David C. Kennedy

Cellular Consequences of Copper Complexes Used To Catalyze Bioorthogonal Click Reactions

Copper toxicity is a critical issue in the development of copper-based catalysts for copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reactions for applications in living systems. The effects and related toxicity of copper on mammalian cells are dependent on the ligand environment. Copper complexes can be highly toxic, can induce changes in cellular metabolism, and can be rapidly taken up by cells, all of which can affect their ability to function as catalysts for CuAAC in living systems. Herein, we have evaluated the effects of a number of copper complexes that are typically used to catalyze CuAAC reactions on four human cell lines by measuring mitochondrial activity based on the metabolism of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to study toxicity, inductively coupled plasma mass spectrometry to study cellular uptake, and coherent anti-Stokes Raman scattering (CARS) microscopy to study effects on lipid metabolism. We find that ligand environment around copper influences all three parameters. Interestingly, for the Cu(II)-bis-L-histidine complex (Cu(his)(2)), cellular uptake and metabolic changes are observed with no toxicity after 72 h at micromolar concentrations. Furthermore, we show that under conditions where other copper complexes kill human hepatoma cells, Cu(I)-L-histidine is an effective catalyst for CuAAC labeling of live cells following metabolic incorporation of an alkyne-labeled sugar (Ac(4)ManNAl) into glycosylated proteins expressed on the cell surface. This result suggests that Cu(his)(2) or derivatives thereof have potential for in vivo applications where toxicity as well as catalytic activity are critical factors for successful bioconjugation reactions.

Chemical contrast for imaging living systems: molecular vibrations drive CARS microscopy

The nonlinear variant of Raman spectroscopy, coherent anti-Stokes Raman scattering (CARS) microscopy, combines powerful Raman signal enhancement with several other advantages such as label-free detection and has been used to image various cellular processes including host-pathogen interactions and lipid metabolism.

Direct imaging of the disruption of hepatitis C virus replication complexes by inhibitors of lipid metabolism.

Here we have simultaneously characterized the influence of inhibitors of peroxisome proliferator-activated receptor alpha (PPARalpha) and the mevalonate pathway on hepatocyte lipid metabolism and the subcellular localization of hepatitis C virus (HCV) RNA using two-photon fluorescence (TPF) and coherent anti-Stokes Raman scattering (CARS) microscopy. Using this approach, we demonstrate that modulators of PPARalpha signaling rapidly cause the dispersion of HCV RNA from replication sites and simultaneously induce lipid storage and increases in lipid droplet size. We demonstrate that reductions in the levels of cholesterol resulting from inhibition of the mevalonate pathway upregulates triglyceride levels. We also show that the rate of dispersion of HCV RNA is very rapid when using a PPARalpha antagonist. This occurs with a faster rate to that of direct inhibition of 3-hydroxy-3-methyglutaryl CoA reductase (HMG-CoA reductase) using lovastatin in living cells, demonstrating the potential therapeutic value of modulating host cell pathways as part of a strategy to eliminate chronic HCV infection.

A novel regulatory metal binding domain is present in the C terminus of Arabidopsis Zn2+-ATPase HMA2

HMA2 is a Zn2+-ATPase from Arabidopsis thaliana. It contributes to the maintenance of metal homeostasis in cells by driving Zn2+ efflux. Distinct from P1B-type ATPases, plant Zn2+-ATPases have long C-terminal sequences rich in Cys and His. Removal of the 244 amino acid C terminus of HMA2 leads to a 43% reduction in enzyme turnover without significant effect on the Zn2+ K½ for enzyme activation. Characterization of the isolated HMA2 C terminus showed that this fragment binds three Zn2+ with high affinity (Kd = 16 ± 3nm). Circular dichroism spectral analysis indicated the presence of 8% α-helix, 45% β-sheet, and 48% random coil in the C-terminal peptide with noticeable structural changes upon metal binding (8% α-helix, 39% β-sheet, and 52% random coil). Zn K-edge XAS of Zn-C-MBD in the presence of one equivalent of Zn2+ shows that the average zinc complex formed is composed of three His and one Cys residues. Upon the addition of two extra Zn2+ ions per C-MBD, these appear coordinated primarily by His residues thus, suggesting that the three Zn2+ binding domains might not be identical. Modification of His residues with diethyl pyrocarbonate completely inhibited Zn2+ binding to the C terminus, pointing out the importance of His residues in Zn2+ coordination. In contrast, alkylation of Cys with iodoacetic acid did not prevent Zn2+ binding to the HMA2 C terminus. Zn K-edge XAS of the Cys-alkylated protein was consistent with (N/O)4 coordination of the zinc site, with three of those ligands fitting for His residues. In summary, plant Zn2+-ATPases contain novel metal binding domains in their cytoplasmic C terminus. Structurally distinct from the well characterized N-terminal metal binding domains present in most P1B-type ATPases, they also appear to regulate enzyme turnover rate.

Nanoscale aggregation of cellular beta2-adrenergic receptors measured by plasmonic interactions of functionalized nanoparticles.

Adrenergic signaling that controls the contraction of cardiac myocyte cells and the beating of the mammalian heart is initiated by ligand binding to adrenergic receptors contained in nanoscale multiprotein complexes at the cellular membrane. Here we demonstrate that the surface-enhanced Raman scattering (SERS) of functionalized silver nanoparticles can be used to report on the receptor aggregation state of specifically label beta(2)-adrenergic receptors on mouse cardiac myocyte cells. Furthermore, multimodal imaging including Raman, Rayleigh scattering, scanning electron microscopy, and luminescence imaging was combined to fully characterize the beta(2)-adrenergic receptor-mediated aggregation of silver nanoparticles on the membrane of cardiac myocytes. Scanning electron microscopy analysis reveals distinct SERS active clusters of between 10 and 70 nanoparticles per signaling domain from ultra-high-resolution images of beta(2)-adrenergic receptor clusters on the cellular membrane. These techniques can be generally applied to study the aggregation of other cell surface receptors and explore their distribution on cell surfaces.

Dynamics of lipid droplets induced by the hepatitis C virus core protein

The hepatitis C virus (HCV) is a global health problem, with limited treatment options and no vaccine available. HCV uses components of the host cell to proliferate, including lipid droplets (LD) onto which HCV core proteins bind and facilitate viral particle assembly. We have measured the dynamics of HCV core protein-mediated changes in LDs and rates of LD movement on microtubules using a combination of coherent anti-Stokes Raman scattering (CARS), two-photon fluorescence (TPF), and differential interference contrast (DIC) microscopies. Results show that the HCV core protein induces rapid increases in LD size. Particle tracking experiments show that HCV core protein slowly affects LD localization by controlling the directionality of LD movement on microtubules. These dynamic processes ultimately aid HCV in propagating and the molecules and interactions involved represent novel targets for potential therapeutic intervention.

Coordination changes and auto-hydroxylation of FIH-1: uncoupled O2-activation in a human hypoxia sensor.

Hypoxia sensing is the generic term for pO2-sensing in humans and other higher organisms. These cellular responses to pO2 are largely controlled by enzymes that belong to the Fe(II) alpha-ketoglutarate (alphaKG) dependent dioxygenase superfamily, including the human enzyme called the factor inhibiting HIF (FIH-1), which couples O2-activation to the hydroxylation of the hypoxia inducible factor alpha (HIFalpha). Uncoupled O2-activation by human FIH-1 was studied by exposing the resting form of FIH-1 (alphaKG + Fe)FIH-1, to air in the absence of HIFalpha. Uncoupling lead to two distinct enzyme oxidations, one a purple chromophore (lambda(max) = 583 nm) arising from enzyme auto-hydroxylation of Trp296, forming an Fe(III)-O-Trp296 chromophore [Y.-H. Chen, L.M. Comeaux, S.J. Eyles, M.J. Knapp, Chem. Commun. (2008), doi:10.1039/B809099H]; the other a yellow chromophore due to Fe(III) in the active site, which under some conditions also contained variable levels of an oxygenated surface residue (oxo)Met275. The kinetics of purple FIH-1 formation were independent of Fe(II) and alphaKG concentrations, however, product yield was saturable with increasing [alphaKG] and required excess Fe(II). Yellow FIH-1 was formed from (succinate+Fe)FIH-1, or by glycerol addition to (alphaKG+Fe)FIH-1, suggesting that glycerol could intercept the active oxidant from the FIH-1 active site and prevent hydroxylation. Both purple and yellow FIH-1 contained high-spin, rhombic Fe(III) centers, as shown by low temperature EPR. XAS indicated distorted octahedral Fe(III) geometries, with subtle differences in inner-shell ligands for yellow and purple FIH-1. EPR of Co(II)-substituted FIH-1 (alphaKG + Co)FIH-1, indicated a mixture of 5-coordinate and 6-coordinate enzyme forms, suggesting that resting FIH-1 can readily undergo uncoupled O2-activation by loss of an H2O ligand from the metal center.

Exploiting plasmonics in biosensing and bioimaging: monitoring cell receptors with surface enhanced spectroscopy and microscopy

The plasmon resonance of noble metal nanoparticles (NP) manifests itself in a variety of extraordinary optical properties. Resonant excitation of the conduction electrons by incident radiation generates a localized surface plasmon resonance (LSPR) that is responsible for a variety of surface enhanced optical phenomena. This unique optical property coupled with well-established surface chemistry allows us to utilize both Ag and Au NP as optical contrasting agents to probe and monitor the surface receptors of cells. We have employed two plasmon-assisted optical techniques (namely, surface enhanced Raman scattering, and resonant Rayleigh scattering) to monitor the adrenergic receptors in mammalian cardiomyocyte cells that have been labeled with functionalized Ag NPs. In this study, a unique Raman reporter molecule, 4-(mercaptomethyl)benzonitrile, was developed to provide an easily identifiable vibration, the C≡N stretch, in a spectral window free from Raman bands of cell constituents and other biomolecules used in receptor crosslinking and surface passivation. Successfully labeled cells were then monitored with both optical techniques. Both techniques are related through the plasmonic properties of the noble metal NP and combined with high resolution imaging techniques; we outline the importance that different NP architectures play in the different imaging techniques. Furthermore, we will discuss the instrumentation and plasmonic implications in the design of NP best suited for such multimodal imaging approaches.