A Novel Construction Strategy And Functional Characterization Of M1-type Ubiquitin Chain Genes

Ubiquitination is one of the most important and widely distributed protein post-translational modification systems in the cell.Modifier,ubiquitin(Ub),is a small protein that has a molecular weight of 8.5 k Da.The 8 residues of an Ub molecule(Met1,Lys6,Lys11,Lys27,Lys29,Lys33,Lys48,or Lys63)can also be modified by other Ub molecules to form 8 types of poly-ubiquitin chains.The ?-carboxyl group of the Gly76 at C-terminal in an Ub molecule can conjugate to ?-amino group of the first Met at N-terminal in another Ub molecule to form peptide bond,there by generating M1-type Ub chains(also known as linear Ub chains).The modification of M1-type Ub chains on various cellular proteins plays an indispensable role in physiological processes,such as innate immunity,tumorigenesis,inflammation development,and neuro degenerative diseases.The M1-type Ub chain proteins are the important material for the investigation in this field.At present,there are three main methods for isolating ubiquitinated proteins: one is to use antibodies against a specific type of Ub chain to isolate ubiquitinated proteins;the second way is that the ubiquitinated proteins are hydrolyzed by trypsin to produce characteristics peptide segment(K-?-G-G),and then isolated using an antibody against this peptide segment;the third is to use the tandem Ub binding domain to isolate ubiquitinated proteins.These methods own advantages and disadvantages,respectively.The main problem of them is that these methods are not able to identify the known length and type of Ub chains directly.To analyze proteins modified by M1-type Ub chains,the current method is mainly carried out through the expression of LUBAC in vivo to generate the M1-type chain modification and the analysis of OTULIN.Ub chains in different lengths can be directly synthesized via an enzyme-catalyzed reactionin vitro.There are also reports that M1-type Ub chains with unnatural linkage could be prepared via genetic engineering method.None of these strategies for preparing M1-type Ub chains can determine the length of the chain,nor do they have a natural linkage.These products cannot truly reflect the natural chain modification mechanism in vivo.Therefore,M1-type Ub chain genes with natural linkage and known length are important to the research in this field.According to the characteristics of Ub gene sequence,in the current study,a novel construction strategy of M1-type Ub chain genes in vitro was invented.The coding sequence of Gly75 and Gly76 residues of Ub is "GGAGGC",and the sequenc CT was added down stream of the first Ub gene to create the Stu I restriction site "AGG?CCT".This modified Ub gene is cut to generate a blunt end of "Ub-GGAGG",which was cloned into p ET22 b vector to construct a "p ET22b-Ub-GGAGG".When designing the second Ub gene,we constructed a mono-ubiquitin gene fragment "C-Ub" with an additional base C added to its 5'end,while its 3'end was still added bases CT to generate Stu I,and we inserted this fragment into the "p ET22b-Ub-GGAGG" to generate M1-type Ub chain genes with an intact reading frame.The results showed that longer M1-type Ub chain genes with the correct molecular weight were obtained after several cycles of "C-Ub" fragment insertion.The constructed M1-type Ub chain genes did not have redundant nucleotides between adjacent Ub genes which conjugate with natural linkage.This study finally constructed the prokaryotic and eukaryotic expression vectors of the M1-type Ub chain genes with 2 to 8 Ub length,which proved the feasibility of this strategy.The constructed M1-type Ub chains were expressed in E coli.cells and further purified.The purified Ub2?8-GST proteins were subjected to specific hydrolysis by OTULIN except for Ub1-GST.The specific antibodies against M1-type Ub chains can also recognize the purified M1-type Ub chains except for Ub1-GST.These results suggested that the M1-type Ub chains prepared through the construction strategy of this study have a natural linkage mode and complete natural activity,and can be used to study the function of intracellular modification.For the functional characterization,the fluorescence detection showed that the M1-type Ub chain genes could be expressed normally in human cells.The Pull-Down experiment using affinity tags showed that the exogenously expressed M1-type Ub chains modified the endogenous proteins.Some modified target proteins were extracted from the total cellular proteins and hydrolyzed by OTULIN.The M1-type Ub chains with different length presented different modification ability.The cell viability test results suggested that the viability of the cells did not change significantly after exogenous expression of different length of M1-type Ub chain genes,and no significant apoptosis occurred compared with control groups.The obtained M1-type Ub chain proteins was further used to screen DNA aptamers.The results showed that aptamer candidates were able to bind to Ub4-GST.In this study,we invented a novel construction strategy of M1-type Ub chain genes.The purified M1-type Ub chain proteins were fully active and linked with natural linkaged mode.M1-type Ub chain genes could express in human cells and functionally modified cellular proteins.The transfected M1-type Ub chain genes did not induce the significant change of cell viability and apoptosis.Aptamers with certain affinity activity were screened for Ub4-GST.Summarily,the M1-type Ub chain genes constructed with our invent stretagy can be functionally used for M1-type Ub chain modification reaserch.The current study provided important tools and techniques to the colleagues in this field.