goblet cells. However; the number was tmchanged in Jawl KO mice. Thus, Jawl is an important element involved in tuft cell mediated-type2 immunity.