Identification of the substrates of the E3 ubiquitin-ligase SCF(Fbxo7) using human proteome microarray

• Grant number: 12/09241-8
• Support type: Scholarships abroad - Research Internship - Post-doctor
• Effective date (Start): November 01, 2012
• Effective date (End): October 31, 2013
• Field of knowledge: Biological Sciences - Biochemistry - Enzymology
• Principal researcher: Marcelo Damário Gomes
• Grantee: Felipe Roberti Teixeira
• Supervisor abroad: Heike Laman
• Home Institution: Faculdade de Medicina de Ribeirão Preto (FMRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
• Research place: University of Cambridge, England
• Associated to the scholarship: 10/16464-8 - Application of protein microarray to identify substrates of SCF1 (FBXO25) E3 ubiquitin ligase, BP.PD

Abstract

Fbxo7 is an unusual protein which has been found to be associated with two major human diseases, cancer and Parkinson's disease (PD). Fbxo7 is one of the 69 human F-box proteins that serve as specificity factors for the largest family of ubiquitin-ligases known as SCF-type E3 ubiquitin ligases. These modular, multi-subunited holoenzymes are composed of S-phase-kinase associated protein 1 (SKP1), really interesting new gene-box 1 (RBX1), Cullin 1, and an F-box protein (FBP). Within this complex, the FBP subunit recruits the usually post-translationally modified, substrate protein thus potentiating its ubiquitination. Fbxo7 mediates the ubiquitination of a mitotic spindle protein, HURP (hepatoma upregulated protein), which is degraded by the 26S proteasome as a consequence. It also ubiquitinates two regulators of NF-ºB signalling, c-IAP1 (inhibitor of apoptosis protein) and TRAF2 (TNF receptor-associated factor 2), but these proteins are not destabilized as a result of this modification. Fbxo7 is, additionally, an unusual FBP because a subset of its interacting proteins, including cell cycle regulatory proteins Cdk6 and p27, and a proteasome regulator PI31, are not ubiquitinated. Through its direct interaction with p27 and Cdk6, Fbxo7 over-expression can enhance the level of cyclin D-Cdk6 complexes, and cause Cdk6-dependent transformation of immortalised fibroblasts. Because of its capacity to stabilise these G1/S phase cell cycle regulators, Fbxo7 is considered a putative oncogene. In addition, in p53 null haematopoietic stem and progenitor cells, exogenous Fbxo7 expression precipitates the development of T cell lymphoma in recipient mice. However, in other cell types, such as pro-B cells and pro-erythroblasts, Fbxo7 is not a transforming gene, but instead has a negative regulatory role in cell proliferation. It has recently been shown that autosomal recessive mutations in Fbxo7, now also called PARK15, cause an atypical PD. Four disease-associated mutations in PARK15/Fbxo7 have been identified, and which based on their location within the protein, would be predicted to interfere with the ubiquitin ligase activity of SCFFbxo7 or to prevent its interaction with it substrates. However, of the substrates described to date, none has been linked to Parkinson's disease. Moreover, it is unlikely that cell cycle regulation is a factor in PD as it is the premature death of terminally differentiated dopaminergic neurons which causes the disease. Thus, there is a pressing need to identify ubiquitinated substrates of Fbxo7 which will explain the underlying defect which causes PD. We propose to apply a powerful, high-throughput proteomic approach, known as proteome microarray, to identify novel ubiquitinated substrates of Fbxo7. This technology has been used to identify substrates of purified E3-ligases through in vitro ubiquitination assays. Thus, we can identify in a single experiment all potential cellular targets of Fbxo7, without consideration for intracellular cell-cycle or cell-lineage-dependent barriers which might limit the functional contact between Fbxo7 and its targets. We will directly identify the ubiquitinated targets of Fbxo7 and thus attain a more systemic view about its cellular function and gain insight into the deregulated functions of Fbxo7's E3 ubiquitin-ligase activity in both diseases. (AU)

Scientific publications

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

MASON, BETHANY; FLACH, SUSANNE; TEIXEIRA, FELIPE R.; GARCIA, RAQUEL MANZANO; RUEDA, OSCAR M.; ABRAHAM, JEAN E.; CALDAS, CARLOS; EDWARDS, PAUL A. W.; LAMAN, HEIKE. Fbxl17 is rearranged in breast cancer and loss of its activity leads to increased globalO-GlcNAcylation. CELLULAR AND MOLECULAR LIFE SCIENCES, v. 77, n. 13, p. 2605-2620, JUL 2020. Web of Science Citations: 0.

TEIXEIRA, FELIPE ROBERTI; RANDLE, SUZANNE J.; PATEL, SHACHI P.; MEVISSEN, TYCHO E. T.; ZENKEVICIUTE, GRASILDA; KOIDE, TIE; KOMANDER, DAVID; LAMAN, HEIKE. Gsk3 beta and Tomm20 are substrates of the SCFFbxo7/PARK15 ubiquitin ligase associated with Parkinson's disease. Biochemical Journal, v. 473, n. 20, p. 3563-3580, OCT 2016. Web of Science Citations: 6.