Seth L. Robia

Loyola University Chicago, Chicago, Illinois, United States

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Publications (59)239.4 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Muscle contractility is regulated by a network of many proteins. In cardiomyocytes, the sarco(endo)plasmic reticulum Ca2+ -ATPase, SERCA, and its regulatory protein, phospholamban are responsible for ~70% of Ca2+ reuptake into the SR. While unphosphorylated, PLN inhibits SERCA by lowering its apparent Ca2+ affinity. Upon phosphorylation by PKA at Ser16, PLN inhibition is relieved. This tightly regulated interaction can be easily disrupted by mutation or changes in protein level, leading to heart disease. Thus, understanding the molecular interactions between SERCA/PLN and possible regulators is essential. Here, we report that ssDNA binds the cytoplasmic domain of PLN with low nanomolar dissociation constants, relieving inhibition of SERCA. The relief of inhibition is length dependent, while affinity is constant for oligonucleotides longer than 10 bases. Solution and solid-state NMR experiments have provided residue specific information that ssDNA targets the cytoplasmic domain of PLN and does not affect SERCA in the absence of PLN. In-cell FRET, and NMR experiments determined that addition of ssDNA does not dissociate PLN from SERCA. SERCA/PLN has become a highly targeted complex for development of small molecule regulators because of its prevalence in many cardiovascular diseases. While some therapies are currently being investigated, none have proceeded past clinical trials. These results provide a promising avenue for development of novel regulators of the SERCA/PLN complex. Additionally, they support previous findings from our group detailing the intricate balance that is necessary for proper cardiac function.
    Biophysical Society National Meeting 2015; 02/2015
  • Neha Abrol, Pieter P de Tombe, Seth L Robia
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    ABSTRACT: A naturally-occurring Arg9Cys mutation (R9C) of phospholamban (PLB) triggers cardiomyopathy and premature death by altering regulation of sarco/endoplasmic reticulum calcium-ATPase (SERCA). The goal of this study was to investigate the acute physiological consequences of R9C-PLB mutation on cardiomyocyte calcium kinetics and contractility. We measured the physiological consequences of R9C-PLB mutation on calcium transients and sarcomere shortening in adult cardiomyocytes. In contrast to studies of chronic R9C-PLB expression in transgenic mice, we found that acute expression of R9C-PLB exerts a positively inotropic and lusitropic effect in cardiomyocytes. Importantly, R9C-PLB exhibited blunted sensitivity to frequency potentiation and β-adrenergic stimulation, two major physiological mechanisms for the regulation of cardiac performance. To identify the molecular mechanism of R9C pathology, we quantified the effect of R9C on PLB oligomerization and PLB-SERCA binding. Fluorescence resonance energy transfer (FRET) measurements in live cells revealed that R9C-PLB exhibited an increased propensity for oligomerization, and this was further increased by oxidative stress. The R9C also decreased PLB binding to SERCA, and altered the structure of the PLB-SERCA regulatory complex. The structure change after oxidative modification of R9C-PLB is similar to that observed after PLB phosphorylation. We conclude that R9C mutation of PLB decreases SERCA inhibition by decreasing the amount of the regulatory complex and altering its conformation. This has an acute inotropic/lusitropic effect, but yields negative consequences of impaired frequency potentiation and blunted β-adrenergic responsiveness. We envision a self-reinforcing mechanism beginning with phosphomimetic R9C-PLB oxidation and loss of SERCA inhibition, leading to impaired calcium regulation and heart failure. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    The Journal of biological chemistry. 01/2015;
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    ABSTRACT: TRIM5α proteins are a potent barrier to the cross-species transmission of retroviruses. TRIM5α proteins exhibit an ability to self-associate at many levels, ultimately leading to the formation of protein assemblies with hexagonal symmetry in vitro and cytoplasmic assemblies when expressed in cells. However, the role of these assemblies in restriction, the determinants that mediate their formation, and the organization of TRIM5α molecules within these assemblies have remained unclear. Here we show that α-helical elements within the Linker2 region of rhesus macaque TRIM5α govern the ability to form cytoplasmic assemblies in cells and restrict HIV-1 infection. Mutations that reduce α-helix formation by the Linker2 region disrupt assembly and restriction. More importantly, mutations that enhance the α-helical content of the Linker2 region, relative to the wild-type protein, also exhibit an increased ability to form cytoplasmic assemblies and restrict HIV-1 infection. Molecular modeling of the TRIM5α dimer suggests a model in which α-helical elements within the Linker2 region dock to α-helices of the coiled-coil domain, likely establishing proper orientation and spacing of protein domains necessary for assembly and restriction. Collectively, these studies provide critical insight into the determinants governing TRIM5α assembly and restriction and demonstrate that the antiviral potency of TRIM5α proteins can be significantly increased without altering the affinity of SPRY/capsid binding. IMPORTANCE: Many members of the tripartite motif (TRIM) family of proteins act as restriction factors that directly inhibit viral infection and activate innate immune signaling pathways. Another common feature of TRIM proteins is the ability to form protein assemblies in the nucleus or the cytoplasm. However, the determinants in TRIM proteins required for assembly and the degree to which assembly affects TRIM protein function have been poorly understood. Here we show that alpha helices in the Linker2 (L2) region of rhesus TRIM5α govern assembly and restriction of HIV-1 infection. Helix-disrupting mutations disrupt the assembly and restriction of HIV-1, while helix-stabilizing mutations enhance assembly and restriction relative to the wild-type protein. Circular dichroism analysis suggests that that the formation of this helical structure is supported by intermolecular interactions with the coiled-coil (CC) domain in the CCL2 dimer. These studies reveal a novel mechanism by which the antiviral activity of TRIM5α proteins can be regulated and provide detailed insight into the assembly determinants of TRIM family proteins.
    Journal of Virology 08/2014; 88(16):8911-23. · 4.65 Impact Factor
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    ABSTRACT: To determine the structural and regulatory role of the C-terminal residues of phospholamban (PLB) in the membranes of living cells, we fused fluorescent protein tags to PLB and sarco/endoplasmic reticulum calcium A TPase (SERCA). Alanine substitution of PLB C-terminal residues significantly altered fluorescence resonance energy transfer (FRET) from PLB to PLB and SERCA to PLB, suggesting a change in quaternary conformation of PLB pentamer and SERCA-PLB regulatory complex. V al to Ala substitution at position 49 (V49A) had particularly large effects on PLB pentamer structure and PLB- SERCA regulatory complex conformation, increasing and decreasing probe separation distance, respectively. We also quantified a decrease in oligomerization affinity, an increase in binding affinity of V49A-PLB for SERCA, and gain of inhibitory function as quantified by calcium-dependent ATPase activity. Notably, deletion of only a few C-terminal residues resulted in significant loss of PLB membrane anchoring and mislocalization to the cytoplasm and nucleus. C-terminal truncations also resulted in progressive loss of PLB-PLB FRET, due to a decrease in the apparent affinity of PLB oligomerization. We quantified a similar decrease in the binding affinity of truncated PLB for SERCA, and loss of inhibitory potency. However, despite decreased SERCA-PLB binding, intermolecular FRET for V al49-stop (V49X) truncation mutant was paradoxically increased as a result of a 11.3 angstrom decrease in the distance between donor and acceptor fluorophores. We conclude that PLB C-terminal residues are critical for localization, oligomerization, and regulatory function. In particular, the PLB C-terminus is an important determinant of the quaternary structure of the SERCA regulatory complex.
    Journal of Biological Chemistry 07/2014; · 4.60 Impact Factor
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    ABSTRACT: We have used a "two-color" SERCA (sarco/endoplasmic reticulum calcium ATPase) biosensor and a unique high-throughput fluorescence lifetime plate reader (FLT-PR) to develop a high-precision live-cell assay designed to screen for small molecules that perturb SERCA structure. A SERCA construct, in which red fluorescent protein (RFP) was fused to the N terminus and green fluorescent protein (GFP) to an interior loop, was stably expressed in an HEK cell line that grows in monolayer or suspension. Fluorescence resonance energy transfer (FRET) from GFP to RFP was measured in the FLT-PR, which increases precision 30-fold over intensity-based plate readers without sacrificing throughput. FRET was highly sensitive to known SERCA modulators. We screened a small chemical library and identified 10 compounds that significantly affected two-color SERCA FLT. Three of these compounds reproducibly lowered FRET and inhibited SERCA in a dose-dependent manner. This assay is ready for large-scale HTS campaigns and is adaptable to many other targets.
    Journal of Biomolecular Screening 02/2014; 19(2):215-22. · 2.01 Impact Factor
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    ABSTRACT: The sarcoendoplasmic reticulum calcium ATPase (SERCA) plays a key role in cardiac calcium handling and is considered a high-value target for the treatment of heart failure. SERCA undergoes conformational changes as it harnesses the chemical energy of ATP for active transport. X-ray crystallography has provided insight into SERCA structural substates, but it is not known how well these static snapshots describe in vivo conformational dynamics. The goals of this work were to quantify the direction and magnitude of SERCA motions as the pump performs work in live cardiac myocytes, and to identify structural determinants of SERCA regulation by phospholamban. We measured intramolecular fluorescence resonance energy transfer (FRET) between fluorescent proteins fused to SERCA cytoplasmic domains. We detected four discrete structural substates for SERCA expressed in cardiac muscle cells. The relative populations of these discrete states oscillated with electrical pacing. Low FRET states were most populated in low Ca (diastole), and were indicative of an open, disordered structure for SERCA in the E2 (Ca-free) enzymatic substate. High FRET states increased with Ca (systole), suggesting rigidly closed conformations for the E1 (Ca-bound) enzymatic substates. Notably, a special compact E1 state was observed after treatment with β-adrenergic agonist or with coexpression of phosphomimetic mutants of phospholamban. The data suggest that SERCA calcium binding induces the pump to undergo a transition from an open, dynamic conformation to a closed, ordered structure. Phosphorylated phospholamban stabilizes a unique conformation of SERCA that is characterized by a compact architecture.
    Biophysical Journal 10/2013; 105(8):1812-21. · 3.83 Impact Factor
  • Biophysical Journal 01/2013; 104(2):153-. · 3.83 Impact Factor
  • Biophysical Journal 01/2013; 104(2):60-. · 3.83 Impact Factor
  • Biophysical Journal 01/2013; 104(2):17a–18a. · 3.83 Impact Factor
  • Nikolai Smolin, Seth L. Robia
    Biophysical Journal 01/2013; 104(2):60-. · 3.83 Impact Factor
  • Biophysical Journal 01/2013; 104(2):71-. · 3.83 Impact Factor
  • Biophysical Journal 01/2013; 104(2):71-. · 3.83 Impact Factor
  • Biophysical Journal 01/2013; 104(2):299-. · 3.83 Impact Factor
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    ABSTRACT: The sarco(endo)plasmic reticulum calcium ATPase (SERCA) undergoes conformational changes while transporting calcium, but the details of the domain motions are still unclear. The objective of the present study was to measure distances between the cytoplasmic domains of SERCA2a in order to reveal the magnitude and direction of conformational changes. Using fluorescence microscopy of live cells, we measured intramolecular fluorescence resonance energy transfer (FRET) from a donor fluorescent protein fused to the SERCA N-terminus to an acceptor fluorescent protein fused to either the N-, P-, or transmembrane domain. The "2-color" SERCA constructs were catalytically active as indicated by ATPase activity in vitro and Ca uptake in live cells. All constructs exhibited dynamic FRET changes in response to the pump ligands calcium and thapsigargin (Tg). These FRET changes were quantified as an index of SERCA conformational changes. Intramolecular FRET decreased with Tg for the two N-domain fusion sites (at residue 509 or 576), while the P- (residue 661) and TM-domain (C-terminus) fusions showed increased FRET with Tg. The magnitude of the Tg-dependent conformational change was not decreased by coexpression of phospholamban (PLB), nor did PLB slow the kinetics of Tg binding. FRET in ionophore-permeabilized cells was lower in EGTA than in saturating calcium for all constructs, indicating a decrease in domain separation distance with the structural transition from E2 (Ca-free) to E1 (Ca-bound). The data suggest closure of the cytoplasmic headpiece with Ca-binding. The present results provide insight into the structural dynamics of the Ca-ATPase. In addition, the 2-color SERCA constructs developed for this study may be useful for evaluating candidate small molecule regulators of Ca uptake activity.
    PLoS ONE 07/2012; 7(7):e40369. · 3.53 Impact Factor
  • Biophysical Journal 01/2012; 102(3):709-. · 3.83 Impact Factor
  • Sandeep Pallikkuth, Seth L. Robia
    Biophysical Journal 01/2012; 102(3):709-. · 3.83 Impact Factor
  • Philip A. Bidwell, Seth L. Robia
    Biophysical Journal 01/2012; 102(3):710-. · 3.83 Impact Factor
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    ABSTRACT: Dysferlin was previously identified as a key player in muscle membrane repair and its deficiency leads to the development of muscular dystrophy and cardiomyopathy. However, little is known about the oligomerization of this protein in the plasma membrane. Here we report for the first time that dysferlin forms a dimer in vitro and in living adult skeletal muscle fibers isolated from mice. Endogenous dysferlin from rabbit skeletal muscle exists primarily as a ∼460 kDa species in detergent-solubilized muscle homogenate, as shown by sucrose gradient fractionation, gel filtration and cross-linking assays. Fluorescent protein (YFP) labeled human dysferlin forms a dimer in vitro, as demonstrated by fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) analyses. Dysferlin also dimerizes in living cells, as probed by fluorescence resonance energy transfer (FRET). Domain mapping FRET experiments showed that dysferlin dimerization is mediated by its transmembrane domain and by multiple C2 domains. However, C2A did not significantly contribute to dimerization; notably, this is the only C2 domain in dysferlin known to engage in a Ca-dependent interaction with cell membranes. Taken together, the data suggest that Ca-insensitive C2 domains mediate high affinity self-association of dysferlin in a parallel homodimer, leaving the Ca-sensitive C2A domain free to interact with membranes.
    PLoS ONE 11/2011; 6(11):e27884. · 3.53 Impact Factor
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    ABSTRACT: The TRIM5 proteins are cellular restriction factors that prevent retroviral infection in a species-specific manner. Multiple experiments indicate that restriction activity requires accessory host factors, including E2-enzymes. To better understand the mechanism of restriction, we conducted yeast-two hybrid screens to identify proteins that bind to two TRIM5 orthologues. The only cDNAs that scored on repeat testing with both TRIM5 orthologues were the proteasome subunit PSMC2 and ubiquitin. Using co-immunoprecipitation assays, we demonstrated an interaction between TRIM5α and PSMC2, as well as numerous other proteasome subunits. Fluorescence microscopy revealed co-localization of proteasomes and TRIM5α cytoplasmic bodies. Forster resonance energy transfer (FRET) analysis indicated that the interaction between TRIM5 and PSMC2 was direct. Previous imaging experiments demonstrated that, when cells are challenged with fluorescently-labeled HIV-1 virions, restrictive TRIM5α orthologues assemble cytoplasmic bodies around incoming virion particles. Following virus challenge, we observed localization of proteasome subunits to rhTRIM5α cytoplasmic bodies that contained fluorescently labeled HIV-1 virions. Taken together, the results presented here suggest that localization of the proteasome to TRIM5α cytoplasmic bodies makes an important contribution to TRIM5α-mediated restriction.
    Retrovirology 11/2011; 8:93. · 4.77 Impact Factor
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    ABSTRACT: Protein kinase D (PKD) is a nodal point in cardiac hypertrophic signaling. It triggers nuclear export of class II histone deacetylase (HDAC) and regulates transcription. Although this pathway is thought to be critical in cardiac hypertrophy and heart failure, little is known about spatiotemporal aspects of PKD activation at the myocyte level. Here, we demonstrate that in adult cardiomyocytes two important neurohumoral stimuli that induce hypertrophy, endothelin-1 (ET1) and phenylephrine (PE), trigger comparable global PKD activation and HDAC5 nuclear export, but via divergent spatiotemporal PKD signals. PE-induced HDAC5 export is entirely PKD-dependent, involving fleeting sarcolemmal PKD translocation (for activation) and very rapid subsequent nuclear import. In contrast, ET1 recruits and activates PKD that remains predominantly sarcolemmal. This explains why PE-induced nuclear HDAC5 export in myocytes is totally PKD-dependent, whereas ET1-induced HDAC5 export depends more prominently on InsP3 and CaMKII signaling. Thus α-adrenergic and ET-1 receptor signaling via PKD in adult myocytes feature dramatic differences in cellular localization and translocation in mediating hypertrophic signaling. This raises new opportunities for targeted therapeutic intervention into distinct limbs of this hypertrophic signaling pathway.
    Journal of Biological Chemistry 09/2011; 286(38):33390-33400. · 4.60 Impact Factor

Publication Stats

497 Citations
239.40 Total Impact Points

Institutions

  • 2007–2013
    • Loyola University Chicago
      • • Department of Cell and Molecular Physiology
      • • Physiology
      Chicago, Illinois, United States
  • 2011
    • University of Minnesota Twin Cities
      • Department of Biochemistry, Molecular Biology and Biophysics (CBS)
      Minneapolis, MN, United States
  • 2010–2011
    • Loyola University Medical Center
      • • Department of Physiology
      • • Cardiovascular Institute
      Maywood, IL, United States
  • 2009–2011
    • University of California, Davis
      • Department of Pharmacology
      Davis, CA, United States
  • 2005
    • University of Minnesota Duluth
      • Department of Chemistry and Biochemistry
      Duluth, MN, United States
  • 2001–2005
    • University of Wisconsin–Madison
      Madison, Wisconsin, United States