[Show abstract][Hide abstract] 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.
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. · 5.08 Impact Factor
[Show abstract][Hide abstract] 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; · 4.65 Impact Factor
[Show abstract][Hide abstract] 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. · 2.21 Impact Factor
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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 01/2012; 7(7):e40369. · 3.53 Impact Factor
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tyrosine-phosphorylated focal adhesion kinase (FAK) is required for the hypertrophic response of cardiomyocytes to growth factors and mechanical load, but the role of FAK serine phosphorylation in this process is unknown. The aims of the present study were to characterize FAK serine phosphorylation in cultured neonatal rat ventricular myocytes (NRVM), analyse its functional significance during hypertrophic signalling, and examine its potential role in the pathogenesis of human dilated cardiomyopathy (DCM).
Endothelin-1 (ET-1) and other hypertrophic factors induced a time- and dose-dependent increase in FAK-S910 phosphorylation. ET-1-induced FAK-S910 phosphorylation required ET(A)R-dependent activation of PKCδ and Src via parallel Raf-1 → MEK1/2 → ERK1/2 and MEK5 → ERK5 signalling pathways. Replication-deficient adenoviruses expressing wild-type (WT) FAK and a non-phosphorylatable, S910A-FAK mutant were then used to examine the functional significance of FAK-S910 phosphorylation. Unlike WT-FAK, S910A-FAK increased the half-life of GFP-tagged paxillin within costameres (as determined by total internal reflection fluorescence microscopy and fluorescence recovery after photobleaching) and increased the steady-state FAK-paxillin interaction (as determined by co-immunoprecipitation and western blotting). These alterations resulted in reduced NRVM sarcomere reorganization and cell spreading. Finally, we found that FAK was serine-phosphorylated at multiple sites in non-failing, human left ventricular tissue. FAK-S910 phosphorylation and ERK5 expression were dramatically reduced in patients undergoing heart transplantation for end-stage DCM.
FAK undergoes S910 phosphorylation via PKCδ and Src-dependent pathways that are important for cell spreading and sarcomere reorganization. Reduced FAK-S910 phosphorylation may contribute to sarcomere disorganization in DCM.
Cardiovascular Research 09/2011; 92(3):409-19. · 5.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Focal adhesion kinase-related nonkinase (FRNK), the C-terminal domain of focal adhesion kinase (FAK), is a tyrosine-phosphorylated, vascular smooth muscle cell (VSMC)-specific inhibitor of cell migration. FRNK inhibits both FAK and proline-rich tyrosine kinase 2 (PYK2) in cultured VSMCs, and both kinases may be involved in VSMC invasion during vascular remodeling.
Adenovirally mediated gene transfer of green fluorescent protein-tagged, wild-type (wt) FRNK into balloon-injured rat carotid arteries confirmed that FRNK overexpression inhibited both FAK and PYK2 phosphorylation and downstream signaling in vivo. To identify which kinase was involved in regulating VSMC invasion, adenovirally mediated expression of specific short hairpin RNAs was used to knock down FAK versus PYK2 in cultured VSMCs, but only FAK short hairpin RNA was effective in reducing VSMC invasion. The role of FRNK tyrosine phosphorylation was then examined using adenoviruses expressing nonphosphorylatable (Tyr168Phe-, Tyr232Phe-, and Tyr168,232Phe-) green fluorescent protein-FRNK mutants. wtFRNK and all FRNK mutants localized to FAs, but only Tyr168 phosphorylation was required for FRNK to inhibit invasion. Preventing Tyr168 phosphorylation also increased FRNK-paxillin interaction, as determined by coimmunoprecipitation, total internal reflection fluorescence microscopy, and fluorescence recovery after photobleaching. Furthermore, wtFRNK competed with FAK for binding to p130(Cas) (a critically important regulator of cell migration) and prevented its phosphorylation. However, Tyr168Phe-FRNK was unable to bind p130(Cas).
We propose a 3-stage mechanism for FRNK inhibition: focal adhesion targeting, Tyr168 phosphorylation, and competition with FAK for p130 binding and phosphorylation, which are all required for FRNK to inhibit VSMC invasion.
[Show abstract][Hide abstract] ABSTRACT: To investigate the mechanism of regulation of sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA) by phospholamban (PLB), we expressed Cerulean-SERCA and yellow fluorescent protein (YFP)-PLB in adult rabbit ventricular myocytes using adenovirus vectors. SERCA and PLB were localized in the sarcoplasmic reticulum and were mobile over multiple sarcomeres on a timescale of tens of seconds. We also observed robust fluorescence resonance energy transfer (FRET) from Cerulean-SERCA to YFP-PLB. Electrical pacing of cardiac myocytes elicited cytoplasmic Ca(2+) elevations, but these increases in Ca(2+) produced only modest changes in SERCA-PLB FRET. The data suggest that the regulatory complex is not disrupted by elevations of cytosolic calcium during cardiac contraction (systole). This conclusion was also supported by parallel experiments in heterologous cells, which showed that FRET was reduced but not abolished by calcium. Thapsigargin also elicited a small decrease in PLB-SERCA binding affinity. We propose that PLB is not displaced from SERCA by high calcium during systole, and relief of functional inhibition does not require dissociation of the regulatory complex. The observed modest reduction in the affinity of the PLB-SERCA complex with Ca(2+) or thapsigargin suggests that the binding interface is altered by SERCA conformational changes. The results are consistent with multiple modes of PLB binding or alternative binding sites.
Journal of Biological Chemistry 08/2011; 286(40):35044-50. · 4.65 Impact Factor