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Browsing by Author "Doud, Emma H."
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Item 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 MedicineSkeletal 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.Item 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 MedicineBackground: 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.Item 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 MedicineProteomics 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.Item 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 MedicinePrion 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.Item Distinguishing post-translational modifications in dominantly inherited frontotemporal dementias: FTLD-TDP Type A (GRN) vs Type B (C9orf72)(Wiley, 2022) Cracco, Laura; Doud, Emma H.; Hallinan, Grace I.; Garringer, Holly J.; Jacobsen, Max H.; Richardson, Rose M.; Buratti, Emanuele; Vidal, Ruben; Ghetti, Bernardino; Newell, Kathy L.; Pathology and Laboratory Medicine, School of MedicineAims: Frontotemporal dementias are neuropathologically characterised by frontotemporal lobar degeneration (FTLD). Intraneuronal inclusions of transactive response DNA-binding protein 43 kDa (TDP-43) are the defining pathological hallmark of approximately half of the FTLD cases, being referred to as FTLD-TDP. The classification of FTLD-TDP into five subtypes (Type A to Type E) is based on pathologic phenotypes; however, the molecular determinants underpinning the phenotypic heterogeneity of FTLD-TDP are not well known. It is currently undetermined whether TDP-43 post-translational modifications (PTMs) may be related to the phenotypic diversity of the FTLDs. Thus, the investigation of FTLD-TDP Type A and Type B, associated with GRN and C9orf72 mutations, becomes essential. Methods: Immunohistochemistry was used to identify and map the intraneuronal inclusions. Sarkosyl-insoluble TDP-43 was extracted from brains of GRN and C9orf72 mutation carriers post-mortem and studied by Western blot analysis, immuno-electron microscopy and mass spectrometry. Results: Filaments of TDP-43 were present in all FTLD-TDP preparations. PTM profiling identified multiple phosphorylated, N-terminal acetylated or otherwise modified residues, several of which have been identified for the first time as related to sarkosyl-insoluble TDP-43. Several PTMs were specific for either Type A or Type B, while others were identified in both types. Conclusions: The current results provide evidence that the intraneuronal inclusions in the two genetic diseases contain TDP-43 filaments. The discovery of novel, potentially type-specific TDP-43 PTMs emphasises the need to determine the mechanisms leading to filament formation and PTMs, and the necessity of exploring the validity and occupancy of PTMs in a prognostic/diagnostic setting.Item Erratum: Skeletal muscle-specific overexpression of miR-486 limits mammary tumor-induced skeletal muscle functional limitations(Elsevier, 2022-08-20) Wang, Ruizhong; Kumar, Brijesh; Doud, Emma H.; Mosley, Amber L.; Alexander, Matthew S.; Kunkel, Louis M.; Nakshatri, Harikrishna; Surgery, School of Medicine[This corrects the article DOI: 10.1016/j.omtn.2022.03.009.].Item Global lysine methylome profiling using systematically characterized affinity reagents(Springer Nature, 2023-01-07) Berryhill, Christine A.; Hanquier, Jocelyne N.; Doud, Emma H.; Cordeiro‑Spinetti, Eric; Dickson, Bradley M.; Rothbart, Scott B.; Mosley, Amber L.; Cornett, Evan M.; Biochemistry and Molecular Biology, School of MedicineLysine methylation modulates the function of histone and non-histone proteins, and the enzymes that add or remove lysine methylation—lysine methyltransferases (KMTs) and lysine demethylases (KDMs), respectively—are frequently mutated and dysregulated in human diseases. Identification of lysine methylation sites proteome-wide has been a critical barrier to identifying the non-histone substrates of KMTs and KDMs and for studying functions of non-histone lysine methylation. Detection of lysine methylation by mass spectrometry (MS) typically relies on the enrichment of methylated peptides by pan-methyllysine antibodies. In this study, we use peptide microarrays to show that pan-methyllysine antibodies have sequence bias, and we evaluate how the differential selectivity of these reagents impacts the detection of methylated peptides in MS-based workflows. We discovered that most commercially available pan-Kme antibodies have an in vitro sequence bias, and multiple enrichment approaches provide the most comprehensive coverage of the lysine methylome. Overall, global lysine methylation proteomics with multiple characterized pan-methyllysine antibodies resulted in the detection of 5089 lysine methylation sites on 2751 proteins from two human cell lines, nearly doubling the number of reported lysine methylation sites in the human proteome.Item Inflammation primes the kidney for recovery by activating AZIN1 A-to-I editing(bioRxiv, 2023-11-09) Heruye, Segewkal; Myslinski, Jered; Zeng, Chao; Zollman, Amy; Makino, Shinichi; Nanamatsu, Azuma; Mir, Quoseena; Janga, Sarath Chandra; Doud, Emma H.; Eadon, Michael T.; Maier, Bernhard; Hamada, Michiaki; Tran, Tuan M.; Dagher, Pierre C.; Hato, Takashi; Biochemistry and Molecular Biology, School of MedicineThe progression of kidney disease varies among individuals, but a general methodology to quantify disease timelines is lacking. Particularly challenging is the task of determining the potential for recovery from acute kidney injury following various insults. Here, we report that quantitation of post-transcriptional adenosine-to-inosine (A-to-I) RNA editing offers a distinct genome-wide signature, enabling the delineation of disease trajectories in the kidney. A well-defined murine model of endotoxemia permitted the identification of the origin and extent of A-to-I editing, along with temporally discrete signatures of double-stranded RNA stress and Adenosine Deaminase isoform switching. We found that A-to-I editing of Antizyme Inhibitor 1 (AZIN1), a positive regulator of polyamine biosynthesis, serves as a particularly useful temporal landmark during endotoxemia. Our data indicate that AZIN1 A-to-I editing, triggered by preceding inflammation, primes the kidney and activates endogenous recovery mechanisms. By comparing genetically modified human cell lines and mice locked in either A-to-I edited or uneditable states, we uncovered that AZIN1 A-to-I editing not only enhances polyamine biosynthesis but also engages glycolysis and nicotinamide biosynthesis to drive the recovery phenotype. Our findings implicate that quantifying AZIN1 A-to-I editing could potentially identify individuals who have transitioned to an endogenous recovery phase. This phase would reflect their past inflammation and indicate their potential for future recovery.Item Loss of succinyl-CoA synthetase in mouse forebrain results in hypersuccinylation with perturbed neuronal transcription and metabolism(Elsevier, 2023) Lancaster, Makayla S.; Kim, Byungwook; Doud, Emma H.; Tate, Mason D.; Sharify, Ahmad D.; Gao, Hongyu; Chen, Duojiao; Simpson, Ed; Gillespie, Patrick; Chu, Xiaona; Miller, Marcus J.; Wang, Yue; Liu, Yunlong; Mosley, Amber L.; Kim, Jungsu; Graham, Brett H.; Medical and Molecular Genetics, School of MedicineLysine succinylation is a subtype of protein acylation associated with metabolic regulation of succinyl-CoA in the tricarboxylic acid cycle. Deficiency of succinyl-CoA synthetase (SCS), the tricarboxylic acid cycle enzyme catalyzing the interconversion of succinyl-CoA to succinate, results in mitochondrial encephalomyopathy in humans. This report presents a conditional forebrain-specific knockout (KO) mouse model of Sucla2, the gene encoding the ATP-specific beta isoform of SCS, resulting in postnatal deficiency of the entire SCS complex. Results demonstrate that accumulation of succinyl-CoA in the absence of SCS leads to hypersuccinylation within the murine cerebral cortex. Specifically, increased succinylation is associated with functionally significant reduced activity of respiratory chain complex I and widescale alterations in chromatin landscape and gene expression. Integrative analysis of the transcriptomic data also reveals perturbations in regulatory networks of neuronal transcription in the KO forebrain. Together, these findings provide evidence that protein succinylation plays a significant role in the pathogenesis of SCS deficiency.Item Mapping the Anti-Cancer Activity of α-Connexin Carboxyl-Terminal (aCT1) Peptide in Resistant HER2+ Breast Cancer(MDPI, 2024-01-19) Baker, Kimberly M.; Abt, Melissa; Doud, Emma H.; Oblak, Adrian L.; Yeh, Elizabeth S.; Pharmacology and Toxicology, School of MedicineConnexin 43 (Cx43) is a protein encoded by the GJA1 gene and is a component of cell membrane structures called gap junctions, which facilitate intercellular communication. Prior evidence indicates that elevated GJA1 expression in the HER2-positive (HER2+) subtype of breast cancer is associated with poor prognosis. Prior evidence also suggests that HER2+ breast cancers that have become refractory to HER2-targeted agents have a loss of Cx43 gap junction intercellular communication (GJIC). In this study, a Cx43-targeted agent called alpha-connexin carboxyl-terminal peptide (aCT1) is examined to determine whether GJIC can be rescued in refractory HER2+ breast cancer cells. A proposed mechanism of action for aCT1 is binding to the tight junction protein Zonal Occludens-1 (ZO-1). However, the true scope of activity for aCT1 has not been explored. In this study, mass spectrometry proteomic analysis is used to determine the breadth of aCT1-interacting proteins. The NanoString nCounter Breast Cancer 360 panel is also used to examine the effect of aCT1 on cancer signaling in HER2+ breast cancer cells. Findings from this study show a dynamic range of binding partners for aCT1, many of which regulate gene expression and RNA biology. nCounter analysis shows that a number of pathways are significantly impacted by aCT1, including upregulation of apoptotic factors, leading to the prediction and demonstration that aCT1 can boost the cell death effects of cisplatin and lapatinib in HER2+ breast cancer cells that have become resistant to HER2-targeted agents.
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