167 results on '"Thomas DD"'
Search Results
2. S100A1 Protein Does Not Compete with Calmodulin for Ryanodine Receptor Binding but Structurally Alters the Ryanodine Receptor center dot Calmodulin Complex
- Author
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Rebbeck, RT, Nitu, FR, Rohde, D, Most, P, Bers, DM, Thomas, DD, and Cornea, RL
- Published
- 2016
3. Kinetics and mapping of Ca-driven calmodulin conformations on skeletal and cardiac muscle ryanodine receptors.
- Author
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Rebbeck RT, Svensson B, Zhang J, Samsó M, Thomas DD, Bers DM, and Cornea RL
- Subjects
- Kinetics, Animals, Humans, Protein Conformation, Protein Binding, Sarcoplasmic Reticulum metabolism, Ryanodine Receptor Calcium Release Channel metabolism, Ryanodine Receptor Calcium Release Channel chemistry, Calmodulin metabolism, Calmodulin chemistry, Calcium metabolism, Myocardium metabolism, Muscle, Skeletal metabolism, Fluorescence Resonance Energy Transfer
- Abstract
Calmodulin transduces [Ca
2+ ] information regulating the rhythmic Ca2+ cycling between the sarcoplasmic reticulum and cytoplasm during contraction and relaxation in cardiac and skeletal muscle. However, the structural dynamics by which calmodulin modulates the sarcoplasmic reticulum Ca2+ release channel, the ryanodine receptor, at physiologically relevant [Ca2+ ] is unknown. Using fluorescence lifetime FRET, we resolve different structural states of calmodulin and Ca2+ -driven shifts in the conformation of calmodulin bound to ryanodine receptor. Skeletal and cardiac ryanodine receptor isoforms show different calmodulin-ryanodine receptor conformations, as well as binding and structural kinetics with 0.2-ms resolution, which reflect different functional roles of calmodulin. These FRET methods provide insight into the physiological calmodulin-ryanodine receptor structural states, revealing additional distinct structural states that complement cryo-EM models that are based on less physiological conditions. This technology will drive future studies on pathological calmodulin-ryanodine receptor interactions and dynamics with other important ryanodine receptor bound modulators., (© 2024. The Author(s).)- Published
- 2024
- Full Text
- View/download PDF
4. Allosteric coupling asymmetry mediates paradoxical activation of BRAF by type II inhibitors.
- Author
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Rasmussen DM, Semonis MM, Greene JT, Muretta JM, Thompson AR, Toledo Ramos S, Thomas DD, Pomerantz WCK, Freedman TS, and Levinson NM
- Subjects
- Allosteric Regulation drug effects, Humans, Protein Conformation, Protein Binding, Models, Molecular, Proto-Oncogene Proteins B-raf metabolism, Proto-Oncogene Proteins B-raf antagonists & inhibitors, Proto-Oncogene Proteins B-raf chemistry, Protein Kinase Inhibitors pharmacology, Protein Kinase Inhibitors metabolism, Protein Multimerization drug effects
- Abstract
The type II class of RAF inhibitors currently in clinical trials paradoxically activate BRAF at subsaturating concentrations. Activation is mediated by induction of BRAF dimers, but why activation rather than inhibition occurs remains unclear. Using biophysical methods tracking BRAF dimerization and conformation, we built an allosteric model of inhibitor-induced dimerization that resolves the allosteric contributions of inhibitor binding to the two active sites of the dimer, revealing key differences between type I and type II RAF inhibitors. For type II inhibitors the allosteric coupling between inhibitor binding and BRAF dimerization is distributed asymmetrically across the two dimer binding sites, with binding to the first site dominating the allostery. This asymmetry results in efficient and selective induction of dimers with one inhibited and one catalytically active subunit. Our allosteric models quantitatively account for paradoxical activation data measured for 11 RAF inhibitors. Unlike type II inhibitors, type I inhibitors lack allosteric asymmetry and do not activate BRAF homodimers. Finally, NMR data reveal that BRAF homodimers are dynamically asymmetric with only one of the subunits locked in the active αC-in state. This provides a structural mechanism for how binding of only a single αC-in inhibitor molecule can induce potent BRAF dimerization and activation., Competing Interests: DR, MS, JG, JM, AT, ST, DT, WP, TF, NL No competing interests declared, (© 2024, Rasmussen et al.)
- Published
- 2024
- Full Text
- View/download PDF
5. Structural Dynamics of Protein Interactions Using Site-Directed Spin Labeling of Cysteines to Measure Distances and Rotational Dynamics with EPR Spectroscopy.
- Author
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Roopnarine O and Thomas DD
- Abstract
Here we review applications of site-directed spin labeling (SDSL) with engineered cysteines in proteins, to study the structural dynamics of muscle and non-muscle proteins, using and developing the electron paramagnetic resonance (EPR) spectroscopic techniques of dipolar EPR, double electron electron resonance (DEER), saturation transfer EPR (STEPR), and orientation measured by EPR. The SDSL technology pioneered by Wayne Hubbell and collaborators has greatly expanded the use of EPR, including the measurement of distances between spin labels covalently attached to proteins and peptides. The Thomas lab and collaborators have applied these techniques to elucidate dynamic interactions in the myosin-actin complex, myosin-binding protein C, calmodulin, ryanodine receptor, phospholamban, utrophin, dystrophin, β-III-spectrin, and Aurora kinase. The ability to design and engineer cysteines in proteins for site-directed covalent labeling has enabled the use of these powerful EPR techniques to measure distances, while showing that they are complementary with optical spectroscopy measurements., Competing Interests: Conflict of interest DDT holds equity in, and serves as executive officer for Photonic Pharma LLC (PP), which had no role in this study. OR is the sole proprietor of Editing Science LLC, which had no role in this study. These relationships have been reviewed and managed by the University of Minnesota.
- Published
- 2024
- Full Text
- View/download PDF
6. Nimesulide, a COX-2 inhibitor, sensitizes pancreatic cancer cells to TRAIL-induced apoptosis by promoting DR5 clustering †.
- Author
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Vunnam N, Young MC, Liao EE, Lo CH, Huber E, Been M, Thomas DD, and Sachs JN
- Subjects
- Humans, Receptors, TNF-Related Apoptosis-Inducing Ligand metabolism, Apoptosis, Cell Line, Tumor, TNF-Related Apoptosis-Inducing Ligand pharmacology, TNF-Related Apoptosis-Inducing Ligand metabolism, Pancreatic Neoplasms, Cyclooxygenase 2 Inhibitors pharmacology, Pancreatic Neoplasms pathology
- Abstract
Nimesulide is a nonsteroidal anti-inflammatory drug and a COX-2 inhibitor with antitumor and antiproliferative activities that induces apoptosis in oral, esophagus, breast, and pancreatic cancer cells. Despite being removed from the market due to hepatotoxicity, nimesulide is still an important research tool being used to develop new anticancer drugs. Multiple studies have been done to modify the nimesulide skeleton to develop more potent anticancer agents and related compounds are promising scaffolds for future development. As such, establishing a mechanism of action for nimesulide remains an important part of realizing its potential. Here, we show that nimesulide enhances TRAIL-induced apoptosis in resistant pancreatic cancer cells by promoting clustering of DR5 in the plasma membrane. In this way, nimesulide acts like a related compound, DuP-697, which sensitizes TRAIL-resistant colon cancer cells in a similar manner. Our approach applies a time-resolved FRET-based biosensor that monitors DR5 clustering and conformational states in the plasma membrane. We show that this tool can be used for future high-throughput screens to identify novel, nontoxic small molecule scaffolds to overcome TRAIL resistance in cancer cells.
- Published
- 2023
- Full Text
- View/download PDF
7. Regulation of cardiac calcium signaling by newly identified calcium pump modulators.
- Author
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Bovo E, Rebbeck RT, Roopnarine O, Cornea RL, Thomas DD, and Zima AV
- Subjects
- Mice, Humans, Animals, HEK293 Cells, Sarcoplasmic Reticulum Calcium-Transporting ATPases metabolism, Myocytes, Cardiac metabolism, Sarcoplasmic Reticulum metabolism, Calcium metabolism, Calcium Signaling
- Abstract
In cardiomyocytes, the sarco/endoplasmic reticulum Ca
2+ -ATPase (SERCA2a) is a central component of intracellular Ca2+ regulation. Several heart diseases, including heart failure, are associated with reduced myocardial contraction due to SERCA2a downregulation. Therefore, the need for developing new drugs that could improve SERCA2a function is high. We have recently identified SERCA2a modulators (Compounds 6 and 8) from our screening campaigns and confirmed activation of biochemical SERCA2a ATPase activity and Ca2+ uptake activity. In this study, confocal microscopy and in-cell Ca2+ imaging were used to characterize the effects of these SERCA2a activators on Ca2+ regulation in mouse ventricular myocytes and endoplasmic reticulum (ER) Ca2+ uptake in a HEK293 cell expressing human SERCA2a. Analysis of cytosolic Ca2+ dynamics in cardiomyocytes revealed that both Compounds (6 and 8) increase the action potential-induced Ca2+ transients and sarcoplasmic reticulum (SR) Ca2+ load. While Compound 6 induced a negligible effect on Ca2+ transients invoked by the L-type Ca2+ channel (LTCC) current, Compound 8 increased Ca2+ transients during LTCC activation, suggesting an off-target protein interaction of Compound 8. Analysis of ER Ca2+ transport by human SERCA2a in HEK cells showed that only Compound 6 increased both ER Ca2+ uptake and ER Ca2+ load significantly, whereas Compound 8 had no effect on SERCA2a Ca2+ transport. This study revealed that Compound 6 exhibits promising characteristics that can improve intracellular Ca2+ dynamics by selectively enhancing SERCA2a Ca2+ uptake., Competing Interests: Declaration of competing interest DDT holds equity in, and serve as an executive officer for, Photonic Pharma LLC, which had no role in this study. OR is the sole proprietor of Editing Science LLC, which had no role in this study. These relationships have been reviewed and managed by the University of Minnesota. The other authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)- Published
- 2023
- Full Text
- View/download PDF
8. Drug discovery for heart failure targeting myosin-binding protein C.
- Author
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Bunch TA, Guhathakurta P, Thompson AR, Lepak VC, Carter AL, Thomas JJ, Thomas DD, and Colson BA
- Subjects
- Humans, Actins metabolism, Myocardium metabolism, Myosins metabolism, Phosphorylation drug effects, Protein Binding drug effects, Drug Evaluation, Preclinical, Biosensing Techniques, Adenosine Triphosphatases metabolism, Muscle, Skeletal metabolism, Recombinant Proteins metabolism, Enzyme Activation drug effects, Fluorescence Resonance Energy Transfer, Drug Discovery methods, Heart Failure drug therapy, Heart Failure metabolism, Small Molecule Libraries pharmacology, Myofibrils drug effects, Carrier Proteins metabolism
- Abstract
Cardiac MyBP-C (cMyBP-C) interacts with actin and myosin to fine-tune cardiac muscle contractility. Phosphorylation of cMyBP-C, which reduces the binding of cMyBP-C to actin and myosin, is often decreased in patients with heart failure (HF) and is cardioprotective in model systems of HF. Therefore, cMyBP-C is a potential target for HF drugs that mimic its phosphorylation and/or perturb its interactions with actin or myosin. We labeled actin with fluorescein-5-maleimide (FMAL) and the C0-C2 fragment of cMyBP-C (cC0-C2) with tetramethylrhodamine (TMR). We performed two complementary high-throughput screens (HTS) on an FDA-approved drug library, to discover small molecules that specifically bind to cMyBP-C and affect its interactions with actin or myosin, using fluorescence lifetime (FLT) detection. We first excited FMAL and detected its FLT, to measure changes in fluorescence resonance energy transfer (FRET) from FMAL (donor) to TMR (acceptor), indicating binding. Using the same samples, we then excited TMR directly, using a longer wavelength laser, to detect the effects of compounds on the environmentally sensitive FLT of TMR, to identify compounds that bind directly to cC0-C2. Secondary assays, performed on selected modulators with the most promising effects in the primary HTS assays, characterized the specificity of these compounds for phosphorylated versus unphosphorylated cC0-C2 and for cC0-C2 versus C1-C2 of fast skeletal muscle (fC1-C2). A subset of identified compounds modulated ATPase activity in cardiac and/or skeletal myofibrils. These assays establish the feasibility of the discovery of small-molecule modulators of the cMyBP-C-actin/myosin interaction, with the ultimate goal of developing therapies for HF., Competing Interests: Conflict of interest D. D. T. holds equity in, and serves as President of, Photonic Pharma LLC. This relationship has been reviewed and managed by the University of Minnesota. The present research is a pre-commercial collaboration between Photonic Pharma, UMN, and the University of Arizona. B. A. C. serves as President of BC Biologics LLC. This relationship has been reviewed and managed by the University of Arizona. BC Biologics had no role in this study. B. A. C. filed a PCT patent application based on this work (patent pending, serial no. PCT/US21/14,142). The other authors declare no competing financial interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2023
- Full Text
- View/download PDF
9. Allosteric coupling asymmetry mediates paradoxical activation of BRAF by type II inhibitors.
- Author
-
Rasmussen DM, Semonis MM, Greene JT, Muretta JM, Thompson AR, Ramos ST, Thomas DD, Pomerantz WCK, Freedman TS, and Levinson NM
- Abstract
The type II class of RAF inhibitors currently in clinical trials paradoxically activate BRAF at subsaturating concentrations. Activation is mediated by induction of BRAF dimers, but why activation rather than inhibition occurs remains unclear. Using biophysical methods tracking BRAF dimerization and conformation we built an allosteric model of inhibitor-induced dimerization that resolves the allosteric contributions of inhibitor binding to the two active sites of the dimer, revealing key differences between type I and type II RAF inhibitors. For type II inhibitors the allosteric coupling between inhibitor binding and BRAF dimerization is distributed asymmetrically across the two dimer binding sites, with binding to the first site dominating the allostery. This asymmetry results in efficient and selective induction of dimers with one inhibited and one catalytically active subunit. Our allosteric models quantitatively account for paradoxical activation data measured for 11 RAF inhibitors. Unlike type II inhibitors, type I inhibitors lack allosteric asymmetry and do not activate BRAF homodimers. Finally, NMR data reveal that BRAF homodimers are dynamically asymmetric with only one of the subunits locked in the active αC-in state. This provides a structural mechanism for how binding of only a single αC-in inhibitor molecule can induce potent BRAF dimerization and activation.
- Published
- 2023
- Full Text
- View/download PDF
10. Nitric oxide inhibits FTO demethylase activity to regulate N 6 -methyladenosine mRNA methylation.
- Author
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Kuschman HP, Palczewski MB, Hoffman B, Menhart M, Wang X, Glynn S, Islam ABMMK, Benevolenskaya EV, and Thomas DD
- Subjects
- Humans, Methylation, RNA, Messenger genetics, RNA, Messenger metabolism, Alpha-Ketoglutarate-Dependent Dioxygenase FTO genetics, Alpha-Ketoglutarate-Dependent Dioxygenase FTO chemistry, Alpha-Ketoglutarate-Dependent Dioxygenase FTO metabolism, Nitric Oxide, Adenosine metabolism
- Abstract
N
6 -methyladenosine (m6 A) is the most abundant internal modification on eukaryotic mRNAs. Demethylation of m6 A on mRNA is catalyzed by the enzyme fat mass and obesity-associated protein (FTO), a member of the nonheme Fe(II) and 2-oxoglutarate (2-OG)-dependent family of dioxygenases. FTO activity and m6 A-mRNA are dysregulated in multiple diseases including cancers, yet endogenous signaling molecules that modulate FTO activity have not been identified. Here we show that nitric oxide (NO) is a potent inhibitor of FTO demethylase activity by directly binding to the catalytic iron center, which causes global m6 A hypermethylation of mRNA in cells and results in gene-specific enrichment of m6 A on mRNA of NO-regulated transcripts. Both cell culture and tumor xenograft models demonstrated that endogenous NO synthesis can regulate m6 A-mRNA levels and transcriptional changes of m6 A-associated genes. These results build a direct link between NO and m6 A-mRNA regulation and reveal a novel signaling mechanism of NO as an endogenous regulator of the epitranscriptome., Competing Interests: Declaration of competing interest None., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)- Published
- 2023
- Full Text
- View/download PDF
11. RyR2 Binding of an Antiarrhythmic Cyclic Depsipeptide Mapped Using Confocal Fluorescence Lifetime Detection of FRET.
- Author
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Šeflová J, Schwarz JA, Smith AN, Svensson B, Blackwell DJ, Phillips TA, Nikolaienko R, Bovo E, Rebbeck RT, Zima AV, Thomas DD, Van Petegem F, Knollmann BC, Johnston JN, Robia SL, and Cornea RL
- Subjects
- Mice, Swine, Humans, Animals, Ryanodine chemistry, Ryanodine metabolism, Ryanodine therapeutic use, Fluorescence Resonance Energy Transfer methods, HEK293 Cells, Arrhythmias, Cardiac drug therapy, Arrhythmias, Cardiac metabolism, Calcium metabolism, Myocytes, Cardiac metabolism, Ryanodine Receptor Calcium Release Channel chemistry, Ryanodine Receptor Calcium Release Channel metabolism, Depsipeptides metabolism
- Abstract
Hyperactivity of cardiac sarcoplasmic reticulum (SR) ryanodine receptor (RyR2) Ca
2+ -release channels contributes to heart failure and arrhythmias. Reducing the RyR2 activity, particularly during cardiac relaxation (diastole), is a desirable therapeutic goal. We previously reported that the unnatural enantiomer ( ent ) of an insect-RyR activator, verticilide, inhibits porcine and mouse RyR2 at diastolic (nanomolar) Ca2+ and has in vivo efficacy against atrial and ventricular arrhythmia. To determine the ent -verticilide structural mode of action on RyR2 and guide its further development via medicinal chemistry structure-activity relationship studies, here, we used fluorescence lifetime (FLT)-measurements of Förster resonance energy transfer (FRET) in HEK293 cells expressing human RyR2. For these studies, we used an RyR-specific FRET molecular-toolkit and computational methods for trilateration (i.e., using distances to locate a point of interest). Multiexponential analysis of FLT-FRET measurements between four donor-labeled FKBP12.6 variants and acceptor-labeled ent -verticilide yielded distance relationships placing the acceptor probe at two candidate loci within the RyR2 cryo-EM map. One locus is within the Ry12 domain (at the corner periphery of the RyR2 tetrameric complex). The other locus is sandwiched at the interface between helical domain 1 and the SPRY3 domain. These findings document RyR2-target engagement by ent -verticilide, reveal new insight into the mechanism of action of this new class of RyR2-targeting drug candidate, and can serve as input in future computational determinations of the ent -verticilide binding site on RyR2 that will inform structure-activity studies for lead optimization.- Published
- 2023
- Full Text
- View/download PDF
12. Cardiomyopathy-associated variants alter the structure and function of the α-actinin-2 actin-binding domain.
- Author
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Atang AE, Rebbeck RT, Thomas DD, and Avery AW
- Subjects
- Humans, Actinin genetics, Actinin metabolism, Actins metabolism, Mutation, Cardiomyopathies, Cardiomyopathy, Dilated genetics, Cardiomyopathy, Hypertrophic genetics
- Abstract
Hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), and restrictive cardiomyopathy (RCM) are characterized by thickening, thinning, or stiffening, respectively, of the ventricular myocardium, resulting in diastolic or systolic dysfunction that can lead to heart failure and sudden cardiac death. Recently, variants in the ACTN2 gene, encoding the protein α-actinin-2, have been reported in HCM, DCM, and RCM patients. However, functional data supporting the pathogenicity of these variants is limited, and potential mechanisms by which these variants cause disease are largely unexplored. Currently, NIH ClinVar lists 34 ACTN2 missense variants, identified in cardiomyopathy patients, which we predict are likely to disrupt actin binding, based on their localization to specific substructures in the α-actinin-2 actin binding domain (ABD). We investigated the molecular consequences of three ABD localized, HCM-associated variants: A119T, M228T and T247 M. Using circular dichroism, we demonstrate that the mutant ABD proteins can attain a well-folded state. However, thermal denaturation studies show that all three mutations are destabilizing, suggesting a structural disruption. Importantly, A119T decreased actin binding, and M228T and T247M cause increased actin binding. We suggest that altered actin binding underlies pathogenesis for cardiomyopathy mutations localizing to the ABD of α-actinin-2., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)
- Published
- 2023
- Full Text
- View/download PDF
13. Molecular Mechanism of a FRET Biosensor for the Cardiac Ryanodine Receptor Pathologically Leaky State.
- Author
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Svensson B, Nitu FR, Rebbeck RT, McGurran LM, Oda T, Thomas DD, Bers DM, and Cornea RL
- Subjects
- Binding Sites, Drug Delivery Systems, Fluorescence Resonance Energy Transfer, Ryanodine Receptor Calcium Release Channel
- Abstract
Ca
2+ leak from cardiomyocyte sarcoplasmic reticulum (SR) via hyperactive resting cardiac ryanodine receptor channels (RyR2) is pro-arrhythmic. An exogenous peptide (DPc10) binding promotes leaky RyR2 in cardiomyocytes and reports on that endogenous state. Conversely, calmodulin (CaM) binding inhibits RyR2 leak and low CaM affinity is diagnostic of leaky RyR2. These observations have led to designing a FRET biosensor for drug discovery targeting RyR2. We used FRET to clarify the molecular mechanism driving the DPc10-CaM interdependence when binding RyR2 in SR vesicles. We used donor-FKBP12.6 (D-FKBP) to resolve RyR2 binding of acceptor-CaM (A-CaM). In low nanomolar Ca2+ , DPc10 decreased both FRETmax (under saturating [A-CaM]) and the CaM/RyR2 binding affinity. In micromolar Ca2+ , DPc10 decreased FRETmax without affecting CaM/RyR2 binding affinity. This correlates with the analysis of fluorescence-lifetime-detected FRET, indicating that DPc10 lowers occupancy of the RyR2 CaM-binding sites in nanomolar (not micromolar) Ca2+ and lengthens D-FKBP/A-CaM distances independent of [Ca2+ ]. To observe DPc10/RyR2 binding, we used acceptor-DPc10 (A-DPc10). CaM weakens A-DPc10/RyR2 binding, with this effect being larger in micromolar versus nanomolar Ca2+ . Moreover, A-DPc10/RyR2 binding is cooperative in a CaM- and FKBP-dependent manner, suggesting that both endogenous modulators promote concerted structural changes between RyR2 protomers for channel regulation. Aided by the analysis of cryo-EM structures, these insights inform further development of the DPc10-CaM paradigm for therapeutic discovery targeting RyR2.- Published
- 2023
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- View/download PDF
14. Fluorescence lifetime FRET assay for live-cell high-throughput screening of the cardiac SERCA pump yields multiple classes of small-molecule allosteric modulators.
- Author
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Roopnarine O, Yuen SL, Thompson AR, Roelike LN, Rebbeck RT, Bidwell PA, Aldrich CC, Cornea RL, and Thomas DD
- Subjects
- Humans, High-Throughput Screening Assays, Heart, Adenosine Triphosphatases, Fluorescence Resonance Energy Transfer, Heart Failure drug therapy
- Abstract
We have used FRET-based biosensors in live cells, in a robust high-throughput screening (HTS) platform, to identify small-molecules that alter the structure and activity of the cardiac sarco/endoplasmic reticulum calcium ATPase (SERCA2a). Our primary aim is to discover drug-like small-molecule activators that improve SERCA's function for the treatment of heart failure. We have previously demonstrated the use of an intramolecular FRET biosensor, based on human SERCA2a, by screening two different small validation libraries using novel microplate readers that detect the fluorescence lifetime or emission spectrum with high speed, precision, and resolution. Here we report results from FRET-HTS of 50,000 compounds using the same biosensor, with hit compounds functionally evaluated using assays for Ca
2+ -ATPase activity and Ca2+ -transport. We focused on 18 hit compounds, from which we identified eight structurally unique scaffolds and four scaffold classes as SERCA modulators, approximately half of which are activators and half are inhibitors. Five of these compounds were identified as promising SERCA activators, one of which activates Ca2+ -transport even more than Ca2+ -ATPase activity thus improving SERCA efficiency. While both activators and inhibitors have therapeutic potential, the activators establish the basis for future testing in heart disease models and lead development, toward pharmaceutical therapy for heart failure., (© 2023. The Author(s).)- Published
- 2023
- Full Text
- View/download PDF
15. Single-cell RNA-seq reveals intratumoral heterogeneity in osteosarcoma patients: A review.
- Author
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Thomas DD, Lacinski RA, and Lindsey BA
- Abstract
While primary bone malignancies make up just 0.2% of all cancers, osteosarcoma (OS) is the third most common cancer in adolescents. Due to its highly complex and heterogeneous tumor microenvironment (TME), OS has proven difficult to treat. There has been little to no improvement in therapy for this disease over the last 40 years. Even the recent success of immunotherapies in other blood-borne and solid malignancies has not translated to OS. With frequent recurrence and lung metastases continuing to pose a challenge in the clinic, recent advancements in molecular profiling, such as single-cell RNA sequencing (scRNA-seq), have proven useful in identifying novel biomarkers of OS tumors while providing new insight into this TME that could potentially lead to new therapeutic options. This review combines the analyses of over 150,000 cells from 18 lesions ranging from primary, recurrent, and metastatic OS lesions, revealing distinct cellular populations and gene signatures that exist between them. Here, we detail these previous findings and ultimately convey the intratumoral heterogeneity that exists within OS tumor specimens., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Author(s).)
- Published
- 2023
- Full Text
- View/download PDF
16. Early-phase drug discovery of β-III-spectrin actin-binding modulators for treatment of spinocerebellar ataxia type 5.
- Author
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Guhathakurta P, Rebbeck RT, Denha SA, Keller AR, Carter AL, Atang AE, Svensson B, Thomas DD, Hays TS, and Avery AW
- Subjects
- Humans, Drug Discovery, Neurons metabolism, Actins genetics, Actins metabolism, Spectrin metabolism, Spinocerebellar Ataxias drug therapy, Spinocerebellar Ataxias genetics, Spinocerebellar Ataxias metabolism
- Abstract
β-III-Spectrin is a key cytoskeletal protein that localizes to the soma and dendrites of cerebellar Purkinje cells and is required for dendritic arborization and signaling. A spinocerebellar ataxia type 5 L253P mutation in the cytoskeletal protein β-III-spectrin causes high-affinity actin binding. Previously we reported a cell-based fluorescence assay for identification of small-molecule actin-binding modulators of the L253P mutant β-III-spectrin. Here we describe a complementary, in vitro, fluorescence resonance energy transfer (FRET) assay that uses purified L253P β-III-spectrin actin-binding domain (ABD) and F-actin. To validate the assay for high-throughput compatibility, we first confirmed that our 50% FRET signal was responsive to swinholide A, an actin-severing compound, and that this yielded excellent assay quality with a Z' value > 0.77. Second, we screened a 2684-compound library of US Food and Drug Administration-approved drugs. Importantly, the screening identified numerous compounds that decreased FRET between fluorescently labeled L253P ABD and F-actin. The activity and target of multiple Hit compounds were confirmed in orthologous cosedimentation actin-binding assays. Through future medicinal chemistry, the Hit compounds can potentially be developed into a spinocerebellar ataxia type 5-specific therapeutic. Furthermore, our validated FRET-based in vitro high-throughput screening platform is poised for screening large compound libraries for β-III-spectrin ABD modulators., Competing Interests: Conflict of interest D. D. T. holds equity in, and serves as an executive officer for, Photonic Pharma LLC. These relationships have been reviewed and managed by the University of Minnesota. Photonic Pharma had no role in this study, except to provide some instrumentation, as stated in Experimental Procedures., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2023
- Full Text
- View/download PDF
17. FRET assay for live-cell high-throughput screening of the cardiac SERCA pump yields multiple classes of small-molecule allosteric modulators.
- Author
-
Roopnarine O, Yuen SL, Thompson AR, Roelike LN, Rebbeck RT, Bidwell PA, Aldrich CC, Cornea RL, and Thomas DD
- Abstract
We have used FRET-based biosensors in live cells, in a robust high-throughput screening (HTS) platform, to identify small-molecules that alter the structure and activity of the cardiac sarco/endoplasmic reticulum calcium ATPase (SERCA2a). Our primary aim is to discover drug-like small-molecule activators that improve SERCA’s function for the treatment of heart failure. We have previously demonstrated the use of an intramolecular FRET biosensor, based on human SERCA2a, by screening a small validation library using novel microplate readers that can detect the fluorescence lifetime or emission spectrum with high speed, precision, and resolution. Here we report results from a 50,000-compound screen using the same biosensor, with hit compounds functionally evaluated using Ca
2+ -ATPase and Ca2+ -transport assays. We focused on 18 hit compounds, from which we identified eight structurally unique compounds and four compound classes as SERCA modulators, approximately half of which are activators and half are inhibitors. While both activators and inhibitors have therapeutic potential, the activators establish the basis for future testing in heart disease models and lead development, toward pharmaceutical therapy for heart failure.- Published
- 2023
- Full Text
- View/download PDF
18. Interaction of DWORF with SERCA and PLB as determined by EPR spectroscopy.
- Author
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Rustad MD, Roopnarine O, Cornea RL, and Thomas DD
- Subjects
- Electron Spin Resonance Spectroscopy methods, Spin Labels, Endoplasmic Reticulum metabolism, Calcium metabolism, Sarcoplasmic Reticulum metabolism, Micropeptides, Sarcoplasmic Reticulum Calcium-Transporting ATPases metabolism, Calcium-Binding Proteins metabolism
- Abstract
Insufficient sarco/endoplasmic reticulum calcium ATPase (SERCA) activity significantly contributes to heart failure, which is a leading cause of death worldwide. A characteristic pathology of cardiac disease is the slow and incomplete Ca
2+ removal from the myocyte cytoplasm in diastole, which is primarily driven by SERCA, the integral transmembrane Ca2+ pump. Phospholamban (PLB) allosterically inhibits SERCA by reducing its apparent Ca2+ affinity. Recently, the 34-codon novel dwarf open reading frame (DWORF) micropeptide has been identified as a muscle-specific SERCA effector, capable of reversing the inhibitory effects of PLB and independently activating SERCA in the absence of PLB. However, the structural basis for these functions has not yet been determined in a system of defined molecular components. We have used electron paramagnetic resonance (EPR) spectroscopy to investigate the protein-protein interactions of DWORF, co-reconstituted in proteoliposomes with SERCA and spin-labeled PLB. We analyzed the change of PLB rotational mobility in response to varying DWORF concentration, to quantify competitive binding of DWORF and PLB. We determined that DWORF competes with PLB for binding to SERCA at low [Ca2+ ], although the measured affinity of DWORF for SERCA is an order of magnitude weaker than that of PLB for SERCA, indicating cooperativity. The sensitivity of EPR to structural dynamics, using stereospecifically attached spin labels, allows us to obtain new information needed to refine the molecular model for regulation of SERCA activity, as needed for development of novel therapeutic remedies against cardiac pathologies., Competing Interests: Declaration of competing interest DDT and RLC hold equity in and serve as executive officers for Photonic Pharma LLC, and OR is the sole proprietor of Editing Science LLC. Neither company had any role in this study. These relationships have been reviewed and managed by the University of Minnesota., (Copyright © 2023 Elsevier Inc. All rights reserved.)- Published
- 2023
- Full Text
- View/download PDF
19. Cardiac calcium regulation in human induced pluripotent stem cell cardiomyocytes: Implications for disease modeling and maturation.
- Author
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Ernst P, Bidwell PA, Dora M, Thomas DD, and Kamdar F
- Abstract
Human induced pluripotent stem cell cardiomyocytes (hiPSC-CMs) are based on ground-breaking technology that has significantly impacted cardiovascular research. They provide a renewable source of human cardiomyocytes for a variety of applications including in vitro disease modeling and drug toxicity testing. Cardiac calcium regulation plays a critical role in the cardiomyocyte and is often dysregulated in cardiovascular disease. Due to the limited availability of human cardiac tissue, calcium handling and its regulation have most commonly been studied in the context of animal models. hiPSC-CMs can provide unique insights into human physiology and pathophysiology, although a remaining limitation is the relative immaturity of these cells compared to adult cardiomyocytes Therefore, this field is rapidly developing techniques to improve the maturity of hiPSC-CMs, further establishing their place in cardiovascular research. This review briefly covers the basics of cardiomyocyte calcium cycling and hiPSC technology, and will provide a detailed description of our current understanding of calcium in hiPSC-CMs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Ernst, Bidwell, Dora, Thomas and Kamdar.)
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- 2023
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20. New N-aryl-N-alkyl-thiophene-2-carboxamide compound enhances intracellular Ca 2+ dynamics by increasing SERCA2a Ca 2+ pumping.
- Author
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Nikolaienko R, Bovo E, Yuen SL, Treinen LM, Berg K, Aldrich CC, Thomas DD, Cornea RL, and Zima AV
- Subjects
- Animals, Humans, Mice, Calcium metabolism, HEK293 Cells, Myocytes, Cardiac metabolism, Sarcoplasmic Reticulum metabolism, Thiophenes pharmacology, Heart Failure metabolism, Sarcoplasmic Reticulum Calcium-Transporting ATPases metabolism
- Abstract
The type 2a sarco/endoplasmic reticulum Ca
2+ -ATPase (SERCA2a) plays a central role in the intracellular Ca2+ homeostasis of cardiac myocytes, pumping Ca2+ from the cytoplasm into the sarcoplasmic reticulum (SR) lumen to maintain relaxation (diastole) and prepare for contraction (systole). Diminished SERCA2a function has been reported in several pathological conditions, including heart failure. Therefore, development of new drugs that improve SERCA2a Ca2+ transport is of great clinical significance. In this study, we characterized the effect of a recently identified N-aryl-N-alkyl-thiophene-2-carboxamide (or compound 1) on SERCA2a Ca2+ -ATPase and Ca2+ transport activities in cardiac SR vesicles, and on Ca2+ regulation in a HEK293 cell expression system and in mouse ventricular myocytes. We found that compound 1 enhances SERCA2a Ca2+ -ATPase and Ca2+ transport in SR vesicles. Fluorescence lifetime measurements of fluorescence resonance energy transfer between SERCA2a and phospholamban indicated that compound 1 interacts with the SERCA-phospholamban complex. Measurement of endoplasmic reticulum Ca2+ dynamics in HEK293 cells expressing human SERCA2a showed that compound 1 increases endoplasmic reticulum Ca2+ load by enhancing SERCA2a-mediated Ca2+ transport. Analysis of cytosolic Ca2+ dynamics in mouse ventricular myocytes revealed that compound 1 increases the action potential-induced Ca2+ transients and SR Ca2+ load, with negligible effects on L-type Ca2+ channels and Na+ /Ca2+ exchanger. However, during adrenergic receptor activation, compound 1 did not further increase Ca2+ transients and SR Ca2+ load, but it decreased the propensity toward Ca2+ waves. Suggestive of concurrent desirable effects of compound 1 on RyR2, [3 H]-ryanodine binding to cardiac SR vesicles shows a small decrease in nM Ca2+ and a small increase in μM Ca2+ . Accordingly, compound 1 slightly decreased Ca2+ sparks in permeabilized myocytes. Thus, this novel compound shows promising characteristics to improve intracellular Ca2+ dynamics in cardiomyocytes that exhibit reduced SERCA2a Ca2+ uptake, as found in failing hearts., Competing Interests: Declaration of interests R.L.C. and D.D.T. hold equity in, and serve as executive officers for, Photonic Pharma LLC, which had no role in this study. These relationships have been reviewed and managed by the University of Minnesota., (Copyright © 2022 Biophysical Society. Published by Elsevier Inc. All rights reserved.)- Published
- 2023
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21. Indirubin Inhibits TRAIL-Induced Activation of Death Receptor 5 in Jurkat Cells.
- Author
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Young MC, Vunnam N, Rebbeck RT, Yuen SL, Thomas DD, and Sachs JN
- Abstract
Death receptor 5 (DR5) is an apoptosis-inducing membrane receptor that mediates cell death in several life-threatening conditions. There is a crucial need for the discovery of DR5 antagonists for the therapeutic intervention of conditions in which the overactivation of DR5 underlies the pathophysiology. DR5 activation mediates cell death in non-alcoholic fatty liver disease (NAFLD) and neurodegenerative processes including amyloid-beta (Aβ) accumulation, spinal cord injury (SCI), and brain ischemia. In the current work, we used fluorescence resonance energy transfer (FRET) to monitor the conformational dynamics of DR5 that mediate death signaling. We used a time-resolved FRET screening platform to screen the Selleck library of 2863 U.S. Food and Drug Administration (FDA)-approved compounds. The high-throughput screen (HTS) identified 13 compounds that modulated the FRET between DR5 monomers beyond 5 median absolute deviations (MADs) from the DMSO controls. Of these 13 compounds, indirubin was identified to specifically inhibit tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced caspase-8 activity without modulating DR5 surface expression or TRAIL binding. Indirubin inhibited Fas-associated death domain (FADD) oligomerization and increased cellular FLICE-inhibitory protein (c-FLIP) expression; both are molecular mechanisms involved in inhibiting the DR5 signaling cascade. This study has elucidated previously unknown properties of indirubin that make it a promising candidate for therapeutic investigation of diseases in which overactivation of DR5 underlies pathology., Competing Interests: Declaration of Conflicting Interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
- Published
- 2023
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22. Advancements in a FRET Biosensor for Live-Cell Fluorescence-Lifetime High-Throughput Screening of Alpha-Synuclein.
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Braun AR, Kochen NN, Yuen SL, Liao EE, Cornea RL, Thomas DD, and Sachs JN
- Subjects
- alpha-Synuclein metabolism, Fluorescence Resonance Energy Transfer methods, High-Throughput Screening Assays, Emodin, Proanthocyanidins, Biosensing Techniques
- Abstract
There is a critical need for small molecules capable of rescuing pathophysiological phenotypes induced by alpha-synuclein (aSyn) misfolding and oligomerization. Building upon our previous aSyn cellular fluorescence lifetime (FLT)-Förster resonance energy transfer (FRET) biosensors, we have developed an inducible cell model incorporating the red-shifted mCyRFP1/mMaroon1 (OFP/MFP) FRET pair. This new aSyn FRET biosensor improves the signal-to-noise ratio, reduces nonspecific background FRET, and results in a 4-fold increase (transient transfection) and 2-fold increase (stable, inducible cell lines) in FRET signal relative to our previous GFP/RFP aSyn biosensors. The inducible system institutes greater temporal control and scalability, allowing for fine-tuning of biosensor expression and minimizes cellular cytotoxicity due to overexpression of aSyn. Using these inducible aSyn-OFP/MFP biosensors, we screened the Selleck library of 2684 commercially available, FDA-approved compounds and identified proanthocyanidins and casanthranol as novel hits. Secondary assays validated the ability of these compounds to modulate aSyn FLT-FRET. Functional assays probing cellular cytotoxicity and aSyn fibrillization demonstrated their capability to inhibit seeded aSyn fibrillization. Proanthocyanidins completely rescued aSyn fibril-induced cellular toxicity with EC
50 of 200 nM and casanthranol supported a 85.5% rescue with a projected EC50 of 34.2 μM. Furthermore, proanthocyanidins provide a valuable tool compound to validate our aSyn biosensor performance in future high-throughput screening campaigns of industrial-scale (million-compound) chemical libraries.- Published
- 2023
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23. The Chemical Biology of NO that Regulates Oncogenic Signaling and Metabolism: NOS2 and Its Role in Inflammatory Disease.
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Miranda KM, Ridnour LA, Cheng RYS, Wink DA, and Thomas DD
- Subjects
- Humans, Signal Transduction, Neoplasms genetics, Nitric Oxide metabolism, Nitric Oxide Synthase Type II genetics, Nitric Oxide Synthase Type II metabolism
- Abstract
Nitric oxide (NO) and the enzyme that synthesizes it, nitric oxide synthase 2 (NOS2), have emerged as key players in inflammation and cancer. Expression of NOS2 in tumors has been correlated both with positive outcomes and with poor prognoses. The chemistry of NO is the major determinate to the biological outcome and the concentration of NO, which can range over five orders of magnitude, is critical in determining which pathways are activated. It is the activation of specific oncogenic and immunological mechanisms that shape the outcome. The kinetics of specific reactions determine the mechanisms of action. In this review, the relevant reactions of NO and related species are discussed with respect to these oncogenic and immunological signals.
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- 2023
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24. Enhancing interaction of actin and actin-binding domain 1 of dystrophin with modulators: Toward improved gene therapy for Duchenne muscular dystrophy.
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Guhathakurta P, Carter AL, Thompson AR, Kurila D, LaFrence J, Zhang L, Trask JR, Vanderheyden B, Muretta JM, Ervasti JM, and Thomas DD
- Subjects
- Humans, Genetic Therapy, Protein Binding drug effects, Small Molecule Libraries chemistry, Small Molecule Libraries metabolism, Small Molecule Libraries pharmacology, Protein Domains, Actins metabolism, Dystrophin genetics, Dystrophin chemistry, Muscular Dystrophy, Duchenne genetics, Muscular Dystrophy, Duchenne therapy
- Abstract
Duchenne muscular dystrophy is a lethal muscle disease, caused by mutations in the gene encoding dystrophin, an actin-binding cytoskeletal protein. Absence of functional dystrophin results in muscle weakness and degeneration, eventually leading to cardiac and respiratory failure. Strategies to replace the missing dystrophin via gene therapy have been intensively pursued. However, the dystrophin gene is too large for current gene therapy approaches. Currently available micro-dystrophin constructs lack the actin-binding domain 2 and show decreased actin-binding affinity in vitro compared to full-length dystrophin. Thus, increasing the actin-binding affinity of micro-dystrophin, using small molecules, could be a beneficial therapeutic approach. Here, we have developed and validated a novel high-throughput screening (HTS) assay to discover small molecules that increase the binding affinity of dystrophin's actin-binding domain 1 (ABD1). We engineered a novel FRET biosensor, consisting of the mClover3, fluorescent protein (donor) attached to the C-terminus of dystrophin ABD1, and Alexa Fluor 568 (acceptor) attached to the C-terminal cysteine of actin. We used this biosensor in small-molecule screening, using a unique high-precision, HTS fluorescence lifetime assay, identifying several compounds from an FDA-approved library that significantly increase the binding between actin and ABD1. This HTS assay establishes feasibility for the discovery of small-molecule modulators of the actin-dystrophin interaction, with the ultimate goal of developing therapies for muscular dystrophy., Competing Interests: Conflict of interest D. D. T. holds equity in and serves as President of Photonic Pharma LLC. This relationship has been reviewed and managed by the University of Minnesota. Photonic Pharma had no role in this study, except to provide instrumentation, as stated in Experimental procedures. J. M. M., J. M. E., and D. D. T. are entitled to royalties from Sarepta Therapeutics, the company sponsoring this research project. This royalty interest has been reviewed and managed according to the University of Minnesota's conflict of interest policies. J. M. E. has received compensation for consulting for Sarepta. This relationship has been reviewed and managed by the University of Minnesota in accordance with its conflict of interest polices. All other authors declare no conflicts of interest with the contents of the article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2022
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25. A Large-Scale High-Throughput Screen for Modulators of SERCA Activity.
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Bidwell PA, Yuen SL, Li J, Berg K, Rebbeck RT, Aldrich CC, Roopnarine O, Cornea RL, and Thomas DD
- Subjects
- Animals, Ion Transport, Endoplasmic Reticulum metabolism, Muscle Cells metabolism, Calcium metabolism, Mammals metabolism, Sarcoplasmic Reticulum Calcium-Transporting ATPases metabolism, Sarcoplasmic Reticulum metabolism
- Abstract
The sarco/endoplasmic reticulum Ca-ATPase (SERCA) is a P-type ion pump that transports Ca
2+ from the cytosol into the endoplasmic/sarcoplasmic reticulum (ER/SR) in most mammalian cells. It is critically important in muscle, facilitating relaxation and enabling subsequent contraction. Increasing SERCA expression or specific activity can alleviate muscle dysfunction, most notably in the heart, and we seek to develop small-molecule drug candidates that activate SERCA. Therefore, we adapted an NADH-coupled assay, measuring Ca-dependent ATPase activity of SERCA, to high-throughput screening (HTS) format, and screened a 46,000-compound library of diverse chemical scaffolds. This HTS platform yielded numerous hits that reproducibly alter SERCA Ca-ATPase activity, with few false positives. The top 19 activating hits were further tested for effects on both Ca-ATPase and Ca2+ transport, in both cardiac and skeletal SR. Nearly all hits increased Ca2+ uptake in both cardiac and skeletal SR, with some showing isoform specificity. Furthermore, dual analysis of both activities identified compounds with a range of effects on Ca2+ -uptake and ATPase, which fit into distinct classifications. Further study will be needed to identify which classifications are best suited for therapeutic use. These results reinforce the need for robust secondary assays and criteria for selection of lead compounds, before undergoing HTS on a larger scale.- Published
- 2022
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26. A rare human centenarian variant of SIRT6 enhances genome stability and interaction with Lamin A.
- Author
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Simon M, Yang J, Gigas J, Earley EJ, Hillpot E, Zhang L, Zagorulya M, Tombline G, Gilbert M, Yuen SL, Pope A, Van Meter M, Emmrich S, Firsanov D, Athreya A, Biashad SA, Han J, Ryu S, Tare A, Zhu Y, Hudgins A, Atzmon G, Barzilai N, Wolfe A, Moody K, Garcia BA, Thomas DD, Robbins PD, Vijg J, Seluanov A, Suh Y, and Gorbunova V
- Subjects
- Aged, 80 and over, Humans, Centenarians, Alleles, Genomic Instability, Lamin Type A, Sirtuins
- Abstract
Sirtuin 6 (SIRT6) is a deacylase and mono-ADP ribosyl transferase (mADPr) enzyme involved in multiple cellular pathways implicated in aging and metabolism regulation. Targeted sequencing of SIRT6 locus in a population of 450 Ashkenazi Jewish (AJ) centenarians and 550 AJ individuals without a family history of exceptional longevity identified enrichment of a SIRT6 allele containing two linked substitutions (N308K/A313S) in centenarians compared with AJ control individuals. Characterization of this SIRT6 allele (centSIRT6) demonstrated it to be a stronger suppressor of LINE1 retrotransposons, confer enhanced stimulation of DNA double-strand break repair, and more robustly kill cancer cells compared with wild-type SIRT6. Surprisingly, centSIRT6 displayed weaker deacetylase activity, but stronger mADPr activity, over a range of NAD
+ concentrations and substrates. Additionally, centSIRT6 displayed a stronger interaction with Lamin A/C (LMNA), which was correlated with enhanced ribosylation of LMNA. Our results suggest that enhanced SIRT6 function contributes to human longevity by improving genome maintenance via increased mADPr activity and enhanced interaction with LMNA., (©2022 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)- Published
- 2022
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27. Fluorescence Lifetime Measurement of Prefibrillar Sickle Hemoglobin Oligomers as a Platform for Drug Discovery in Sickle Cell Disease.
- Author
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Vunnam N, Hansen S, Williams DC, Been MO, Lo CH, Pandey AK, Paulson CN, Rohde JA, Thomas DD, Sachs JN, and Wood DK
- Subjects
- Drug Discovery, Hemoglobins, Humans, Oxygen metabolism, Anemia, Sickle Cell drug therapy, Anemia, Sickle Cell metabolism, Hemoglobin, Sickle chemistry, Hemoglobin, Sickle metabolism
- Abstract
The molecular origin of sickle cell disease (SCD) has been known since 1949, but treatments remain limited. We present the first high-throughput screening (HTS) platform for discovering small molecules that directly inhibit sickle hemoglobin (HbS) oligomerization and improve blood flow, potentially overcoming a long-standing bottleneck in SCD drug discovery. We show that at concentrations far below the threshold for nucleation and rapid polymerization, deoxygenated HbS forms small assemblies of multiple α
2 β2 tetramers. Our HTS platform leverages high-sensitivity fluorescence lifetime measurements that monitor these temporally stable prefibrillar HbS oligomers. We show that this approach is sensitive to compounds that inhibit HbS polymerization with or without modulating hemoglobin oxygen binding affinity. We also report the results of a pilot small-molecule screen in which we discovered and validated several novel inhibitors of HbS oligomerization.- Published
- 2022
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28. Synergistic FRET assays for drug discovery targeting RyR2 channels.
- Author
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Rebbeck R, Ginsburg KS, Ko CY, Fasoli A, Rusch K, Cai GF, Dong X, Thomas DD, Bers DM, and Cornea RL
- Subjects
- Animals, Calcium metabolism, Calmodulin metabolism, Drug Discovery, Myocytes, Cardiac metabolism, Rats, Sarcoplasmic Reticulum metabolism, Tacrolimus Binding Proteins metabolism, Fluorescence Resonance Energy Transfer methods, Ryanodine Receptor Calcium Release Channel metabolism
- Abstract
A key therapeutic target for heart failure and arrhythmia is the deleterious leak through sarcoplasmic reticulum (SR) ryanodine receptor 2 (RyR2) calcium release channels. We have previously developed methods to detect the pathologically leaky state of RyR2 in adult cardiomyocytes by monitoring RyR2 binding to either calmodulin (CaM) or a biosensor peptide (DPc10). Here, we test whether these complementary binding measurements are effective as high-throughput screening (HTS) assays to discover small molecules that target leaky RyR2. Using FRET, we developed and validated HTS procedures under conditions that mimic a pathological state, to screen the library of 1280 pharmaceutically active compounds (LOPAC) for modulators of RyR2 in cardiac SR membrane preparations. Complementary FRET assays with acceptor-labeled CaM and DPc10 were used for Hit prioritization based on the opposing binding properties of CaM vs. DPc10. This approach narrowed the Hit list to one compound, Ro 90-7501, which altered FRET to suggest increased RyR2-CaM binding and decreased DPc10 binding. Follow-up studies revealed that Ro 90-7501 does not detrimentally affect myocyte Ca
2+ transients. Moreover, Ro 90-7501 partially inhibits overall Ca2+ leak, as assessed by Ca2+ sparks in permeabilized rat cardiomyocytes. Together, these results demonstrate (1) the effectiveness of our HTS approach where two complementary assays synergize for Hit ranking and (2) a drug discovery process that combines high-throughput, high-precision in vitro structural assays with in situ myocyte assays of the pathologic RyR2 leak. These provide a drug discovery platform compatible with large-scale HTS campaigns, to identify agents that inhibit RyR2 for therapeutic development., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)- Published
- 2022
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29. Methods to the madness - Fundamental techniques in NO research.
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Thomas DD and Shiva S
- Published
- 2022
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30. Cardiac ryanodine receptor N-terminal region biosensors identify novel inhibitors via FRET-based high-throughput screening.
- Author
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Zhang J, Singh DP, Ko CY, Nikolaienko R, Wong King Yuen SM, Schwarz JA, Treinen LM, Tung CC, Rožman K, Svensson B, Aldrich CC, Zima AV, Thomas DD, Bers DM, Launikonis BS, Van Petegem F, and Cornea RL
- Subjects
- Animals, Calcium metabolism, Fluorescence Resonance Energy Transfer, High-Throughput Screening Assays, Mice, Muscle, Skeletal chemistry, Muscle, Skeletal metabolism, Biosensing Techniques, Ryanodine Receptor Calcium Release Channel analysis, Ryanodine Receptor Calcium Release Channel metabolism
- Abstract
The N-terminal region (NTR) of ryanodine receptor (RyR) channels is critical for the regulation of Ca
2+ release during excitation-contraction (EC) coupling in muscle. The NTR hosts numerous mutations linked to skeletal (RyR1) and cardiac (RyR2) myopathies, highlighting its potential as a therapeutic target. Here, we constructed two biosensors by labeling the mouse RyR2 NTR at domains A, B, and C with FRET pairs. Using fluorescence lifetime (FLT) detection of intramolecular FRET signal, we developed high-throughput screening (HTS) assays with these biosensors to identify small-molecule RyR modulators. We then screened a small validation library and identified several hits. Hits with saturable FRET dose-response profiles and previously unreported effects on RyR were further tested using [3 H]ryanodine binding to isolated sarcoplasmic reticulum vesicles to determine effects on intact RyR opening in its natural membrane. We identified three novel inhibitors of both RyR1 and RyR2 and two RyR1-selective inhibitors effective at nanomolar Ca2+ . Two of these hits activated RyR1 only at micromolar Ca2+ , highlighting them as potential enhancers of excitation-contraction coupling. To determine whether such hits can inhibit RyR leak in muscle, we further focused on one, an FDA-approved natural antibiotic, fusidic acid (FA). In skinned skeletal myofibers and permeabilized cardiomyocytes, FA inhibited RyR leak with no detrimental effect on skeletal myofiber excitation-contraction coupling. However, in intact cardiomyocytes, FA induced arrhythmogenic Ca2+ transients, a cautionary observation for a compound with an otherwise solid safety record. These results indicate that HTS campaigns using the NTR biosensor can identify compounds with therapeutic potential., Competing Interests: Conflict of interest D. D. T. and R. L. C. hold equity in and serve as executive officers for Photonic Pharma LLC. These relationships have been reviewed and managed by the University of Minnesota. Photonic Pharma had no role in this study., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)- Published
- 2022
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31. Sarcoplasmic Reticulum from Horse Gluteal Muscle Is Poised for Enhanced Calcium Transport.
- Author
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Autry JM, Svensson B, Carlson SF, Chen Z, Cornea RL, Thomas DD, and Valberg SJ
- Abstract
We have analyzed the enzymatic activity of the sarcoplasmic reticulum (SR) Ca
2+ -transporting ATPase (SERCA) from the horse gluteal muscle. Horses are bred for peak athletic performance yet exhibit a high incidence of exertional rhabdomyolysis, with elevated levels of cytosolic Ca2+ proposed as a correlative linkage. We recently reported an improved protocol for isolating SR vesicles from horse muscle; these horse SR vesicles contain an abundant level of SERCA and only trace-levels of sarcolipin (SLN), the inhibitory peptide subunit of SERCA in mammalian fast-twitch skeletal muscle. Here, we report that the in vitro Ca2+ transport rate of horse SR vesicles is 2.3 ± 0.7-fold greater than rabbit SR vesicles, which express close to equimolar levels of SERCA and SLN. This suggests that horse myofibers exhibit an enhanced SR Ca2+ transport rate and increased luminal Ca2+ stores in vivo. Using the densitometry of Coomassie-stained SDS-PAGE gels, we determined that horse SR vesicles express an abundant level of the luminal SR Ca2+ storage protein calsequestrin (CASQ), with a CASQ-to-SERCA ratio about double that in rabbit SR vesicles. Thus, we propose that SR Ca2+ cycling in horse myofibers is enhanced by a reduced SLN inhibition of SERCA and by an abundant expression of CASQ. Together, these results suggest that horse muscle contractility and susceptibility to exertional rhabdomyolysis are promoted by enhanced SR Ca2+ uptake and luminal Ca2+ storage.- Published
- 2021
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32. Direct detection of the myosin super-relaxed state and interacting-heads motif in solution.
- Author
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Chu S, Muretta JM, and Thomas DD
- Subjects
- Amino Acid Motifs, Animals, Benzylamines therapeutic use, Cardiomyopathy, Hypertrophic drug therapy, Cardiomyopathy, Hypertrophic metabolism, Cattle, Kinetics, Myosins metabolism, Uracil chemistry, Uracil therapeutic use, Benzylamines chemistry, Fluorescence Resonance Energy Transfer, Myosins chemistry, Uracil analogs & derivatives
- Abstract
The interacting-heads motif (IHM) is a structure of myosin that has been proposed to modulate cardiac output by occluding myosin molecules from undergoing the force-generating cycle. It is hypothesized to be the structural basis for the super-relaxed state (SRX), a low-ATPase kinetic state thought to be cardioprotective. The goal of the present study was to test this hypothesis by determining directly and quantitatively the fractions of myosin in the IHM and SRX under the same conditions in solution. To detect the structural IHM, we used time-resolved fluorescence resonance energy transfer to quantitate two distinct populations. One population was observed at a center distance of 2.0 nm, whereas the other was not detectable by fluorescence resonance energy transfer, implying a distance greater than 4 nm. We confirmed the IHM assignment to the 2.0-nm population by applying the same cross-linking protocol used previously to image the IHM by electron microscopy. Under the same conditions, we also measured the fraction of myosin in the SRX using stopped-flow kinetics. Our results show that the populations of SRX and IHM myosin were similar, unless treated with mavacamten, a drug that recently completed phase III clinical trials to treat hypertrophic cardiomyopathy and is proposed to act by stabilizing both the SRX and IHM. However, we found that mavacamten had a much greater effect on the SRX (55% increase) than on the IHM (4% increase). We conclude that the IHM structure is sufficient but not necessary to produce the SRX kinetic state., Competing Interests: Conflict of interest D. D. T. holds equity in, and serves as an executive officer for, Photonic Pharma LLC. These relationships have been reviewed and managed by the University of Minnesota. Photonic Pharma had no role in this study. All other authors declare that they have no conflicts of interest related to the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2021
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33. Integrated Phosphoproteomics for Identifying Substrates of Human Protein Kinase A ( PRKACA ) and Its Oncogenic Mutant DNAJB 1 -PRKACA .
- Author
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Karamafrooz A, Brennan J, Thomas DD, and Parker LL
- Subjects
- Adolescent, Cyclic AMP-Dependent Protein Kinase Catalytic Subunits genetics, Cyclic AMP-Dependent Protein Kinase Catalytic Subunits metabolism, Gene Expression Regulation, Neoplastic, HSP40 Heat-Shock Proteins genetics, Humans, Oncogenes, Carcinoma, Hepatocellular genetics, Liver Neoplasms genetics
- Abstract
The DNAJB1-PRKACA fusion is the signature genetic event of fibrolamellar hepatocellular carcinoma (FL-HCC), a rare but lethal liver cancer that primarily affects adolescents and young adults. A deletion fuses the first exon of the HSP40 gene ( DNAJB1 ), with exons 2-10 of protein kinase A ( PRKACA ), producing the chimeric kinase DNAJB1-PKA
ca (J-PKAca ). The HSP40 portion's scaffolding/chaperone function has been implicated in redirecting substrate recognition to upregulate oncogenic pathways, but the direct substrates of this fusion are not fully known. We integrated cell-based and in vitro phosphoproteomics to identify substrates targeted directly by PKA and J-PKAca , comparing phosphoproteome profiles from cells with in vitro rephosphorylation of peptides and proteins from lysates using recombinant enzymes. We identified a subset of phosphorylation sites in both cell-based and in vitro experiments, as well as altered pathways and proteins consistent with observations from related studies. We also treated cells with PKA inhibitors that function by two different mechanisms (rpcAMPs and PKI) and examined phosphoproteome profiles, finding some substrates that persisted in the presence of inhibitors and revealing differences between WT and chimera. Overall, these results provide potential insights into J-PKAca 's oncogenic activity in a complex cellular system and may provide candidate targets for therapeutic follow-up.- Published
- 2021
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34. Nitric oxide and hydrogen sulfide: Sibling rivalry in the family of epigenetic regulators.
- Author
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Kuschman HP, Palczewski MB, and Thomas DD
- Subjects
- Epigenesis, Genetic, Humans, Nitric Oxide metabolism, Siblings, Gasotransmitters metabolism, Hydrogen Sulfide metabolism
- Abstract
Nitric oxide (NO) and hydrogen sulfide (H
2 S) were previously only known for their toxic properties. Now they are regarded as potent gaseous messenger molecules (gasotransmitters) that rapidly transverse cell membranes and transduce cellular signals through their chemical reactions and modifications to protein targets. Both are known to regulate numerous physiological functions including angiogenesis, vascular tone, and immune response, to name a few. NO and H2 S often work synergistically and in competition to regulate each other's synthesis, target protein activity via posttranslational modifications (PTMs), and chemical interactions. In addition to their canonical modes of action, increasing evidence has demonstrated that NO and H2 S share another signaling mechanism: epigenetic regulation. This review will compare and contrast biosynthesis and metabolism of NO and H2 S, their individual and shared interactions, and the growing body of evidence for their roles as endogenous epigenetic regulatory molecules., (Copyright © 2021 Elsevier Inc. All rights reserved.)- Published
- 2021
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35. Cardiac myosin-binding protein C interaction with actin is inhibited by compounds identified in a high-throughput fluorescence lifetime screen.
- Author
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Bunch TA, Guhathakurta P, Lepak VC, Thompson AR, Kanassatega RS, Wilson A, Thomas DD, and Colson BA
- Subjects
- Actins chemistry, Animals, Biosensing Techniques, Calorimetry, Fluorescence, Fluorescence Resonance Energy Transfer, Humans, Protein Binding, Rabbits, Sarcomeres metabolism, Time Factors, Actins metabolism, Carrier Proteins metabolism, High-Throughput Screening Assays, Myocardium metabolism
- Abstract
Cardiac myosin-binding protein C (cMyBP-C) interacts with actin and myosin to modulate cardiac muscle contractility. These interactions are disfavored by cMyBP-C phosphorylation. Heart failure patients often display decreased cMyBP-C phosphorylation, and phosphorylation in model systems has been shown to be cardioprotective against heart failure. Therefore, cMyBP-C is a potential target for heart failure drugs that mimic phosphorylation or perturb its interactions with actin/myosin. Here we have used a novel fluorescence lifetime-based assay to identify small-molecule inhibitors of actin-cMyBP-C binding. Actin was labeled with a fluorescent dye (Alexa Fluor 568, AF568) near its cMyBP-C binding sites; when combined with the cMyBP-C N-terminal fragment, C0-C2, the fluorescence lifetime of AF568-actin decreases. Using this reduction in lifetime as a readout of actin binding, a high-throughput screen of a 1280-compound library identified three reproducible hit compounds (suramin, NF023, and aurintricarboxylic acid) that reduced C0-C2 binding to actin in the micromolar range. Binding of phosphorylated C0-C2 was also blocked by these compounds. That they specifically block binding was confirmed by an actin-C0-C2 time-resolved FRET (TR-FRET) binding assay. Isothermal titration calorimetry (ITC) and transient phosphorescence anisotropy (TPA) confirmed that these compounds bind to cMyBP-C, but not to actin. TPA results were also consistent with these compounds inhibiting C0-C2 binding to actin. We conclude that the actin-cMyBP-C fluorescence lifetime assay permits detection of pharmacologically active compounds that affect cMyBP-C-actin binding. We now have, for the first time, a validated high-throughput screen focused on cMyBP-C, a regulator of cardiac muscle contractility and known key factor in heart failure., Competing Interests: Conflict of interest D. D. T. holds equity in and serves as President of Photonic Pharma LLC. This relationship has been reviewed and managed by the University of Minnesota. Photonic Pharma had no role in this study, except to provide some instrumentation, as stated in Experimental procedures. B. A. C. filed a PCT patent application based on this work (patent pending, serial no. PCT/US21/14142). The other authors declare no competing financial interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2021
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36. Nitric Oxide Modulates Metabolic Processes in the Tumor Immune Microenvironment.
- Author
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McGinity CL, Palmieri EM, Somasundaram V, Bhattacharyya DD, Ridnour LA, Cheng RYS, Ryan AE, Glynn SA, Thomas DD, Miranda KM, Anderson SK, Lockett SJ, McVicar DW, and Wink DA
- Subjects
- Animals, Humans, Neoplasms pathology, Neoplasms immunology, Nitric Oxide immunology, Signal Transduction immunology, Tumor Microenvironment immunology
- Abstract
The metabolic requirements and functions of cancer and normal tissues are vastly different. Due to the rapid growth of cancer cells in the tumor microenvironment, distorted vasculature is commonly observed, which creates harsh environments that require rigorous and constantly evolving cellular adaption. A common hallmark of aggressive and therapeutically resistant tumors is hypoxia and hypoxia-induced stress markers. However, recent studies have identified alterations in a wide spectrum of metabolic pathways that dictate tumor behavior and response to therapy. Accordingly, it is becoming clear that metabolic processes are not uniform throughout the tumor microenvironment. Metabolic processes differ and are cell type specific where various factors promote metabolic heterogeneity within the tumor microenvironment. Furthermore, within the tumor, these metabolically distinct cell types can organize to form cellular neighborhoods that serve to establish a pro-tumor milieu in which distant and spatially distinct cellular neighborhoods can communicate via signaling metabolites from stroma, immune and tumor cells. In this review, we will discuss how biochemical interactions of various metabolic pathways influence cancer and immune microenvironments, as well as associated mechanisms that lead to good or poor clinical outcomes.
- Published
- 2021
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37. Potent inhibitors of toxic alpha-synuclein identified via cellular time-resolved FRET biosensors.
- Author
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Braun AR, Liao EE, Horvath M, Kalra P, Acosta K, Young MC, Kochen NN, Lo CH, Brown R, Evans MD, Pomerantz WCK, Rhoades E, Luk K, Cornea RL, Thomas DD, and Sachs JN
- Abstract
We have developed a high-throughput drug discovery platform, measuring fluorescence resonance energy transfer (FRET) with fluorescent alpha-synuclein (αSN) biosensors, to detect spontaneous pre-fibrillar oligomers in living cells. Our two αSN FRET biosensors provide complementary insight into αSN oligomerization and conformation in order to improve the success of drug discovery campaigns for the treatment of Parkinson's disease. We measure FRET by fluorescence lifetime, rather than traditional fluorescence intensity, providing a structural readout with greater resolution and precision. This facilitates identification of compounds that cause subtle but significant conformational changes in the ensemble of oligomeric states that are easily missed using intensity-based FRET. We screened a 1280-compound small-molecule library and identified 21 compounds that changed the lifetime by >5 SD. Two of these compounds have nanomolar potency in protecting SH-SY5Y cells from αSN-induced death, providing a nearly tenfold improvement over known inhibitors. We tested the efficacy of several compounds in a primary mouse neuron assay of αSN pathology (phosphorylation of mouse αSN pre-formed fibrils) and show rescue of pathology for two of them. These hits were further characterized with biophysical and biochemical assays to explore potential mechanisms of action. In vitro αSN oligomerization, single-molecule FRET, and protein-observed fluorine NMR experiments demonstrate that these compounds modulate αSN oligomers but not monomers. Subsequent aggregation assays further show that these compounds also deter or block αSN fibril assembly.
- Published
- 2021
- Full Text
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38. Structural basis for allosteric control of the SERCA-Phospholamban membrane complex by Ca 2+ and phosphorylation.
- Author
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Weber DK, Reddy UV, Wang S, Larsen EK, Gopinath T, Gustavsson MB, Cornea RL, Thomas DD, De Simone A, and Veglia G
- Subjects
- Animals, Calcium metabolism, Calcium-Binding Proteins metabolism, Escherichia coli, Magnetic Resonance Spectroscopy, Membrane Proteins metabolism, Molecular Structure, Protein Conformation, Rabbits, Sarcoplasmic Reticulum, Sarcoplasmic Reticulum Calcium-Transporting ATPases metabolism, Signal Transduction, Allosteric Regulation, Calcium-Binding Proteins chemistry, Phosphorylation, Sarcoplasmic Reticulum Calcium-Transporting ATPases chemistry
- Abstract
Phospholamban (PLN) is a mini-membrane protein that directly controls the cardiac Ca
2+ -transport response to β-adrenergic stimulation, thus modulating cardiac output during the fight-or-flight response. In the sarcoplasmic reticulum membrane, PLN binds to the sarco(endo)plasmic reticulum Ca2+ -ATPase (SERCA), keeping this enzyme's function within a narrow physiological window. PLN phosphorylation by cAMP-dependent protein kinase A or increase in Ca2+ concentration reverses the inhibitory effects through an unknown mechanism. Using oriented-sample solid-state NMR spectroscopy and replica-averaged NMR-restrained structural refinement, we reveal that phosphorylation of PLN's cytoplasmic regulatory domain signals the disruption of several inhibitory contacts at the transmembrane binding interface of the SERCA-PLN complex that are propagated to the enzyme's active site, augmenting Ca2+ transport. Our findings address long-standing questions about SERCA regulation, epitomizing a signal transduction mechanism operated by posttranslationally modified bitopic membrane proteins., Competing Interests: DW, UR, SW, EL, TG, MG, RC, DT, AD, GV No competing interests declared, (© 2021, Weber et al.)- Published
- 2021
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39. Author Correction: Defective internal allosteric network imparts dysfunctional ATP/substrate-binding cooperativity in oncogenic chimera of protein kinase A.
- Author
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Olivieri C, Walker C, Karamafrooz A, Wang Y, Manu VS, Porcelli F, Blumenthal DK, Thomas DD, Bernlohr DA, Simon SM, Taylor SS, and Veglia G
- Published
- 2021
- Full Text
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40. Allostery governs Cdk2 activation and differential recognition of CDK inhibitors.
- Author
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Majumdar A, Burban DJ, Muretta JM, Thompson AR, Engel TA, Rasmussen DM, Subrahmanian MV, Veglia G, Thomas DD, and Levinson NM
- Subjects
- Allosteric Site genetics, Cell Cycle physiology, Cell Cycle Proteins metabolism, Cyclin A metabolism, Cyclin-Dependent Kinase 2 antagonists & inhibitors, Cyclin-Dependent Kinases antagonists & inhibitors, Cyclin-Dependent Kinases metabolism, Cyclins metabolism, Humans, Microtubule-Associated Proteins metabolism, Models, Biological, Phosphorylation physiology, Protein Kinase Inhibitors metabolism, Protein Kinase Inhibitors pharmacology, Proto-Oncogene Proteins metabolism, Tumor Suppressor Proteins metabolism, Allosteric Regulation physiology, Cyclin-Dependent Kinase 2 metabolism
- Abstract
Cyclin-dependent kinases (CDKs) are the master regulators of the eukaryotic cell cycle. To become activated, CDKs require both regulatory phosphorylation and binding of a cognate cyclin subunit. We studied the activation process of the G1/S kinase Cdk2 in solution and developed a thermodynamic model that describes the allosteric coupling between regulatory phosphorylation, cyclin binding and inhibitor binding. The results explain why monomeric Cdk2 lacks activity despite sampling an active-like state, reveal that regulatory phosphorylation enhances allosteric coupling with the cyclin subunit and show that this coupling underlies differential recognition of Cdk2 and Cdk4 inhibitors. We identify an allosteric hub that has diverged between Cdk2 and Cdk4 and show that this hub controls the strength of allosteric coupling. The altered allosteric wiring of Cdk4 leads to compromised activity toward generic peptide substrates and comparative specialization toward its primary substrate retinoblastoma (RB).
- Published
- 2021
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41. Defective internal allosteric network imparts dysfunctional ATP/substrate-binding cooperativity in oncogenic chimera of protein kinase A.
- Author
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Olivieri C, Walker C, Karamafrooz A, Wang Y, Manu VS, Porcelli F, Blumenthal DK, Thomas DD, Bernlohr DA, Simon SM, Taylor SS, and Veglia G
- Subjects
- Allosteric Regulation, Binding Sites, Catalytic Domain, Cyclic AMP metabolism, Cyclic AMP-Dependent Protein Kinase Catalytic Subunits genetics, HSP40 Heat-Shock Proteins genetics, Humans, Ligands, Molecular Dynamics Simulation, Peptide Fragments genetics, Phosphorylation, Protein Binding, Recombinant Fusion Proteins metabolism, Adenosine Triphosphate metabolism, Cyclic AMP-Dependent Protein Kinase Catalytic Subunits metabolism, HSP40 Heat-Shock Proteins metabolism, Peptide Fragments metabolism
- Abstract
An aberrant fusion of the DNAJB1 and PRKACA genes generates a chimeric protein kinase (PKA-C
DNAJB1 ) in which the J-domain of the heat shock protein 40 is fused to the catalytic α subunit of cAMP-dependent protein kinase A (PKA-C). Deceivingly, this chimeric construct appears to be fully functional, as it phosphorylates canonical substrates, forms holoenzymes, responds to cAMP activation, and recognizes the endogenous inhibitor PKI. Nonetheless, PKA-CDNAJB1 has been recognized as the primary driver of fibrolamellar hepatocellular carcinoma and is implicated in other neoplasms for which the molecular mechanisms remain elusive. Here we determined the chimera's allosteric response to nucleotide and pseudo-substrate binding. We found that the fusion of the dynamic J-domain to PKA-C disrupts the internal allosteric network, causing dramatic attenuation of the nucleotide/PKI binding cooperativity. Our findings suggest that the reduced allosteric cooperativity exhibited by PKA-CDNAJB1 alters specific recognitions and interactions between substrates and regulatory partners contributing to dysregulation.- Published
- 2021
- Full Text
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42. Vorinostat exhibits anticancer effects in triple-negative breast cancer cells by preventing nitric oxide-driven histone deacetylation.
- Author
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Palczewski MB, Kuschman HP, Bovee R, Hickok JR, and Thomas DD
- Subjects
- Acetylation drug effects, Antineoplastic Agents chemistry, Cell Line, Tumor, Cell Proliferation drug effects, Cell Survival drug effects, Dose-Response Relationship, Drug, Drug Screening Assays, Antitumor, Histone Deacetylase Inhibitors chemistry, Humans, Triple Negative Breast Neoplasms metabolism, Triple Negative Breast Neoplasms pathology, Vorinostat chemistry, Antineoplastic Agents pharmacology, Histone Deacetylase Inhibitors pharmacology, Histones metabolism, Nitric Oxide metabolism, Triple Negative Breast Neoplasms drug therapy, Vorinostat pharmacology
- Abstract
Triple-negative breast cancers (TNBC) that produce nitric oxide (NO) are more aggressive, and the expression of the inducible form of nitric oxide synthase (NOS2) is a negative prognostic indicator. In these studies, we set out to investigate potential therapeutic strategies to counter the tumor-permissive properties of NO. We found that exposure to NO increased proliferation of TNBC cells and that treatment with the histone deacetylase inhibitor Vorinostat (SAHA) prevented this proliferation. When histone acetylation was measured in response to NO and/or SAHA, NO significantly decreased acetylation on histone 3 lysine 9 (H3K9ac) and SAHA increased H3K9ac. If NO and SAHA were sequentially administered to cells (in either order), an increase in acetylation was observed in all cases. Mechanistic studies suggest that the "deacetylase" activity of NO does not involve S -nitrosothiols or soluble guanylyl cyclase activation. The observed decrease in histone acetylation by NO required the interaction of NO with cellular iron pools and may be an overriding effect of NO-mediated increases in histone methylation at the same lysine residues. Our data revealed a novel pathway interaction of Vorinostat and provides new insight in therapeutic strategy for aggressive TNBCs., (© 2021 Walter de Gruyter GmbH, Berlin/Boston.)
- Published
- 2021
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43. Novel drug discovery platform for spinocerebellar ataxia, using fluorescence technology targeting β-III-spectrin.
- Author
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Rebbeck RT, Andrick AK, Denha SA, Svensson B, Guhathakurta P, Thomas DD, Hays TS, and Avery AW
- Subjects
- Actins chemistry, Actins genetics, Fluorescence Resonance Energy Transfer, Gene Expression, Green Fluorescent Proteins genetics, Green Fluorescent Proteins metabolism, HEK293 Cells, Humans, Kinetics, Luminescent Proteins genetics, Luminescent Proteins metabolism, Marine Toxins pharmacology, Models, Biological, Models, Molecular, Mutation, Neuroprotective Agents pharmacology, Protein Binding drug effects, Protein Conformation, alpha-Helical, Protein Conformation, beta-Strand, Protein Interaction Domains and Motifs, Recombinant Fusion Proteins chemistry, Recombinant Fusion Proteins genetics, Reproducibility of Results, Spectrin chemistry, Spectrin genetics, Spinocerebellar Ataxias genetics, Spinocerebellar Ataxias metabolism, Spinocerebellar Ataxias pathology, Red Fluorescent Protein, Actins metabolism, Binding Sites drug effects, High-Throughput Screening Assays, Recombinant Fusion Proteins metabolism, Spectrin metabolism
- Abstract
Numerous diseases are linked to mutations in the actin-binding domains (ABDs) of conserved cytoskeletal proteins, including β-III-spectrin, α-actinin, filamin, and dystrophin. A β-III-spectrin ABD mutation (L253P) linked to spinocerebellar ataxia type 5 (SCA5) causes a dramatic increase in actin binding. Reducing actin binding of L253P is thus a potential therapeutic approach for SCA5 pathogenesis. Here, we validate a high-throughput screening (HTS) assay to discover potential disrupters of the interaction between the mutant β-III-spectrin ABD and actin in live cells. This assay monitors FRET between fluorescent proteins fused to the mutant ABD and the actin-binding peptide Lifeact, in HEK293-6E cells. Using a specific and high-affinity actin-binding tool compound, swinholide A, we demonstrate HTS compatibility with an excellent Z'-factor of 0.67 ± 0.03. Screening a library of 1280 pharmacologically active compounds in 1536-well plates to determine assay robustness, we demonstrate high reproducibility across plates and across days. We identified nine Hits that reduced FRET between Lifeact and ABD. Four of those Hits were found to reduce Lifeact cosedimentation with actin, thus establishing the potential of our assay for detection of actin-binding modulators. Concurrent to our primary FRET assay, we also developed a high-throughput compatible counter screen to remove undesirable FRET Hits. Using the FRET Hits, we show that our counter screen is sensitive to undesirable compounds that cause cell toxicity or ABD aggregation. Overall, our FRET-based HTS platform sets the stage to screen large compound libraries for modulators of β-III-spectrin, or disease-linked spectrin-related proteins, for therapeutic development., Competing Interests: Conflict of interest D. D. T. holds equity in and serves as an executive officer for Photonic Pharma LLC. These relationships have been reviewed and managed by the University of Minnesota. Photonic Pharma had no role in this study, except to provide some instrumentation, as stated in Experimental Procedures. R. T. R., A. K. A., S. A. D., B. S., P. G., T. S. H., and A. W. A. have no conflict of interest to disclose., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2021
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44. Fluorescence-Based TNFR1 Biosensor for Monitoring Receptor Structural and Conformational Dynamics and Discovery of Small Molecule Modulators.
- Author
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Lo CH, Schaaf TM, Thomas DD, and Sachs JN
- Subjects
- Cell Line, Computational Biology methods, Drug Discovery methods, Fluorescence Resonance Energy Transfer, Fluorescent Antibody Technique, Gene Expression, Genes, Reporter, High-Throughput Nucleotide Sequencing, Humans, Ligands, Microscopy, Fluorescence, Protein Binding, Receptors, Tumor Necrosis Factor, Type I genetics, Receptors, Tumor Necrosis Factor, Type I metabolism, Small Molecule Libraries, Software, Structure-Activity Relationship, Biosensing Techniques, Molecular Conformation, Molecular Docking Simulation, Molecular Dynamics Simulation, Receptors, Tumor Necrosis Factor, Type I chemistry
- Abstract
Inhibition of tumor necrosis factor receptor 1 (TNFR1) is a billion-dollar industry for treatment of autoimmune and inflammatory diseases. As current therapeutics of anti-TNF leads to dangerous side effects due to global inhibition of the ligand, receptor-specific inhibition of TNFR1 signaling is an intensely pursued strategy. To monitor directly the structural changes of the receptor in living cells, we engineered a fluorescence resonance energy transfer (FRET) biosensor by fusing green and red fluorescent proteins to TNFR1. Expression of the FRET biosensor in living cells allows for detection of receptor-receptor interactions and receptor structural dynamics. Using the TNFR1 FRET biosensor, in conjunction with a high-precision and high-throughput fluorescence lifetime detection technology, we developed a time-resolved FRET-based high-throughput screening platform to discover small molecules that directly target and modulate TNFR1 functions. Using this method in screening multiple pharmaceutical libraries, we have discovered a competitive inhibitor that disrupts receptor-receptor interactions, and allosteric modulators that alter the structural states of the receptor. This enables scientists to conduct high-throughput screening through a biophysical approach, with relevance to compound perturbation of receptor structure, for the discovery of novel lead compounds with high specificity for modulation of TNFR1 signaling.
- Published
- 2021
- Full Text
- View/download PDF
45. Mechanistic analysis of actin-binding compounds that affect the kinetics of cardiac myosin-actin interaction.
- Author
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Roopnarine O and Thomas DD
- Subjects
- Actins drug effects, Adenosine Triphosphatases drug effects, Adenosine Triphosphatases metabolism, Animals, Cardiac Myosins drug effects, Cardiac Myosins physiology, Cattle, Fluorescence, High-Throughput Screening Assays methods, Kinetics, Muscle Contraction physiology, Myosin Subfragments drug effects, Myosin Subfragments metabolism, Myosins drug effects, Myosins metabolism, Physics, Protein Binding, Pyrenes chemistry, Rabbits, Small Molecule Libraries pharmacology, Actins chemistry, Actins metabolism, Cardiac Myosins metabolism
- Abstract
Actin-myosin mediated contractile forces are crucial for many cellular functions, including cell motility, cytokinesis, and muscle contraction. We determined the effects of ten actin-binding compounds on the interaction of cardiac myosin subfragment 1 (S1) with pyrene-labeled F-actin (PFA). These compounds, previously identified from a small-molecule high-throughput screen (HTS), perturb the structural dynamics of actin and the steady-state actin-activated myosin ATPase activity. However, the mechanisms underpinning these perturbations remain unclear. Here we further characterize them by measuring their effects on PFA fluorescence, which is decreased specifically by the strong binding of myosin to actin. We measured these effects under equilibrium and steady-state conditions, and under transient conditions, in stopped-flow experiments following addition of ATP to S1-bound PFA. We observed that these compounds affect early steps of the myosin ATPase cycle to different extents. They increased the association equilibrium constant K
1 for the formation of the strongly bound collision complex, indicating increased ATP affinity for actin-bound myosin, and decreased the rate constant k+2 for subsequent isomerization to the weakly bound ternary complex, thus slowing the strong-to-weak transition that actin-myosin interaction undergoes early in the ATPase cycle. The compounds' effects on actin structure allosterically inhibit the kinetics of the actin-myosin interaction in ways that may be desirable for treatment of hypercontractile forms of cardiomyopathy. This work helps to elucidate the mechanisms of action for these compounds, several of which are currently used therapeutically, and sets the stage for future HTS campaigns that aim to discover new drugs for treatment of heart failure., Competing Interests: Conflict of interest D. D. T. holds equity in, and serves as President of, Photonic Pharma LLC. This relationship has been reviewed and managed by the University of Minnesota. Photonic Pharma had no role in this study. The authors declare no conflicts of interest in regard to this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)- Published
- 2021
- Full Text
- View/download PDF
46. The transmembrane peptide DWORF activates SERCA2a via dual mechanisms.
- Author
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Li A, Yuen SL, Stroik DR, Kleinboehl E, Cornea RL, and Thomas DD
- Subjects
- Calcium metabolism, Calcium-Binding Proteins metabolism, Cell Line, HEK293 Cells, Heart Failure metabolism, Humans, Myocardium metabolism, Myocytes, Cardiac metabolism, Peptides physiology, Sarcoplasmic Reticulum metabolism, Sarcoplasmic Reticulum Calcium-Transporting ATPases physiology, Peptides metabolism, Sarcoplasmic Reticulum Calcium-Transporting ATPases metabolism
- Abstract
The Ca-ATPase isoform 2a (SERCA2a) pumps cytosolic Ca
2+ into the sarcoplasmic reticulum (SR) of cardiac myocytes, enabling muscle relaxation during diastole. Abnormally high cytosolic [Ca2+ ] is a central factor in heart failure, suggesting that augmentation of SERCA2a Ca2+ transport activity could be a promising therapeutic approach. SERCA2a is inhibited by the protein phospholamban (PLB), and a novel transmembrane peptide, dwarf open reading frame (DWORF), is proposed to enhance SR Ca2+ uptake and myocyte contractility by displacing PLB from binding to SERCA2a. However, establishing DWORF's precise physiological role requires further investigation. In the present study, we developed cell-based FRET biosensor systems that can report on protein-protein interactions and structural changes in SERCA2a complexes with PLB and/or DWORF. To test the hypothesis that DWORF competes with PLB to occupy the SERCA2a-binding site, we transiently transfected DWORF into a stable HEK cell line expressing SERCA2a labeled with a FRET donor and PLB labeled with a FRET acceptor. We observed a significant decrease in FRET efficiency, consistent with a decrease in the fraction of SERCA2a bound to PLB. Surprisingly, we also found that DWORF also activates SERCA's enzymatic activity directly in the absence of PLB at subsaturating calcium levels. Using site-directed mutagenesis, we generated DWORF variants that do not activate SERCA, thus identifying residues P15 and W22 as necessary for functional SERCA2a-DWORF interactions. This work advances our mechanistic understanding of the regulation of SERCA2a by small transmembrane proteins and sets the stage for future therapeutic development in heart failure research., Competing Interests: Conflict of interest D. D. T. and R. L. C. hold equity in, and serve as executive officers of, Photonic Pharma LLC. These relationships have been reviewed and managed by the University of Minnesota in accordance with its conflict-of-interest policies. Photonic Pharma had no role in this study. The authors declare no conflicts of interest with regard to this article., (Published by Elsevier Inc.)- Published
- 2021
- Full Text
- View/download PDF
47. FRET and optical trapping reveal mechanisms of actin activation of the power stroke and phosphate release in myosin V.
- Author
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Gunther LK, Rohde JA, Tang W, Cirilo JA Jr, Marang CP, Scott BD, Thomas DD, Debold EP, and Yengo CM
- Subjects
- Adenosine Triphosphatases metabolism, Adenosine Triphosphate metabolism, Animals, Chickens, Kinetics, Models, Molecular, Mutation, Myosin Type V genetics, Actins metabolism, Fluorescence Resonance Energy Transfer methods, Myosin Type V metabolism, Optical Imaging methods, Phosphates metabolism
- Abstract
Myosins generate force and motion by precisely coordinating their mechanical and chemical cycles, but the nature and timing of this coordination remains controversial. We utilized a FRET approach to examine the kinetics of structural changes in the force-generating lever arm in myosin V. We directly compared the FRET results with single-molecule mechanical events examined by optical trapping. We introduced a mutation (S217A) in the conserved switch I region of the active site to examine how myosin couples structural changes in the actin- and nucleotide-binding regions with force generation. Specifically, S217A enhanced the maximum rate of lever arm priming (recovery stroke) while slowing ATP hydrolysis, demonstrating that it uncouples these two steps. We determined that the mutation dramatically slows both actin-induced rotation of the lever arm (power stroke) and phosphate release (≥10-fold), whereas our simulations suggest that the maximum rate of both steps is unchanged by the mutation. Time-resolved FRET revealed that the structure of the pre- and post-power stroke conformations and mole fractions of these conformations were not altered by the mutation. Optical trapping results demonstrated that S217A does not dramatically alter unitary displacements or slow the working stroke rate constant, consistent with the mutation disrupting an actin-induced conformational change prior to the power stroke. We propose that communication between the actin- and nucleotide-binding regions of myosin assures a proper actin-binding interface and active site have formed before producing a power stroke. Variability in this coupling is likely crucial for mediating motor-based functions such as muscle contraction and intracellular transport., (Copyright © 2020 © 2020 Gunther et al. Published by Elsevier Inc. All rights reserved.)
- Published
- 2020
- Full Text
- View/download PDF
48. Met125 is essential for maintaining the structural integrity of calmodulin's C-terminal domain.
- Author
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Nelson SED, Weber DK, Rebbeck RT, Cornea RL, Veglia G, and Thomas DD
- Subjects
- Binding Sites, Calcium metabolism, Circular Dichroism, Humans, Magnetic Resonance Spectroscopy, Methionine metabolism, Mutation genetics, Oxidation-Reduction, Protein Binding, Ryanodine Receptor Calcium Release Channel chemistry, Ryanodine Receptor Calcium Release Channel metabolism, Calmodulin chemistry, Calmodulin metabolism
- Abstract
We have used NMR and circular dichroism spectroscopy to investigate the structural and dynamic effects of oxidation on calmodulin (CaM), using peroxide and the Met to Gln oximimetic mutations. CaM is a Ca
2+ -sensitive regulatory protein that interacts with numerous targets. Due to its high methionine content, CaM is highly susceptible to oxidation by reactive oxygen species under conditions of cell stress and age-related muscle degeneration. CaM oxidation alters regulation of a host of CaM's protein targets, emphasizing the importance of understanding the mechanism of CaM oxidation in muscle degeneration and overall physiology. It has been shown that the M125Q CaM mutant can mimic the functional effects of methionine oxidation on CaM's regulation of the calcium release channel, ryanodine receptor (RyR). We report here that the M125Q mutation causes a localized unfolding of the C-terminal lobe of CaM, preventing the formation of a hydrophobic cluster of residues near the EF-hand Ca2+ binding sites. NMR analysis of CaM oxidation by peroxide offers further insights into the susceptibility of CaM's Met residues to oxidation and the resulting structural effects. These results further resolve oxidation-driven structural perturbation of CaM, with implications for RyR regulation and the decay of muscle function in aging.- Published
- 2020
- Full Text
- View/download PDF
49. Looking for the Outsider.
- Author
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Vodopivec DM, Thomas DD, Palermo NE, Steenkamp DW, and Lee SL
- Subjects
- Adult, Choristoma surgery, Fractures, Bone diagnostic imaging, Humans, Hypercalcemia etiology, Hyperparathyroidism, Primary therapy, Hypocalcemia drug therapy, Hypocalcemia etiology, Male, Parathyroid Glands diagnostic imaging, Parathyroid Neoplasms blood, Parathyroid Neoplasms complications, Parathyroid Neoplasms surgery, Positron-Emission Tomography, Shoulder diagnostic imaging, Technetium Tc 99m Sestamibi, Tomography, X-Ray Computed, Clavicle injuries, Fractures, Bone etiology, Hyperparathyroidism, Primary etiology, Parathyroid Glands surgery, Parathyroid Hormone blood, Parathyroid Neoplasms diagnosis
- Published
- 2020
- Full Text
- View/download PDF
50. Purification of sarcoplasmic reticulum vesicles from horse gluteal muscle.
- Author
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Autry JM, Karim CB, Cocco M, Carlson SF, Thomas DD, and Valberg SJ
- Subjects
- Animals, Calcium metabolism, Centrifugation, Electrophoresis, Agar Gel, Extracellular Vesicles metabolism, Glycogen Phosphorylase metabolism, Horses, Protein Isoforms metabolism, Sarcoplasmic Reticulum metabolism, Sarcoplasmic Reticulum Calcium-Transporting ATPases metabolism, Extracellular Vesicles chemistry, Muscle, Skeletal metabolism
- Abstract
We have analyzed protein expression and enzyme activity of the sarcoplasmic reticulum Ca
2+ -transporting ATPase (SERCA) in horse gluteal muscle. Horses exhibit a high incidence of recurrent exertional rhabdomyolysis, with myosolic Ca2+ proposed, but yet to be established, as the underlying cause. To better assess Ca2+ regulatory mechanisms, we developed an improved protocol for isolating sarcoplasmic reticulum (SR) vesicles from horse skeletal muscle, based on mechanical homogenization and optimized parameters for differential centrifugation. Immunoblotting identified the peak subcellular fraction containing the SERCA1 protein (fast-twitch isoform). Gel analysis using the Stains-all dye demonstrated that calsequestrin (CASQ) and phospholipids are highly enriched in the SERCA-containing subcellular fraction isolated from horse gluteus. Immunoblotting also demonstrated that these horse SR vesicles show low content of glycogen phosphorylase (GP), which is likely an abundant contaminating protein of traditional horse SR preps. The maximal Ca2+ -activated ATPase activity (Vmax ) of SERCA in horse SR vesicles isolated using this protocol is 5‒25-fold greater than previously-reported SERCA activity in SR preps from horse skeletal muscle. We propose that this new protocol for isolating SR vesicles will be useful for determining enzymatic parameters of horse SERCA with high fidelity, plus assessing regulatory effect of SERCA peptide subunit(s) expressed in horse muscle., (Copyright © 2020 Elsevier Inc. All rights reserved.)- Published
- 2020
- Full Text
- View/download PDF
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