Sivaraj Sivaramakrishnan - US grants

Abstract Kinase mediated phosphorylation of proteins broadly regulates cellular responses in normal and disease states. Kinases in cellular signaling networks play the role of `micro-processors' that couple different stimuli to distinct signaling outputs. The versatility and specificity of their cellular function arise from the coordination of several intra-molecular and inter-molecular protein interactions. However, current approaches to probe kinases treat them as simple `on-off' switches and do not address their complex spatial and temporal regulation in cells. We have developed a technology, termed the kinase toolbox, which monitors and/or controls these protein interactions to provide a detailed mechanistic understanding of the cellular function of any kinase. In addition, the kinase toolbox overcomes the limitations of existing techniques to identify small molecules/therapeutics that differentiate between closely related kinases. We have developed and tested kinase toolboxes for focal adhesion kinase (FAK) and protein kinase C (PKC). We propose to pursue three complementary and parallel goals in order to realize the transformative potential of this new technology, while distributing risk. Our first goal is to use the PKC toolbox to map the spatial and temporal regulation of two closely related PKC isoforms in cellular models of cardiac hypertrophy and diabetic retinopathy. In addition to proof-of-concept, the PKC toolbox has already provided us with new conceptual insights that broadly apply to the AGC kinase superfamily (60 members). Our second goal is to use these insights to understand the similarities and differences in the regulation of five closely related AGC kinases (PKA, Akt/PKB, PKC, PDK1 and S6K1). Our third goal is to conduct pilot studies of three new approaches, based on the kinase toolbox, to design isoform-specific inhibitors of AGC kinases. Taken together, the proposed research is an essential first step towards our long- term goal of designing and characterizing high-specificity inhibitors of AGC kinases, which are important drug targets in disease states such as diabetes, heart failure and cancer. Successful completion of the outlined studies will transform our understanding of kinases in general, while providing researchers with new tools and a roadmap to study their cellular function.

DESCRIPTION (provided by applicant): Kinase mediated phosphorylation of proteins broadly regulates cellular responses in normal and disease states. Kinases in cellular signaling networks play the role of 'micro-processors' that couple different stimuli to distinct signaling outputs. The versatility and specificity of their cellular function arise from the coordination of several intra-molecular and inter-molecular protein interactions. However, current approaches to probe kinases treat them as simple 'on-off' switches and do not address their complex spatial and temporal regulation in cells. We have developed a technology, termed the kinase toolbox, which monitors and/or controls these protein interactions to provide a detailed mechanistic understanding of the cellular function of any kinase. In addition, the kinase toolbox overcomes the limitations of existing techniques to identify small molecules/therapeutics that differentiate between closely related kinases. We have developed and tested kinase toolboxes for focal adhesion kinase (FAK) and protein kinase C (PKC). We propose to pursue three complementary and parallel goals in order to realize the transformative potential of this new technology, while distributing risk. Our first goal is to use the PKC toolbox to map the spatial an temporal regulation of two closely related PKC isoforms in cellular models of cardiac hypertrophy and diabetic retinopathy. In addition to proof-of-concept, the PKC toolbox has already provided us with new conceptual insights that broadly apply to the AGC kinase superfamily (60 members). Our second goal is to use these insights to understand the similarities and differences in the regulation of five closely related AGC kinases (PKA, Akt/PKB, PKC, PDK1 and S6K1). Our third goal is to conduct pilot studies of three new approaches, based on the kinase toolbox, to design isoform-specific inhibitors of AGC kinases. Taken together, the proposed research is an essential first step towards our long-term goal of designing and characterizing high-specificity inhibitors of AGC kinases, which are important drug targets in disease states such as diabetes, heart failure and cancer. Successful completion of the outlined studies will transform our understanding of kinases in general, while providing researchers with new tools and a roadmap to study their cellular function.

DESCRIPTION (provided by applicant): While several recent studies have reported on distinct ligand-dependent GPCR conformations, it remains unclear how these conformations elicit differential downstream responses. In contrast, deciphering G protein selection from functional response is complicated by the myriad of factors that regulate GPCR signaling, including relative abundance and availability of GPCR, G proteins, localization to different membrane surfaces or micro-domains, and the influence of regulatory proteins such as scaffolds, kinases, arrestins and the cellular endocytic apparatus. The proposed research addresses these limitations with a novel class of FRET-based SPASM sensors developed by the PI to systematically modulate protein-protein interactions in live cells. Research will focus o four GPCRs, b2-adrenoceptor (b2-AR), a2A-adrenoceptor, melanocortin4 receptor and b3-adrenoceptor, whose differential G protein selection has important functional consequences in the progression and treatment of heart failure, obesity and diabetes. Studies in aim 1 utilize sensors that detect the interaction between a GPCR and a peptide derived from the Ga subunit c-terminus, a known determinant of GPCR-G protein pairing. FRET-based measurements will be combined with established approaches to test the hypothesis that GPCRs adopt functionally distinct conformations in a ligand-dependent manner to trigger differential downstream responses. In preliminary studies, specificity of sensor measurements was tested with b2-AR and led to the identification of a functional Gi conformation triggered by the beta-blocker metoprolol. Studies are structured to dissect distinct events in the GPCR-G protein interaction followed by stimulation with canonical and biased agonists. A clear understanding of the GPCR-G protein interaction and G protein selection from FRET/BRET studies is limited by the lack of control over relative concentration. Studies in aim 2 will leverage the SPASM technique to delineate the sequence of events prior to and after ligand stimulation. Key questions addressed are: (1) is the ligand-free GPCR basally associated or pre-coupled to a G protein? (2) Does the GPCR dissociate from the G protein following ligand-stimulation? (3) Does G protein selection correlate with the inherent binding affinity of a GPCR for a G protein? (4) To what extent is biased signaling influenced by the local concentration of GPCR and G protein? Systematic variation of the concentration of the GPCR-G protein interaction using the SPASM technique will be used to assess the specificity of FRET and downstream responses. The proposed research has the potential to create a new technical and conceptual platform for future studies on ligand-driven signaling initiated by any GPCR. The extensive validation of the SPASM sensors is an integral part of this study, which in turn will pave the way for their use in live-cell drug screens to identify small molecules that bias GPCR signaling towards therapeutically desired outcomes.

Title: Development of scanning optofluidic cell lasers for highly sensitive cellular and tissue analysis

The goal of the project: Develop a novel laser based scanning microscope to study cells and tissues.


Non-Technical Abstract:
The traditional microscope relies on fluorescence to study cells and tissues. However, any small change in cellular activities and tissue structures may not be picked up by the microscope. The proposed project is aimed to develop a laser-based microscope in which laser emission instead of fluorescence is used for detection. Laser emission is much more sensitive than fluorescence. Therefore, small changes in cellular/tissue activities will cause large signals in laser emission, which allows us to study cell and tissues at a more detailed level.

The proposed research will provide a new, powerful, and sensitive platform technology complementary to conventional fluorescence based methods for cell/tissue analysis. The detection principle developed through the project can broadly be applicable to any types of laser cavities and bio-species. The proposed research will further provide in-depth understanding of how light interacts with living organisms and biological materials, which will be significant for the development of novel photonic devices. The students in the project will receive interdisciplinary training in optofluidics, photonics, nano/microfabrication, biochemistry, cell biology, and biosensing. The outreach program will educate the general public and train students/teachers from local high schools and non-research-intensive colleges.


Technical Abstract:
The proposed project is to systematically investigate the biofunctional scanning optofluidic cell laser technology platform and apply it in highly sensitive cell/tissue analysis. The optofluidic laser is an emerging sensing technology that integrates microfluidics, optical microcavity, and gain medium in liquid environment. It employs laser emission as the sensing signal. A small modulation in the gain medium induced by a small change in the underlying biological activities can result in a significant change in the laser output due to optical feedback provided by the laser cavity. Thus, the optofluidic laser based detection is orders of magnitude more sensitive than the fluorescence counterpart. The proposed scanning optofluidic laser is based on a high-performance laser cavity and cells/tissues containing gain media. The laser output will be recorded for detailed understanding of cellular/tissue activities.

The proposed research will provide a new, powerful, and sensitive platform technology complementary to conventional fluorescence based methods for cell/tissue analysis. The detection principle developed through the project can broadly be applicable to any types of laser cavities and bio-species. Furthermore, study of the optofluidic cell lasers will provide in-depth understanding of how light interacts with living organisms and biological materials, which will be significant for the development of novel photonic devices. The students in the project will receive interdisciplinary training in optofluidics, photonics, nano/microfabrication, biochemistry, cell biology, and biosensing. The outreach program will educate the general public and train students/teachers from local high schools and non-research-intensive colleges.

Summary: Title: Structural Basis of G-protein selectivity in GPCRs using Multiscale Dynamics Upon binding to agonists G protein-coupled receptors (GPCRs) mediate multiple signaling pathways by coupling to intracellular transducer proteins such as G proteins and/or ?-arrestins. Certain agonists exhibit selectivity in their efficacy to specific G-protein signaling pathways. Such selective ligands provide precise therapeutic benefits with fewer side effects as drugs compared to GPCR-targeted drugs in the market. There are very few G-protein selective GPCR agonists known to date, because designing G-protein selective agonists is a daunting experimental challenge. Additionally, there is a serious lack of understanding of structural information on how GPCRs modulate their functional selectivity for their cognate G-protein in cells. There are several contributing factors to how an agonist- GPCR pair shows selectivity to specific G-protein. These factors include, nature of conformational ensembles of the agonist-GPCR-G-protein complexes, and several cellular factors. Delineating the contribution from the structural ensemble of the agonist-GPCR-G-protein complexes has been sparse due to huge experimental challenges in crystallography and NMR of these complexes. We propose to combine two state-of-the-art dynamics techniques, such as ensemble based multi-resolution molecular dynamics method tightly integrated in an iterative fashion with scalable genetically coded FRET sensor biophysical measurements in live cells, to probe the structural basis of G-protein selectivity. The scalability of these two techniques is a huge advantage to probe the functional selectivity of several agonist-GPCR pairings. We propose to use the combination of these two techniques to (a) identify the structural determinants in the agonist-GPCR complex that contribute significantly to G-protein selectivity in nine different agonist-GPCR pairs. (b) We also propose to delineate the structural determinants that contribute to functional selectivity when the GPCR is bound to a partial agonist as opposed to a full agonist, and when the agonist-GPCR is also bound to an allosteric modulator. The outcome of the proposed work will enable structure based design of selective agonists for class A GPCRs, and also provide an understanding of the biological process of how GPCRs recognize their cognate G-proteins in live cells.

PROJECT SUMMARY Cellular processes such as signaling and membrane traffic emerge from an ensemble of dynamic, transient protein-protein interactions in crowded cellular environments. Aberrant protein-protein interactions are frequently implicated in debilitating or fatal diseases such as diabetes, neurodegenerative diseases, and cancer. Established structural and cell biological techniques are mostly limited to dissecting the function of stable protein complexes, and do not investigate emergent behavior stemming from multiple transient interactions. To address this challenge, we use DNA nanotechnology scaffolds to pattern macromolecules in vitro and a novel genetically encoded ER/K linker to probe and modulate protein interactions in live cells. Together, we leverage these technologies to dissect the molecular mechanisms of multiplicity in G protein- coupled receptor (GPCR) signaling and biophysical regulation of unconventional myosin function in cells. We have successfully investigated two distinct aspects of GPCR signaling specificity in cells using biosensors engineered by linking GPCR and G protein elements with an ER/K linker. First, we hypothesize that ligands stabilize GPCR conformational sub-states that selectively interface with one or more G? C-termini, to tune ligand efficacy and potency for downstream pathways. Our goal is to use a combination of GPCR biosensors and multi-scale molecular dynamics simulations to define hot-spot residues, structural motifs, and allosteric pathways in both the GPCR and G protein that drive signaling specificity. Second, our research has advanced a role for concurrent and sequential interactions between GPCR and effectors on signaling specificity. Our goal is to combine GPCR biosensors and traditional pharmacology approaches to dissect the synergistic effects of G proteins on GPCR signaling. Together, our research provides insights into GPCR signaling specificity that can be leveraged in structure-based drug discovery efforts to identify functionally selective GPCR ligands. Unconventional myosins are essential in numerous cellular processes including membrane traffic, contractility, and cell division. Defining the motile function of myosins in cells is challenged by the myriad geometries of both actin networks and cargo, paired with a diversity of motor-cargo interfaces. We use cargo-mimetic DNA nanotechnology scaffolds combined with computational modeling to successfully dissect the mechanical coordination in myosin ensembles. We will use these approaches to gain insight into the biophysical regulation of myosin function through the motor-cargo interface. We hypothesize that cargo interfaces act as molecular modules that tune myosin function to match the functional requirements of individual cellular processes. Our goal is to dissect myosin regulation through interactions with distinct cargo adaptors, Rab GTPases, and cell- derived cargo complexes. Our research will advance our understanding of emergent myosin function in cells, while providing a broad theoretical framework for the cargo-mediated regulation of cytoskeletal motors.

Cardiac myosin-binding protein C (cMyBP-C) is a sarcomeric thick filament associated protein that is essential to normal cardiac structure and function. The importance of cMyBP-C is emphasized by mutations to cMyBP-C being a leading cause of hypertrophic cardiomyopathy. Despite being a key regulator of cardiac contractility, the molecular mechanism by which cMyBP-C modulates actomyosin force and motion generation is far from certain. Although cMyBP-C's N-terminal domains can bind to actin and the myosin head region, it is not known which of these binding partners is physiologically relevant and whether these binding partner interactions modulate cardiac contractility by directly affecting actomyosin power generation or indirectly by altering Ca2+- dependent thin filament activation. With phosphorylation of cMyBP-C's N terminus occurring in response to ?- adrenergic stimulation, phosphorylation may offer a measure of cMyBP-C functional tunability in order to enhance cardiac contractility. To address these questions, we propose two specific aims. Aim 1 will test the hypothesis that phosphorylation modulates cMyBP-C's N-terminal domain structure to influence its binding partner interactions (i.e. thin filament and myosin head region). We will use a novel mass-spectrometry technique and atomic force microscopy to characterize the molecular mechanics of cMyBP-C's N terminus that has been structurally altered due to phosphorylation or mutagenesis. The functional impact of these structural perturbations will be characterized in the context of cardiac myofibrils and native thick filaments to determine if cMyBP-C operates only where it exist in the thick filament and whether it can sequester cardiac myosin into a reserve pool of super-relaxed myosin heads. Thus, we will measure the location and time course of fluorescent-ATP turnover in single cardiac myofibrils and the force generated by native thick filaments in the laser trap in preparations from transgenic mice expressing phosphorylation and binding partner ablated mutant cMyBP-C. In Aim 2 we will create DNA-based ?designer? thick filament nanotubes to define how the spatial relationships that normally exist in the thick filament between cMyBP-C and its myosin and actin binding partners are critical determinants of cMyBP-C's modes of operation. These DNA-nanotubes will allow exquisite nanometer spatial positioning of expressed cMyBP-C and human ?-cardiac myosin on the nanotube surface relative to each other. By this novel approach we can assign cMyBP C's modulation of actomyosin motility to binding of the myosin head and/or thin filament, as assessed by both thin filament motility and force generation using the laser trap. With the knowledge and understanding of cMyBP-C function derived from these collective studies, targeted therapies directed at cMyBP-C binding partner interactions may be developed to help modulate and to improve cardiac performance in the failing heart.

Abstract The goal of this research supplement is to complement the aims of the parent R01 by directly examining the formation of the inhibited state of cardiac myosin, super-relaxed state (SRX), and to examine the influence of cMyBP-C and disease mutations on the formation/stabilization of this key structural state. The importance of understanding the role of cMyBP-C in cardiac contractility is highlighted by work demonstrating that its phosphorylation state plays a role in enhancing contractility and during heart failure decreased phosphorylation likely contributes to contractile defects. cMyBP-C is proposed to influence the cardiac myosin SRX in a phosphorylation dependent manner. In this proposal we will design a FRET biosensor of the SRX which will allow direct examination of the influence of cMyBP-C on this crucially important conformation of myosin. We will also examine hypertrophic cardiomyopathy (HCM) mutations in the cardiac myosin S2 region known to interact with cMyBP-C. In Aim 1 we will characterize the FRET biosensor by correlating the FRET signal with other measurements of the SRX, such as actin-activated ATPase and single turnover ATPase assays. We will vary the ionic strength and examine the drug Omecamtiv Mercarbil to demonstrate that the FRET sensor can be used to measure the mole fraction of cardiac myosin in the SRX conformation. In Aim 2 we will introduce HCM mutations into the S2 region and examine their impact on the formation/stabilization of the SRX. Finally we will also examine if cMyBP-C can alter the HCM mutants response to formation/stabilization of the SRX. Overall, the proposal will greatly complement the parent R01 by providing direct measurements of myosin structure which will be crucial for interpreting the studies of cMyBP-C role in thick and thin filament regulation of muscle contraction. AIM #1. Examine the formation of the super-relaxed state (SRX) by FRET in human cardiac myosin. AIM #2. Examine the functional impact of HCM mutations in the S2 region of cardiac myosin.