Elucidating Proteasome Catalytic Subunit Composition and Its Role in Proteasome Inhibitor Resistance

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

Lipid Targets of the Antimalarial Trioxanes in \u3ci\u3ePlasmodium falciparum\u3c/i\u3e

Malaria is among the most debilitating diseases of man. The protozoan parasite, Plasmodium falciparum, causes over a million annual fatalities. The antimalarial trioxanes, exemplified by artemisinin, are among the few pharmaceuticals for which clinical resistance has not become widespread. Artemisinin is a naturally occurring sesquiterpene lactone, containing a unique endoperoxide pharmacophore. Despite extensive study, the precise antimalarial mechanism of action of trioxanes remains elusive. Heme iron-mediated cleavage of the endoperoxide within the parasite digestive vacuole is hypothesized to generate cytotoxic metabolites capable of alkylating heme and damaging cellular macromolecules. The hypothesis of this research is that the endoperoxide pharmacophore underlies the intracellular distribution of trioxanes; thereby identifying cellular targets of the artemisinin-based antimalarials. We therefore identified trioxane cellular accumulation patterns within laboratory cultures of P. falciparum using microscopic visualization of fluorescently-labeled artemisinin derivatives. We further determined the ability of artemisinin to generate reactive oxygen species in vitro and in vivo. Microscopic imaging of a fluorescent trioxane-CH2CH2 O-dansyl demonstrated rapid and specific accumulation of drug within neutral lipid bodies. Fluorescent labeling was competitive with artemisinin. Experiments with inactive artemisinin derivatives and the iron chelator, desferrioxamine, demonstrated that localization within lipid bodies was both iron- and endoperoxide-dependent. An oxidation-sensitive BODIPY lipid probe showed artemisinin-mediated peroxyl radical formation in vivo that was inhibited by lipid body depletion. Moreover, lipid extracts from artemisinin-exposed parasites contained increased fatty acids and a cholesteryl ester. in vitro reactions of artemisinin and heme in the presence of digestive vacuole-associated lipids generated fluorescent peroxidation end-products whose formation was inhibited by α-tocopherol. We found that novel, fluorescently-labeled artemisinin derivatives actively localize to digestive vacuole-associated neutral lipid bodies in P. falciparum. Further, trioxanes altered parasite lipid composition and therefore may be crucial to their antiparasitic mechanism

A model racing plant : founding and economic history of Keeneland racetrack.

This thesis is an economic and historical examination of Keeneland racetrack in Lexington, Kentucky. The material commences with a historical overview of the role of sport and recreational activities in the United States. Putting sport and leisure activity in the context of mirroring changes in society is the focus of the section. Furthermore, this material explores the general sporting environment in United States history, moves to horseracing\u27s place within that history, then concludes with the scope of the horse and horseracing in Kentucky, and particularly, the Bluegrass region of central Kentucky. The following material focuses on the unique and remarkable history and economic survival of Keeneland racetrack in Lexington, Kentucky. The historiography of United States sport and leisure activities, horseracing, and the horse industry in Kentucky is surveyed in the material. The thesis is divided into five sections, covering the history of sports and horseracing in the United States, the founding of Keeneland racetrack, and the survival of Keeneland as a small, cosmopolitan track amid the larger corporate tracks prevalent in the horse industry of the twenty-first century. The Introduction and Chapter One gives an overview of the history of sports and recreational history and horseracing in the United States. Chapter One concludes with the history of the horse and horseracing in Kentucky and the Bluegrass region of central Kentucky. Chapter Two focuses on the horsemen of central Kentucky\u27s founding and opening of the Keeneland Association and Keeneland racetrack. Chapter Three explores the economic survival of the small, cosmopolitan track within the context of the multi-billion dollar horse industry of the twentieth-century, continuing in the first years of the twenty-first century. The Conclusion highlights the main thesis and arguments for each chapter. This material links sport and leisure activity, through the oldest sporting event in the United States (horseracing), within the context of a comparison between sport and change in society

Aurora Volume 26

College formerly located at Olivet, Illinois and known as Olivet University, 1912-1923; Olivet College, 1923-1939, Olivet Nazarene College, 1940-1986, Olivet Nazarene University, 1986-https://digitalcommons.olivet.edu/arch_yrbks/1092/thumbnail.jp

Aurora Volume 26

College formerly located at Olivet, Illinois and known as Olivet University, 1912-1923; Olivet College, 1923-1939, Olivet Nazarene College, 1940-1986, Olivet Nazarene University, 1986-https://digitalcommons.olivet.edu/arch_yrbks/1092/thumbnail.jp

High-Resolution Snapshots of Proteasome Inhibitors In Action Revise Inhibition Paradigms and Inspire Next-Generation Inhibitor Design

New high-resolution crystal structures reported by Schrader and colleagues refine our understanding of how peptide epoxyketone anticancer drugs inactivate their target: the human proteasome. These findings provide important clues for the design of next-generation proteasome inhibitor drugs.OAIID:RECH_ACHV_DSTSH_NO:T201616321RECH_ACHV_FG:RR00200001ADJUST_YN:EMP_ID:A079739CITE_RATE:2.85FILENAME:Carmony_et_al-2016-ChemBioChem.pdfDEPT_NM:제약학과EMAIL:[email protected]_YN:YFILEURL:https://srnd.snu.ac.kr/eXrepEIR/fws/file/cc8c4840-372e-45ca-b9e3-85b0151a0515/linkCONFIRM:

Detection of Panulirus argus Virus 1 (PaV1) in the Caribbean spiny lobster using fluorescence in situ hybridization (FISH)

Panulirus argus Virus 1 (PaV1) is the first virus known to be pathogenic to a wild lobster. It infects the Caribbean spiny lobster P. argus from the Florida Keys, and has a predilection for juveniles. The monitoring of the virus in wild populations and study of its behavior in the laboratory require the development of reliable diagnostic tools. A sensitive and specific fluorescence in situ hybridization (FISH) assay was developed for detection of PaV1. The lower detection limit using a 110 bp DNA probe in a dot-blot hybridization for PaV1 DNA was 10 pg of cloned template PaV1 DNA and 10 ng of genomic DNA extracted from the hemolymph of diseased spiny lobster. The fluorescein (FITC)-labeled probe specifically hybridized to PaV1-infected cells in the hepatopancreas, hindgut, gills, heart, foregut, and nerve tissues. FITC staining was observed around the inner periphery of the nuclear membrane, with lighter staining in a more dispersed pattern within the nucleus. The probe did not hybridize with host tissues of uninfected spiny lobsters, nor did it cross-react with 4 other virus samples tested. This assay will facilitate our understanding of the pathogenesis of the viral disease and help in monitoring efforts directed at determining the prevalence of PaV1 in juvenile nurseries for this lobster

Detection of Panulirus Argus Virus 1 (PaV1) in the Caribbean Spiny Lobster Using Fluorescence in situ Hybridization (FISH)

Panulirus argus Virus 1 (PaV1) is the first virus known to be pathogenic to a wild lobster. It infects the Caribbean spiny lobster P. argus from the Florida Keys, and has a predilection for juveniles. The monitoring of the virus in wild populations and study of its behavior in the laboratory require the development of reliable diagnostic tools. A sensitive and specific fluorescence in situ hybridization (FISH) assay was developed for detection of PaV1. The lower detection limit using a 110 bp DNA probe in a dot-blot hybridization for PaV1 DNA was 10 pg of cloned template PaV1 DNA and 10 ng of genomic DNA extracted from the hemolymph of diseased spiny lobster. The fluorescein (FITC)-labeled probe specifically hybridized to PaV1-infected cells in the hepatopancreas, hindgut, gills, heart, foregut, and nerve tissues. FITC staining was observed around the inner periphery of the nuclear membrane, with lighter staining in a more dispersed pattern within the nucleus. The probe did not hybridize with host tissues of uninfected spiny lobsters, nor did it cross-react with 4 other virus samples tested. This assay will facilitate our understanding of the pathogenesis of the viral disease and help in monitoring efforts directed at determining the prevalence of PaV1 in juvenile nurseries for this lobster