Fbxl13 (F-box and leucine-rich repeat protein 13) is an orphan F-box protein. Fbox proteins are a family of substrate-targeting specificity factors for the SCF superfamily of E3 ubiquitin ligases. Since their discovery, many F-box proteins have been shown to have oncogenic and tumour suppressive roles. The importance of Fbxl13 itself in tumourigenesis is reflected in several genome-wide shRNA screens. Fbxl13 depletion in human cancer cells correlates with increased ionising radiation sensitivity and increased genomic instability. Furthermore, Fbxl13 depletion reduces proliferation in mouse embryonic epidermis. Conversely, Fbxl13 amplification is frequently observed in several cancer patient cohorts. However, the main function of Fbxl13 is unknown and its biochemical mechanism of action remains uncharacterised. The aim of this study was to identify the interactors, substrates, and functions of Fbxl13, in order to elucidate its role in tumourigenesis. In this study, I identify and validate Fbxl13 interactors Centrin-2, Centrin-3, Cep152, and Cep192. I show that Fbxl13 is enriched at the centrosome, and present evidence that Fbxl13 targets Cep192-3 for ubiquitin mediated proteolysis. In line with this, Fbxl13 overexpression downregulated centrosomal Cep192 and γ-tubulin, and disrupted the microtubule nucleation activity at the centrosome. Finally, Fbxl13 amplification in U2OS cells is associated with increased cell motility. Thus, we propose that Fbxl13 is a novel regulator of centrosome microtubule nucleation activity.

Apolipoprotein E (APOE) is one of the most important genetic risk factors for late-onset sporadic Alzheimer’s disease (LOAD). APOE is a 35 kDa glycoprotein and ligand known to bind to members of the low-density lipoprotein receptor (LDLR) family, including APOE receptor 2 (apoER2; official gene name LRP8). ApoER2 is a type I transmembrane protein with a large extracellular domain (ECD) and a short cytoplasmic tail that can be proteolytic cleaved. In addition, apoER2 is enriched in the brain and plays an important role in synaptic function and plasticity. Interestingly, the ECD of apoER2 contains several ligand binding repeats that are organized into exons with aligning phase junctions, which allows exon skipping during alternative splicing to retain protein fidelity. The amount of alternative spliced isoforms distinguishes apoER2 from the rest of the LDLR family members. In fact, mouse Apoer2 has been identified as one of the top ten neuronal genes related to cell-type exon skipping events. Regarding human APOER2, we have identified over 40 different APOER2 isoforms from human brain using gene-specific primers and amplifying the N- and C-terminal open reading frame of APOER2. The majority of APOER2 variants consist of a diverse array of exon skipping events within the ligand binding domain (LBD). We therefore, hypothesized that human APOER2 splice variants act as functionally divergent isoforms that can influence ligand binding properties, receptor proteolysis and changes to synaptic function. ApoER2 undergoes sequential proteolytic cleavage in response to ligand binding, resulting in the release of C-terminal fragments (CTFs) and transcriptionally active intracellular domain (ICD). We therefore, systematically tested whether the diversity of human N-terminal APOER2 splice variants lacking various LBDs affects APOER2 cleavage and signaling events. We found that alternative splicing of certain APOER2 exons generated different amounts of CTFs compared to full-length APOER2 (APOER2-FL). The pattern was not simply based on the number of ligand binding domains suggesting that excision of certain exons may alter the tertiary structure of the receptor sufficiently to make the receptor more or less accessible to cleavage and generation of CTFs. To further characterize APOER2 splice variants, we specifically examined APOER2 splice variants that generated the highest and lowest amounts of CTF generation compared to APOER2-FL and focused on APOER2 splice variant lacking exons 5-8 (Δ5-8) and lacking exons 4-6 (Δ4-6), respectively. The differential CTF generation of APOER2 Δ5-8 and Δ4-6 reflects the proteolytic release of the APOER2-ICD. This APOER2-ICD mediates transcriptional activation, facilitated by the Mint1 adaptor protein. To investigate whether human N-terminal APOER2 splice variants influence APOE binding and receptor cleavage properties, we used microscale thermophoresis and tested the well-validated human APOE mimetic peptide. We found that specific exons or ligand-binding cassettes differentially affect APOE peptide binding to APOER2 splice variants. In addition, APOE peptide induces generation of APOER2-CTF acutely within one hour. Functionally, we demonstrated that APOER2 is required for spontaneous neurotransmitter release in mature neurons. Loss of mouse Apoer2 robustly decreased miniature event frequency in excitatory synapses compared to heterozygous Apoer2 neurons. We found APOER2-FL fully restored the miniature event frequency in excitatory synapses but not APOER2 Δ5-8. APOER2 Δ4-6 restored the miniature event frequency similar to heterozygous Apoer2 neurons. These results suggest that different human N-terminal APOER2 splice variants have distinct and differential synaptic properties signifying a role of APOER2 splice variants as regulators of synaptic function.

Cell cycle progression in Caulobacter crescentus depends on the master regulator, CtrA. During the transition from swarmer to stalk cell (G1 to S phase), CtrA is degraded by the AAA+ protease ClpXP and levels rise again in the predivisional stage. The focus of this work is to explore how cyclic, regulated degradation is controlled. CtrA is known to bind to the origin of replication, thereby suppressing replication, so we first asked if DNA binding had an effect on CtrA stability. CtrA is readily degraded by ClpXP on its own, but when bound to DNA containing the proper binding sites, degradation is inhibited. Stabilization is dependent on DNA binding, as CtrA mutants deficient in DNA binding show the same degradation regardless of addition of DNA, as does CtrA in the presence of a mutant origin sequence lacking CtrA binding sites. Looking closely at CtrA degradation in the presence of auxiliary factors suggests that higher order complex formation may be a mechanism of protecting critical cell cycle regulators from premature proteolysis. In vivo study of over-expression of CtrA mutants revealed that accumulation of non-degradable CtrA, CtrA-DD, perturbs the cell cycle, leading to filamentation and a G2 arrest. Over-expression of the DNA-binding domain alone showed filamentation but no G2 arrest, suggesting that CtrA-DD is detrimental for reasons including, but likely not limited to, its ability to bind DNA. Exogenously expressing other domains of CtrA may further elucidate the mechanism of its regulated degradation in vivo.

No description available.

Delta-Notch signaling is crucial for development of nearly every tissue in metazoans. Signals received by the Notch receptor influence transcription of select target genes that ultimately restrict the developmental fate of the signal receiving cell with respect to its neighbors. The Notch pathway also functions in contexts of abnormal proliferation and differentiation, e.g. cancer and inflammation. Therefore, understanding the regulation of signaling through the Notch receptor protein at the cellular and molecular level is of great significance. In this dissertation, I investigated three ways in which Notch signaling is regulated, namely (1) proteolysis of the Delta ligand; (2) endocytosis of the Delta ligand; and (3) proteolysis of the Notch receptor.. The Delta protein has three functions. First, Delta is a ligand for Notch when bound to it from an adjacent cell. Second, Delta is an inhibitor of Notch when coexpressed with it in the same cell. Third, Delta is hypothesized to be a receptor and, upon binding to Notch, signals to nucleus. Delta undergoes proteolysis by ADAM proteases and there are two contradictory models for the role of Delta cleavage: (1) cleavage disables Delta function; and (2) cleavage activates Delta function. Overall, the results presented in this dissertation strengthen the first model and weaken the second one. Consistent with the first model, we showed that preventing Delta cleavage strengthens its ligand function. As well, when co-expressed in the same with Notch, Delta cleavage is upregulated therefore disabling Delta function as inhibitor of Notch. In contrast to the second model, we showed that Delta proteolysis does not follow a previously established pattern of cleavages typical of cell surface proteins that are activated by proteolysis. Delta also undergoes endocytosis. Two general models have emerged that are again contradictory: (1) endocytosis downregulates cell surface expression of Delta and therefore diminishes its ability to bind Notch; (2) endocytosis of Delta invokes activation of Notch signaling. Overall, our results strengthen the first model and weaken the second one. In support of the first model, we first demonstrated that Notch activation shows a linear relationship to the amount of Delta ligand present on the cell surface and that subsequent inhibition of cell surface expression of Delta leads to its loss of function. In contrast to the second model, we showed that endocytosis of Delta is not required to activate Notch. We also resolved that earlier evidence in support for this model stemmed from misinterpretations of the properties of a Delta mutant protein. Proteolysis of Notch activates the signaling cascade. Binding of Delta to Notch was previously regarded as a requisite regulatory step to invoke receptor proteolysis. We identified the ability of Kuzbanian and TACE, ADAM proteases that cleave Notch in response to Delta stimulation, to activate Notch in a ligand-independent manner. Altogether, our results demonstrate that proteolysis and endocytosis of Delta are independent mechanisms that act to downregulate Delta function and are therefore an important means of attenuating the Notch signal. Alternatively, we find a novel means of enhancing Notch signals in specific contexts, namely through ligand-independent Notch activation by the ADAMs Kuzbanian and TACE. With respect to the latter observation, Kuzbanian and TACE expression is known to be elevated in several human diseases, and thus predicts that engagement of Notch signaling is a contributing factor in these pathologies.

Mixed-function oxidases play a major role in the metabolism of xenobiotics. The main component of this system is the cytochrome P450, it oxidizes substrates coming into our body to more polar products. Another component of mixed-function system - the cytochrome b5 (cyt b5) is able to modulate the function of cytochrome P450, the mechanism of this modulation is yet unknown. However, it is believed that it could be mediated via transfer of electron or allosteric modulation of cytochrome P450 caused by interaction with cyt b5. The aim of this thesis was to find and prepare analogs of cyt b5, which are unable to transfer electrons to cytochrome P450 and simultaneously are structurally very similar to native cyt b5. The conformational stability of cyt b5 and its analogs was monitored using pulse proteolysis. This method employs proteases to cleave the evaluated protein at varying concentration of a denaturant. For soluble proteins, urea is typically used as denaturant in combination with thermolysin as protease. While for membrane proteins, sodium dodecyl sulfate (SDS) is usually used as denaturant together with subtilisin as protease. The aim of this thesis was to use these methods to compare a conformational stability of the native human cyt b5 with apo-cyt b5 and analogs of the cyt b5 reconstituted...

O proteassomo é o componente do sistema Ubiquitina-Proteassomo (UPS), responsável pela degradação de proteínas intracelulares marcadas com cauda de ubiquitina. No entanto, a unidade catalítica do proteassomo (20SPT), destituída de unidades regulatórias, é capaz de degradar proteínas de maneira ubiquitina-independente. Diversas modificações pós-traducionais já foram descritas para o 20SPT, incluindo a S-glutatiolação. De acordo com Demasi e col., (2003) o 20SPT da levedura Saccharomyces cerevisiae possui a atividade tipo-quimiotripsina modulada por glutationa e o mecanismo de glutatiolação implica na formação do intermediário ácido sulfênico. No presente trabalho, identificamos por espectrometria de massas (MS/MS) um total de sete resíduos diferentes de cisteína glutatiolados no 20SPT, sendo seis in vitro por incubação com GSH e três in vivo, extraído de células crescidas até atingir fase estacionária tardia em meio rico. Analisando a estrutura 3D do 20SPT, observou-se que os resíduos de cisteína glutatiolados não estão localizados na entrada da câmara catalítica nem próximos aos sítios-ativos, indicando um mecanismo alostérico da modulação da atividade proteassomal. O proteassomo glutatiolado extraído de leveduras é capaz de degradar proteínas oxidadas de maneira mais eficiente que o proteassomo reduzido por DTT, e ainda, esta degradação gera perfis peptídicos diferenciados por utilizar distintamente as atividades sítio-especificas, como visualizado por análises de HPLC e MS/MS. Por microscopia eletrônica verificamos a conformação aberta da câmara catalítica do proteassomo glutatiolado, sendo esta imediatamente fechada pela remoção da glutationa do 20SPT na presença de DTT. Caracterizamos ainda, enzimas reponsáveis pela desglutatiolação do 20SPT, capazes de recuperar as atividades proteassomais que haviam sido diminuídas pela glutatiolação: as oxidoredutases glutarredoxina 2 e as tiorredoxinas citosólicas. O mecanismo ainda inclui a hidrólise dessas oxidorredutases, fenômeno também verificado para diversas proteínas da suprafamília tiorredoxina, provavelmente devido a propriedades estruturais desta família. A glutatiolação do proteassomo apresenta-se como uma nova modificação pós-traducional de ocorrência fisiológica dependente do estado redox celular. Esta modificação promove aumento da atividade proteolítica, sugerindo uma função antioxidante atuante na remoção de proteínas oxidadas durante desafios oxidativos / The proteasome is the protease of the Ubiquitin-Proteasome System (UPS) responsible for the breakdown of intracellular ubiquitin-tagged proteins. However, the catalytic particle of the proteasome (20SPT) is capable of hydrolyzing some substrates in an ubiquitin-independent fashion. The S-glutathiolation of the 20SPT was described among several post-translational modifications and according to Demasi et. al. (2003), the chymotrypsin-like activity of proteasome from yeast Saccharomyces cerevisiae is regulated by glutathione. The mechanism of S-glutathiolation is dependent on the formation of the sulfenic acid intermediate in the cisteine residues of the 20SPT. In this present work, we identified in vitro and in vivo, a total of seven different S-glutathiolated proteasomal cysteine residues by mass spectrometry studies (MS/MS) and, by analyzing the 3D structure of the 20SPT, the modified cysteine residues are not located either on the entrance of the catalytic core or near to the active sites, indicating an allosteric mechanism of proteasomal modulation. During protein degradation, the natively S-glutathiolated 20SPT produces different patterns of peptide products when compared to the DTT-reduced particle through distinct site-specific cleavage of the protein substrates, as herein demonstrated by HPLC and MS/MS analyses. Furthermore, by electron microscopy, we showed that the entrance of the natively glutathiolated 20SPT is in the open conformation that immediately shifts to the closed conformation in the presence of DTT. We have also characterized the deglutathiolase role of the oxidoreductases Glutaredoxin 2 and Citosolic Thioredoxins 1 and 2 which recover the partially inhibited 20SPT activities. The deglutathiolation mechanism also includes the oxidoreductase degradation dependent on the 20SPT activation. The proteasome Sglutathiolation emerges as a new physiological post-translational modification correlated to the cellular redox state. Moreover, the S-glutathiolation of the 20SPT increases its proteolytic activity suggesting an antioxidant role by removing oxidized proteins generated during oxidative challenges.

La maladie de Huntington (MH) est une maladie neurodégénérative héréditaire autosomique dominante. Elle est due à l’expansion anormale de polyglutamine dans la partie N-terminal de la protéine huntingtine (HTT). Une des étapes clés de la pathologie est le clivage de la HTT pleine longueur en fragments N-terminaux plus petits, contenant l’expansion de polyglutamine, et qui sont toxiques pour les neurones. En effet, les clivages de la HTT mutée génère des fragments N-terminaux (N-ter) de tailles comprises entre les acides aminés 1-105 et 1-586 observés dans des extraits de cerveaux de patients MH post-mortem et dont l’implication dans la mort neuronal est bien caractérisée. Mes travaux de thèse ont visé à modéliser le clivage de la HTT et à évaluer les conséquences sur la survie neuronale.Au cours de ma thèse, j’ai développé un outil permettant de contrôler le clivage de la HTT dans le temps et à des sites spécifiques. J’ai étudié le clivage de la HTT à deux sites stratégiques : les positions clivées par la caspase-6 et par la bléomycine hydrolase/cathepsine Z. A l’aide de cet outil, j’ai montré que le clivage de la HTT confère une toxicité cellulaire qui dépend du profil du clivage. Plus précisément, J’ai décrit une interaction intramoléculaire au sein des domaines de la HTT. Mes résultats indiquent que cette interaction protège les cellules de la toxicité induite par le clivage de la HTT mutée. En effet, les clivages successifs de la HTT annulent cette interaction, ce qui induit la libération des fragments N-ter mutants et provoque la mort cellulaire à l’issue de leur translocation nucléaire. Pour conclure, au cours de ma thèse, j’ai montré que la protéolyse successive de la HTT induit des processus cytotoxiques différents. / Huntington’s disease (HD) is an autosomal dominant inherited neurodegenerative disorder caused by an abnormal polyglutamine (polyQ) expansion in the N-terminus of the protein huntingtin (HTT). A crucial step in HD pathogenesis is the cleavage of full-length HTT into smaller N-terminal (N-ter) fragments that contain the polyQ stretch and that are toxic to neurons. HTT cleavage generates short N-ter fragments whose amino-acid positions range from 1-105 to 1-586. These fragments are observed in HD post mortem brain samples and their participation in neuronal death in HD is well characterized. During my PhD research, I investigated the consequences of full-length mutant HTT proteolysis by developing a time and site-specific controlled system for HTT proteolysis. I have assessed HTT cleavage on two sites caspase-6 and cathepsin Z. My results show that HTT cleavage induces neurotoxicity in vitro as well as in vivo, toxicity which depends on HTT proteolysis pattern. Briefly, we described an intramolecular interaction within the HTT domains which is impaired upon successive proteolysis of HTT. We found that HTT intramolecular interaction buffer mutant N-ter HTT-induced toxicity. Moreover, specific cleavages of the mutant HTT generated toxic N-ter fragments as they translocate into the nucleus. To conclude, my PhD work has shown that additional cleavage of mutant HTT induces cytotoxicity by different mechanisms.

Dementia, the most common cause of which is Alzheimer's disease (AD), currently affects 850,000 people in the UK, a figure set to rise to over 1 million by 2025. There is currently no disease modifying therapy available to slow or halt this progressive disease. Current understanding of AD implicates the neurotoxic amyloid-β (Aβ) peptide as the primary initiator in a cascade of events leading to the neuronal cell death and brain atrophy associated with the disease. Therefore, inhibiting the production or enhancing the clearance of Aβ within the brain has become a major target for the production of disease modifying therapeutics. Aβ is produced by brain cells through the sequential proteolytic cleavage of a larger transmembrane protein known as the amyloid precursor protein (APP) by β- and γ-secretases. Several aspects of APP physiology can influence its proteolysis, and thus Aβ production, including the isoform of APP which is expressed, its trafficking and subcellular location and its physical interactions with other proteins in the cellular environment. Here we have investigated the influence of subcellular trafficking and location and protein-protein interactions on the differential proteolysis of two APP isoforms, APP695 and APP751 in a neuroblastoma cell line. We have shown that APP751 undergoes less amyloidogenic proteolysis than APP695 and that retention within the early secretory pathway may contribute to this difference. APP751 shows higher co-localisation to the trans-Golgi network than APP695 in immunofluorescence microscopy studies, while addition of a mutation which causes APP proteolysis in the secretory pathway reduces the large difference in amyloidogenic proteolysis of these two isoforms. Targeting APP endocytosis from the cell surface, thought to be a key determinant in Aβ generation, effects APP isoform proteolysis and Aβ production to a similar extent in both the APP isoforms suggesting differences in proteolysis occur before this trafficking event. We also show by immunoblot analysis that the APP isoforms may be differentially cleaved by proteases other than β- and γ-secretase to produce recently identified proteolytic fragments. Using a liquid chromatography - tandem mass spectrometry approach coupled to prior stable isotope labelling of amino acids in cell culture (SILAC), we have identified the interactomes of the two APP isoforms in our model system. Gene ontology analysis identified enrichment of nuclear and mitochondrial proteins specifically in the APP695 interactome. Using siRNA mediated protein knockdown, we have shown interactions with Fe65 and ataxin-10 specifically influence Aβ generation from the APP695 isoform. Fe65 alters proteolysis at the rate limiting β-secretase cleavage step, while ataxin-10 alters proteolysis by γ-secretase. Interaction with growth-associated protein 43 specifically influences Aβ generation from the APP751 isoform, altering proteolysis at the γ-secretase step. Finally we have shown that recently discovered familial AD-linked mutation and protective mutation within the Aβ region of the APP protein have consistent effects on APP proteolysis in both the APP isoforms.

Health consciousness drives people to drink skim milk. Yet, improving the blue-white appearance and watery texture of skim milk is necessary to make consumers happy with skim milk. In this study, the influence of limited proteolysis with soluble or immobilized proteases, heat treatment, and pH on the whiteness of skim milk were examined to meet this goal. Limited proteolysis with milk-clotting enzymes increased the whiteness of skim milk. the proteases porcine pepsin and chymosin were immobilized onto nonporous ceramic, glass, and controlled pore glass (CPG) beads. The enzymes were coupled to beads either directly or via crosslinker proteins. Pepsin, immobilized onto CPG beads via crosslinker proteins, exhibited the best properties with respect to enzymatic activity, stability, and whitening efficiency. The L value (whiteness) of this immobilized enzyme-treated skim milk was 79.5, which approached the whiteness of 1% fat milk. Immobilized proteases whitened skim milk more effectively than did soluble proteases. The whiteness of skim milk was determined at various temperatures from 4 to 90°C. The L value increased with increasing temperature throughout the range tested. For samples not heated above 50°C, the increases in L values were completely reversible on cooling. Partial reversibility was observed with samples heated at 70°C and above. Milk whiteness was also determined at different pH values ranging from 5.0 to 8.2 at temperatures of 4, 20, and 30°C. The L value increased with decreasing pH and increasing temperature. A maximum L value of 80.0 was obtained at pH 5.0 and 30°C, which is higher than the L value of skim milk at its natural pH at room temperature. The temperature-dependent dissociation of major caseins was investigated by size exclusion chromatography at temperatures from 10 to 40°C. Free soluble β-casein and κ-casein were found only at 10°C.