Identification Of Linear Ubiquitination Substrates In Cancer Pathogenesis

Ubiquitin is a highly conserved polypeptide,which is comprised of 76 amino acids.Protein ubiquitination is one of the most common and important post-translational modifications in eukaryotes.It can be connected to proteins in seven different ways,on the basis of lysine residues internally connected to ubiquitins?namely K6,K11,K27,K29,K33,K48 and K63?.On the other hands,ubiquitination modifications can be either monoubiquitination or polyubiquitination by the length.The protein ubiquitination reaction is a complex enzyme cascade reaction,which concludes a ubiquitin activating enzyme?E1?,a ubiquitin conjugating enzyme?E2?and a ubiquitin ligase?E3?.Firstly,the ubiquitin is activated by the ubiquitin activating enzyme and then is subsequently transferred to the ubiquitin conjugating enzyme.Finally,the ubiquitin ligase connect ubiquitin with the substrate protein,which is essential to the whole process.Many biological process are participated in ubiquitinations,such as K48-linked protein degradation,immune response and programmed cell death^[1-2].In recent years,a new type of ubiquitination modification has gradually come into people's eyes with the further development of ubiquitination research,in which the glycine at the N-terminal of the ubiquitin monomer is connected with the methionine at the C-terminal of another ubiquitin monomer.Unlike the known lysine-dependent connection within the ubiquitins,the eighth ubiquitination modification,which occurs when the ubiquitin molecules are joined end to end,is known as linear ubiquitination^[3].The linear ubiquitin chain is crucial for NF-?B signaling and mainly participated in immunity response or cell death pathways.Its abnormal regulation can cause many diseases,such as cancer and neurodegeneration^[4],and become a public health problem that seriously threatens people's health and social economic development.NEMO?NF-kappa-B essential modulator?is one of the members of IKK complex in NF-?B signaling pathway and is the first substrate to be reported of linear ubiquitination.Its molecular structure is characterized by containing a UBAN?Ubiquitin binding in ABIN and NEMO proteins?domain which can recruit to and combine with linear ubiquitination chains^[5].In addition,we know little about other substrates modified by linear ubiquitination and their biological functions.And the exploration of more unknown linear ubiquitination substrates is particularly important for further exploration of its biological functions.However,it is greatly difficult to identify the related substrates and explore the function of them due to the special connection manner and the extremely low content.Therefore,one of the most critical problems is the lack of a systematic system for screening and identifying linear ubiquitination substrates.In this study,the UBAN domain of NEMO was used as a protein probe to screen potent linear ubiquitination substrates in cell lysis^[6].And in use of the above strategy,Aurora kinase A?Aurora-A?was identified as a potential substrate of linear ubiquitination by mass spectrometry.Aurora kinase?AKs?belongs to the family of serine/threonine kinases family,including Aurora-A,Aurora kinase B?Aurora-B?and Aurora kinase C?Aurora-C?three member,with greatly overlapping catalytic structural domain and functions.The AKs is pivotal to the regulation of cell cycle checkpoint,maturation and assembly of spindle and centrosome,chromosome separation and cytokinesis^[8-9].Abnormal expression of AKs can trigger the dysfunction of chromosomal and promote the formation of various malignant tumors,as well as the poor prognosis of tumors.Therefore,it has become one of the hot spots in the cancer-related public health field to develop anticancer drugs targeting AKs^[10].Aurora-A is the first and most important member of AKs and plays a preferential role in cell proliferation.It has been reported in the literature that the phosphorylation modification on Aurora-A can be used as an indicator of its activation^[11],and its own activity reaches the peak during the transition from G2 phase to M phase.In other phases,the kinase activity of Aurora A is maintained at a low level through the ubiquitin-proteasome pathway^[12].It has been reported that Aurora-A is overexpressed in a variety of malignant tumors and its kinase activity plays all important role in tumorigenesis and metastasis,in which it is becoming a novel target of anticancer drugs.In this study,we confirmed the mass spectrometry identification results through the ubiquitination experiment,namely,Aurora-A has the ability to bind to the linear ubiquitination chain.Further,the interference of linear ubiquitination E3 ligase LUBAC?Linear ubiquitination assembly complex?can up-regulate the phosphorylation level of Aurora kinase A,which is closely related to the kinase activity.These results suggest that linear ubiquitination may regulate the activity of Aurora-A by down-regulating its phosphorylation level,thereby participating in the regulation of mitotic cycle progression.Summarily,in this research,the UBAN domain of NEMO was used as a protein probe to screen potent linear ubiquitination substrates.Adopting the proteomics strategy,the putative substrates Aurora-A was identified.And the experiment was designed to verify that Aurora-A can bind to the linear ubiquitin chain.Further,phosphorylation level of Aurora-A was up-regulated and self-activated by interfering with LUBAC.The finding suggests that linear ubiquitination may be involved in the regulation of mitotic process.Significantly,the strategy of identifying the linear ubiquitination substrates established in this paper provides the novel idea and method for further exploring more biological functions of linear ubiquitination.Also,it is becoming the biological basis for further overcoming the public health challenge of tumor treatment.