John H. Connor

Seeing protein monolayers with naked eye through plasmonic Fano resonances

We introduce an ultrasensitive label-free detection technique based on asymmetric Fano resonances in plasmonic nanoholes with far reaching implications for point-of-care diagnostics. By exploiting extraordinary light transmission phenomena through high-quality factor (Qsolution ∼ 200) subradiant dark modes, we experimentally demonstrate record high figures of merits (FOMs as high as 162) for intrinsic detection limits surpassing that of the gold standard prism coupled surface-plasmon sensors (Kretschmann configuration). Our experimental record high sensitivities are attributed to the nearly complete suppression of the radiative losses that are made possible by the high structural quality of the fabricated devices as well as the subradiant nature of the resonances. Steep dispersion of the plasmonic Fano resonance profiles in high-quality plasmonic sensors exhibit dramatic light intensity changes to the slightest perturbations within their local environment. As a spectacular demonstration of the extraordinary sensitivity and the quality of the fabricated biosensors, we show direct detection of a single monolayer of biomolecules with naked eye using these Fano resonances and the associated Wood’s anomalies. To fabricate high optical-quality sensors, we introduce a high-throughput lift-off free evaporation fabrication technique with extremely uniform and precisely controlled nanofeatures over large areas, leading to resonance line-widths comparable to that of the ideally uniform structures as confirmed by our time-domain simulations. The demonstrated label-free sensing platform offers unique opportunities for point-of-care diagnostics in resource poor settings by eliminating the need for fluorescent labeling and optical detection instrumentation (camera, spectrometer, etc.) as well as mechanical and light isolation.

An Optofluidic Nanoplasmonic Biosensor for Direct Detection of Live Viruses from Biological Media

Fast and sensitive virus detection techniques, which can be rapidly deployed at multiple sites, are essential to prevent and control future epidemics and bioterrorism threats. In this Letter, we demonstrate a label-free optofluidic nanoplasmonic sensor that can directly detect intact viruses from biological media at clinically relevant concentrations with little to no sample preparation. Our sensing platform is based on an extraordinary light transmission effect in plasmonic nanoholes and utilizes group-specific antibodies for highly divergent strains of rapidly evolving viruses. So far, the questions remain for the possible limitations of this technique for virus detection, as the penetration depths of the surface plasmon polaritons are comparable to the dimensions of the pathogens. Here, we demonstrate detection and recognition of small enveloped RNA viruses (vesicular stomatitis virus and pseudotyped Ebola) as well as large enveloped DNA viruses (vaccinia virus) within a dynamic range spanning 3 orders of magnitude. Our platform, by enabling high signal to noise measurements without any mechanical or optical isolation, opens up opportunities for detection of a broad range of pathogens in typical biology laboratory settings.

Growth Arrest and DNA Damage-Inducible Protein GADD34 Assembles a Novel Signaling Complex Containing Protein Phosphatase 1 and Inhibitor 1

ABSTRACT The growth arrest and DNA damage-inducible protein, GADD34, was identified by its interaction with human inhibitor 1 (I-1), a protein kinase A (PKA)-activated inhibitor of type 1 protein serine/threonine phosphatase (PP1), in a yeast two-hybrid screen of a human brain cDNA library. Recombinant GADD34 (amino acids 233 to 674) bound both PKA-phosphorylated and unphosphorylated I-1(1–171). Serial truncations mapped the C terminus of I-1 (amino acids 142 to 171) as essential for GADD34 binding. In contrast, PKA phosphorylation was required for PP1 binding and inhibition by the N-terminal I-1(1–80) fragment. Pulldowns of GADD34 proteins expressed in HEK293T cells showed that I-1 bound the central domain of GADD34 (amino acids 180 to 483). By comparison, affinity isolation of cellular GADD34/PP1 complexes showed that PP1 bound near the C terminus of GADD34 (amino acids 483 to 619), a region that shows sequence homology with the virulence factors ICP34.5 of herpes simplex virus and NL-S of avian sarcoma virus. While GADD34 inhibited PP1-catalyzed dephosphorylation of phosphorylase a, the GADD34-bound PP1 was an active eIF-2α phosphatase. In brain extracts from active ground squirrels, GADD34 bound both I-1 and PP1 and eIF-2α was largely dephosphorylated. In contrast, the I-1/GADD34 and PP1/GADD34 interactions were disrupted in brain from hibernating animals, in which eIF-2α was highly phosphorylated at serine-51 and protein synthesis was inhibited. These studies suggested that modification of the I-1/GADD34/PP1 signaling complex regulates the initiation of protein translation in mammalian tissues.

Molecular memory by reversible translocation of calcium/calmodulin-dependent protein kinase II.

Synaptic plasticity is thought to be a key process for learning, memory and other cognitive functions of the nervous system. The initial events of plasticity require the conversion of brief electrical signals into alterations of the biochemical properties of synapses that last for much longer than the initial stimuli. Here we show that a regulator of synaptic plasticity, calcium/calmodulin-dependent protein kinase IIα (CaMKII), sequentially translocates to postsynaptic sites, undergoes autophosphorylation and gets trapped for several minutes until its dissociation is induced by secondary autophosphorylation and phosphatase 1 action. Once dissociated, CaMKII shows facilitated translocation for several minutes. This suggests that trapping of CaMKII by its targets and priming of CaMKII translocation may function as biochemical memory mechanisms that change the signaling capacity of synapses.

Regulation of Synaptic Strength by Protein Phosphatase 1

We investigated the role of postsynaptic protein phosphatase 1 (PP1) in regulating synaptic strength by loading CA1 pyramidal cells either with peptides that disrupt PP1 binding to synaptic targeting proteins or with active PP1. The peptides blocked synaptically evoked LTD but had no effect on basal synaptic currents mediated by either AMPA or NMDA receptors. They did, however, cause an increase in synaptic strength following the induction of LTD. Similarly, PP1 had no effect on basal synaptic strength but enhanced LTD. In cultured neurons, synaptic activation of NMDA receptors increased the proportion of PP1 localized to synapses. These results suggest that PP1 does not significantly regulate basal synaptic strength. Appropriate NMDA receptor activation, however, allows PP1 to gain access to synaptic substrates and be recruited to synapses where its activity is necessary for sustaining LTD.

Capture and transfer of HIV-1 particles by mature dendritic cells converges with the exosome-dissemination pathway

Exosomes are secreted cellular vesicles that can be internalized by dendritic cells (DCs), contributing to antigen-specific naive CD4(+) T-cell activation. Here, we demonstrate that human immunodeficiency virus type 1 (HIV-1) can exploit this exosome antigen-dissemination pathway intrinsic to mature DCs (mDCs) for mediating trans-infection of T lymphocytes. Capture of HIV-1, HIV-1 Gag-enhanced green fluorescent protein (eGFP) viral-like particles (VLPs), and exosomes by DCs was up-regulated upon maturation, resulting in localization within a CD81(+) compartment. Uptake of VLPs or exosomes could be inhibited by a challenge with either particle, suggesting that the expression of common determinant(s) on VLP or exosome surface is necessary for internalization by mDCs. Capture by mDCs was insensitive to proteolysis but blocked when virus, VLPs, or exosomes were produced from cells treated with sphingolipid biosynthesis inhibitors that modulate the lipid composition of the budding particles. Finally, VLPs and exosomes captured by mDCs were transmitted to T lymphocytes in an envelope glycoprotein-independent manner, underscoring a new potential viral dissemination pathway.

Vesicular Stomatitis Virus Infection Alters the eIF4F Translation Initiation Complex and Causes Dephosphorylation of the eIF4E Binding Protein 4E-BP1

ABSTRACT Vesicular stomatitis virus (VSV) modulates protein synthesis in infected cells in a way that allows the translation of its own 5′-capped mRNA but inhibits the translation of host mRNA. Previous data have shown that inactivation of eIF2α is important for VSV-induced inhibition of host protein synthesis. We tested whether there is a role for eIF4F in this inhibition. The multisubunit eIF4F complex is involved in the regulation of protein synthesis via phosphorylation of cap-binding protein eIF4E, a subunit of eIF4F. Translation of host mRNA is significantly reduced under conditions in which eIF4E is dephosphorylated. To determine whether VSV infection alters the eIF4F complex, we analyzed eIF4E phosphorylation and the association of eIF4E with other translation initiation factors, such as eIF4G and the translation inhibitor 4E-BP1. VSV infection of HeLa cells resulted in the dephosphorylation of eIF4E at serine 209 between 3 and 6 h postinfection. This time course corresponded well to that of the inhibition of host protein synthesis induced by VSV infection. Cells infected with a VSV mutant that is delayed in the ability to inhibit host protein synthesis were also delayed in dephosphorylation of eIF4E. In addition to decreasing eIF4E phosphorylation, VSV infection also resulted in the dephosphorylation and activation of eIF4E-binding protein 4E-BP1 between 3 and 6 h postinfection. Analysis of cap-binding complexes showed that VSV infection reduced the association of eIF4E with the eIF4G scaffolding subunit at the same time as its association with 4E-BP1 increased and that these time courses correlated with the dephosphorylation of eIF4E. These changes in the eIF4F complex occurred over the same time period as the onset of viral protein synthesis, suggesting that activation of 4E-BP1 does not inhibit translation of viral mRNAs. In support of this idea, VSV protein synthesis was not affected by the presence of rapamycin, a drug that blocks 4E-BP1 phosphorylation. These data show that VSV infection results in modifications of the eIF4F complex that are correlated with the inhibition of host protein synthesis and that translation of VSV mRNAs occurs despite lowered concentrations of the active cap-binding eIF4F complex. This is the first noted modification of both eIF4E and 4E-BP1 phosphorylation levels among viruses that produce capped mRNA for protein translation.

Deactylase Inhibitors Disrupt Cellular Complexes Containing Protein Phosphatases and Deacetylases

Affinity isolation of protein serine/threonine phosphatases on the immobilized phosphatase inhibitor microcystin-LR identified histone deacetylase 1(HDAC1), HDAC6, and HDAC10 as novel components of cellular phosphatase complexes. Other HDACs, specifically HDAC2, -3, -4, and -5, were excluded from such complexes. In vitro biochemical studies showed that recombinant HDAC6, but not HDAC4, bound directly to the protein phosphatase (PP)1 catalytic subunit. No association was observed between HDAC6 and PP2A, another major protein phosphatase. PP1 binding was mapped to the second catalytic domain and adjacent C-terminal sequences in HDAC6, and treatment of cells with trichostatin A (TSA) disrupted endogenous HDAC6·PP1 complexes. Consistent with the inhibition of tubulin deactylase activity of HDAC6, TSA enhanced cellular tubulin acetylation, and acetylated tubulin was present in the PP1 complexes from TSA-treated cells. Trapoxin B, a weak HDAC6 inhibitor, and calyculin A, a cell-permeable phosphatase inhibitor, had no effect on the stability of the HDAC6·PP1 complexes or on tubulin acetylation. Mutations that inactivated HDAC6 prevented its incorporation into cellular PP1 complexes and suggested that when bound together both enzymes were active. Interestingly, TSA disrupted all the cellular HDAC·phosphatase complexes analyzed. This study provided new insight into the mechanism by which HDAC inhibitors elicited coordinate changes in cellular protein phosphorylation and acetylation and suggested that changes in these protein modifications at multiple subcellular sites may contribute to the known ability of HDAC inhibitors to suppress cell growth and transformation.

Molecular determinants of nuclear protein phosphatase-1 regulation by NIPP-1.

NIPP-1 is a subunit of the major nuclear protein phosphatase-1 (PP-1) in mammalian cells and potently inhibits PP-1 activity in vitro. Using yeast two-hybrid and co-sedimentation assays, we mapped a PP-1-binding site and the inhibition function to the central one-third domain of NIPP-1. Full-length NIPP-1 (351 residues) and the central domain, NIPP-1143–217, were equally potent PP-1 inhibitors (IC50 = 0.3 nm). Synthetic peptides spanning the central domain of NIPP-1 further narrowed the PP-1 inhibitory function to residues 191–200. A second, noninhibitory PP-1-binding site was identified by far-Western assays with digoxygenin-conjugated catalytic subunit (PP-1C) and included a consensus RVXF motif (residues 200–203) found in many other PP-1-binding proteins. The substitutions, V201A and/or F203A, in the RVXF motif, or phosphorylation of Ser199 or Ser204, which are established phosphorylation sites for protein kinase A and protein kinase CK2, respectively, prevented PP-1C-binding by NIPP-1191–210 in the far-Western assay. NIPP-1191–210 competed for PP-1 inhibition by full-length NIPP-11–351, inhibitor-1 and inhibitor-2, and dissociated PP-1C from inhibitor-1- and NIPP-1143–217-Sepharose but not from full-length NIPP-11–351-Sepharose. Together, these data identified some of the key elements in the central domain of NIPP-1 that regulate PP-1 activity and suggested that the flanking sequences stabilize the association of NIPP-1 with PP-1C.

Importance of the β12-β13 Loop in Protein Phosphatase-1 Catalytic Subunit for Inhibition by Toxins and Mammalian Protein Inhibitors

Type-1 protein serine/threonine phosphatases (PP1) are uniquely inhibited by the mammalian proteins, inhibitor-1 (I-1), inhibitor-2 (I-2), and nuclear inhibitor of PP1 (NIPP-1). In addition, several natural compounds inhibit both PP1 and the type-2 phosphatase, PP2A. Deletion of C-terminal sequences that included the β12-β13 loop attenuated the inhibition of the resulting PP1α catalytic core by I-1, I-2, NIPP-1, and several toxins, including tautomycin, microcystin-LR, calyculin A, and okadaic acid. Substitution of C-terminal sequences from the PP2A catalytic subunit produced a chimeric enzyme, CRHM2, that was inhibited by toxins with dose-response characteristics of PP1 and not PP2A. However, CRHM2 was insensitive to the PP1-specific inhibitors, I-1, I-2, and NIPP-1. The anticancer compound, fostriecin, differed from other phosphatase inhibitors in that it inhibited wild-type PP1α, the PP1α catalytic core, and CRHM2 with identical IC50. Binding of wild-type and mutant phosphatases to immobilized microcystin-LR, NIPP-1, and I-2 established that the β12-β13 loop was essential for the association of PP1 with toxins and the protein inhibitors. These studies point to the importance of the β12-β13 loop structure and conformation for the control of PP1 functions by toxins and endogenous proteins.