The Regulation Of A Unique Leucine-rich Repeat Protein33in TLR Signaling And Smoothened Ubiquitination By A Ubiquitin Ligase Complex

Part I:The regulation of a unique leucine-rich repeat protein33(LRRC33) in TLR signalingToll-like receptors (TLRs) play an important role in innate immune responses against pathogenic microbes such as viruses, intracellular bacteria or parasites [1]. Although the innate immune system is designed to fight infectious pathogens, excessive activation of TLR signaling may lead to unwarranted inflammation with hazardous outcomes. For this reason, mechanisms of restraining excessive inflammation and controlling homeostasis for innate immunity are the focus of intense study.Experimental strategies:We first performed RT-PCR and in situ hybridization to examine the tissue distributions of LRRC33, and then analyzed whether LRRC33regulated NF-κB activities and the relationship with TLRs by NF-κB reporter assays, co-IP, ELISA and the translocation of p65(subunit of NF-κB). We finally analyzed the function of LRRC33in TLR signaling pathway by knockdown techniques in the monocyte-derived dendritic cells.Major findings:1. Using either SMART or Pfarm with several transmembrane-predicition and motif-detection programs, we identified that LRRC33, a novel member of leucine-rich repeat (LRR) protein family, played a critical role in desensitizing TLR signaling. LRRC33is TLR homolog that contains17putative LRRs in the extracellular region but lacks a cytoplasmic Toll/IL-1receptor (TIR) domain. Expression of LRRC33appears to be ubiquitous with high level of expression found in bone marrow, thymus, liver, lung, intestine and spleen. We also analyzed LRRC33expression in different cell lines. LRRC33could be detected in U937and also in monocytes; however, its level gradually decreased during GM-CSF-mediated cellular differentiation from monocytes to matured dendritic cells (mDCs). These data imply that LRRC33is important in cellular differentaion.2. The co-immunoprecipitation analysis showed that LRRC33interacted with TLR2/TLR3/TLR4/TLR9. In contrast with TLRs, LRRC33showed no association with MyD88. For example, functional analysis showed that LRRC33could inhibit TLR-mediated basal NF-κB activation, and this inhibitory effect was significantly enhanced when cells were stimulated with PGN and LPS, which are ligands for TLR2and TLR4respectively. Consistent with the inhibitory effect of LRRC33, the production of IL-8, a major cytokine induced by the TLR-mediated NF-κB activation, was greatly reduced when TLRs were co-expressed with LRRC33. Meanwhile, LRRC33also inhibits the nuclear translocation of p65(subunit of NF-κB).3. We found that the extracellular domain of LRRC33(EC) other than the intracellular domain of LRRC33(IC) retained the ability to interact with TLR2. Consistent with this observation, EC but not IC of LRRC33maintained the ability to inhibit NF-κB activation as well as IL-8production in cells. To further define the region required for the interaction of LRRC33with TLRs, we systematically deleted LRRs in LRRC33by constructing several deletion mutants, including D1(delete1-9LRRs), D2(delete10-14LRRs), D3(delete15LRRs), D4(delete16-17LRRs). The results suggest that these LRRs have no structural specificity in their association with TLRs. In addition, these mutants maintained, at least partially, the ability to inhibit NF-κB activation and the suppression of IL-8production.4. Silencing LRRC33in the dendritic cells (DCs) greatly increased NF-κB activation. Consistent with the finding, knockdown of endogenous LRRC33could drastically elevate the secretion of TNF-a by LPS stimulation. Interestingly, knockdown of LRRC33also induced the duration of LPS-mediated JNK and p38activation in DCs.Main conclusions:LRRC33plays a widespread and important role in controlling the homeostasis of innate immunity. LRRC33may interact with multiple TLRs to inhibit TLR-mediated NF-κB and AP-1activation, and decrease the production of cytokines. Thus, our study sheds a new insight into the understanding of the regulation of TLR signaling and inflammatory response. More importantly, LRRC33may potentially act as a target for the future treatment of human associated disease. Part II:Smoothened ubiquitination by a ubiquitin ligase complexHedgehog (Hh) protein function as morphogens and play critical roles in pattern formation and cell growth control [26]. Hh signaling has also been implicated in tissue repair and stem cell maintenance. Malfunction of Hh signaling causes birth defects as well as several types of cancers [27,35]. The basic scheme of Hh signal transduction is conserved from Drosophila to mammals. The receptor complex contains Patched (Ptc) and Smoothened (Smo). Ptc is the receptor. Smo is a signal tranducer which plays a critical role in regulating Hh signaling pathway. Although Smo has been extensively studied and some of the molecular mechanisms have been uncovered, there are still critical questions remaining. It has been reported that Hh and phorphorylation promote Smo accumulation and activation [68]. In addition, RNAi of E1(ubiquitin-activating enzyme) Ubal can upregulate Smo accumulation. Ubiquitination is normally involved in protein modification that leads to protein degradation, endocytosis, sorting and trafficking of transmembrane proteins [28,33]. This project is to identify and characterize the ubiquitination machinery for Smo, which will let us know how the levels of Smo is controlled and how ubiquitination regulates Smo intracellular trafficking.Experimental strategies:we performed RNAi screen of E3ligases for Smo, a genetic modifier screen to examine adult wing phenotypes, which is a more sensitive readout and the ubiqutination assay to directly detect Smo ubiquitnation in cultured S2cells. We then demonstrated the effects of E2-Eff and E3-CG9153RNAi on Smo ubiquitination by knocking down endogenous Eff/CG9153or overexpressing Eff/CG9153in S2cells. To test whether CG9135regulates Smo ubiquitiantion by interacting with Smo, we carried out co-immunoprecipitation experiments. To further map the CG9153domain responsible for interaction with Smo, we made CG9153trunctions (including Nl, N2and HECT). To determine the pathway for Smo degradation by CG9153, we treated S2cells with ammonium chloride (NH4CI), a lysosomal inhibitor or MG132, a proteasomal inhibitor. We also detected whether Cullins were also involved in Smo ubiquitiation by in vivo ubiquitination assays. We finally performed both loss-and gain-of function studies in wing discs to examine the regulation of Smo accumulation by Ubal, Eff, CG9153and Cul4.Major findings:1. CG9153which belongs to HECT E3and Eff may work cooperatively as the ubiquitin-protein ligase (E3) and ubiquitin-conjugating enzyme (E2) for Smo. Sliencing Eff or CG9153downregulates Smo ubiquitination, whereas overexpression of E3upregulates Smo ubiquitination, but the overexpression of Eff did not. Eff likely needs E3in order to transfer Ub to Smo. In addition, CG9153interacts with Smo, which is downregulated by Hh stimulation. At the same time, Hh upregualtes CG9153ubiquitination. Furthermore, Cu14is required for CG9153to enhance Smo ubiquitianion.2. CG9153may regulate the Smo stability either by the proteasomal degradation pathway or by the lysosomal degradation pathway.3. In wing discs, silencing Eff/Ubal increases Smo and Ci. The overexpression analysis demonstrates that CG9153downregulates Smo accumulation in a dose dependent manner.Major conclusions:CG9153regulates Smo ubiquitination by interacting with Smo. Ubal, Eff, CG9153and Cu14can form a complex to regulate Smo. All of these offer a new insight into the understanding of molecular mechanism of Smo regulation.