This work examines skeletal muscle catabolism in cancer and its attenuation by Eicosapentaenoic Acid (EPA). In vivo studies in mice bearing a cachexia inducing murine colon adenocarcinoma - MAC16, demonstrated an elevation in the gastrocnemius muscle in the activity and expression of regulatory components of the ubiquitin-proteasome proteolytic pathway. This was accompanied by an accelerated loss of muscle tissue correlating with an increase in overall weight loss, all of which were attenuated by prior daily dosing with EPA. Recently a proteolysis inducing factor (PIF) has been isolated from the MAC16 tumour, and from the serum and urine of cachectic cancer patients. Previous studies have shown that PIF induces protein degradation in vitro, and that this is possibly mediated through 15-hydroxyeicosatetraenoic acid (15-HETE), a metabolite of the n-6 polyunsaturated fatty acid- arachidonate. Employing the murine myoblast cell line C2C12, it was shown that both PIF and 15-HETE increased protein degradation and expression of proteasome subunits, processes which were again attenuated by prior incubation in EPA. Similarly, in NMRI mice which had been fasted for 24hours, EPA and the lipoxygenase inhibitor CV-6504 (but not structurally related fatty acids) inhibited skeletal muscle proteolysis and expression of various proteasome subunits, showing that firstly, EPA may be anti-cachexic partly through its ability to influence 15-HETE production; and secondly that the effect is specific for EPA as other fatty acids had no effect. Previous studies have suggested the involvement of the signal transduction family NFKB in response to PIF in the liver. It has been demonstrated here that both PIF and 15-HETE increased nuclear translocation of NFKB in the skeletal muscle of tumour bearing mice and that EPA inhibited this process by its ability to prevent the degradation of the NFKB inhibitor protein IKB. When an NFKB inhibitor was added to C2C12 myotubes, prior to the addition of PIF, proteasome activity and protein degradation was inhibited, showing that NFKB is responsible for the increased proteasome activity and muscle catabolism induced by PIF. Taken together this work suggests that 15-hydroxyeicosatetraenoic acid is the intracellular mediator for PIF induced protein degradation in skeletal muscle and that elevated muscle catabolism is accomplished through an increased functioning of the ubiquitin-proteasome pathway, a process possibly mediated through an NFKB dependent mechanism. The anticachectic (and possibly the anti-tumourigenic) effects of EPA appear to be achieved in part by its ability to inhibit the degradation of IKB and possibly by its ability to interfere with 15-HETE production.

Friedreich's ataxia (FRDA) is a progressive neurodegenerative disorder caused by a homozygous GAA repeat expansion mutation in intron 1 of the frataxin gene (FXN), which instigates transcriptional issues. As a consequence, reduced levels of frataxin protein lead to mitochondrial iron accumulation, oxidative stress and ultimately cell death; particularly in dorsal root ganglia (DRG) sensory neurons and the dentate nucleus of the cerebellum. In addition to neurological disability, FRDA is associated with cardiomyopathy, diabetes mellitus and skeletal deformities. Currently there is no effective treatment for FRDA and patients die prematurely. Recent findings suggest that abnormal GAA expansion plays a role in histone modification, subjecting the FXN gene to heterochromatin silencing. Therefore, as an epigenetic-based therapy, I investigated the efficacy and tolerability of two histone methyltransferase (HMTase) inhibitor compounds, BIX0194 (G9a-inhibitor) and GSK126 (EZH2-inhibitor), to specifically target and reduce H3K9me2/3 and H3K27me3 levels, respectively, in FRDA human and mouse primary fibroblasts. We show that a combination treatment of BIX0194 and GSK126, significantly increased FXN gene expression levels and reduced the repressive histone marks. However, no increase in frataxin expression was seen. Nevertheless, our results are still promising and may encourage to investigate HMTase inhibitors with other synergistic epigenetic-based therapies for further preliminary studies. Additionally, it has been reported that ubiquitin-proteasome pathway (UPP) controls frataxin stability, thus leading to the development of new therapeutic approaches aimed at preventing the degradation of frataxin. Here we investigated the efficacy of various proteasome inhibitors (MG132, Bortezomib, Salinosporamide A and Ixazomib) using human primary fibroblasts. Only treatments using ixazomib indicated a small increase in frataxin protein; II however, an increase in the cell cycle stress modulator, p27Kip1, was also observed. Therefore, at this stage the use of proteasome inhibitor compounds cannot be advocated for FRDA therapy. Moreover, a study has proposed that increased degradation of D-serine by D-amino acid oxidase (DAO), may lead to low NMDA functioning and impair neural signalling, causing ataxia. Therefore, we investigated a DAO inhibitor, TAK-831, on the YG8sR FRDA mouse model, and detected a significant improvement in ataxia motor coordination deficits. TAK 831 is now proposed for further studies and is currently undergoing randomized Phase 2 clinical trials for FRDA in USA.

Angelman syndrome (AS), a genetic disorder occurring in approximately one in every 15,000 births, is characterized by severe mental retardation, seizures, difficulty speaking and ataxia. The gene responsible for AS was discovered to be UBE3A and encodes an E6-AP ubiquitin ligase. A unique feature of this gene is that it undergoes maternal imprinting in a neuron-specific manner. In the majority of AS cases, there is a mutation or deletion in the maternally inherited UBE3A gene, although other cases are the result of uniparental disomy or mismethylation of the maternal gene. While most human disorders characterized by severe mental retardation involve abnormalities in brain structure, no gross anatomical changes are associated with AS. Although it was previously believed that UBE3A was imprinted in a brain region-specific manner, primarily in the hippocampus and cortex, recent evidence indicates that there is a widespread knockdown of Ube3a protein throughout the AS mouse brain. As a result, it became necessary to evaluate AS human brain samples to verify the relevance and accuracy of the AS mouse model. It was determined that Ube3a is deficient throughout all major brain regions in humans with AS. The remainder of this dissertation work was focused on determining if increased UBE3A expression in the AS mouse brain would be sufficient to rescue the AS phenotype. The results show that adeno-associated virus-mediated UBE3A delivery is not effective in the AS neonatal brain. In the adult AS mouse brain, however, it increased Ube3a in the hippocampus to near wild-type levels. This was sufficient to rescue the associative fear conditioning learning deficit in the AS mouse and improve learning and memory in the Morris water maze. These studies are the first to demonstrate that increased protein production in the adult AS mouse is sufficient to improve the AS phenotype, indicating that the symptoms of AS are not necessarily embryonic developmental.

Ubiquitin proteasome pathway is an important cellular pathway that affects the fate of almost all intracellular proteins. Misregulation of this pathway has been found to be associated with a broad range of human diseases, such as cancer, neurodegenerative diseases, as well as viral infections. Ubiquitin recognition by the proteasome is of central importance to this pathway. So far, two proteasome subunits, Rpn10 and Rpn13, have been identified as ubiquitin receptors. An alternative pathway is mediated by shuttling factors. In yeast, three shuttling factors, known as UBL-UBA proteins, have been found. A UBL receptor activity of the proteasome has been attributed to Rpn1. However, yeast cell mutated all five proteasomal ubiquitin receptors is still viable. To identify the additional proteasomal ubiquitin receptor in cells, I first obtained and characterized a new Rpn13 mutant allele. This Rpn13 mutant completely abolished its ubiquitin binding activity, and functionally resembles a null allele. Rpn13 substrate pool has also been sought in this mutant cells. In the second part of this dissertation, I reported a novel ubiquitin binding site on proteasomal subunit Rpn1. With the help of NMR analysis, Rpn1's ubiquitin and UBL binding surfaces were resolved at high resolution and found to substantially overlap. A specific Rpn1 mutation that disrupts both ubiquitin and UBL binding while not compromising the folding of Rpn1 was obtained. This mutant allele shows a pleiotropic proteasomal defect in vivo. Moreover, I found that the dual ubiquitin/UBL binding activity is not unique in Rpn1, but a common feature in all three proteasomal ubiquitin receptors. In summary, the proteasome adopts a multilayer ubiquitin/UBL binding surface to ensure flexible substrate recognition.

No description available.

The significance of poxvirus manipulation of the host ubiquitin proteasome system has become increasingly apparent. Ubiquitin is post-translationally added to target proteins by a highly conserved enzymatic cascade, typically resulting in protein degradation via the 26S proteasome. The highly conserved poxvirus protein, p28, is a functional ubiquitin ligase and a critical virulence factor. Here, we investigate the relationship between p28 and ubiquitination. We observed that the KilA-N DNA binding domain in p28 targeted p28 to viral factories, where p28 co-localized with conjugated ubiquitin. Furthermore, we determined that p28 is highly regulated by ubiquitination and proteasomal degradation. Disruption of p28 ubiquitin ligase activity revealed that p28 is regulated through auto-ubiquitination and ubiquitination by an additional unknown ubiquitin ligase. Moreover, we observed Lysine-48 ubiquitin linkages, Lysine-63 ubiquitin linkages and a proteasomal subunit co-localizing with p28 at the viral factory, suggesting an intricate relationship between p28 and proteasomal degradation. / Virology

<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.

Proteasome inhibitors bortezomib and carfilzomib are FDA-approved anticancer agents that have contributed to significant improvements in treatment outcomes. However, the eventual onset of acquired resistance continues to limit their clinical utility, yet a clear consensus regarding the underlying mechanisms has not been reached. Bortezomib and carfilzomib are known to target both the constitutive proteasome and the immunoproteasome, two conventional proteasome subtypes comprising distinctive sets of catalytic subunits. While it has become increasingly evident that additional, ‘intermediate’ proteasome subtypes, which harbor non-standard mixtures of constitutive proteasome and immunoproteasome catalytic subunits, represent a considerable proportion of the proteasome population in many cell types, less is known regarding their contribution to cellular responses to proteasome inhibitors. Importantly, previous studies in murine models have shown that individual proteasome subtypes differ in sensitivity to specific proteasome inhibitors. Furthermore, research efforts in our laboratory and others have revealed that proteasome catalytic subunit expression levels and activity profiles are altered when human cancer cells acquire resistance to proteasome inhibitors. We therefore hypothesized that changes in the relative abundances of individual proteasome subtypes contribute to the acquired resistance of cancer cells to bortezomib and carfilzomib. A major obstacle in testing our hypothesis was a lack of chemical probes suitable for use in identifying distinct proteasome subtypes. We addressed this by developing a series of bifunctional proteasome probes capable of crosslinking specific pairs of catalytic subunits colocalized within individual proteasome complexes and compatible with immunoblotting-based detection of the crosslinked subunit pairs. We confirmed the utility of these probes in discerning the identities of individual proteasome subtypes in a multiple myeloma cell line that abundantly expresses catalytic subunits of both the constitutive proteasome and immunoproteasome. Our findings indicate that constitutive proteasomes, immunoproteasomes, and intermediate proteasomes co-exist within these cells and support conclusions drawn from previous studies in other cell types. We also established non-small cell lung cancer cell line models of acquired bortezomib and carfilzomib resistance in which to test our hypothesis. Using immunoblotting and proteasome activity assays, we discovered that changes in the expression levels and activities of individual catalytic proteasome subunits were associated with the emergence of acquired resistance to bortezomib or carfilzomib. These changes were inhibitor-dependent and persisted after the selective pressure of the inhibitor was removed. Finally, results obtained using our bifunctional proteasome probes suggest that the altered abundance of an intermediate proteasome subtype is associated with acquired proteasome inhibitor resistance. Collectively, our results provide evidence linking changes proteasome composition with acquired proteasome inhibitor resistance and support the hypothesis that such changes are involved in resistance mechanisms to these inhibitors.

Doctor of Philosophy / Division of Biology / Jeroen Roelofs / The ubiquitin-proteasome pathway is the major pathway of selective protein degradation in the cell. Disruption of this pathway affects cellular protein homeostasis and contributes to diseases like cancer, and neurodegeneration. The end point of this pathway is the proteasome, a complex protease formed by 66 polypeptides. Structurally, it can be subdivided into the Core Particle (CP) and the Regulatory Particle (RP). The CP harbors the proteolytic sites, whereas, the RP contains six orthologous AAA-ATPases, the Rpt proteins. These Rpt’s are essential for proteasome function and are at the interface between RP and CP. The work in this thesis focuses on the Rpt subunit Rpt5 from yeast. The C-terminal tail of Rpt5 has been shown to contribute to the binding with the CP. However, our study showed it is also essential for the interaction with Nas2, one of nine proteasome-specific chaperones. Thus, Nas2 might function as a regulator of the Rpt5-CP interaction. Further analyses suggested that Nas2 has an additional function in assembly, and that mutating the tail of Rpt5 results in increased binding of the proteasome-associated protein Ecm29 to the proteasome. We showed that Ecm29 binds Rpt5 directly, thereby inducing a closed conformation of the CP substrate entry channel, and inhibiting proteasomal ATPase activity. Consistent with these activities, several proteasome mutant strains showed Ecm29-dependent accumulation of unstable substrates. Thus, Ecm29 is an inhibitor of the proteasome in vivo and in vitro. Interestingly, besides the Rpt5 mutants, several other proteasome mutants show increased levels of Ecm29, suggesting Ecm29 has a role in quality control. Consistent with this, we observed that Ecm29 associates preferably with specific mutants and nucleotide-depleted proteasomes. Based on our data we propose a model, where early in assembly Nas2 binds to the Rpt5 tail inhibiting the Rpt5-CP interaction directly. Later in assembly Ecm29 performs a quality control function, where it recognizes and remains bound to defective proteasomes. By inhibiting these proteasomes Ecm29 prevents the aberrant degradation of proteins.

BACKGROUND: Carfilzomib (CFZ) is a proteasome inhibitor that selectively and irreversibly binds to its target and has been approved in the US for treatment of relapsed and refractory multiple myeloma. Phase 1B studies of CFZ reported signals of clinical activity in solid tumors, including small cell lung cancer (SCLC). The aim of this study was to investigate the activity of CFZ in lung cancer models. METHODS: A diverse panel of human lung cancer cell lines and a SHP77 small cell lung cancer xenograft model were used to investigate the anti-tumor activity of CFZ. RESULTS: CFZ treatment inhibited both the constitutive proteasome and the immunoproteasome in lung cancer cell lines. CFZ had marked anti-proliferative activity in A549, H1993, H520, H460, and H1299 non-small cell lung cancer (NSCLC) cell lines, with IC₅₀ values after 96 hour exposure from <1.0 nM to 36 nM. CFZ had more variable effects in the SHP77 and DMS114 SCLC cell lines, with IC₅₀ values at 96 hours from <1 nM to 203 nM. Western blot analysis of CFZ-treated H1993 and SHP77 cells showed cleavage of poly ADP ribose polymerase (PARP) and caspase-3, indicative of apoptosis, and induction of microtubule-associated protein-1 light chain-3B (LC3B), indicative of autophagy. In SHP77 flank xenograft tumors, CFZ monotherapy inhibited tumor growth and prolonged survival, while no additive or synergistic anti-tumor efficacy was observed for CFZ + cisplatin (CDDP). CONCLUSIONS: CFZ demonstrated anti-proliferative activity in lung cancer cell lines in vitro and resulted in a significant survival advantage in mice with SHP77 SCLC xenografts, supporting further pre-clinical and clinical investigations of CFZ in NSCLC and SCLC.