- Browse by Author
Browsing by Author "Wek, Ronald"
Now showing 1 - 10 of 10
Results Per Page
Sort Options
Item Characterization of a Putative Acid Phosphatase in Toxoplasma Gondii and Its Role in Parasite Propagation(2020-11) Blakely, William James; Arrizabalaga, Gustavo; Gilk, Stacey; Meroueh, Samy; Wek, RonaldThe parasite Toxoplasma gondii infects approximately one-third of people worldwide. Infection can lead to severe disease in those with a compromised immune system and primary infection during pregnancy can lead to severe birth defects or miscarriage. Treatment options are limited, have significant side effects, and are ineffective for all infection stages. Imperative to the discovery of novel therapeutic targets is a thorough understanding of how Toxoplasma propagates within a host. To replicate, the parasite must enter the cells of an infected organism where, during the invasion process, it surrounds itself with host cell membrane to form a parasitophorous vacuole (PV), within which it freely divides. To endure the intracellular environment of a host cell, Toxoplasma secretes a large repertoire of proteins beyond the PV to manipulate important host cellular functions. How these Toxoplasma proteins transit from parasites to host cell is not well understood. Protein translocation into the host cell is mediated by three proteins hypothesized to function as a putative translocon complex inside the PV, but whether other proteins are involved in the structure or regulation of this putative translocon remains unknown. The secreted protein GRA44, which contains a putative acid phosphatase domain, has been discovered to interact with members of this translocon and is required for downstream alteration of host cells. GRA44 was found to be post-translationally cleaved in a region homologous to sequences targeted by protozoan proteases of the secretory pathway with both major cleavage products secreted to the PV. Conditional knockdown of GRA44 resulted in loss of host cell cMyc upregulation, a phenotype also seen in translocon member disruption. Therefore, the putative acid phosphatase GRA44, in association with the translocon complex, is critical for host cell manipulation during infection, a process Toxoplasma relies upon for successful propagation as an intracellular pathogen.Item Endoplasmic reticulum calcium dynamics and insulin secretion in pancreatic β cells(2017-08-15) Yamamoto, Wataru; Evans-Molina, Carmella; Day, Richard; Sturek, Michael; Obukhov, Alexander; Wek, RonaldUnder normal conditions, ER Ca2+ levels are estimated to be at least three orders of magnitude higher than intracellular Ca2+. This steep Ca2+ concentration gradient is maintained by the balance of Ca2+ uptake into the ER via the sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) pump and ER Ca2+ release through Ryanodine receptors (RyR) and Inositol 1,4,5-triphosphate (IP3) receptors (IP3R). Emerging data suggest that alterations in β cell ER Ca2+ levels lead to diminished insulin secretion and reduced β cell survival in both type 1 and type 2 diabetes. However, the mechanisms leading to β cell ER Ca2+ loss remain incompletely understood, and a specific role for either RyR or IP3R dysfunction in diabetes has been largely untested. To this end, we applied intracellular and ER-Ca2+ imaging techniques in INS-1 β cells and isolated mouse and human islets to define whether RyR or IP3R activity were altered under diabetogenic conditions. Results revealed preferential alterations in RyR function in response to ER stress, while pro-inflammatory cytokine stress primarily impacted IP3R activity. Consistent with this, pharmacological inhibition of RyR and IP3Rs prevented ER Ca2+ loss under ER and pro-inflammatory stress, respectively. However, RyR inhibition was unique in its ability to prevent β cell death, delayed initiation of the unfolded protein response (UPR), and dysfunctional glucose-induced Ca2+ oscillations in tunicamycin treated INS-1 β cells and islets from Akita mice. Monitoring at the single cell level revealed that ER stress acutely increased intracellular Ca2+ transients and this was dependent on both ER Ca2+ leak from the RyR and plasma membrane depolarization, suggesting ER Ca2+ dynamics regulate cellular excitability. Collectively, our findings suggest that ER-stress induced RyR dysfunction regulates β cell ER Ca2+ dynamics, propagation of the UPR, insulin secretion, and cell survival. These data indicate that RyR-mediated loss of ER Ca2+ and β cell hyperexcitability may be early pathogenic events in diabetes.Item FANCA maintains genomic stability through regulating BUBR1 acetylation(2017-08) Abdul Sater, Zahi Abass; Nalepa, Grzegorz; Clapp, Wade; Goebl, Mark; Wek, RonaldFanconi 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.Item The impact of the termination override mutation on the activity of SSU72(2016-12-19) McCracken, Neil Andrew; Mosley, Amber; Wek, Ronald; Goebl, MarkSsu72, 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.Item The Mechanisms by Which Small Molecules Modulate the HSP60/10 Chaperonin System to Elicit Antimicrobial Effects(2023-06) Stevens, Mckayla Marie; Johnson, Steven; Turchi, John; Hoang, Quyen; Wek, Ronald; Absalon, SabrinaHeat Shock Protein 60/10 (HSP60/10, or GroEL/ES in bacteria) chaperonin systems play a critical role in protein homeostasis through facilitating proper folding of misfolded or partially folded polypeptides that are otherwise prone to aggregation. HSP60 chaperonins are highly conserved and essential in nearly all organisms studied thus far, making them a promising target for antibiotic development. Early high-throughput screens in the Johnson lab have identified five main scaffolds that, though hit-to-lead development, have been optimized for chaperonin inhibition and antimicrobial effects. While these initial studies have shown promising evidence to support the viability of a chaperonin-targeting antibiotic strategy, it was unclear whether the conservation of human HSP60 (48% identity to bacterial GroEL) would hinder this therapeutic strategy from advancing due to potential toxicity associated with off-target inhibition of the human homolog. Additionally, while chaperonin inhibition often correlated with cytotoxicity to the various pathogens studied, there was a clear need to investigate inhibitor mechanisms to 1) verify on-target effects, and 2) guide future development of more potent and selective chaperonin-targeting antibiotic candidates. Herein, we conduct a medium-throughput screening of known bioactive molecules, approved drugs, and natural products against both bacterial GroEL and human HSP60, demonstrating that most molecules exhibited low-to-no toxicity to human cells in culture, despite being near equipotent inhibitors of human HSP60 and E. coli GroEL in our refolding assays. Thus, sequence conservation between human HSP60 and bacterial GroELs does not necessarily predict toxicity in vivo. We then investigate inhibitory mechanisms of our most well-established inhibitor series, the phenylbenzoxazole (PBZ) series, identifying three binding sites whereby PBZ molecules modulate GroEL folding and ATPase functions in a site-specific manner, predominately through its ability to interact with its co-chaperone GroES. Finally, we demonstrate that two standard of care drugs for T. brucei infections, suramin and nifurtimox, may elicit their trypanocidal effects through inhibiting HSP60. Due to structural similarities, we then screened our N-acylhydrazone (NAH) and α,β-unsaturated ketone (ABK) series of HSP60 inhibitors against T. brucei, finding that they are highly potent and selective trypanocidal agents. Together, these studies further support HSP60 as a viable drug target for antibiotic development.Item Nmp4 Suppresses Osteoanabolic Potency(2023-07) Heim, Crystal Noelle; Bidwell, Joseph; Wek, Ronald; White, Kenneth; Robling, Alexander; Plotkin, LilianTreating severe osteoporosis is limited to two strategies: 1. Stimulation of the parathyroid hormone receptor with analogs for parathyroid hormone (PTH) or parathyroid hormone related peptide, and 2. Stimulation of Wnt signaling via neutralization of sclerostin, a natural inhibitor of this pathway, with a monoclonal antibody (romosozumab-aqqg, Scl-mAb). Despite mobilizing distinct molecular and cellular pathways to stimulate bone gain, all their efficacies rapidly diminish. Identifying the barrier to enhancing potency is a clinical priority. We recently reported that mice harboring the conditional loss of the transcription factor Nmp4 (Nuclear Matrix Protein 4) in mesenchymal stem/progenitor cells (MSPCs) exhibited no measurable baseline effect on the skeleton but showed a significantly enhanced increase in bone formation during PTH therapy. Remarkably, (and unexpectedly) skeletal response to PTH therapy was not improved when Nmp4 was conditionally disabled at the osteoblast or osteocyte stages. For the present study, we hypothesized that the potency of any osteoanabolic drug is pre-programmed (and can be re-programmed) in osteoprogenitors. To test this hypothesis, we treated our global Nmp4-/- mice, various conditional knockout mice, and their controls with Scl-mAb. We observed a similar pattern of improved bone response in our mouse models, which we previously observed with the PTH therapy. That is, removal of Nmp4 early in osteoblast differentiation (MSPC) was required for an exaggerated bone-formation response to Scl-mAb therapy. Disabling Nmp4 later in osteogenic differentiation did not increase the potency of Scl-mAb. These data suggest that Nmp4 is part of a common barrier to improving the efficacy of any osteoanabolic. Potential pathways and actors that comprise the re-programming of Nmp4-/- MSPCs to support the exaggerated osteoanabolic effect on the skeleton are discussed.Item Pathophysiological role of MicroRNA-29 in pancreatic ductal adenocarcinoma(2018-05-23) Kwon, Jason Jae-Hyuk; Kota, Janaiah; Korc, Murray; Liu, Yunlong; Wek, RonaldPancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy and responds poorly to current therapies. Thus, it is imperative to develop novel treatments for PDAC. Dense fibrotic stroma associated with PDAC abrogates drug perfusion into the tumor, and pancreatic stellate cells (PSCs) are the major stromal cells responsible for fibrosis. Activated PSCs produce pro-inflammatory factors and secrete an excessive amount of extracellular matrix (ECM) proteins, the major stromal proteins in PDAC. MicroRNAs (miRNAs) are conserved small non-coding RNAs that regulate gene expression by binding to the 3′UTR of target mRNA transcripts, causing translational repression or degradation. A single miRNA regulates several targets within intracellular networks and can have a profound impact on normal physiology. miR-29 has been previously reported to have anti-fibrotic and tumor suppressive roles in various cancers. We found miR-29 expression was significantly decreased in activated PSCs and pancreatic cancer cells in vitro, in vivo models, as well as in PDAC patient biopsies. Through in vitro studies in activated PSC, we found that miR-29 inhibited the expression of ECM proteins and reduced cancer growth when co-cultured with pancreatic cancer cells. miR-29 overexpression in pancreatic cancer cells decreased their invasive potential and sensitized chemoresistant cancer cells to gemcitabine treatment by inhibiting autophagy through the direct targeting of two essential, autophagy related genes, TFEB and ATG9A. In developing therapies and for in vivo functional studies, viral-based gene delivery is a powerful tool to target the pancreas. We tested various self-complementary recombinant adeno-associated virus (scAAV) serotypes in normal mice (C57BL/6) and in a KrasG12D-driven pancreatic cancer mouse model via systemic and intraductal delivery methods. We found that retrograde intraductal delivery of scAAV6 safely targeted the pancreas/neoplasm with the greatest efficiency. Our findings provide a better understanding of miR-29 in pancreatic cancer and demonstrates its potential therapeutic use to target PDAC.Item Restriction of Glioma Progression and Mesenchymal Characteristics by Angiomotin-like 1(2020-10) Lange, Kevin Clayton; Wells, Clark D.; Dong, X. Charlie; Ivan, Mircea; Mayo, Lindsey; Wek, RonaldAngiomotin-like 1 (AmotL1) serves as a scaffold for protein complexes that promote cell polarity and HIPPO signaling to enable their suppression of oncogenic phenotypes in multiple epithelial-derived cancers. In this study, an analysis of multiple tumor databases revealed that AmotL1 transcript levels associate with positive survival and reduced tumor grade in astrocytomas. The suppression of AmotL1 transcript levels was most prevalent in in glioblastoma tumor regions that are associated with invasion and mesenchymal-like transcriptional profiles. Factors associated with tumor progression were consequently causally linked to AmotL1 expression in normal astrocytes and glioblastoma cells. While most tumor suppressive effects of AmotL1 are related to its regulation of YAP and TAZ, the potent effects of AmotL1 down-regulation were found to be independent of these two oncoproteins. Further, AmotL1 was shown to inhibit Wnt signaling through binding of the Fzd4 receptor via MAGI-3. Such binding was associated with an ability by AmotL1 to redistribute Fzd4 from the cell surface to intracellular complexes with AmotL1 and MAGI-3. AmotL1 was also shown to be transcriptionally suppressed under hypoxia by HIF2α. This suppression was found to promote invasion by increasing levels of c-MET. These results show that hypoxia suppresses AmotL1 to promote a likely mesenchymal transition. These effects help to explain the association of AmotL1 down-regulation in glioblastomas with increased tumor grade and poor patient survival.Item The Roles of Danio Rerio Nrf2 Paralogs in Response to Oxidative Stress in the Pancreatic Beta Cell(2020-06) Doszpoly, Agnes; Linnemann, Amelia; Anderson, Ryan; Wek, RonaldOxidative stress can disrupt cellular homeostasis, leading to cellular dysfunction and apoptosis. The Nrf2 transcription factor regulates the antioxidant response in cells by binding to antioxidant response elements (ARE) in DNA and activating genes of enzymes that combat oxidative stress. During the pathogenesis of diabetes mellitus (DM), β-cells are exposed to increased amounts of reactive oxygen species (ROS) that cause oxidative stress. Zebrafish (ZF) are excellent models for studying the dynamic mechanisms associated with DM pathogenesis, and we recently developed a ZF model of β-cell apoptosis caused by ROS. Two paralogs of Nrf2 have been identified in ZF, Nrf2a and Nrf2b, but their roles in pancreas development and/or β-cell survival are unknown. To investigate their roles, Nrf2a and Nrf2b antisense morpholinos (MO) were injected into Day 0 ZF embryos and analyzed over time. While Nrf2a MO showed no obvious phenotypes compared to WT, Nrf2b MO exhibited reduced pancreas size and islets with disrupted morphology. Ins:NTR Nrf2a MO showed reduced β-cell loss upon exposure to Metronidazole (MTZ) under generation of ROS compared to WT. Sequence analysis of ZF nrf2b in 3-day post-fertilization (dpf) embryos revealed a novel splice variant containing an additional exon that has not been described. Further investigation of Nrf2a and Nrf2b is likely to yield additional insights regarding the function and regulation of the NRF2-signaling pathway and their roles in β-cell protection under oxidative stress.Item Using Chemical Probes to Define the Role of Aldehyde Dehydrogenase 1A in a Breast Cancer Model(2022-09) Takahashi, Cyrus; Hurley, Thomas; Georgiadis, Millie; Harrington, Maureen; Hawkins, Shannon; Wek, RonaldThe aldehyde dehydrogenase (ALDH) superfamily comprises a group of NAD(P)+-dependent enzymes that catalyze the conversion of aldehydes to their corresponding carboxylic acids. Of the nineteen human ALDH enzymes, members of the ALDH1A subfamily consisting of ALDH1A1, ALDH1A2, and ALDH1A3 have attracted interest as markers of cancer stem cells (CSCs) in several cancer types including lung, breast, and ovarian. CSCs represent a distinct subpopulation of highly tumorigenic cells that promote metastasis, recurrence, and resistance to conventional cancer therapies. The increased expression and activity of ALDH1A in CSCs is well-documented, as is the correlation between ALDH1A and a more aggressive cancer phenotype with poorer treatment outcomes. However, the actual functional role of ALDH1A in the context of CSCs has yet to be clearly defined. Elucidating this role will lead to a greater understanding of CSC biology and evaluate ALDH1A as a potential anti-CSC therapeutic target. In this study, previously developed and characterized selective small-molecule inhibitors of ALDH1A were used in conjunction with global transcriptomic, proteomic, and metabolomic analyses to identify pathways that could potentially establish a link between ALDH1A activity and early events in CSC formation in a triple-negative breast cancer (TNBC) model. These approaches revealed that ALDH1A inhibition is associated with mitochondrial and metabolic dysfunction and perturbation of the electron transport chain. ALDH1A inhibition also resulted in an increase in markers of endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR), specifically mediated through the Protein kinase RNA-like endoplasmic reticulum kinase (PERK) pathway. These effects appear to occur independently of both the canonical function of ALDH1A in detoxifying reactive aldehydes as well as its potential metabolic contribution through the generation of NADH. Together, these results suggest a separate role for ALDH1A in TNBC CSCs in protecting against ER stress that warrants further study.