Raman spectroscopy offers much better spectral selectivity but its usage has been limited by its poor sensitivity. The discovery of surface-enhanced Raman scattering (SERS) effect, which results in increased sensitivities of up to 108-fold for some compounds, has eliminated this drawback. A new SERS active substrate was developed in this study. Silver nanoparticle-doped polyvinyl alcohol (PVA) coated SERS substrate prepared through chemical and electrochemical reduction of silver particles dispersed in the polymer matrix. Performances of the substrates were evaluated with some biologically important compounds. The specific detection of DNA has gained significance in recent years since increasingly DNA sequences of different organisms are being assigned. Such sequence knowledge can be employed for identification of the genes of microorganisms or diseases. In this study, specific proteasome gene sequences were detected both label free spectrophotometric detection and SERS detection. In label free spectrophotometic detection, proteasome gene probe and complementary target gene sequence were attached to the gold nanoparticles separately. Then, the target and probe oligonucleotide-modified gold solutions were mixed for hybridization and the shift in the surface plasmon absorption band of gold nanoparticles were followed. SERS detection of specific nucleic acid sequences are mainly based on hybridization of DNA targets to complementary probe sequences, which are labelled with SERS active dyes. In this study, to show correlation between circulating proteasome levels and disease state we suggest a Raman spectroscopic technique that uses SERGen probes. This novel approach deals with specific detection of elevated or decreased levels of proteasome genes&rsquo / transcription in patients as an alternative to available enzyme activity measurement methods. First, SERGen probes were prepared using SERS active labels and specific proteasome gene sequences. Then DNA targets to complementary SERGen probe sequences were hybridized and SERS active label peak was followed.

Aryl hydrocarbon receptor (AHR) is a sensor protein, activated by aromatic chemical species for transcriptionally regulating xenobiotic metabolizing enzymes. AHR is also known to be involved in a variety of pathogenesis such as cancer, diabetes mellitus, cirrhosis, asthma, etc. The AHR signaling induced by xenobiotics has been intensively studied whereas its physiological role in the absence of xenobiotics is poorly understood. Despite a number of ligands of AHR have been reported thus far, further applications are still hampered by the lack of specificity and/or the partially agonistic activity. Thus, a pure AHR antagonist is needed for deciphering the AHR cryptic as well as potential therapeutic agent. The Proteolysis Targeting Chimera (PROTAC) is a bi-functional small molecule containing a ligand and proteolysis inducer. PROTAC recruits the target protein to proteolysis machinery and elicits proteolysis. Thus far, a number of PROTAC have been prepared and demonstrated to effectively induce the degradation of targeted protein in cultured cells, validating PROTAC as a useful research tool. In the present study, PROTACs based on apigenin was prepared and demonstrated to induce the degradation of AHR, providing the proof of concept. To improve activity, a synthetic structure, CH-223191, was optimized for antagonistic activity by positional scanning identifying several AHR antagonists. PROTACs based on the optimal structure were prepared and assessed their biological activity. The products and synthetic scheme described hereby will be helpful for the further understanding on AHR biology as well as for developing therapeutic agents targeting AHR.

The diverse outcomes of ubiquitination primarily relate to the flexibility of ubiquitin in forming homo- or heterotypic chains on each of its seven lysine residues which in turn stimulate distinct downstream signaling pathways. These ubiquitin signals must be selectively initiated on the substrate protein and subsequently decoded to facilitate the desired cellular function. These initiation and decoding steps often involve additional post-translational modifications and ubiquitin receptor proteins, but the enzymes and ubiquitin chains involved for many ubiquitinated substrates are not clear. Here, I have explored the initiation and decoding of ubiquitin signals, focusing on lysine-11 (K11) linked polyubiquitin chains and their role in protein degradation. I established in vitro assays to understand how K11-chains are decoded and whether these chains act as a signal for proteasome-mediated degradation. Pure homotypic K11-chains did not bind the proteasome or its associated ubiquitin binding proteins, but did bind to the mitophagy ubiquitin receptors, MyosinVI and TAX1BP1. Heterotypic K11/K48 linkages not only bound the proteasome but also stimulated degradation of the cell cycle substrate, cyclin B1. To further explore the functions of K11-chains I focused on the hypoxia inducible transcription factor (HIF) pathway, as K11-ubiquitination had been implicated in proteasome-independent degradation of the transcription factor. I established an in vitro assay to initiate HIF ubiquitination, via prolyl hydroxylation, and determine the type of ubiquitin chains involved. Recombinant HIF isoforms were rapidly hydroxylated when incubated with cell extracts. Moreover, the levels of iron and small molecule metabolites within the lysates regulated HIF hydroxylation. However, this hydroxylation was insufficient to reproducibly promote HIF ubiquitination or determine the ubiquitin chains involved. While the nature of the polyubiquitin chains formed in the HIF pathway remain elusive, my studies identify distinct roles for homotypic and heterotypic K11-polyubiquitination in proteasome-mediated degradation.

Most patients diagnosed with cancer will receive systematic chemotherapy at some point during their illness, which almost always cause severe side effects for the patients such as, anemia, nausea and vomiting. The problems with today’s chemotherapy is not only that it cause severe side effects, but also that the cancer may develop resistance to the therapy, which is why the development of a new type of therapeutic agent is in dire need. The ubiquitin proteasome system (UPS) is a vital machinery for the cancer cells to maintain protein homeostasis, which also make them vulnerable to any disruption of this system. In recent years, a new technology has been developed that utilize the UPS by chemically bringing an E3 ubiquitin ligase into close proximity of a protein of choice and tagging the protein with ubiquitin for degradation. This technology is called proteolysis targeting chimera (PROTAC). In this project, we managed to theoretically develop a new type of cancer therapeutic agent, that utilize the PROTAC system together with the first-in-class proteasome regulatory subunit, POH1, inhibitor Capzimin as a warhead. By using Capzimin as a warhead it should be possible to polyubiquitinate POH1, and thus induce proteotoxic stress in the cancer cells to trigger apoptosis. This theoretically developed drug is therefore called Capzimin-PROTAC, which should be able to trigger apoptosis in cancer cells, and at the same time being relatively safe to normal healthy cells.

and keywords Abstract and keywords The ubiquitin-proteasome system (UPS) is a tightly and specifically regulated system of protein degradation in eukaryotic cells. Inhibition of an UPS component might represent a strategy to control human diseases, including cancer. Modulation of the UPS can also be employed in basic research strategies. This thesis deals with two independent yet methodologically connected research aims - first, to search for the target of the newly identified UPS inhibitor CBU79, and second, to develop a fluorescent cell-based reporter exploiting proteasomal degradation. In the first part of my work, previous findings regarding the molecular mechanisms of CBU79 inhibiton on the UPS were confirmed. In the next step, I characterized how the UPS inhibitor CBU79 affects protein synthesis using the metabolic labelling of proteins based on click chemistry. I also examined the cytotoxic effect of CBU79 treatment on different cell lines. Finally, I performed a CRISPR/Cas9 whole-genome enrichment screen with the aim to find a potential target of the inhibitor. I found out that CBU79 probably decreases levels of protein synthesis by triggering cellular signalling via the unfolded protein response (UPR). Using the screen, I found 22 potential targets of the CBU79 inhibitor that will be...

Cullin-dependent ubiquitin ligases are responsible for the regulation of most cellular processes. Despite their mutated forms being the cause of many human diseases, their physiological roles are not sufficiently described. In the presented results, we focused on the physiological role of ubiquitin ligase SCFFBXO38 (SKP1-CULLIN1-FBXO38), whose mutated forms are responsible for the progression of distal neuropathy. Preparation of mouse model deficient in FBXO38 revealed that homozygous pups were born in a lower than expected ratio. Animals were growth-retarded, both at the level of the whole organism and individual organs, especially the liver and testes. Males with a deletion in the Fbxo38 gene had significantly lower reproductive capacity, which was associated with lower production of mature sperm and pathological changes in the structure of seminiferous tubules. We found that the FBXO38 protein is functionally expressed in Sertoli cells responsible for regulating spermatogenesis and seminiferous tubules integrity. Detailed analysis of spermatogenic populations revealed a defect at the level of spermatocyte differentiation. The dynamics of this differentiation depend on the hematotesticular barrier functional integrity formed by the intercellular junctions of Sertoli cells. We confirmed that the...

Dilated (DCM) and ischemic cardiomyopathies (ICM) are associated with cardiac remodeling, where the ubiquitin–proteasome system (UPS) holds a central role. Little is known about the UPS and its alterations in patients suffering from DCM or ICM. The aim of this study is to characterize the UPS activity in human heart tissue from cardiomyopathy patients. Myocardial tissue from ICM (n = 23), DCM (n = 28), and control (n = 14) patients were used to quantify ubiquitinylated proteins, E3-ubiquitin-ligases muscle-atrophy-F-box (MAFbx)/atrogin-1, muscle-RING-finger-1 (MuRF1), and eukaryotic-translation-initiation-factor-4E (eIF4E), by Western blot. Furthermore, the proteasomal chymotrypsin-like and trypsin-like peptidase activities were determined fluorometrically. Enzyme activity of NAD(P)H oxidase was assessed as an index of reactive oxygen species production. The chymotrypsin- (p = 0.71) and caspase-like proteasomal activity (p = 0.93) was similar between the groups. Trypsin-like proteasomal activity was lower in ICM (0.78 ± 0.11 µU/mg) compared to DCM (1.06 ± 0.08 µU/mg) and control (1.00 ± 0.06 µU/mg; p = 0.06) samples. Decreased ubiquitin expression in both cardiomyopathy groups (ICM vs. control: p < 0.001; DCM vs. control: p < 0.001), as well as less ubiquitin-positive deposits in ICM-damaged tissue (ICM: 4.19% ± 0.60%, control: 6.28% ± 0.40%, p = 0.022), were detected. E3-ligase MuRF1 protein expression (p = 0.62), NADPH-oxidase activity (p = 0.63), and AIF-positive cells (p = 0.50). Statistical trends were detected for reduced MAFbx protein expression in the DCM-group (p = 0.07). Different levels of UPS components, E3 ligases, and UPS activation markers were observed in myocardial tissue from patients affected by DCM and ICM, suggesting differential involvement of the UPS in the underlying pathologies.

No description available.

Le VIH-1 a développé plusieurs mécanismes menant à la dégradation de son récepteur cellulaire, la molécule CD4, dans le but d’augmenter la relâche de particules virales infectieuses et d’éviter que la cellule soit surinfectée. L’un de ces mécanismes est la dégradation, induite par la protéine virale Vpu, du CD4 nouvellement synthétisé au niveau du réticulum endoplasmique (RE). Vpu doit lier CD4 et recruter l’ubiquitine ligase cellulaire SCFβ-TrCP, via sa liaison à β-TrCP, afin de dégrader CD4. Puisque CD4 doit être retenu au RE pour permettre à Vpu d’induire sa dégradation via le système ubiquitine-protéasome, il a été suggéré que ce processus implique un mécanisme semblable à une voie cellulaire de dégradation des protéines mal-repliées appelée ERAD (« endoplasmic reticulum-associated degradation »). La dégradation par ERAD implique généralement la dislocation des protéines du RE vers le cytoplasme afin de permettre leur poly-ubiquitination et leur dégradation par le protéasome. Nous avons démontré que Vpu induit la poly-ubiquitination de CD4 dans des cellules humaines. Nos résultats suggèrent aussi que CD4 doit subir une dislocation afin d’être dégradé par le protéasome en présence de Vpu. De plus, un mutant transdominant négatif de l’ATPase p97, qui est impliquée dans la dislocation des substrats ERAD, inhibe complètement la dégradation de CD4 par Vpu. Enfin, nos résultats ont montré que l’ubiquitination sur des résidus accepteurs de l’ubiquitine (lysines) de la queue cytoplasmique de CD4 n’était pas essentielle, mais que la mutation des lysines ralentit le processus de dégradation de CD4. Ce résultat suggère que l’ubiquitination de la queue cytosolique de CD4 pourrait représenter un événement important dans le processus de dégradation induit par Vpu. L’attachement de l’ubiquitine a généralement lieu sur les lysines de la protéine ciblée. Toutefois, l’ubiquitination sur des résidus non-lysine (sérine, thréonine et cystéine) a aussi été démontrée. Nous avons démontré que la mutation de tous les sites potentiels d’ubiquitination cytoplasmiques de CD4 (K, C, S et T) inhibe la dégradation par Vpu. De plus, la présence de cystéines dans la queue cytoplasmique apparaît suffisante pour rendre CD4 sensible à Vpu en absence de lysine, sérine et thréonine. Afin d’expliquer ces résultats, nous proposons un modèle dans lequel l’ubiquitination de la queue cytosolique de CD4 serait nécessaire à sa dégradation et où les sites d’ubiquitination de CD4 seraient sélectionnés de façon non spécifique par l’ubiquitine ligase recrutée par Vpu. Enfin, nous avons observé que la co-expression d’une protéine Vpu incapable de recruter β-TrCP (Vpu S52,56/D) semble stabiliser le CD4 qui est retenu au RE. De plus, d’autres mutants de Vpu qui semblent capables de recruter β-TrCP et CD4 sont toutefois incapables d’induire sa dégradation. Ces résultats suggèrent que l’association de Vpu à CD4 et β-TrCP est essentielle mais pas suffisante pour induire la dégradation de CD4. Par conséquent, ces résultats soulèvent la possibilité que Vpu puisse recruter d’autres facteurs cellulaires pour induire la dégradation de CD4. Les résultats présentés ont permis de mieux définir le mécanisme de dégradation de CD4 par Vpu dans des cellules humaines. De plus, ces résultats nous ont permis d’élaborer un modèle dans lequel l’ubiquitine ligase cellulaire SCFβ-TrCP démontre de la flexibilité dans le choix des résidus à ubiquitiner afin d’induire la dégradation de CD4. Enfin, ces études jettent un oeil nouveau sur le rôle de Vpu dans ce processus puisque nos résultats suggèrent que Vpu doive recruter d’autres partenaires cellulaires, mis à part β-TrCP, pour induire la dégradation de CD4. / HIV-1 has developed many mechanisms leading to the down-regulation of its cellular receptor, the CD4 molecule, in order to increase the release of infectious viral particles and to inhibit superinfection of the target cell. One of these mechanisms is the HIV-1 Vpu-mediated degradation of newly synthesized CD4 at the level of endoplasmic reticulum (ER). Vpu must interact with CD4 and recruit the cellular ubiquitin ligase SCFβ-TrCP, via its binding to β-TrCP, in order to induce CD4 degradation. Because CD4 has to be retained in the ER to allow Vpu to induce its degradation via the ubiquitin-proteasome system, it has been suggested that this process involves a mechanism reminiscent of a cellular degradation pathway involved in the proteolysis of unfolded proteins called ERAD (endoplasmic reticulum-associated degradation). The ERAD degradation usually involves the dislocation of proteins from the ER to the cytoplasm in order to induce their poly-ubiquitination and subsequent degradation by the proteasome. We demonstrated that Vpu induces the poly-ubiquitination of CD4 in human cells. Our results also suggest that CD4 has to be dislocated in order to be degraded by the proteasome in presence of Vpu. Furthermore, the expression of a transdominant negative mutant of the ATPase p97, that is involved in the dislocation of ERAD substrates, inhibits completely the Vpu-mediated CD4 degradation process. Finally, our results demonstrated that the ubiquitination of putative ubiquitin acceptor residues (lysines) in the cytosolic tail of CD4 is not essential but the mutation of these lysines slowed down the process of CD4 degradation induced by Vpu. This results suggests that ubiquitination of CD4 cytosolic tail could represent an important step during Vpu-mediated CD4 degradation. Ubiquitin is usually attached on lysine residues in the targetted protein. However, the ubiquitination on non-lysine residues (S, T and C) has also been demonstrated. We demonstrated that the mutation of all cytosolic potential ubiquitination sites (K, C, S and T) of CD4 abolishes Vpu-mediated degradation. In addition, the presence of cysteines in the cytosolic tail of CD4 appeared sufficient to render CD4 sensitive to Vpu in absence of lysine, serine or threonine. In order to explain these results, we propose a model in which CD4 cytosolic tail ubiquitination is necessary for its degradation and where ubiquitination sites are selected non specifically by the ubiquitin ligase recruited by Vpu. Finally, we observed that co-expression of a phosphorylation mutant of Vpu unable to interact with β-TrCP (Vpu S52,56/D) appears to stabilize ER-retained CD4 molecules. In addition, other Vpu mutants seem able to recruit β-TrCP and CD4 without inducing CD4 degradation. These results suggest that Vpu association with CD4 and β-TrCP is essential but not sufficient for CD4 degradation. Consequently, these results raised the possibility that other cellular factors could be recruited by Vpu in order to induce CD4 degradation. The results presented here allowed us to better define the mechanism underlying Vpu-mediated CD4 degradation. In addition, these results allowed us to elaborate a model in which the ubiquitin ligase SCFβ-TrCP show some flexibility in the choice of ubiquitination sites in order to induce CD4 degradation. Finally, theses studies shed a new light on the role of Vpu in the CD4 degradation process because our results suggest that Vpu could recruit, in addition of β-TrCP, other cellular partners in order to induce CD4 degradation.

Cell homeostasis is maintained via strictly regulated processes. One of the important regulation systems is ubiquitin-proteasome proteolytic pathway. Proteins to be degraded are posttranslationally modified with polyubiquitin chains and targeted to the proteasome for degradation. Ubiquitin-proteasome system consists of several processes: ubiquitination of target substrates via set of enzymes, substrate transfer and degradation in the 26S proteasome. There are two ways of ubiquitinated substrate recognition via proteasome. It is either directly by proteasomal receptors or by protein shuttles. Shuttling factors bind polyubiquitinated target substrate and transfer it to the entrance of proteasomal cavity thanks to their typical domain architecture. The N-terminal ubiquitin-like domain binds to regulatory particle of the proteasome and the C-terminal ubiquitin-associated domain binds polyubiqitinated chains on substrates. This thesis focuses on the human DNA damage-inducible protein homolog 2 (Ddi2), a potential member of protein shuttles of humans, and on the interaction of its ubiquitin-like domain with its putative interaction partner, a proteasomal subunit PSMD2. PSMD2 has been cloned, expressed and purified in sufficient yields for further experiments. "Cold" as well as isotopically labeled UBL domain of...