Developmentally regulated GTP-binding protein 2 ameliorates EAE by suppressing the development of T H 17 cells 울산대학교생명과학부박정우

Developmentally regulated GTP-binding protein 2 ameliorates EAE by suppressing the development of T H 17 cells 울산대학교생명과학부박정우

DRG2 DRG: developmentally regulated GTP-binding protein Upstream activators P GAP G protein Inactive GDP GTP GEF G protein Active GTP GDP Downstream effectors (Kumar et al. 1992; Sazuka et al. 1992).

GTP-binding protein (G protein)

DRG - Different from well-known G protein subfamily (trimeric G, monomeric G, EF-TU) in size and amino acid sequences (5-10% aa identity) Novel subfamily of G protein 1 50 100 150 200 250 300 350 405 aa EF-TU Trimeric Gα ras DRG2 171 350 384 Ras -----MTEYKLVVVGAVG-VGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQY--------------MRTGEGFLCVFAINNTKSFEDIHQYREQI 100 EF-TU AKGEFIRTKPHVNVGTIGHVDHGKTTLTAALTFVTAAENPNVEVKDYGDIDKAPEERARGITINTAHVEYETAKRHYSHVDCPGHADYIKNMITGAAQMDGAILVVSAADGPMPQTRE HI 120 Gα -----MGAGASAEEKHSRELEKKLKEDAEKDARTVKLLLLGAGESGKSTIVKQMKIIHQDGYSLEECLEFIAIIYGN--------------TLQSILAIVRAMTTLNIQYGDSARQDDAR 101 DRG2 MGILEKISEIEKEIARTQKNKATEYHLGLLKAKLAKYRAQLLEPSKSASSKGEGFDVMKSGDARVALIGFPSVGKSTFLS ---------LMTSTASEAASYEFTTLTCIPGVIEYKGANI 111... : :.. Ras KRVKDSDDVPMVLVGNKCDLAAR----TVESRQAQDLARSYGIP----------------YIETSAKTRQG--------------VEDAFYTLVREIRQHKLRKL--------------- 171 EF-TU LLARQVGVPYIVVFMNKVDMVDDPELLDLVEMEVRDLLNQYEFPGDEVPVIRGSALLALEQMHRNPKTRRGENEWVDKIWELLDAIDEYIPTPVRDVDKPFLMPVEDVFTITGRGTVA TG 240 Gα KLMHMADTIEEGTMPKEMSDIIQRLWKDSGIQACFERASEYQLNDSAG-----------YYLSDLERLVTPG------------YVPTEQDVLRSRVKTTGIIETQFSFKDLNFRMFDVG 198 DRG2 QLLDLPGIIEGAAQGKGRGRQVIAVARTADVIIMMLDATKGEVQRSLLEK----------ELESVGIRLNKHKPNIYFKPKKGGGISFNSTVMLTQCSEKLVQLILHEYKIFNAEVLFRE 221. :... : :. : Ras ------------------------------------------------------------------------------------------------------------------------ EF-TU --RIERGKVKVGDEVEIVGLAPETRKTVVTGVEMHRKTLQEGIAGDNVGVLLRGVSREEVERGQVLAKPGSITPHTKFEASVYVLKKEEGGRHTGFFSGYRPQFYFRTTDVTGVVQLP PG 358 Gα GQRSERKKWIHCFEGVTCIIFIAALSAYDMVLVEDDEVNRMHESLHLFNSICNHRYFATTSIVLFLNKKDVFFEKIKKAHLSICFPDYDGPNTYEDAGNYIKVQFLELNMRRDVKEIYSH 318 DRG2 DCSPDEFIDVIVGNRVYMPCLYVYNKIDQISMEEVDRLARKPNSVVISCGMKLNLDYLLEMLWEYLALTCIYTKKRGQRPDFTDAIILRKGASVEHVCHRIHRSLASQFKYALVW GTSTK 341 Ras ----------------------------------------------- EF-TU VEMVMPGDNVTFTVELIKPVALEEGLRFAIREGGRTVGAGVVTKILE 405 Gα MTCATDTQNVKFVFDAVTDIIIKENLKDCGLF--------------- 350 DRG2 YSPQRVGLTHTMEHEDVIQIVKKIQLLDLPGIIEGAAQGKGRG---- 384

DRG - Belongs to Obg family - Five subfamily: DRG, Obg, YyaF/YchF, Ygr210, NOG1 J. Mol. Biol. 2002 317: 41-72 Obg: spo0b-associated GTP-binding protein

DRG - OBG family - Involved in initiation of sporulation and assembly of the 50S ribosome subunit in bacteria PLoS Biol 2014 12: e1001866

DRG - DRG has been found in a wide variety of organisms (from bacteria to human cells) - High sequence identity aa sequence identity (%) Mouse Bovine Xenopus Zebrafish Human DRG2 98 98 95 91 Important roles in biological process. 7

DRG: Published articles Mouse DRG1 was identified by a subtractive cdna cloning approach that is expressed at high levels in developing mouse brain (Kumar et al. 1992; Sazuka et al. 1992). Human DRG2 was originally identified due to its down-regulation in fibroblasts transformed by SV40 (Schenker et al. 1994). Fish DRG2 was increased by rhabdovirus infection (Lee et al. 1998). Mouse and human DRG1 was found to interact specifically with the SCL (TAL-1) oncogene (Mahajan et al. 1996; Zhao et al. 1998). The DRG1 and the DRG2 mrna are widely expressed in human and mouse tissues and show a very similar distribution pattern (Li & Trueb 2000). Overexpression of DRG2 increases G2/M phase cells and decreases sensitivity to nocodazole-induced apoptosis. (Song et al. 2004; Ko et al. 2004) DRG family regulatory protein (DFRP) 1 and DFRP2, which regulate expression of DRG proteins through specific binding (Ishikawa et al. 2005) Transcription factor Sp1 regulates basal transcription of the human DRG2 gene. (Kim et al. 2011) Overexpression of developmentally regulated GTP-binding protein 2 (DRG2) increases bone loss. (Ke et al. 2013)

Multiple sclerosis

Multiple sclerosis: autoimmune disease mediated by T H 1 and/or T H 17 Immunity 2011 34: 3 5

DRG2 overexpression attenuates EAE EAE (experimental autoimmune encephalomyelitis): animal model for multiple sclerosis MOG (Myelin Oligodendrocyte Glycoprotein) C57BL/6 Wild type DRG2-TG

Delayed onset and lower severity of EAE in DRG2 TG mice

DRG2 TG mice develop mild EAE. Reduced immune cells infiltration and demyelination in DRG2 TG mice spinal cord No Treatment MOG treatment H&E Luxol fast blue H&E Luxol fast blue WT DRG TG Luxol fast blue: stain the myelin sheaths

DRG2 TG mice develop mild EAE. Reduced immune cell infiltration in the spinal cord of DRG2 TG mice

DRG2 TG mice develop mild EAE. Reduced T H 17 cell infiltration in the spinal cord of DRG2 TG mice. CD4 + IL-17 + : T H 17 CD4 + IFNγ + : T H 1

Immune cell subsets in lymphoid organs of naïve WT and DRG2 TG mice. Bone marrow Thymus Naïve mice Spleen Lymph node

Activated T cells in spleen and lymph nodes of naïve WT and DRG2 TG mice. Total cell Total cell Naïve mice

No difference in the T H 1 and T H 17 population in lymph nodes of naïve WT and DRG2 TG mice. Naïve mice

DRG2 overexpression does not affect the level of IL-10 in peripheral lymphoid organs.

Decrease in the T H 17 population in the peripheral lymphoid organs of DRG2 TG mice EAE-induced mice Spleen Lymph node

Decrease in the proinflammatory cytokines in peripheral immune cells of DRG2 TG mice EAE-induced mice

IL-6 is essential for T H 17 differentiation APC Nature 2008 453: 1051-1057

Effects of DRG2 on the T H 17 differentiation program Normal differentiation into T H 17 cells under T H 17-inducing conditions Naïve CD4 + T cells WT TG Anti-TGFβ + anti-il4, anti-il12 for T H 0 IL-12 + anti-il4 for T H 1 TGF-β + IL6 for T H 17 TGF-β for T reg FACS analysis for T H differentiation ELISA FACS analysis for IL-17 and IFN-γ

Effects of DRG2 on the IL-6 production in APCs APCs from DRG2 TG mice are defective in IL-6 production and induction of T H 17 differentiation APC WT Naïve CD4 + T WT H37 Ra TG WT H37 Ra: M. tuberculosis extract

NF-κB activation is essential for IL6 production Cell Metabolism 2006 4: 417 419

Effects of DRG2 on NF-kB binding to the IL-6 promoter DRG2 suppresses binding of NF-κB to the IL-6 promoter RAW 264.7 cells ChIP EMSA

Effect of DRG2 on the NF-κB activation pathway Exogenous or endogenous ligands TLR DRG2 does not affect the IkB degradation in the NF-κB pathway NEMO/ IKKγ IKKα IKKβ IκB p50 p65 IL-6 p50 p65

ProtoArray Human Protein Microarray nc v3.0 Add DRG2 Protein, wash and add DRG2 antibody (chicken) Wash and add anti-chicken Alexa-647 antibody Wash, dry, And scan 5,000 proteins Control DRG2

Protein Chip: DRG2 interacts with ADA3 Gene symbol Z-score coiled-coil domain containing 97 (CCDC97) 13.00817 TSR2, 20S rrna accumulation, homolog (S. cerevisiae) (TSR2) 11.16517 ubiquitin carboxyl-terminal esterase L3 (ubiquitin thiolesterase) (UCHL3) 11.14548 mitogen-activated protein kinase kinase kinase 11 (MAP3K11) 10.4947 serologically defined colon cancer antigen 3 (SDCCAG3), transcript variant 2 9.98918 RAB24, member RAS oncogene family, 8.53845 minichromosome maintenance complex component 6 (MCM6) 7.87113 c-src tyrosine kinase (CSK) 7.59655 transcriptional adaptor 3 (NGG1 homolog, yeast)-like (TADA3L), transcript variant 2 7.56097 WEE1 homolog (S. pombe) (WEE1) 7.55001 c-mer proto-oncogene tyrosine kinase (MERTK) 7.34578 Rho GTPase activating protein 15 7.19904 mitogen-activated protein kinase 7 (MAPK7), transcript variant 3 7.05786 calumenin (CALU) 7.02515 RNA-binding region (RNP1, RRM) containing 3 6.42103 cellular retinoic acid binding protein 2 (CRABP2) 6.15518 mitogen-activated protein kinase kinase kinase 9 (MAP3K9) 6.11786 coatomer protein complex, subunit zeta 1 (COPZ1) 6.10786 CDC-like kinase 1 5.83851 fms-related tyrosine kinase 1 (FLT1) 5.66684 GTP binding protein, alpha inhibiting activity polypeptide 2 (GNAI2) 5.39448 zinc finger protein 740 5.18279 FYN oncogene related to SRC, FGR, YES (FYN) 5.00762 catenin (cadherin-associated protein), delta 1 4.97077 CDC-like kinase 3 4.92392 ribosomal protein S6 kinase, 90kDa, polypeptide 5 (RPS6KA5) 4.86469 insulin receptor (INSR) 4.86167 NEDD8-specific protease 1 (SENP8) 4.84579 protein tyrosine phosphatase, non-receptor type 5 (striatum-enriched) (PTPN5) 4.77957 ribosomal protein L41 (RPL41), transcript variant 1 4.74066 phosphodiesterase 4A, (PDE4A) 4.69873 coilin (COIL) 4.53166 DRG2 ADA3

DRG2 interacts with PPARγ and enhances PPARγ activity DRG2 ADA3 PPARγ RXR Rosiglitazone: PPARγ agonist

PPARγ mediates inhibition of the NF-κB activity PPARγ activation induces PPARγ sumoylation activated PPARγ stabilizes NCoR binding to kb binding sites inhibits the NF-kB binding to the kb site IL-6 Trends in Mol Medicine 2012 18: 283 291

DRG2 stabilizes NCoR and inhibits NF-κB activity and IL-6 production PPARγ sumoylation ChIP NCoR stabilization GW9662: PPARγ antagonist

DRG2 suppresses inhibits IL-6 production from U937 human monocytes Fig. S9

Proposed model for DRG2 action mechanisms ADA3 Exogenous or endogenous ligands TLR Brain inflammation IL-17 RXR PPARγ T H 17 CNS DRG2 p50 p65 Blood-brain barrier ADA3 DRG2 ADA3 DRG2 RXR PPARγ SUMO T H 17 Periphery RXR PPARγ NCoR p50 p65 IL-6 T H 0

Inactive GDP DRG2 Upstream activators GEF GAP GTP DRG2 Active? Female fertility Aniridia Hair growth DRG2 DRG2 DRG2 DRG2 DRG2 ADA3/ PPAR Rac1 -synuclein DFRP Wee1 NF-kB Endosome and cell migration Mitochondria Protein synthesis Cell cycle Multiple sclerosis Inflammation Tumor metastasis Infection: Virus and Bacteria Parkinson s Obesity