The Establishment Of System For Identification Of Linear Ubiquitin Ligase LUBAC High-throughput Substrates

Ubiquitination is a post-translational modification involved in the regulation of a variety of cellular functions, such as cell cycle、DNA repair、signal transduction and proteasomal degradation[1]. Ubiquitin can be linked to target proteins in different ways,as either a monomer or polyubiquitin chains, which are linked through seven internal Lys residues（K6、K11、K27、K29、K33、K48 and K63）[2]. These eight different types of polyubiquitin chains participate in different biological phenomena. Monoubiquitination is involved in membrane trafficking and endocytosis[1]. Among polyubiquitination, the K48-linked polyubiquitinchain, the most dominant polyubiquitin chain, serves as a degradation signal for the 26 S proteasome, and the K63-linked polyubiquitin chain is involved in signal transduction and DNA repair. The K11-linked polyubiquitin chain participates in degradation during cell cycle progression and NF-"B signaling[1]. The protein ubiquitination reaction is catalysed by three enzymes: a ubiquitin-activating enzyme（E1）, a ubiquitin-conjugating enzyme（E2） and a ubiquitin ligase（E3）. First, the C-terminal carboxyl group of ubiquitin is activated by E1 through ATP to AMP hydrolysis and is then conjugated to the active site Cys residue. The ubiquitin is subsequently transferred to an active Cys of E2. E3 selectively recognizes both the substrates and E2, and catalyses ubiquitin transfer from E2 to the target proteins. So the E3 is critical in this reaction. In 2006 a new type of N-terminal M1-linked linear polyubiquitination was found by professor Kazuhiro Iwai in Osaka City University.M1-linked linear polyubiquitination is not linked through internal Lys residues of ubiquitin,but the N-terminal Met1[3].The human genome encodes more than 600 E3 s while LUBAC is the only E3 known to generate an M1-linked linear polyubiquitin chain.LUBAC is composed of HOIP（HOIL-1-interacting protein,also known as RNF31、ZIBRA and PAUL）, HOIL-1L（longer isoform of haem-oxidizediron-regulatory protein ubiquitin ligase 1）and SHARPIN（ Shank-associated RH domain-interacting protein）.HOIP is responsible for linear ubiquitin formation,while HOIL-1L or Sharpin can’t generate an linear polyubiquitin chain only when HOIP is recruited.Since the discovery of linear polyubiquitin and LUBAC, people are getting more and more interesting in them.It’s very important to identify the substrates of LUBAC for linear ubiquitination regulates a multitude of critical cellular processes through linking the linear polyubiquitin chain tosubstrates.Identification of LUBAC substrates will help to illuminate how linear ubiquitination regulates cellular processes.So far the known substrates include NEMO（NF-"B essential modulator）[4]、TRIM25（ISG15 ligase TRIM25）[5]、RIPK2（Receptor-interacting serine/threonine-protein kinase 2）[6]and ASC（Apoptosis-associated speck-like protein containing a CARD）[7]. Linear polyubiquitin mainly plays a viral role in innate immunity and inflamematory response suppression.It’s significant to identify the substrates exactly and systematic.CRISPR/Cas9（clustered regularly interspaced short palindromic repeat sequences） is wildly used as a newly tool for genome editing,which can recognize and edit DNA through RNA. CRISPR/Cas9 system uses a pair of single guide-RNA（sgRNA）to target the upstream and downstream of gene, Cas9 endonuclease recruited to cut off DNA double-strand, then DNA repair beginning with non-homologous end joining which is imprecise,knock out the target gene at last. Comparing with Zinc-finger nuclease（ZFN）and transcription activator-like effector nuclease（TALEN）,CRISPR/Cas9 is more easy and available,making it a more and more important technology for gene editing.As a crucial tool of proteomics study,mass spectrometry plays an indispensable role in recognize of protein posttranslational modification,identifying sites,structure solution and quantitative determination. Development of mass spectrometry promotesstudy of protein posttranslational modification,increasing identify efficiency,coverage ratio,location accuracy and quantitative accuracy of modification sites. Currently in platform for proteomic analysis centered on mass spectrometry technology,multiple posttranslational modification study including ubiquitination is developing in a more fining,large scale,coordinating andquantifying direction. Protein posttranslational modification in complex biological system is charactered by low abundance,large dynamic range and heterogeneity. Enrichment and purification methods are developed for high identify efficiency and accuracy.So far, enrichment methods of ubiquitination include antibody purification specific to K-diGly residue at peptide level and affinity purification specific to UBD domains at protein level[12].These methods offer a favorable condition for much deeper identification of scale protein ubiquitination at protein level.Except to be linear polyubiquitin E3, the other compents of LUBAC（Sharpin and HOIL-IL）can generate K48、K63 polyubiquitin chain.In order to specially identify linear polyubiquitin, we plan to knock out endogenous HOIP using CRISPR/Cas9 system in HeLa cell,then overexpress exogenous HOIP^WT and HOIP^CS instantaneously. Linear polyubiquitin of NEMO which is the known substrate of LUBAC as the positive control,via affinity mass spectrometry,we can compare sites and ubiquitin protein which may be the new substrate of LUBAC between the two groups. Firstly We chose tow CRISPR knockout sequences referred to CRISPR design website,build them into lenti-CRISPRv2 vector, established the stable HeLa cell line with knocking out HOIP gene. On this basis,we overexpressed exogenous HOIP^WT and HOIP^CS instantaneously,then we observed the ubiquitin of NEMO in this cell line,verifying the relationship between enzyme HOIP and substrate NEMO.From the above,we established the cell system which overexpress exogenous HOIP^WT and HOIP^CS instantaneouslyon the basis of knocking out endogenous HOIP gene to identify new substrates of LUBAC. The new substrates will help us find new function of LUBAC,explore specific mechanism of linear polyubiquitinationinvolved in various biological regulation and extend understanding of linear polyubiquitination in regulation of cell function.