Julien Franck (Presenter)
PRISM INSERM U1192 Lab
Bio: Since 2012, i have integrated the team of Pr. Isabelle Fournier in the PRISM Lab at university of Lille (France) as associate professor. My projects are mainly dedicated to MALDI mass spectrometry imaging and microproteomics. Recently, i started a new project about the detection of alternative proteins and the study of their molecular functions.
Authorship: Julien Franck (1), Vivian Delcourt (1,2), Michel Salzet (1), Xavier Roucou (2), Isabelle Fournier (1)
(1) PRISM INSERM U1192 lab, University Lille1, Villeneuve d'Ascq, France (2) Biochemistry Department, Sherbrooke University, Canada
A mature mRNA is a molecule presenting three different ORFs displaying several alternative ORFs. Recently, alternative proteins were detected in human cells although such a mechanism was considered as anecdotal in eukaryotes. AKT2 protein and its associated alternative proteins altAKT2 were identified from SCOV3 cells. AKT has strong oncogenic function and is a key mediator of PI3K pathway function. AKT is highly activated in the late stage of ovarian cancer and it is possible to assume that AltAkt2 plays a major role in the progression of tumors.
A mature mRNA is a molecule presenting three different ORFs displaying several alternative ORFs. Recently, alternative proteins were detected in human cells although such a mechanism was considered as anecdotal in eukaryotes. Alternative proteins were described by our team in the case of cancer. In collaboration with the group of Dr Roucou, we identified an alternative protein altMRVI1 encoded by the mRNA transcripted from the MRVI1 gene which presented the same amino acid sequence as the BRCA1 IP3 protein1. altMRVI1 one was identified by Liu et al 2 and was described as an interacting protein of the breast cancer susceptibility protein 1 (BRCA1) which is known to be involved in breast and ovarian cancer. The BRCA1 gene belongs to the tumor suppressor gene class and the associated BRCA1 protein is directly involved in the repair of damaged DNA 3,4. If the BRCA1 (or BRCA2) gene is mutated, the associated protein translated is no longer active leading to the proliferation of abnormal cells and therefore growth of tumors. BRCA1 and altMRVI1 were found to be co-expressed in the nucleus and their interaction was clearly demonstrated by co-immunoprecipitation. These results suggest that the proteome is more complex and many proteins not considered so far remain to be identified and their molecular functions elucidated.
A phosphoproteome enrichment5,6, a co-immunoprecipitation experiment7 and a full proteome extract8 available on the public data repository PRIDE (https://www.ebi.ac.uk/pride/archive/) were re-analyzed as well as a full proteome extract form SKOV3 cell lines. Tryptic peptides from SKOV3 were analyzed by nanoLC-HR-MS/MS and identified using Peptideshaker software and the human ORF/AltORF database.
To increase the number of potentially identified alternative proteins, a combination of multiple algorithms was used using the peptideshaker software. We were able to identify 10364 alternative proteins and 857 phosphorylated alternative proteins (1 peptide/protein). Re-analysis of data coming from co-immunoprecipitation of GFP tagged proteins from hela cells allows the identification of reference proteins as well as alternative proteins such as altSRRM2 translated from the SSRM2 gene in the CDS domain according to a +2 reading frame. Considering the SKOV3 cells, we identified the AKT2 protein as well as its associated alternative protein altAkt2. AKT has strong oncogenic function and is a key mediator of PI3K pathway function. AKT is highly activated in the late stage of ovarian cancer and it is possible to assume that AltAkt2 plays a major role in the progression of tumors.
AKT2 was detected in our experiment and was previously detected as overexpressed in human breast cancer and OC 9–12. The description of AKT2 gene involved in these cancers is continuously rising with 131 publications in 2015 (pubmed resource). AKT has strong oncogenic function and is a key mediator of PI3K pathway function. AKT isoforms are phosphorylated at high levels in ovarian cancers. AKT is highly activated in the late stage of OC and therefore it is possible to assume that AKT plays a major role in the progression of tumors. The detection of AKT2 in our experiment is of great importance but the most interesting part remains the identification of an associated alternative protein altAkt2. This protein was found to be translated in the CDS domain or 5’UTR domain following the isoforms according to a +3 reading frame
Alternative proteins not considered so far, can be now described as new potential biomarkers of pathologies.
References & Acknowledgements:
1.Vanderperre, B. et al. Direct detection of alternative open reading frames translation products in human significantly expands the proteome. PLoS One 8, e70698 (2013).
2.Liu, Y. et al. Yeast two-hybrid junk sequences contain selected linear motifs. Nucleic Acids Res. 39, e128 (2011).
3.Hedgepeth, S. C. et al. The BRCA1 tumor suppressor binds to inositol 1,4,5-trisphosphate receptors to stimulate apoptotic calcium release. J. Biol. Chem. 290, 7304–13 (2015).
4.Strickland, K. C. et al. Association and prognostic significance of BRCA1/2-mutation status with neoantigen load, number of tumor-infiltrating lymphocytes and expression of PD-1/PD-L1 in high grade serous ovarian cancer. Oncotarget (2016). doi:10.18632/oncotarget.7277
5.Sharma, K. et al. Ultradeep Human Phosphoproteome Reveals a Distinct Regulatory Nature of Tyr and Ser/Thr-Based Signaling. Cell Rep. 8, 1583–94 (2014).
6.Tong, J., Taylor, P. & Moran, M. F. Proteomic analysis of the epidermal growth factor receptor (EGFR) interactome and post-translational modifications associated with receptor endocytosis in response to EGF and stress. Mol. Cell. Proteomics 13, 1644–58 (2014).
7.Hein, M. Y. et al. A Human Interactome in Three Quantitative Dimensions Organized by Stoichiometries and Abundances. Cell 163, 712–723 (2015).
8.Rosenberger, G. et al. A repository of assays to quantify 10,000 human proteins by SWATH-MS. Sci. data 1, 140031 (2014).
9.Nakayama, K. et al. Sequence mutations and amplification of PIK3CA and AKT2 genes in purified ovarian serous neoplasms. Cancer Biol. Ther. 5, 779–85 (2006).
10.Arboleda, M. J. et al. Overexpression of AKT2/protein kinase Bbeta leads to up-regulation of beta1 integrins, increased invasion, and metastasis of human breast and ovarian cancer cells. Cancer Res. 63, 196–206 (2003).
11.Yuan, Z. Q. et al. Frequent activation of AKT2 and induction of apoptosis by inhibition of phosphoinositide-3-OH kinase/Akt pathway in human ovarian cancer. Oncogene 19, 2324–30 (2000).
12.Cheng, J. Q. et al. AKT2, a putative oncogene encoding a member of a subfamily of protein-serine/threonine kinases, is amplified in human ovarian carcinomas. Proc. Natl. Acad. Sci. U. S. A. 89, 9267–71 (1992).
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