IU Indianapolis ScholarWorks :: Browsing by Author "Goebl, Mark"

Browsing by Author "Goebl, Mark"

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12-lipoxygenase Promotes Macrophage Infiltration and Pancreatic Islet Dysfunction in the Vertebrate Models of Diabetes Pathogenesis
(2020-05) Kulkarni, Abhishek Anant; Harrington, Maureen; Mirmira, Raghavendra; Anderson, Ryan; Goebl, Mark; Mosley, Amber; Marrs, James
Diabetes is a morbid metabolic disorder that affects almost 500 million people worldwide. Although multiple factors contribute to diabetes pathogenesis, pancreatic islet inflammation and dysfunction are shared characteristics of its major forms. 12- lipoxygenase (12-LOX), an enzyme involved in lipid metabolism, has been implicated in islet inflammation. 12-LOX generates reactive oxygen species (ROS) that activate inflammation and serve as major contributors to islet dysfunction. Importantly, since ROS are transient moieties, they are challenging to study in vivo. Hence, establishing better animal models of ROS-mediated stress is critical to facilitate the discovery and preclinical testing of novel diabetes therapeutics. Here, I have adapted a zebrafish model of conditional β-cell injury, which is regulated by the administration of the prodrug metronidazole (MTZ), to study responses to ROS in vivo. I demonstrate that with MTZ treatment, ROS are generated within β-cells and subsequently exhibit recruitment of macrophages into the islet and induction of β-cell death. I utilized this model to uncover roles for macrophages and 12-LOX during islet injury. Excessive macrophage infiltration exacerbates islet inflammation and dysfunction. Interestingly, on the depletion of macrophages in zebrafish, I observed that β-cells recovered normal function upon cessation of prodrug treatment. This suggests that infiltrating macrophages promote maladaptive inflammation and premature removal of damaged β-cells. Thus, limiting the macrophage infiltration may be a therapeutic approach to restoring β-cell function. Based on the established roles of 12-LOX in other contexts, I hypothesized that its inhibition would prevent the localized infiltration of proinflammatory macrophages. To test this, I used both zebrafish and mouse models and observed a significant reduction in macrophage migration upon loss of 12- LOX activity. Furthermore, I found that expression of CXCR3, a crucial receptor regulating migration, was significantly reduced in 12-LOX loss-of-function macrophages. These data suggest a role for 12-LOX in macrophages, which is conserved across species. Collectively, my study reveals novel roles for 12-LOX in macrophage function and provides testable therapeutic targets for the resolution of inflammation-induced damage in the pancreatic islets.
Deep Proteome Profiling in the Progression of Pancreatic Ductal Adenocarcinoma-Associated Cachexia
(2021-09) Umberger, Tara; Mosley, Amber L.; Zimmers, Teresa; Goebl, Mark; Doud, Emma
Cachexia is a devastating muscle wasting syndrome affecting multiple biochemical pathways and is a comorbidity of many diseases including pancreatic ductal adenocarcinoma (PDAC). PDAC patients with cachexia commonly experience systemic inflammation, progressive loss of lean muscle and adipose tissue, and cardiac dysfunction. The present workflow identifies proteins and their post-translational modifications extracted from both cardiac and skeletal muscle tissue isolated from a murine model of PDAC-associated cachexia. Reported here are differentially occurring post-translational modifications found on the most abundant contractile proteins. Tissue from mouse muscle samples were collected two weeks after either receiving a sham surgery or orthotopically implanted with PDAC tumor cells, with or without a follow-up chemotherapy treatment of the standard of care agent gemcitabine with nab-Paclitaxel. Whole tissue blocks of gastrocnemius or heart were either flash frozen and pulverized or homogenized in denaturing lysis buffer and then sonicated to facilitate protein extraction. After disulfide bond reduction, cysteine alkylation, and trypsin digestion, the resultant peptides were subjected to molecular barcoding using tandem mass tag isobaric labeling reagents to facilitate multiplexing. The first and second dimension of peptide separation in the multiplexed sample is accomplished with an offline, high pH, reverse phase (RP)-LC fractionation followed by an online RP-LC at lower pH. The use of high-field asymmetric-waveform ion mobility spectrometry provided a last dimension of separation before MSn analyses. This novel, proteomic workflow enables deep proteome profiling in the progression of cancer-induced cachexia. The use of multi-dimensional chromatographic separation and differential ion mobility technique have allowed us to identify almost 4,500 proteins groups of gastrocnemius muscle tissue and nearly 7,100 protein groups of myocardium taken from the murine PDAC model of pancreatic cancer. A comprehensive analysis of the data collected from this workflow was used to calculate differential post-translational modifications on major contractile proteins isolated from PDAC model muscle tissue, with or without chemotherapy, when compared to sham surgery controls. Differential post-translational modifications and protein expression changes found to contribute to cancer cachexia may elucidate novel molecular mechanisms and cellular signaling that drive cachexia progression.
Erratum to: The loop-less tmCdc34 E2 mutant defective polyubiquitination in vitro and in vivo supports yeast growth in a manner dependent on Ubp14 and Cka2.
(BMC, 2016) Lass, Agnieszka; Cocklin, Ross; Scaglione, Kenneth M.; Skowyra, Michael; Korolev, Sergey; Goebl, Mark; Skowyra, Dorota; Department of Biochemistry and Molecular Biology, IU School of Medicine
FANCA maintains genomic stability through regulating BUBR1 acetylation
(2017-08) Abdul Sater, Zahi Abass; Nalepa, Grzegorz; Clapp, Wade; Goebl, Mark; Wek, Ronald
Fanconi Anemia (FA), a chromosomal instability syndrome, is characterized by bone marrow failure, genetic malformations, and predisposition to malignancies like acute myeloid leukemia (AML) and solid tumors. FA is caused by germline bi-allelic mutations in one of 21 known FA pathway genes and somatic mutations in FA genes are also found in a variety of sporadic cancers. Recently, numerous reports have discovered that the protective function of the FA pathway extends beyond its canonical role in regulation of DNA repair in interphase. In particular, the FA pathway has been shown to function in essential mitotic processes including spindle assembly checkpoint (SAC), cytokinesis, and centrosome maintenance. Understanding of the mechanistic origins of genomic instability leading to carcinogenesis and bone marrow failure has important scientific and clinical implications. To this end, using a micronucleus assay, we showed that both interphase DNA damage and mitotic errors contribute to genomic instability in FA ex vivo and in vivo. Functional studies of primary FA patient cells coupled with super-resolution microscopy revealed that FANCA is important for centrosome dependent spindle assembly supporting the protective role of FA pathway in mitotic processes. Furthermore, we dissected the interactions between the FA pathway and cellular kinase networks by employing a synthetic lethality sh-RNA screen targeting all human kinases. We mapped kinases that were synthetically lethal upon loss of FANCA, particularly those involved in highly conserved signal transduction pathways governing proliferation and cell cycle homeostasis. We mechanistically show that loss of FANCA, the most abundant FA subtype, results in in premature degradation of the mitotic kinase BUBR1 and faster mitotic exit. We further demonstrate that FANCA is important for PCAF-dependent acetylation of BUBR1 to prevent its premature degradation. Our results deepen our understanding of the molecular functions of the FA pathway in mitosis and uncover a mechanistic connection between FANCA and SAC phosphosignaling networks. These findings support the notion that further weakening the SAC through targeting kinases like BUBR1 in FA-deficient cancers may prove to be a rational therapeutic strategy.
The impact of the termination override mutation on the activity of SSU72
(2016-12-19) McCracken, Neil Andrew; Mosley, Amber; Wek, Ronald; Goebl, Mark
Ssu72, an RNA Pol II CTD phosphatase that is conserved across eukaryotes, has been reported to have a wide array of genetic and physical associations with transcription factors and complexes in RNA transcription. Catalytic mutants of Ssu72 are lethal across many eukaryotes, and mutations to non-catalytic sites in SSU72 phosphatase have been shown to lower function. One spontaneous mutation of the SSU72 gene in Saccharomyces cerevisiae (A to C nucleotide mutation resulting in an L84F mutation in the coded protein) was shown to have transcription termination deficiency (termination override or TOV). This SSU72 mutation was suggested by Loya et al. to cause a lowering of the phosphatase activity of the protein and consequently affect proper termination. In research reported herein, an investigation was completed through in-vitro and ex-vivo approaches with the goal of understanding the impact of the SSU72 TOV mutation on the observed phenotype in S. cerevisiae. It can be concluded from work presented in this report that the SSU72 TOV mutation does not cause a decrease in in-vitro phosphatase activity as compared to wild type. Evidence presented even suggests an increase in phosphatase activity as compared to wild type Ssu72. One model for the observed responses in transcription termination is that the phenylalanine substitution in Ssu72 leads to cooperative interactions with proline residues in the CTD. It is proposed that the corresponding increase in Ssu72 phosphatase activity limits RNA Pol II CTD association with termination factors, such as Nrd1, thus causing deficient transcription termination.
In-depth bioinformatics analysis of the phosphoproteome of triple negative breast cancer treated with a tumor selective NQO1 bioactivatable drug
(2021-01) Roy, Gitanjali; Mosley, Amber L.; Georgiadis, Millie M.; Goebl, Mark
The main focus of this study is to elucidate changes in the proteome of triple negative breast cancer cells in response to a novel bioactivated anti-cancer agent IB-DNQ (isobutyl-deoxyniboquinone). NQO1 or NADPH:quinone oxidoreductase-1 is a detoxifying enzyme overexpressed in many solid tumors and low expression in normal cells. IB-DNQ is bio-activated by NQO1 enzyme via a futile redox cycling, producing large amounts of reactive oxygen species (ROS) in the process, which causes DNA lesions in cancer cells. The status of NQO1 is important for the IB-DNQ mediated cancer cell death. IB-DNQ mediated therapy has great potency in killing breast cancer cells compared to PARP inhibitor Rucaparib. From this proteomics study, large changes in phosphorylation are observed in utilizing a combination therapy with low dose of IB-DNQ and PARP inhibitor Rucaparib. Protein phosphorylation events within the transcription machinery and DNA damage repair pathways are changed upon drug treatment. Computational and bioinformatic analysis of kinases involved through kinase substrate enrichment analysis revealed changes in downstream signaling of cell cycle checkpoint proteins. CDK1 and CDK2 substrate phosphorylation was decreased in response to combination drug therapy. Based on the differential kinase activity as determined by substrate abundance, we hypothesize that since CDK1/2 plays an important role in DNA damage repair via the homologous recombination pathway, its downregulation further abrogated double stranded break repair in BRCA deficient cells creating a state of “BRCAness”, leading to heightened sensitivity to the PARP inhibitor Rucaparib. These studies give insight into the mechanism of IB-DNQ action as an anticancer agent.
An Integrative Genome-Based Metabolic Network Map of Saccharomyces Cerevisiae on Cytoscape: Toward Developing A Comprehensive Model
(2022-03) Hamidi, Aram; Goebl, Mark; Cocklin, Ross; Wells, Clark; Harrington, Maureen
Metabolic flux analyses and their more comprehensive forms, genome-scale metabolic networks (GSMNs), have gained tremendous attention in industrial and medical research. Saccharomyces cerevisiae (S. cerevisiae) is one of the organisms that has had its GSMN subjected to multiple frequent updates. The objective of this study is to develop a visualization tool for the GSMN of S. cerevisiae for educational and research purposes. This visualization tool is called the Master Metabolic Map of Saccharomyces cerevisiae (MMMSC). In this study, a metabolic database of S. cerevisiae developed by us was transferred to Cytoscape, a useful and efficient bioinformatics software platform for visualizing molecular networks. After the MMMSC was created, nodes, representing metabolites and enzymes, and edges, representing the chemical reactions that connect the nodes, were curated manually to develop a metabolic visualization map of the whole metabolic system of S. cerevisiae (Figure 4). In the discussion, examples are provided regarding possible applications of MMMSC to predict possible effects of the manipulation of the S. cerevisiae metabolome for industrial and medical purposes. Ultimately, it is concluded that further work is needed to complete the metabolic database of S. cerevisiae and the related MMMSC. In future studies, these tools may be integrated with other omics and other approaches, especially the directed-evolution approach, to increase cost and time efficiency of future research and to find solutions for complex and, thus far, poorly managed environmental and health problems.
Loss of SIMPL increases TNFα sensitivity during hematopoiesis
(2008-10) Benson, Eric Ashley; Harrington, Maureen A.; Goebl, Mark; Clapp, Wade; Skalnik, David
The innate and adaptive immune responses are critical for host survival. The TNFα/NF-κB signaling pathway is a major regulator of the immune response. The TNFα/NF-κB signaling pathway has also been proposed to play a role in the regulation of hematopoiesis. In the TNFα signaling pathway, full induction of NF-κB (specifically the p65 subunit) dependent transcription is regulated by a co-activator SIMPL. The biological significance of SIMPL in TNFα dependent responses is poorly understood. To study SIMPL in vitro and in vivo in mammalian cells, a knockdown system utilizing shRNA (short hairpin RNA) was used. Analysis of hematopoietic progenitor cells infected with a retrovirus encoding the SIMPL shRNA was used to study the role of SIMPL in hematopoiesis. The ability of progenitor cells lacking SIMPL to grow and differentiate was not compromised. In contrast in the progenitors cells lacking SIMPL, TNFα mediated inhibition of colony formation was significantly enhanced. These growth inhibitory effects of SIMPL were not due to an increase in apoptosis. The enhanced inhibitory affects were specific for TNFα and not found in other common hematopoietic inhibitors (TGF-β1 and IFNγ). Results of this work reveal that SIMPL is a component of the hematopoiesis that is required for TNFα dependent effects upon myeloid progenitors.
Mutations in the CDP-Choline Pathway for Phospholipid Biosynthesis Bypass the Requirement for an Essential Phospholipid Transfer Protein
(Cell Press, 1991) Cleves, Ann E.; McGee, Todd P.; Whitters, Eric A.; Champion, Kathleen M.; Aitken, Jacqueline R.; Dowhan, William; Goebl, Mark; Bankaitis, Vytas A.; Biochemistry and Molecular Biology, School of Medicine
SEC14p is the yeast phosphatidylinositol (PI)/phosphatidylcholine (PC) transfer protein, and it effects an essential stimulation of yeast Golgi secretory function. We now report that the SEC14p localizes to the yeast Golgi and that the SEC14p requirement can be specifically and efficiently bypassed by mutations in any one of at least six genes. One of these suppressor genes was the structural gene for yeast choline kinase (CKI), disruption of which rendered the cell independent of the normally essential SEC14p requirement. The antagonistic action of the CKI gene product on SEC14p function revealed a previously unsuspected influence of biosynthetic activities of the CDP-choline pathway for PC biosynthesis on yeast Golgi function and indicated that SEC14p controls the phospholipid content of yeast Golgi membranes in vivo.
Research at the Richard G. Lugar Center for Renewable Energy
(Office of the Vice Chancellor for Research, 2010-04-09) Chen, Rongrong; Goebl, Mark; Cline, Kyle
The Richard G. Lugar Center for Renewable Energy was established at IUPUI in March of 2007. The Center is a focal point for scientific research on renewable energy technologies, as well as policy, law and social issues related to energy. The Center strives to be a leader in many fields of renewable energy and has already proved itself in the areas of fuel cell and battery research and bio-fuels research. An interdisciplinary team of researchers, faculty and graduate students work on finding fuel cell technologies that are more efficient and affordable. Faculty and graduate student research in the School of Medicine, in conjunction with the Lugar Center, has led to the development of a new type of yeast that will improve the production of ethanol. The Lugar Center will continue to promote renewable energy applications through teaching, learning, civic engagement, and synergistic partnerships with industry, government labs and local communities.

Browsing by Author "Goebl, Mark"

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