Regulation Of Ubiquitin Ligase Smurfs And TGF β/BMP Signalling By The Linear Ubquitin Chain Assembly Complex(LUBAC)

Protein ubiquitination plays vital roles in regulating various cytobiological processes such as cell cycle progression, DNA damage repair, signal transduction and membrane localization of various proteins. Moreover,proteins can be modified by single ubiquitin molecules(monoubiquitination) or ubiquitin chains(polyubiquitination). Polyubiquitination regulates protein function by linking different types of polyubiquitin chains to substrates. All the 7 known linkage types of polyubiquitination are inter-ubiquitin linkages formed through lysine residues. In recent years, the eighth ubiquitin linkage type, linear ubiquitination in which the linkages are formed between the amino group of methionine residues of ubiquitin and the carboxy group of glycine residues of another, has been identified.In the year of 2006, professor Iwai Kazuhiro of the Osaka University laboratory in Japanese,first discovered linear ubiquitin complex(Linear ubiquitin chain assembly complex, LUBAC) exist, it is the E3 of linear ubiquitination. The LUBACcomplex is composed of HOIL-1L, HOIP, Sharpin.The deubiquitinating enzyme belongs to the key regulator of Ub signaling.It currently known linear ubiquitin specific DUB OTULIN(OTU domain-containing deubiquitinase includes with linear linkage specificity), CYLD. Cleaving the linear ubiquitination enzyme OTULIN play an important role in the regulation of Wnt signaling pathway and neural development, angiogenesis and other biological functions.Linear ubiquitin complex is playing an important role in the research field of immune and so on. At present, the known linear ubiquitin s Ubstrates are mainly NEMO, RIP2, RIP1 etc., in the process of their activation of the signal transduction pathway. The activation of LUBAC is involved in multiple signaling pathways.LUBACcan specifically activate the NF-κB signaling pathway and mediated ERK activation.ubiquitination system timely and selectively regulate cell function in its substrates with ubiquitin chains, so the identification of linear ubiquitination substrate is very necessary, it will help to elucidate the linear ubiquitin to participate in the specific mechanism of regulation of various cellular functions. Looking for new connections and participate in linear ubiquitin recognition protein complex, it has become a hot research field of linear ubiquitin. It is important to know whether the linear ubiquitin mediated by LUBAC is involved in other signaling pathways as well as new substrates of LUBAC.Smurf1/2 is a leading member of the E3 Nedd4 HECT family and is currently reported to be involved in a variety of cell biology functions. Smurf1 has been found in Smad5 yeast two hybrid studies and Smad5 was the first reported E3, Smurf1 through degradation of classical BMP pathway receptor Smads(e.g. Smad1 and Smad5) and BMP receptor, negative regulation of the BMP signaling pathway and TGFβsignalling. An important substrate protein Smurf1 include Rho A and Prickle1 which regulate cell polarity of Cells, by targeting their ubiquitination. Smurf1 regulates cell polarity, cell migration, Wnt signalling pathway functions.In the process of the research of the regulator mechanism of Smurfs, using yeast two hybrid technology of the spleen library screening by Smurf2, Our group found HOIP which as a core of linear ubiquitin complex molecules.We suspect the Smurfs and HOIP might have a real interaction between them, at the same time what’s the relationship between the Smurfs and LUBAC linear complex, the Smurfs in the regulation of linear ubiquitin, play an important role in the process of LUBAC linear ubiquitin, whether to participate in the new signaling pathways regulating exert important function, these are worthy of our in-depth study.We find that smurfs can interact with HOIP bothe in vivo and in vitro. Other components of LUBAC complex HOIL-1L, Sharpin can not directly interact with Smurfs but after addition of HOIP, Sharpin and HOIL-1L can interact with Smurfs. In vivo CO-IP experiments it showed that HOIP, HOIL-1L, Sharpin can interact with HOIP. HOIP is a protein with a multi domain molecular weight of 120 KD, through the study we found that the C-terminal LDD domain of the HOIP can be combined with Smurf1-HECT-N-Lobe. LDD is known as the linear ubiquitin domain, in all RBR E3 ligase, LDD domain is specific to HOIP, and the specificity of theLUBACfunction is determined by the linear ubiquitin complex. Smurfs as regulatory relationship exists between a ubiquitin ligase and linear ubiquitin complex LUBAC,the Smurfs are involved in the linear ubiquitination.We found that single expression LUBAC complex components, the Smurfs expression levels were unchanged,co-expression LUBAC complex three member HOIP, HOIL-1L and Sharpin, Smurfs protein expression levels were downregulated, LUBAC did not affect the protein level of Smurfs E3 ligase activity of mutant C699 A and C716 A. Detection ubiquitination level of Smurfs, we found that only over-expression of HOIP, the level of Smurfs ubiquitination have no change, contrarily, over-expression LUBAC complex, the level of ubiquitination of Smurfs significantly raised. When knock down the compents of LUBAC complex such as HOIP/HOIL-1L/Sharpin, the level of Smurfs was down regulated, and the same conclusion was obtained in vitro. HOIP C885 is a LUBAC E3 activity center, the C885 A mutant inhibits the upregulation of Smurfs in vitro.OTULIN, as a linear deubiquitinase, can inhibit the increasing of the level of ubiquitination. Smurfs ubiquitination levels are up-regulated and it promoted by LUBAC, that may be Smurfs occurs linear ubiquitination modification. LUBAC enhance E3 activity of Smurf, the reason is that HOIP breaks the Smurf1 to form dimers. We also find that LUBACcan inhibit the activity of TGF- beta /BMP signaling pathway. After knocking down HOIP, CAGA12-luc activity was up regulated, and the target genes SNON, CTGF, PAI-1, Smad7 which were related to the TGFβ signal pathway, were also up-regulated. At the same time, the activation of BRE-luc signal pathway activity was inhibited by LUBAC. What is the real mechanism by which LUBACinhibits the activity of the TGF- beta /BMP signaling pathway? LUBACcomplex can enhance the Smurfs ubiquitin levels, then for the Smurfs important s Ubstrate Rsmads, we found LUBACcomplex can accelerate Smurfs on degradation of Smads. This process is dependent on the Smurfs E3 activity.TGF- beta promotes cell migration, invasion, and metastasis, then what is the role of the LUBACcomplex and OTULIN in cell migration? By Transwell assay, we found that knockdown hoip can reduced the ability of cell migration, relatively, knocking down OTULIN. It is found in RT-PCR experiments, on cell migration, invasion and transfer the TGF beta signaling pathway related target genes PTHr P, MMP2, MMP9 also occurred corresponding up-regulated and down regulated in hoip knock down and OTULIN knock down cells. This topic research we found that through interaction with linear Ubiquitylation complex LUBAC core molecule HOIP, the Smurfs E3 activity was enhanced and accelerated Smurfs substrate degradation and inhibition of the activity of TGF beta signaling pathway, relatively, we also found that linear ubiquitin conjugating enzyme OTULIN and lack of LUBACthe E3 activity of mutants could be abolished the Smurfs ubiquitination enhancement effect and the activity regulation of TGFβ/BMP signaling pathway. This is the first time discovered the linear ubiquitin chemical complex and the linear ubiquitin- chemical modification are involved in the regulation of TGFβ/BMP signaling pathway. And it also has the new regulating mechanism of Smurfs E3 activity.Nedd8(neural precursor cell-expressed developmentally downregulated 8) is a kind of molecular which structure similar to ubiquitin, participate in posttranslational protein modification, this process called Neddylation.Mechanism of Neddylation and ubiquitination is similar, need a series of enzymes E1, E2, E3 mediated enzymatic reaction. Neddylation modified vital activity regulation in the Cullin-Roc ubiquitin ligase in action. Compared with the ubiquitination research present in eukaryotic cells,it has been only rarely Neddylation modify substrate found, the physiological function of Neddylation also needs further research. The Study of Ub and substrate combination is more clearly,It have been found closely to 20 domains(UBD) which can bind to ubiquitin, but for ubiquitin like protein Nedd8 of non covalent Neddylation is rarely.Smurf1 is an important member of the HECT Nedd4 ubiquitin ligase family,plays an important role in the process of bone formation, including embryonic development, cell polarity and in other physiological functions. Smurf2 and Smurf1 are highly similar, both play a very important role in the study of TGFβand BMP signaling pathways.This topic is using the Smurf1 bait yeast two hybrid screening and find the ubiquitin like protein Nedd8. By using GST-Pull down and Co-IP experiments,Smurf1 and Smurf2 were proved could be interacted with Nedd8 in vivo. We use a series of Smurf truncated domain in the interaction experiemts, found Smurf1 WW domain,N-lobe Small domain of HECT domain, C-lobe domain mediated by Nedd8 binding. Smurf2 and Smurf1 are the same, in the HECT domain of N-lobe Small domain and C-lobe mediated binding with Nedd8, but not found with WW domain.NEDL1 and NEDL2 like Smurfs also has been found that mediate binding. Then,in order to find the binding motif,we use the Nedd8 crystal structure and crystal structure of Smurf2-HECT by structure docking and simulate interaction. Throughsequence alignment, we found that the sequence L(X7)R(X4) F(X)ALQ is conservative. Mutant interaction experiments show that this motif mediated interaction. Mutants with the the Nedd8, the Neddylation of Smurf is weakened,stabilize the protein level, and change Smurf form Ub thioester intermediates but not Nedd8. Nedd8 interacting with Smurf plays a key role in ubiquitin level for Smurf E3 activity and substrates.