Thiopeptides are posttranslationally-processed macrocyclic peptide metabolites, characterized by extensive backbone and side chain modifications that include a six-membered nitrogenous ring, thioazol(in)e/oxazol(in)e rings, and dehydrated amino acid residues. Thiostrepton A, produced by Streptomyces laurentii ATCC 31255, is one of the more structurally complex thiopeptides, containing a second macrocycle bearing a quinaldic acid. Thiostrepton A and other thiopeptides are of great interest due to their potent activities against emerging antibiotic-resistant Gram-positive pathogens, in addition to their antimalarial and anticancer properties. The ribosomal origins for thiopeptides have been established, however, few details are known concerning the posttranslational modification steps. Alteration to the primary amino acid sequence of the precursor peptide provides an avenue to probe the substrate specificity of the thiostrepton A posttranslational machinery. The information gathered from current studies can also be used to refine thiostrepton’s structure-activity relationship, providing insight into the key features of its scaffold that impart specificity toward each biological target. A fosmid-dependent biosynthetic engineering platform for thiostrepton A was developed and a series of thiostrepton analogs were successfully produced adapting this method. The seventh residue of thiostrepton A is predicted to be critical for the metabolite’s antibacterial activity. Our results were consistent this hypothesis and demonstrated that substitution of Thr7 in the thiostrepton A precursor peptide disrupts both biological activity and successful biosynthesis of the analogs. The thiostrepton biosynthetic machinery’s tolerances toward structural variation at the second and fourth positions of the TsrA core peptide were probed by the saturation mutagenesis of Ala2 and Ala4, respectively. Eight thiostrepton Ala2 variants were isolated with two analogs truncated at the N-terminus by one amino acid, bearing a shortened quinaldic acid-containing macrocycle. Our results suggested that the identity of the core peptide second residue influences the biosynthesis of a thiostrepton analog, however, not essential for the antibacterial and proteasome inhibitory activities of the full-length variants. Additionally, the quinaldic acid loop size affects thiostrepton’s antibacterial potency, but is not critical for the proteasome inhibitory activity. Sixteen thiostrepton analogs were isolated from Ala4 mutagenesis studies. We demonstrated that the identity of the amino acid residue at the fourth position in the thiostrepton scaffold is not critical to inhibit either the ribosome or the proteasome in vitro.

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Jana Kroupová Supervisor: PharmDr. Anna Jirkovská, Ph.D. Title of diploma thesis: The effect of the proteasome inhibition of the antiproliferative effect of anthracycline antibiotics Anthracycline antibiotics (daunorubicin, doxorubicin) belong to the most effective antitumor drugs. In current clinical practice they are used mostly in the combinations with either "classical" or new targeted antitumor drugs. The proteasome inhibitors (bortezomib and carfilzomib) are also viewed as a part of new "targeted" antitumor drugs. The proteasome is a multienzyme complex in eukaryotic cells which is responsible for intracellular degradation of proteins. The proteasome inhibitors have been largely used in the therapy of multiple myeloma, but their potential has been also studied in the case of other malignancies. Their use in the combination with anthracyclines could be a possible alternative in the therapy of some tumor illnesses, but the effect of combination of anthracyclines and proteasome inhibitors on tumor cells have not been sufficiently explained. The anthracycline therapy is also accompanied by serious adverse side effect - the cardiotoxicity, which potential could be influenced by the...

The problem of finding targeted medicine is a central problem in chemotherapy. From this point of view the ubiquitin-proteasome system is a highly promising object in the pharmaceutical approach. Proteasome plays a critical role in cellular protein degradation, cell cycle and apoptosis regulation. Proteasome inhibitors are substances blocking the actions of proteasome. Cancer cells are more sensitive to inhibition of the ubiquitin-proteasome system than normal cells. Therefore proteasome inhibitors have the potential to be successfully used in the cancer treatment. The study aimed to test various substances to identify possible proteasome inhibitors with the IncuCyteTM FLR image system and fluorometric microculture cytotoxicity assay. Using the IncuCyte FLR method allows for detecting changes in the molecular processes of living cells. To make proteasome inhibition visible the model cell line MelJuSoUbG76V-YFP is used which helps to detect alterations in proteasome activity by means of the yellow fluorescent protein enrichment in cells as a response to proteasome inhibition. Fluorometric microculture cytotoxicity assay is a method for the determination of cytotoxicity in human tumor cells. The study showed that substance #25 possessed a proteasome inhibitory capacity in a dose-dependent manner as demonstrated with the IncuCyte FLR image system. According to the fluorometric microculture cytotoxicity assay, substance #1 was the most stable and toxic. Substances #2 and #185 had selective toxicity against cancer cells and lower effects against normal cells. Combining IncuCyte FLR and fluorometric microculture cytotoxicity assay allows finding substances which act as proteasome inhibitors with high toxic effect.

Heat shock proteins (HSPs) are molecular chaperones that assist in protein synthesis, folding and degradation and prevent stress-induced protein aggregation. The present study examined the pattern of accumulation of HSP30 and HSP70 in cells recovering from heat shock as well as the effect of proteasome inhibition on cytoplasmic/nuclear and endoplasmic reticulum (ER) molecular chaperone accumulation, large multimeric HSP30 complexes, stress granule and aggresome formation in Xenopus laevis A6 kidney epithelial cells. Initial immunoblot analysis revealed the presence of elevated levels of HSP30 after 72 h of recovery. However, the relative levels of HSP70 declined to near control levels after 24 h. The relative levels of both hsp30 and hsp70 mRNA were reduced to low levels after 24 h of recovery from heat shock. Pretreatment of cells with cycloheximide, a translational inhibitor, produced a rapid decline in HSP70 but not HSP30. The cycloheximide-associated decline of HSP70 was blocked by the proteasomal inhibitor, MG132, but had little effect on the relative level of HSP30. Also, treatment of cells with the phosphorylation inhibitor, SB203580, in addition to cycloheximide treatment enhanced the stability of HSP30 compared to cycloheximide alone. Immunocytochemical studies detected the presence of HSP30 accumulation in a granular pattern in the cytoplasm of recovering cells and its association with aggresome-like structures, which was enhanced in the presence of SB203580. To verify if proteasome inhibition in A6 cells induced the formation of similar HSP30 granules, immunoblot and immunocytochemical analyses were performed. MG132, celastrol and withaferin A enhanced ubiquitinated proteins, inhibited chymotrypsin-like activity of the proteasome and induced the accumulation of cytoplasmic/nuclear HSPs, HSP30 and HSP70 as well as ER chaperones, BiP and GRP94 and heme oxygenase-1. Northern blot experiments determined that proteasome inhibitors induced an accumulation in hsp30, hsp70 and bip mRNA but not eIF1α. The final part of this study demonstrated that treatment of A6 cells with proteasome inhibitors or sodium arsenite or cadmium chloride induced HSP30 multimeric complex formation primarily in the cytoplasm. Moreover, these stressors also induced the formation of RNA stress granules, pre-stalled translational complexes, which were detected via TIA1 and polyA binding protein (PABP), which are known stress granule markers. These stress granules, however, did not co-localize with large HSP30 multimeric complexes. In comparison, proteasome inhibition or treatment with sodium arsenite or cadmium chloride also induced the formation of aggresome-like structures, which are proteinaceous inclusion bodies formed as a result of an abundance of aggregated protein. Aggresome formation was identified by monitoring the presence of vimentin and γ-tubulin, both of which are cytoskeletal proteins and serve as markers of aggresome detection. Aggresome formation, which was also verified using the ProteoStat assay, co-localized with large HSP30 multimeric complexes. Co-immunoprecipitation experiments revealed that HSP30 associated with γ-tubulin and β-actin in cells treated with proteasome inhibitors or sodium arsenite or cadmium chloride suggesting a possible role in aggresome formation. In conclusion, this study has shown that the relative levels of heat shock-induced HSP30 persist during recovery in contrast to HSP70. While HSP70 is degraded by the ubiquitin-proteasome system, it is likely that the presence of HSP30 multimeric complexes that are known to associate with unfolded protein as well as its association with aggresome-like structures may delay its degradation. Finally, proteasome inhibition, sodium arsenite and cadmium chloride treatment of A6 cells induced cytoplasmic/nuclear and ER chaperones as well as resulting in the formation stress granules and aggresome-like structures which associated with large HSP30 multimeric complexes.

<p>The ubiquitin-proteasome system is responsible for cellular protein recycling, and it is a crucial system to maintain proper protein balances in cells. Proteasome is the main component of the system, and the system is tightly related to multiple cellular processes. Malfunction of the proteasome could lead to various diseases including cancer, neurodegenerative diseases and autoimmune diseases. As a result, researchers have been developing small molecules to target the proteasome to regulate its function. Currently, three small molecules have been approved by FDA as proteasome inhibitors to treat hematological cancer multiple myeloma. However, these small molecules inhibit the same enzymatic subunit on the proteasome and drug resistance has been observed among patients administrating these proteasome inhibitors. To develop new small molecules to target the proteasome, we started to investigate the 19S regulatory particle of the proteasome. In this work, we presented a workflow of discovering a small molecule selective binder, TXS-8, to 19S regulatory particle subunit Rpn-6. We also developed a series of assays to investigate the impact of small molecule on proteasome activity. At last, we introduced the binding site study of TXS-8, development of TXS-8-based PROTAC and new proteasome probe development.</p> <p>We first developed a one-bead-one-compound (OBOC) library to screen with Rpn-6 to discover potential binders to Rpn-6. After careful evaluation and validation, TXS-8 was discovered as the best hit from the screening. Our covalent pull-down experiment with cell lysate later confirmed TXS-8 as a selective binder of Rpn-6 and proteomic analysis of the pulled down protein also validated Rpn-6 as the major target of TXS-8.</p> <p>We then investigated the impact of TXS-8 in Rpn-6 overexpressed cancer cells like Ramos B-cell and multiple myeloma. TXS-8 was four-fold more toxic in these cells comparing to our control HEK-293T cells. To understand the cause of cell death when dosed with TXS-8, we began to investigate the impact of TXS-8 on proteasome activity, but some preliminary results were inconsistent. By the same time, there is also lack of a general workflow to investigate the impact of small molecules on proteasome activity. Therefore, we developed a three-step process to illustrate the general workflow using TXS-8 as an example. We first knocked down Rpn-6 in HEK-293T cells and monitored proteasome activity changes with a cell permeable probe our lab developed. We then transfected HEK-293T cells with a full-length foreign protein and knocked down Rpn-6 in these cells. We later monitored the degradation of the foreign protein when dosed with TXS-8. In the last step, we monitored the proteasome activity changes in primary cell lines when dosed with TXS-8. From these three steps, we successfully demonstrated a general workflow to investigate if a small molecule can affect proteasome activity. We also concluded that TXS-8 was unable to affect proteasome activity at non-lethal concentration.</p> <p> To further investigate TXS-8 and provide guidance for future structural optimization to improve potency, we proposed two methods on investigating the general binding site of TXS-8 on Rpn-6 using cross-linking techniques that is currently ongoing. We also modified TXS-8 into proteolysis targeting chimeras (PROTACs) to investigate if TXS-8-based PROTAC can improve toxicity and selectively induce Rpn-6 degradation in cells. However, no significant cell toxicity or Rpn-6 degradation was observed when dosed with TXS-8-based PROTACs.</p> Finally, Due to limitation of cell permeable probes, we were unable to investigate the impact of TXS-8 on the caspase-like β1 and trypsin-like β2 subunit of the proteasome in our previous studies. Although TXS-8 did not alter the chymotrypsin-like activity at non-lethal concentration, examining the effect of TXS-8 on the caspase-like and trypsin-like activity could still benefit our research. Besides, we also desire to expand our proteasome activity toolbox by developing more sensitive proteasome probes. Therefore, by analyzing and combing the commercially available proteasome probes and LLVY-Rh probes, we decided to develop selective proteasome probes for the β1 and β2 subunit to provide useful tools for future potential small molecule proteasome regulator characterization.

No description available.

Das Ubiquitin-Proteasom-System (UPS) ist das wichtigste System für den Abbau von nicht mehr benötigten oder beschädigten Proteinen innerhalb der eukaryotischen Zelle und ist somit an der Aufrechterhaltung der zellulären Homöostase beteiligt. Ein Abfall der proteasomalen Aktivität führt zu intrazellulärem Stress. Die Zelle wirkt diesem Abfall entgegen, indem sie die proteasomalen Gene verstärkt exprimiert und dadurch die Neubildung von 26S Proteasomen bewirkt. Während in der Bäckerhefe Saccharomyces cerevisiae mit Rpn4 der Transkriptionsfaktor für die verstärkte Expression identifiziert wurde, war dieser in Säugetieren noch nicht bekannt. In der vorliegenden Arbeit konnte TCF11 (transcription factor 11) als der verantwortliche Transkriptionsfaktor identifiziert werden, der in der humanen Endothelzelllinie Ea.hy926 die Transkription der proteasomalen Gene nach Proteasominhibition induziert. Unter physiologischen Bedingungen ist TCF11 ein N-glykosyliertes ER-ständiges Membranprotein, welches durch die ER-assoziierte Protein Degradation, unter der Mitwirkung des E3-Enzyms HRD1 und der AAA-ATPase p97, schnell abgebaut wird. Nach der Proteasominhibition kommt es zur Akkumulation von oxidierten Proteinen, und TCF11 wird aktiviert und in den Zellkern transportiert. Im Zellkern bindet TCF11 an AREs (antioxidant response element) in den proteasomalen Promotoren und aktiviert dadurch die Transkription der proteasomalen Gene. Darüber hinaus reguliert TCF11 auch die Expression von zahlreichen Enzymen, die die Ubiquitinierung von Proteinen katalysieren. Dadurch wird die zelluläre Homöostase wiederhergestellt und TCF11 sehr wahrscheinlich durch die neu gebildeten Proteasomen abgebaut. Die Ergebnisse der vorliegenden Arbeit zeigen auf, dass die Integrität des UPS nach Proteasominhibition in der humanen Endolthelzelllinie Ea.hy926 über einen TCF11 abhängigen Rückkopplungsmechanismus aufrechterhalten wird. / The ubiquitin-proteasome-system (UPS) is the most important system for regulated protein degradation in eukaryotes. Therefore it is involved in the regulation of cellular homeostasis. Reduced proteasome activity results in proteotoxic stress. To counteract for reduced proteasome activity, eukaryotic cells enhance proteasome gene expression, which results in formation of new 26S proteasomes and recovery of physiological conditions. While in bakers yeast Saccharomyces cerevisiae the transcription factor Rpn4 is responsible for enhanced proteasome gene expression in response to proteasome inhibition, in mammals the responsible transcription factor was unknown. In this thesis, transcription factor TCF11 (transcription factor 11) was identified as a key regulator for 26S-proteasome formation in the human cell line Ea.hy926 to compensate for reduced proteolytic activity. Under non-inducing conditions N-glycosylated TCF11 resides in the endoplasmic reticulum (ER) membrane, where TCF11 is targeted to ER-associated protein degradation system requiring the E3-ubiquitin ligase HRD1 and the AAA-ATPase p97. Proteasome inhibitors trigger the accumulation of oxidant-damaged proteins, and promote the nuclear translocation of TCF11 from the ER, permitting activation of proteasome gene expression by binding of TCF11 to antioxidant response elements (ARE) in their promoter regions. Furthermore TCF11 controlls the expression of additional UPS-related genes. Thus the transcriptional feedback loop regulating human proteasome dependent protein degradation to counteract proteotoxic stress caused by proteasome inhibition was uncovered.

For all living systems, there is a requirement to recycle and regulate proteins. In eukaryotic organisms this is accomplished by the proteasome. The p97 ATPase is another highly conserved and essential complex present throughout the eukaryotic cell. In Paper I we utilized UFD fluorescent substrates to address the role of p97 and cofactors in soluble proteasome degradation. Results using RNAi and Drosophila p97 mutants propose p97 to function upstream of the proteasome on cytosolic proteasome targets as an important unfoldase together with its Ufd1/Npl4 cofactors. The results implicate p97 to be important for degradation of proteasome substrates lacking natural extended peptide regions. In Paper II we focused on identifying transcription factors essential for production of proteasomal subunits and associated proteins in Drosophila S2 cells. We utilized an RNA library targeting 993 known or candidate transcription factors and monitored RNAi depleted Drosophila S2 cells expressing the UFD reporter UbG76VGFP. We identified a range of potential candidates and focused on the bZIP transcription factor Cnc-C. RNAi and qrt-PCR experiments implicated Cnc-C to be involved in transcription of proteasomal subunits. In Paper III we applied our knowledge gained from Paper I about p97 dependent substrates and set up a high-throughput microscopy screening method to potentially find inhibitors specifically targeting the p97 proteasomal sub-pathway. Utilizing UFD substrates with and without C-terminal peptide tails we determined if compounds inhibited the core proteasomal machinery or the p97 pathway specifically. Through a primary and secondary round of screening we identified several new compounds inhibiting the ubiquitin-proteasome pathway though none from our initial screening had specificity for p97. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 3: Manuscript.

<p>The proteasome is a multi-catalytic, multiprotein enzymatic machinery that is responsible for most of the protein degradation in the cell. Cellular protein homeostasis through the proteasome is regulated through the ubiquitin-independent or ubiquitin-dependent degradation pathway, which both utilize different isoforms of the enzymatic machinery. Over the past twenty years, the proteasome has been a well-validated therapeutic target by inhibition of its catalytic particle function, and more recently, through targeted protein degradation with the use of proteolysis targeting chimeras (PROTACs). Inhibition of the proteasome’s catalytic function has been previously shown to be therapeutically advantageous due to the need for high proteasomal activity for the survival of hematological cancer cells, which produce an overabundance of misfolded and unwanted proteins. Despite this success, off-target toxicities and drug-resistant mechanisms remain as dose-limiting factors for proteasome catalytic inhibition. Herein, we describe a variety of peptidomimetic (or “peptide-like”) approaches that target the proteasome beyond standard catalytic inhibition to serve as alternative therapies for hematological cancer. We investigate <em>(1)</em> the preferential structural properties of peptide-conjugated unnatural substrates for different proteasome isoforms’ substrate channels, <em>(2)</em> the effectiveness of an immunoproteasome-targeting peptide-conjugated prodrug strategy, and <em>(3)</em> the unknown binding site of a peptoid probe on the proteasome’s non-catalytic ubiquitin receptor, Rpn-13. This work not only showcases novel strategies to target the proteasome system but also describes methods that could be applied to other challenging enzymes or non-catalytic protein targets.</p>

Das Ubiquitin-Proteasom-System (UPS) stellt eines der wichtigsten zellulären Abbausysteme dar. Es vermittelt die Degradation fehlgefalteter, beschädigter sowie regulatorischer Proteine. Folglich ist es essentiell für die Proteinqualitätskontrolle und für eine Vielzahl zellulärer Prozesse. Eine Störung des UPS steht im engen Zusammenhang mit neurodegenerativen Erkrankungen und malignen Tumoren. Adaptive Mechanismen ermöglichen es der Zelle das UPS an den stetig schwankenden Bedarf proteolytischer Aktivität anzupassen. So wirkt eine erhöhte Expression proteasomaler Gene einem Abfall der proteasomalen Aktivität entgegen. Der Transkriptionsfaktor TCF11/Nrf1 wurde hierbei als Hauptregulator identifiziert. Unter physiologischen Bedingungen ist TCF11/Nrf1 in der ER-Membran lokalisiert und wird über das ER-assoziierte Degradationssystem (ERAD) abgebaut. In Antwort auf Proteasominhibition wird der Transkriptionsfaktor aktiviert und in den Nukleus transferiert. Hier vermittelt er durch Bindung der regulatorischen antioxidative response elements die Genexpression proteasomaler Untereinheiten. Die Ergebnisse dieser Arbeit zeigten, dass es sich bei diesem autoregulatorischen Rückkopplungsmechanismus um einen generellen adaptiven Regulationsmechanismus in Mammalia handelt. Zudem ergaben weitere Untersuchungen, dass der durch Proteasominhibition hervorgerufene oxidative Stress, die TCF11/Nrf1-vermittelte Aktivierung der Genexpression fördert. Die induzierende Wirkung von oxidativem Stress wurde ferner unter Verwendung des Pro-Oxidans Rotenon bekräftigt. Dieses Neurotoxin induziert die TCF11/Nrf1-abhängige Transkription proteasomaler Untereinheiten und folglich die Neubildung aktiver Proteasomkomplexe. Der Transkriptionsfaktor förderte ferner die Zellviabilität Rotenon-behandelter SH-SY5Y Zellen. Diese Ergebnisse demonstrieren, dass die TCF11/Nrf1-vermittelte Genexpression proteasomaler Untereinheiten bedeutend für die Aufrechterhaltung der Redox- sowie der Protein Homöostase ist. / The ubiquitin proteasome system (UPS) represents a major protein degradation machinery. It facilitates the degradation of misfolded and damaged as well as regulatory proteins, thereby ensuring protein quality control and regulation of various cellular processes. Disturbances of the UPS are strongly associated with neurodegeneration and cancer. Adaptive mechanisms enable the cell to deal with changing demand in proteolytic activity. A rise in proteasomal gene expression compensates for decreased proteasomal activity. This adaption is mainly regulated by the transcription factor TCF11/Nrf1. Under unstressed conditions TCF11/Nrf1 resides in the ER-membrane where it is degraded via the ER-associated protein degradation system (ERAD). Proteasome inhibition causes the nuclear translocation of TCF11/Nrf1. In the nucleus, it mediates the gene expression of proteasomal subunits by interacting with their regulatory antioxidant response elements. Within this thesis, it was shown, that this autoregulatory feedback loop represents a general adaptive mechanism in mammalian cells. Moreover, experiments using antioxidative compounds revealed, that the oxidative stress induced by proteasomal inhibition promotes the TCF11/Nrf1-dependent proteasomal gene expression. The inducing effect of oxidative stress was verified using the pro-oxidant rotenone. This neurotoxin activates the transcription of the proteasomal genes resulting in the formation of newly synthesised, active proteasome complexes. Thus, TCF11/Nrf1 exerts a cytoprotective function in response to oxidative and proteotoxic stress in SH-SY5Y cells. In conclusion, this thesis revealed that TCF11/Nrf1-dependent induction of the proteasome expression promotes the maintenance of the redox as well as protein homeostasis.