Browsing by Author "Wek, Ronald"

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    Big Discoveries in Small Parasites: Expansion Microscopy Reveals Plasmodium's Hidden Biology
    (2025-04) Blauwkamp, James Alan; Absalon, Sabrina; Arrizabalaga, Gustavo; Bidwell, Joseph; Wek, Ronald; Yeh, Elizabeth
    Malaria, caused by the intracellular Plasmodium parasites, remains a global health crisis, affecting an estimated 263 million people and causing 597,000 deaths in 2023. The efficacy of current antimalarial strategies is diminishing due to rising resistance to all major drugs developed over the past six decades and the absence of a highly effective, affordable vaccine. My work employs Ultrastructure Expansion Microscopy (U-ExM) to investigate critical aspects of organelle biogenesis, spatial organization, and dynamics during the Plasmodium blood-stage life cycle. Through U-ExM, I have gained insights into the biogenesis, fission, and spatial organization of the mitochondrion and the apicoplast, two essential metabolic organelles. Further, analysis of invasion organelles revealed the function of PfRON11 in the formation of rhoptry organelles. Investigating the mode of action of a novel antimalarial candidate, GNF179, revealed that treated parasites display an expansion of ER folds and a mislocalization of the Golgi Apparatus, leading to rapid parasite death. U-ExM analysis showed similar ER expansion in PfSEY1 knockdown parasites, suggesting that GNF179 targets this protein. Further, I analyzed the assembly and dynamics of the essential nuclear pore complexes (NPCs). U-ExM enabled the visualization and quantification of NPCs during the blood-stage life cycle. Employing a recombination-induced tag exchange (RITE) system, I demonstrated the dynamics of NPC assembly and recycling. Finally, I characterized a previously unstudied protein, PfAnchor, establishing its essential role in apicoplast fission during the blood stage. Using U-EXM and biochemical assays, I demonstrated that PfAnchor associates with the cytoplasmic side of the apicoplast membrane. Live microscopy of PfAnchor-deficient parasites revealed daughter parasites remained clustered together, and U-ExM analysis confirmed a failure in apicoplast fission. Importantly, treatment with azithromycin disrupted the apicoplast's branching structure, resulting in a vesiculated morphology, which rescued growth defects in PfAnchor-deficient parasites. This finding underscores PfAnchor's essential role in apicoplast fission and its impact on parasite replication. Protein pulldown assays showed an interaction between PfAnchor and PfDyn2, recently implicated in apicoplast fission. Collectively, my work advances our understanding of Plasmodium cell biology and establishes U-ExM as a transformative tool for malaria research. These findings can open new avenues for therapeutic interventions against Plasmodium parasites.
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    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, Ronald
    The 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.
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    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, Ronald
    Under 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.
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    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.
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    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.
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    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, Sabrina
    Heat 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.
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    Nmp4 Suppresses Osteoanabolic Potency
    (2023-07) Heim, Crystal Noelle; Bidwell, Joseph; Wek, Ronald; White, Kenneth; Robling, Alexander; Plotkin, Lilian
    Treating 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.
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    Pathophysiological role of MicroRNA-29 in pancreatic ductal adenocarcinoma
    (2018-05-23) Kwon, Jason Jae-Hyuk; Kota, Janaiah; Korc, Murray; Liu, Yunlong; Wek, Ronald
    Pancreatic 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.
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    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, Ronald
    Angiomotin-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.
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    Role of Polycomb Group Protein Mel18 in Hematopoietic Stem Cell Maintenance
    (2025-05) Cai, Wenjie; Wek, Ronald; Zhang, Ji; Capitano, Maegan; Wan, Jun; Ren, Hongxia; Liu, Yan
    Polycomb group (PcG) proteins are epigenetic gene silencers that have been implicated in stem cell maintenance and cancer development. Genetic and biochemical studies indicate that Polycomb group proteins exist in at least two protein complexes, Polycomb repressive complex 2 (PRC2) and Polycomb repressive complex 1 (PRC1). PRC2 complex deposits the mono-, di-, and tri- methylation on histone 3 lysine 27, whereas PRC1 introduces mono-ubiquitination on histone 2A lysine 119 (H2AK119ub1). PRC1 can also regulate 3D chromatin structure. Bmi1 (PCGF4) and Mel18 (PCGF2) are two major homologs of the PCGF subunit within the canonical PRC1 complex. While Bmi1 is a positive regulator of hematopoietic stem cells (HSCs) and leukemia stem cells (LSCs) self-renewal, the role of Mel18 in normal and malignant hematopoiesis is not fully understood. Based upon our previous studies and the literature, I hypothesized that Mel18 inhibits HSC self-renewal and proliferation but promotes HSC senescence. To test my hypothesis, I examined HSC behavior in Mel18 conditional knockout mice (Mel18f/f-Mx1Cre+). I found that acute deletion of Mel18 enhances the repopulating potential of HSCs and increases the number of functional HSCs, without affecting HSC homing. Loss of Mel18 decreased hematopoietic stem and progenitor cell (HSPCs) senescence. In addition, loss of Mel18 promotes cell cycle progression in HSPCs. Therefore, I demonstrated that loss of Mel18 enhances the repopulating potential of HSCs, promotes cell cycle progression in HSCs, but reduces HSC senescence. Mechanistically, loss of Mel18 increases the chromatin accessibility to genes important for HSC self-renewal and ex vivo expansion such as homeobox gene Hoxb4. CUT&RUN sequencing assays revealed that loss of Mel18 reduces the H2AK119ub1 enrichment at the promoter regions of Cdk4 and Cdk6, leading to their enhanced expression in HSPCs. Furthermore, I identified a Mel18-specific chromatin loop at the S100a9 locus, a gene important for senescence and inflammation, using Hi-C assays, Collectively, these findings demonstrated that Mel18 plays an important role in hematopoietic stem cell maintenance. Mel18 inhibits HSC self-renewal and proliferation but promotes HSC senescence through modulating histone modifications, chromatin accessibility, and 3D chromatin structure in HSCs.