IU Indianapolis ScholarWorks :: Browsing by Author "Doud, Emma H."

Browsing by Author "Doud, Emma H."

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A human skeletal muscle stem/myotube model reveals multiple signaling targets of cancer secretome in skeletal muscle
(Elsevier, 2023-03-31) Wang, Ruizhong; Kumar, Brijesh; Bhat-Nakshatri, Poornima; Khatpe, Aditi S.; Murphy, Michael P.; Wanczyk, Kristen E.; Simpson, Edward; Chen, Duojiao; Gao, Hongyu; Liu, Yunlong; Doud, Emma H.; Mosley, Amber L.; Nakshatri, Harikrishna; Surgery, School of Medicine
Skeletal muscle dysfunction or reprogramming due to the effects of the cancer secretome is observed in multiple malignancies. Although mouse models are routinely used to study skeletal muscle defects in cancer, because of species specificity of certain cytokines/chemokines in the secretome, a human model system is required. Here, we establish simplified multiple skeletal muscle stem cell lines (hMuSCs), which can be differentiated into myotubes. Using single nuclei ATAC-seq (snATAC-seq) and RNA-seq (snRNA-seq), we document chromatin accessibility and transcriptomic changes associated with the transition of hMuSCs to myotubes. Cancer secretome accelerated stem to myotube differentiation, altered the alternative splicing machinery and increased inflammatory, glucocorticoid receptor, and wound healing pathways in hMuSCs. Additionally, cancer secretome reduced metabolic and survival pathway associated miR-486, AKT, and p53 signaling in hMuSCs. hMuSCs underwent myotube differentiation when engrafted into NSG mice and thus providing a humanized in vivo skeletal muscle model system to study cancer cachexia.
Alternative splicing of uromodulin enhances mitochondrial metabolism for adaptation to stress in kidney epithelial cells
(American Society for Clinical Investigation, 2025-04-08) Nanamatsu, Azuma; Rhodes, George J.; LaFavers, Kaice A.; Micanovic, Radmila; Lazar, Virginie; Khan, Shehnaz; Barwinska, Daria; Makino, Shinichi; Zollman, Amy; Cheng, Ying-Hua; Doud, Emma H.; Mosley, Amber L.; Repass, Matthew J.; Kamocka, Malgorzata M.; Baride, Aravind; Phillips, Carrie L.; Kelly, Katherine J.; Eadon, Michael T.; Himmelfarb, Jonathan; Kretzler, Matthias; Bacallao, Robert L.; Dagher, Pierre C.; Hato, Takashi; El-Achkar, Tarek M.; Medicine, School of Medicine
In the kidney, cells of thick ascending limb of the loop of Henle (TAL) are resistant to ischemic injury, despite high energy demands. This adaptive metabolic response is not fully understood even though the integrity of TAL cells is essential for recovery from acute kidney injury (AKI). TAL cells uniquely express uromodulin, the most abundant protein secreted in healthy urine. Here, we demonstrate that alternative splicing generates a conserved intracellular isoform of uromodulin, which contributes to metabolic adaptation of TAL cells. This splice variant was induced by oxidative stress and was upregulated by AKI that is associated with recovery, but not by severe AKI and chronic kidney disease (CKD). This intracellular variant was targeted to the mitochondria, increased NAD+ and ATP levels, and protected TAL cells from hypoxic injury. Augmentation of this variant using antisense oligonucleotides after severe AKI improved the course of injury. These findings underscore an important role of condition-specific alternative splicing in adaptive energy metabolism to hypoxic stress. Enhancing this protective splice variant in TAL cells could become a therapeutic intervention for AKI.
An engineered glutamic acid tRNA for efficient suppression of pathogenic nonsense mutations
(Oxford University Press, 2025) Specht, Caitlin; Tapia, Alejandro; Penrod, Sarah; Soriano, Gabriela A.; Awawdeh, Aya; Alshawi, Sarah A.; White, Cody A.; Beaudoin, Jean-Denis; Doud, Emma H.; Vargas-Rodriguez, Oscar; Huang, Yunjie; Tharp, Jeffery M.; Biochemistry and Molecular Biology, School of Medicine
Nonsense mutations that introduce premature termination codons (PTCs) into protein-coding genes are responsible for numerous genetic diseases; however, there are currently no effective treatment options for individuals affected by these mutations. One approach to combat nonsense-related diseases relies on the use of engineered suppressor transfer RNAs (sup-tRNAs) that facilitate translational stop codon readthrough, thereby restoring full-length protein synthesis. While several sup-tRNAs have shown promising results in preclinical models, many exhibit low PTC suppression efficiency, precluding their use as therapeutics. For example, glutamic acid (Glu) codons represent one of the most common sites for nonsense mutations, yet existing sup-tRNAs are ineffective at suppressing Glu-to-Stop mutations. To address this limitation, here we describe a rationally designed sup-tRNA (tRNAGluV13) with greatly improved ability to suppress PTCs occurring at Glu codons. We demonstrate that tRNAGluV13 efficiently restores protein synthesis from multiple nonsense-containing reporter genes, faithfully installing Glu in response to PTCs. Additionally, we demonstrate that tRNAGluV13 can functionally rescue pathogenic PTCs that cause hereditary breast and ovarian cancer syndrome and cystic fibrosis. The ability of tRNAGluV13 to effectively suppress one of the most common PTC mutations should greatly expand the potential of sup-tRNA-based therapeutics.
An Essential Adaptor for Apicoplast Fission and Inheritance in Malaria Parasites
(Research Square, 2025-05-05) Blauwkamp, James; Rajaram, Krithika; Staggers, Sophia R.; Harrigan, Oliver; Doud, Emma H.; Prigge, Sean T.; Sun, Stella Y.; Absalon, Sabrina; Pharmacology and Toxicology, School of Medicine
Blood-stage Plasmodium falciparum parasites rely on a non-photosynthetic plastid, the apicoplast, for survival, making it an attractive target for antimalarial intervention. Like the mitochondrion, the apicoplast cannot be generated de novo and must be inherited by daughter parasites during cell division. This inheritance relies on coordinated apicoplast positioning and fission, but the molecular mechanisms controlling these processes remain poorly understood. Here, we identify a previously uncharacterized P. falciparum protein (Pf3D7_0613600), which we name PfAnchor, as a key regulator of apicoplast fission. Using Ultrastructure Expansion Microscopy (U-ExM), we show that PfAnchor localizes to the apicoplast throughout the asexual blood-stage. Conditional depletion disrupts apicoplast fission, leading to incomplete cytokinesis and parasite death. Notably, loss of the apicoplast's elongated branched structure via azithromycin treatment rescues these defects, underscoring Anchor's specific role in apicoplast fission. Immunoprecipitation identified an interaction with the dynamin-like GTPase PfDyn2, a key mediator of both apicoplast and mitochondrial fission, establishing PfAnchor as the first apicoplast-specific dynamin adaptor protein. Our findings define PfAnchor as an essential factor for apicoplast fission and inheritance in P. falciparum blood-stage parasites, highlighting parasite-specific organelle division as a potential vulnerability for therapeutic intervention.
Bis-aryl-α,β-unsaturated ketone (ABK) chaperonin inhibitors exhibit selective cytotoxicity to colorectal cancer cells that correlates with levels of aberrant HSP60 in the cytosol
(Elsevier, 2022) Chitre, Siddhi; Ray, Anne-Marie; Stevens, Mckayla; Doud, Emma H.; Liechty, Hope; Washburn, Alex; Tepper, Katelyn; Sivinski, Jared; O’Hagan, Heather M.; Georgiadis, Millie M.; Chapman, Eli; Johnson, Steven M.; Biochemistry and Molecular Biology, School of Medicine
While many studies have established the importance of protein homeostasis in tumor progression, little effort has been made to examine the therapeutic potential of targeting the HSP60 chaperonin system. In healthy cells, HSP60 is localized to the mitochondrial matrix; however, emerging evidence indicates HSP60 can be over-expressed and mis-localized to the cytosol of cancer cells, which is hypothesized to promote tumor cell survival and proliferation. This opens a potential avenue to selectively target the aberrant HSP60 in the cytosol as a chemotherapeutic strategy. In the present work, we examined a series of bis-aryl-α,β-unsaturated ketone (ABK) HSP60 inhibitors for their ability to selectively target cancerous vs non-cancerous colon and intestine cells. We found that lead analogs inhibited migration and clonogenicity of cancer cells, with cytotoxicity correlating with the level of aberrant HSP60 in the cytosol.
Characterization of BioID tagging systems in budding yeast and exploring the interactome of the Ccr4-Not complex
(Oxford University Press, 2024) Pfannenstein, Jeffrey; Tyryshkin, Misha; Gulden, Moira E.; Doud, Emma H.; Mosley, Amber L.; Reese, Joseph C.; Biochemistry and Molecular Biology, School of Medicine
The modified Escherichia coli biotin ligase BirA* was the first developed for proximity labeling of proteins (BioID). However, it has low activity at temperatures below 37°C, which reduces its effectiveness in organisms growing at lower temperatures, such as budding yeast. Multiple derivatives of the enzymes have been engineered, but a thorough comparison of these variations of biotin ligases and the development of versatile tools for conducting these experiments in Saccharomyces cerevisiae would benefit the community. Here, we designed a suite of vectors to compare the activities of biotin ligase enzymes in yeast. We found that the newer TurboID versions were the most effective at labeling proteins, but they displayed low constitutive labeling of proteins even in the absence of exogenous biotin, due to biotin contained in the culture medium. We describe a simple strategy to express free BioID enzymes in cells that can be used as an appropriate control in BioID studies to account for the promiscuous labeling of proteins caused by random interactions between bait-BioID enzymes in cells. We also describe chemically induced BioID systems exploiting the rapamycin-stabilized FRB-FKBP interaction. Finally, we used the TurboID version of the enzyme to explore the interactome of different subunits of the Ccr4-Not gene regulatory complex. We find that Ccr4-Not predominantly labeled cytoplasmic mRNA regulators, consistent with its function in mRNA decay and translation quality control in this cell compartment.
Characterization of endoplasmic reticulum-associated degradation in the human fungal pathogen Candida albicans
(PeerJ, 2023-08-25) Doss, Ellen M.; Moore, Joshua M.; Harman, Bryce H.; Doud, Emma H.; Rubenstein, Eric M.; Bernstein, Douglas A.; Biochemistry and Molecular Biology, School of Medicine
Background: Candida albicans is the most prevalent human fungal pathogen. In immunocompromised individuals, C. albicans can cause serious systemic disease, and patients infected with drug-resistant isolates have few treatment options. The ubiquitin-proteasome system has not been thoroughly characterized in C. albicans. Research from other organisms has shown ubiquitination is important for protein quality control and regulated protein degradation at the endoplasmic reticulum (ER) via ER-associated protein degradation (ERAD). Methods: Here we perform the first characterization, to our knowledge, of ERAD in a human fungal pathogen. We generated functional knockouts of C. albicans genes encoding three proteins predicted to play roles in ERAD, the ubiquitin ligases Hrd1 and Doa10 and the ubiquitin-conjugating enzyme Ubc7. We assessed the fitness of each mutant in the presence of proteotoxic stress, and we used quantitative tandem mass tag mass spectrometry to characterize proteomic alterations in yeast lacking each gene. Results: Consistent with a role in protein quality control, yeast lacking proteins thought to contribute to ERAD displayed hypersensitivity to proteotoxic stress. Furthermore, each mutant displayed distinct proteomic profiles, revealing potential physiological ERAD substrates, co-factors, and compensatory stress response factors. Among candidate ERAD substrates are enzymes contributing to ergosterol synthesis, a known therapeutic vulnerability of C. albicans. Together, our results provide the first description of ERAD function in C. albicans, and, to our knowledge, any pathogenic fungus.
Comprehensive Overview of Bottom-Up Proteomics Using Mass Spectrometry
(American Chemical Society, 2024-06-04) Jiang, Yuming; Rex, Devasahayam Arokia Balaya; Schuster, Dina; Neely, Benjamin A.; Rosano, Germán L.; Volkmar, Norbert; Momenzadeh, Amanda; Peters-Clarke, Trenton M.; Egbert, Susan B.; Kreimer, Simion; Doud, Emma H.; Crook, Oliver M.; Yadav, Amit Kumar; Vanuopadath, Muralidharan; Hegeman, Adrian D.; Mayta, Martín L.; Duboff, Anna G.; Riley, Nicholas M.; Moritz, Robert L.; Meyer, Jesse G.; Biochemistry and Molecular Biology, School of Medicine
Proteomics is the large scale study of protein structure and function from biological systems through protein identification and quantification. "Shotgun proteomics" or "bottom-up proteomics" is the prevailing strategy, in which proteins are hydrolyzed into peptides that are analyzed by mass spectrometry. Proteomics studies can be applied to diverse studies ranging from simple protein identification to studies of proteoforms, protein-protein interactions, protein structural alterations, absolute and relative protein quantification, post-translational modifications, and protein stability. To enable this range of different experiments, there are diverse strategies for proteome analysis. The nuances of how proteomic workflows differ may be challenging to understand for new practitioners. Here, we provide a comprehensive overview of different proteomics methods. We cover from biochemistry basics and protein extraction to biological interpretation and orthogonal validation. We expect this Review will serve as a handbook for researchers who are new to the field of bottom-up proteomics.
Comprehensive Overview of Bottom-Up Proteomics using Mass Spectrometry
(ArXiv, 2023-11-13) Jiang, Yuming; Rex, Devasahayam Arokia Balaya; Schuster, Dina; Neely, Benjamin A.; Rosano, Germán L.; Volkmar, Norbert; Momenzadeh, Amanda; Peters-Clarke, Trenton M.; Egbert, Susan B.; Kreimer, Simion; Doud, Emma H.; Crook, Oliver M.; Yadav, Amit Kumar; Vanuopadath, Muralidharan; Mayta, Martín L.; Duboff, Anna G.; Riley, Nicholas M.; Moritz, Robert L.; Meyer, Jesse G.; Biochemistry and Molecular Biology, School of Medicine
Proteomics is the large scale study of protein structure and function from biological systems through protein identification and quantification. "Shotgun proteomics" or "bottom-up proteomics" is the prevailing strategy, in which proteins are hydrolyzed into peptides that are analyzed by mass spectrometry. Proteomics studies can be applied to diverse studies ranging from simple protein identification to studies of proteoforms, protein-protein interactions, protein structural alterations, absolute and relative protein quantification, post-translational modifications, and protein stability. To enable this range of different experiments, there are diverse strategies for proteome analysis. The nuances of how proteomic workflows differ may be challenging to understand for new practitioners. Here, we provide a comprehensive overview of different proteomics methods to aid the novice and experienced researcher. We cover from biochemistry basics and protein extraction to biological interpretation and orthogonal validation. We expect this work to serve as a basic resource for new practitioners in the field of shotgun or bottom-up proteomics.
Cryo-EM structures of prion protein filaments from Gerstmann-Sträussler-Scheinker disease
(Springer, 2022) Hallinan, Grace I.; Ozcan, Kadir A.; Hoq, Md Rejaul; Cracco, Laura; Vago, Frank S.; Bharath, Sakshibeedu R.; Li, Daoyi; Jacobsen, Max; Doud, Emma H.; Mosley, Amber L.; Fernandez, Anllely; Garringer, Holly J.; Jiang, Wen; Ghetti, Bernardino; Vidal, Ruben; Pathology and Laboratory Medicine, School of Medicine
Prion protein (PrP) aggregation and formation of PrP amyloid (APrP) are central events in the pathogenesis of prion diseases. In the dominantly inherited prion protein amyloidosis known as Gerstmann-Sträussler-Scheinker (GSS) disease, plaques made of PrP amyloid are present throughout the brain. The c.593t > c mutation in the prion protein gene (PRNP) results in a phenylalanine to serine amino acid substitution at PrP residue 198 (F198S) and causes the most severe amyloidosis among GSS variants. It has been shown that neurodegeneration in this disease is associated with the presence of extracellular APrP plaques and neuronal intracytoplasmic Tau inclusions, that have been shown to contain paired helical filaments identical to those found in Alzheimer disease. Using cryogenic electron microscopy (cryo-EM), we determined for the first time the structures of filaments of human APrP, isolated post-mortem from the brain of two symptomatic PRNP F198S mutation carriers. We report that in GSS (F198S) APrP filaments are composed of dimeric, trimeric and tetrameric left-handed protofilaments with their protomers sharing a common protein fold. The protomers in the cross-β spines consist of 62 amino acids and span from glycine 80 to phenylalanine 141, adopting a previously unseen spiral fold with a thicker outer layer and a thinner inner layer. Each protomer comprises nine short β-strands, with the β1 and β8 strands, as well as the β4 and β9 strands, forming a steric zipper. The data obtained by cryo-EM provide insights into the structural complexity of the PrP filament in a dominantly inherited human PrP amyloidosis. The novel findings highlight the urgency of extending our knowledge of the filaments' structures that may underlie distinct clinical and pathologic phenotypes of human neurodegenerative diseases.

Browsing by Author "Doud, Emma H."

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