Parkinson’s Disease (PD) is the second most common neurodegenerative disorder after Alzheimer’s and old age is the strongest risk factor for developing PD. PD has traditionally been seen as a motor disorder, but its non-motor symptoms such as dysautonomia, sensory dysfunction, sleeping problems and neuropsychiatric features equally add to the disease burden. There is no cure for PD and this is probably a reflection of our poor understanding of the disease pathogenesis. One way of tackling this is to focus on the small, but significant number of PD patients with a family history compatible with Mendelian autosomal inheritance (10-15%). Hereditary and sporadic PD share important clinical and neuropathological features, and there is reasonable hope that dissecting molecular pathways of PD gene products will have more general implications for the pathophysiology of PD associated neurodegeneration and help device new treatment strategies. Mutations in the FBXO7 gene have recently been shown to cause an autosomal recessive early onset Parkinsonian-pyramidal syndrome and FBXO7 has been designated as PARK 15 (Di Fonzo et al., 2009). FBXO7 is a member of the F-box protein family, which functions as the variable subunit of Skp1-Cullin1-F-box protein (SCF) E3 ubiquitin ligase complexes and as such dictate substrate specificity. The canonical outcome of ubiquitylation is proteasomal degradation and my working hypothesis is that FBXO7 may be involved in protein quality control in the brain. A perturbation thereof may be a first step towards FBXO7 dependent disease. At the time of starting with my PhD project, little was known about the molecular function of FBXO7 and how mutations in FBXO7 result in neurodegeneration. In order to learn more and dissect the signalling pathway of FBXO7 I have used tagged stable overexpression cell lines of the FBXO7 wildtype as well as human disease mutant proteins for tag-pulldowns followed by mass-spectrometry to identify interacting partners and possible substrates. With this approach I have been able to confirm the interaction between FBXO7 and its core SCF E3 ligase partners as well as some of the previously reported interacting partners. I have been able to show that not only the FBXO7 wildytpe protein, but also all of the so far reported human disease mutants are able to assemble into an SCF complex. Hence, my fist conclusion is that the human disease mutants do not exert their pathogenicity by SCF complex disruption. Next, a knock-in (KI) mouse model of one of the pathogenic FBXO7 mutations (R378G) was generated and evaluated by molecular and biochemical approaches as well as motor and behaviour phenotyping. In particular, I have used the Fbxo7 mouse model for extensive proteomic screens to identify wildtype (wt) and KI Fbxo7 interactors: endogenous Fbxo7 immunoprecipitations from mouse brain lysates and subsequent fingerprint mass-spectrometry; differential whole proteome: ex vivo differential dimethyl labelling of wt and KI brain samples, and Fbxo7-dependent ubiquitinome analysis: quantitative di-GLY capture proteomics combining in vivo SILAC labelling with antibody-based affinity enrichment of “di-GLY remnant motifs”- containing peptides prior to proteomic profiling of the wild-type in comparison to the homozygous R379G Fbxo7 KI ubiquitinome in MEF lysates. The di-GLY remnant motif is the signature peptide of ubiquitinylated protein sites at peptide level after tryptic digestions. Some of my findings are: • For the first time I show that endogenous Fbxo7 actually assembles into an Skp1-Cullin1-Fbxo7 complex and that the pathogenic R378G does not disrupt SCFFbxo7-KI complex formation in vivo. This is true for the Fbxo7 KI mouse model, but also for patient derived immortalized cell lines carrying the R378G FBXO7 mutation.• Endogenous Fbxo7 interacts with the Sumo E3 ligase complex RanBP2/ RanGAP1*Sumo1/Ubc9 complex. • In the differential enrichment of ubiquitylated protein species in SILAC labelled wild-type and homozygous R379G Fbxo7 KI MEFs, I have clearly identifies 2 highly conserved lysine residues, which are conserved amongst VDAC 1, 2, and 3 in mouse as well as human homologous, to be preferentially ubiquitinylated in a Fbxo7 wild-type background (in collaboration with Dr. Patrick Pedrioli, MRC Programme leader).• There is a significant difference in motor performance between wildtype and homozygous R379G KI Fbxo7 mice at 10 months of age (in collaboration with Dr. Steve Martin, Neuroscience Division, Dundee). • Furthermore, I have successfully set up an in vitro FBXO7 dependent ubiquitinylation assays.

No description available.

Parkinson's disease is a progressive neurodegenerative disorder of the central nervous system, manifesting with both motor and non-motor symptoms. Autosomal recessive mutations in the FBXO7 gene have been identified to cause a rapidly progressing early-onset form of PD. Canonically, FBXO7 functions as a substrate-recruiting subunit of the SCF-type E3 ubiquitin ligase. However, it also has a variety of other atypical functions, such as cell cycle regulation, proteasome regulation, and mitophagy. The overall aim of this research was to characterise the functional role of FBXO7 in various in vitro and in vivo PD models. The models examined included FBXO7 shRNA knockdown SH-SY5Y cell lines, FBXO7 CRISPR knockout SH-SY5Y cell lines, primary patient fibroblasts with a FBXO7 mutation, and MEFs and tissues from a Fbxo7 KO mouse. My analysis of fibroblasts from a patient without FBXO7 expression revealed several interesting phenotypes. Briefly, the patient fibroblasts proliferated slower due to increased apoptosis and lower CDK6 and cyclin D1 expression, which led to fewer cells progressing through the G1 phase of the cell cycle. My experiments showed that these cells also had mitochondrial respiration defects, exhibiting lower basal respiration, ATP production, maximal respiration and spare capacity, in addition to complex I, III and IV deficiencies. Patient fibroblasts also had significantly lower levels of 12S and 16S ribosomal mRNA transcripts, which are necessary for the translation of mitochondrially encoded subunits of complexes I, III, and IV. Similar phenotypes were also observed in MEFs from a Fbxo7 KO mouse model, indicating conservation between human and mouse FBXO7 in regulating mitochondria, cell death and proliferation. In a tissue-specific KO mouse model of PD, where FBXO7 expression was ablated in the dopaminergic neurons, I analysed proteins regulated by FBXO7 which might be responsible for cell loss in the substantia nigra. I discovered that RPL23, a regulator of MDM2, was ubiquitinated by SCFFbxo7 using K48 chain linkages, promoting its degradation by the proteasome. This suggests that misregulation of the MDM2:p53 axis may underlie the cell loss observed in this conditional Fbxo7 KO mouse model. In conclusion, these results elaborate on the role of FBXO7 in mitochondrial biology, and identify a new ubiquitination substrate of FBXO7 in a mouse model of PD. It is hoped that by elucidating the potential pathogenic mechanisms of FBXO7 in rare familial forms of the disease, it will be possible to translate findings to the more prevalent sporadic forms of Parkinson's disease as well.

No description available.

F-box only protein 7 (Fbxo7), a member of the F-box-only subfamily of FBPs, is a biologically and pathophysiologically important human protein that assumes many critical functions. The different functions of Fbxo7 depend on the formation of various multi-protein complexes. Possible interplay between different Fbxo7 functions further complicate the protein-protein interaction networks involved in Fbxo7 biology. Although significant progresses have been made to understand the functions, regulation, specificity, and protein interaction network of Fbxo7, a myriad of questions remain to be answered. The objectives of the work presented in this dissertation are to elucidate the molecular structures underlying the functions of Fbxo7 and the interaction with its protein partners, such as proteasome inhibitor PI31. The best known biological function of Fbxo7 is its role as the substrate-recognition subunit of the SCFFbxo7 (Skp1-Cul1-F-box protein) E3 ubiquitin ligase that catalyzes the ubiquitination of hepatoma up-regulated protein (HURP) and inhibitor of apoptosis protein (IAP). Fbxo7 also assumes various SCF-independent functions through interact with its protein partners that are not the substrates of the ubiquitin proteasome system, such as PI31, Cdk6, p27, PINK1 (PTEN-induced kinase 1), and Parkin. PI31 is a known proteasome regulator which was initially characterized as a proteasome inhibitor in vitro. The binding affinity between Fbxo7 and PI31 is very strong, and The Fbxo7-PI31 interaction is mediated by heterodimerization of the FP domains of the two proteins. This work is focus on study the protein structure of the two FP domains in Fbxo7 and PI3. Chapter 1 reviewed the F-box-only protein Fbxo7 biology including the function of Fbxo7 protein in ubiquitination proteasome pathway and some SCF-independent functions which are relate to human disease. Chapter 2 discussed the function of proteasome inhibitor PI31. With the many important biological functions, Fbxo7 is clearly an extraordinary important protein, but the lack of structural knowledge has hampered efforts to achieve a better understanding of Fbxo7 biology. In this work, we have determined the crystal structure of Fbxo7 FP domain (residues 181-335) and the crystal structure of the PI31 FP domain (residues 1-161) using a longer protein construct both at 2.0Å resolution. The Fbxo7 FP domain adopts an α/β-fold similar to that of the PI31 FP domain and the secondary structure elements of the two FP domains are comparable including the C-terminal helix, indicating that the two FP domains share the same overall global fold. However, an α helix and three β strands in the Fbxo7 are longer than their counterparts in the PI31 FP domain. The two FP domains also differ substantially in the length and conformation of the longest connecting loop. More importantly, structural differences between the two FP domains lead to drastically different modes of inter-domain protein–protein interaction: the PI31 FP domain utilizes either an α interface or β interface for homodimeric interaction, whereas the Fbxo7 FP domain utilizes an αβ interface. We have note that the inter-domain interaction of the Fbxo7 FP domain is much more extensive, featuring a larger contact surface area, better shape complementarity and more hydrophobic and hydrogen-bonding interactions. The results of this structural study provide critical insights into how Fbxo7 may dimerize (or multimerize) and interact with PI31 via the FP domain. Chapter 4 and Chapter 5 discussed the structure determinations, structure features and detail of protein-protein interactions of Fbxo7 and PI31 FP domains. Chapter 2 reviewed the corresponding fundamental biochemical techniques that been used in this study. Chapter 3 discussed protein structure determination by X-ray crystallography in structural biology studies. It was believed that the FP domains of Fbxo7 and PI31 mediate homodimerization and heterodimerization of the proteins and the FP domain is not present in other human proteins. In order to study the Fbxo7-PI31 heterodimerization protein-protein interactions, we performed modeling studies. Chapter 6 discussed the model building and binding studies. Based on the result of model building studies, we propose that an interaction between the two FP domains of Fbxo7 and PI31 should be mediated by a αβ interface using the α-helical surface of the Fbxo7 FP domain and the β-sheet surface of the PI31 FP domain. According to the result of pull down assay, the PI31 FP domain may complete with Skp1 for the binding with Fbxo7. It is possible that the formation of heterodimer between the Fbxo7 and PI31 mediate by FP domains may lead to the Fbxo7 dissociation from SCFFbxo7 complex which might reveal a new regulation mechanism.

No description available.

Das Ubiquitin-Proteasom-System eukaryotischer Zellen spielt eine zentrale Rolle beim Abbau von fehlgefalteten und nicht mehr benötigten Proteinen. Damit erfüllt es regulatorische Funktionen bei zellulären Prozessen wie z.B. dem Zellzyklus und der Transkription. Das Protein Proteasominhibitor 31 (PI31) wurde als Inhibitor des Proteasoms in vitro charakterisiert. Des weiteren wurde gezeigt, daß überexprimiertes PI31 im murinen System ein Modulator der Assemblierung des Immunoproteasoms (i20S) ist. Über die Funktion und Regulation von PI31 im humanen System war bisher nichts bekannt und wurde deshalb in dieser Arbeit untersucht. Es konnte gezeigt werden, daß neben dem PI31-Transkript mindestens neun weitere alternative Spleißvarianten des humanen PI31 Gens PSMF1 existieren. Die PI31-Isoformen V2 bis V10 unterscheiden sich von PI31 (V1) teils durch eine fehlende N-terminale Domäne oder einen veränderten C-Terminus. Die Isoform V5 wird als einzige gewebespezifisch in Testikeln exprimiert und ist im Zellkern lokalisiert. Ausschließlich die Überexpression der Isoform V3 führt zur Inhibition der proteasomalen Aktivität in vivo. Ein modulatorischer Einfluß von PI31 oder einer der Isoformen auf die Assemblierung des humanen i20S bestätigte sich dagegen nicht. Die Überexpression von PI31 und V3 in humanen Zellen führte indes zu einer Akkumulation und verzögerten Degradation von proteasomalen Substraten. Es wurde außerdem gezeigt, daß die Expression von humanem PI31 durch virusassoziierte Stimuli wie dsRNA und Typ I-Interferone induziert werden kann. Für die 3kb lange 3’UTR der PI31-mRNA konnte zusätzlich nachgewiesen werden, daß sie inhibitorisch auf die Expression wirkt und somit eine regulatorische Funktion besitzt. In Zusammenhang mit der von Kirk et al. (2008) gezeigten Heterodimerisierung von PI31 mit dem F-Box Protein Fbxo7, weisen die hier vorgestellten Ergebnisse auf eine Funktion von PI31 und dessen Isoformen bei der Ubiquitinierung von proteasomalen Substraten hin. / The ubiquitin–proteasome pathway is the major intracellular system for protein degradation. It plays an important role in the regulation of cellular processes like cell cycle control, signal transduction and gene transcription. The protein proteasome inhibitor 31 (PI31) was initially characterized as a potent inhibitor of proteasomal activity in vitro. Furthermore it was shown that PI31 modulates the assembly of the murine immunoproteasome (i20S). The function and regulation of PI31 in the human system is so far unexplored and therefore the topic of this study. It was shown that at least nine alternatively spliced variants of the PI31 gene PSMF1 exist additionally to the PI31 transcript. The PI31 isoforms V2 to V10 differ from PI31 (V1) in parts of the N-terminus and in a modified C-terminus. Only the isoform V5 is tissue specific expressed in testis and localized in the nucleus. After overexpression only the isoform V3 has the ability to inhibit the proteasomal activity in vivo. In contrast to the murine system neither PI31 nor the isoforms showed a modulatory effect on the assembly of the i20S. The overexpression of PI31 and V3 in human cells results instead in the accumulation and delayed degradation of proteasomal substrates. Furthermore the expression of human PI31 can be induced by virus associated stimuli like dsRNA and type I interferones. In addition, for the 3kb long 3’UTR of the PI31-mRNA an inhibitory effect on the expression and therefore a regulatory role was shown. Together with data from Kirk et al. (2008), who show the heterodimerization of PI31 with the F-box protein Fbxo7, the presented results suggest a function of PI31 and its isoforms in the process of ubiquitination of proteasomal substrates.