Polymeric assembly of endogenous Tuberous Sclerosis Protein Complex

AbstractTuberous Sclerosis protein complex (pTSC) nucleates a proteinaceous signaling hub that integrates information about the internal and external energy status of the cell in regulation of growth and energy consumption. Biochemical and electron cryomicroscopy (cryoEM) studies of recombinant pTSC have revealed the structure and stoichiometry of the pTSC and have hinted at the possibility that the complex form large oligomers. Here, we have partially purified endogenous pTSC from fasted mammalian brains of rat and pig by leveraging a recombinant antigen binding fragment (Fab) specific for the TSC2 subunit of pTSC. We demonstrate Fab dependent purification of pTSC from membrane solubilized fractions of the brain homogenates. Negative stain electron microscopy of the samples purified from pig brain demonstrates rod-shaped protein particles with a width of 10 nm, a variable length as small as 40 nm and a high degree of conformational flexibility. Larger filaments are evident with a similar 10 nm width and up to 1 μm in length in linear and web-like organizations prepared from pig brain. These observations suggest polymerization of endogenous pTSC into filamentous super-structures.

Download Full-text

Second messenger Ap4A polymerizes target protein HINT1 to transduce signals in FcεRI-activated mast cells

Nature Communications ◽

10.1038/s41467-019-12710-8 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 1

Author(s):

Jing Yu ◽

Zaizhou Liu ◽

Yuanyuan Liang ◽

Feng Luo ◽

Jie Zhang ◽

...

Keyword(s):

Mast Cells ◽

Transcriptional Activation ◽

Second Messenger ◽

Signaling Mechanism ◽

Cellular Processes ◽

Negative Stain ◽

Competitive Mechanism ◽

Rat Basophilic Leukemia Cells ◽

Negative Stain Electron Microscopy ◽

Basophilic Leukemia

Abstract Signal transduction systems enable organisms to monitor their external environments and accordingly adjust the cellular processes. In mast cells, the second messenger Ap4A binds to the histidine triad nucleotide-binding protein 1 (HINT1), disrupts its interaction with the microphthalmia-associated transcription factor (MITF), and eventually activates the transcription of genes downstream of MITF in response to immunostimulation. How the HINT1 protein recognizes and is regulated by Ap4A remain unclear. Here, using eight crystal structures, biochemical experiments, negative stain electron microscopy, and cellular experiments, we report that Ap4A specifically polymerizes HINT1 in solution and in activated rat basophilic leukemia cells. The polymerization interface overlaps with the area on HINT1 for MITF interaction, suggesting a possible competitive mechanism to release MITF for transcriptional activation. The mechanism depends precisely on the length of the phosphodiester linkage of Ap4A. These results highlight a direct polymerization signaling mechanism by the second messenger.

Download Full-text

Structure–function mapping of a heptameric module in the nuclear pore complex

The Journal of Cell Biology ◽

10.1083/jcb.201109008 ◽

2012 ◽

Vol 196 (4) ◽

pp. 419-434 ◽

Cited By ~ 87

Author(s):

Javier Fernandez-Martinez ◽

Jeremy Phillips ◽

Matthew D. Sekedat ◽

Ruben Diaz-Avalos ◽

Javier Velazquez-Muriel ◽

...

Keyword(s):

Nuclear Pore Complex ◽

Protein Domain ◽

Integrative Approach ◽

Nuclear Pore ◽

Mapping Data ◽

Phenotypic Data ◽

Negative Stain ◽

Domain Mapping ◽

Gene Duplication And Loss ◽

Negative Stain Electron Microscopy

The nuclear pore complex (NPC) is a multiprotein assembly that serves as the sole mediator of nucleocytoplasmic exchange in eukaryotic cells. In this paper, we use an integrative approach to determine the structure of an essential component of the yeast NPC, the ∼600-kD heptameric Nup84 complex, to a precision of ∼1.5 nm. The configuration of the subunit structures was determined by satisfaction of spatial restraints derived from a diverse set of negative-stain electron microscopy and protein domain–mapping data. Phenotypic data were mapped onto the complex, allowing us to identify regions that stabilize the NPC’s interaction with the nuclear envelope membrane and connect the complex to the rest of the NPC. Our data allow us to suggest how the Nup84 complex is assembled into the NPC and propose a scenario for the evolution of the Nup84 complex through a series of gene duplication and loss events. This work demonstrates that integrative approaches based on low-resolution data of sufficient quality can generate functionally informative structures at intermediate resolution.

Download Full-text

Faculty Opinions recommendation of Architecture of the tuberous sclerosis protein complex.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.739197469.793583245 ◽

2021 ◽

Author(s):

Daniel Kümmel

Keyword(s):

Tuberous Sclerosis ◽

Protein Complex

Download Full-text

Temperature-dependent reversible assembly of taxol-treated microtubules.

The Journal of Cell Biology ◽

10.1083/jcb.105.6.2847 ◽

1987 ◽

Vol 105 (6) ◽

pp. 2847-2854 ◽

Cited By ~ 50

Author(s):

C A Collins ◽

R B Vallee

Keyword(s):

Electron Microscopy ◽

Self Assembly ◽

Temperature Dependent ◽

Reverse Phase Hplc ◽

Reverse Phase ◽

Microtubule Associated Proteins ◽

Negative Stain ◽

Microtubule Protein ◽

Associated Proteins ◽

Negative Stain Electron Microscopy

Taxol is a plant alkaloid that binds to and strongly stabilizes microtubules. Taxol-treated microtubules resist depolymerization under a variety of conditions that readily disassemble untreated microtubules. We report here that taxol-treated microtubules can be induced to disassemble by a combination of depolymerizating conditions. Reversible cycles of disassembly and reassembly were carried out using taxol-containing microtubules from calf brain and sea urchin eggs by shifting temperature in the presence of millimolar levels of Ca2+. Microtubules depolymerized completely, yielding dimers and ring-shaped oligomers as revealed by negative stain electron microscopy and Bio-Gel A-15m chromatography, and reassembled into well-formed microtubule polymer structures. Microtubule-associated proteins (MAPs), including species previously identified only by taxol-based purification such as MAP 1B and kinesin, were found to copurify with tubulin through reversible assembly cycles. To determine whether taxol remained bound to tubulin subunits, we subjected depolymerized taxol-treated microtubule protein to Sephadex G-25 chromatography, and the fractions were assayed for taxol content by reverse-phase HPLC. Taxol was found to be dissociated from the depolymerized microtubules. Protein treated in this way was found to be competent to reassemble, but now required conditions comparable with those for protein that had never been exposed to taxol. Thus, the binding of taxol to tubulin can be reversed. This has implications for the mechanism of taxol action and for the purification of microtubules from a wide variety of sources for use in self-assembly experiments.

Download Full-text

A site of varicella-zoster virus vulnerability identified by structural studies of neutralizing antibodies bound to the glycoprotein complex gHgL

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1501176112 ◽

2015 ◽

Vol 112 (19) ◽

pp. 6056-6061 ◽

Cited By ~ 33

Author(s):

Yi Xing ◽

Stefan L. Oliver ◽

TuongVi Nguyen ◽

Claudio Ciferri ◽

Avishek Nandi ◽

...

Keyword(s):

Varicella Zoster Virus ◽

Neutralizing Antibodies ◽

Mutational Analysis ◽

Natural Immunity ◽

Varicella Zoster ◽

Glycoprotein Complex ◽

Negative Stain ◽

Virion Envelope ◽

Negative Stain Electron Microscopy ◽

A Site

Varicella-zoster virus (VZV), of the familyAlphaherpesvirinae, causes varicella in children and young adults, potentially leading to herpes zoster later in life on reactivation from latency. The conserved herpesvirus glycoprotein gB and the heterodimer gHgL mediate virion envelope fusion with cell membranes during virus entry. Naturally occurring neutralizing antibodies against herpesviruses target these entry proteins. To determine the molecular basis for VZV neutralization, crystal structures of gHgL were determined in complex with fragments of antigen binding (Fabs) from two human monoclonal antibodies, IgG-94 and IgG-RC, isolated from seropositive subjects. These structures reveal that the antibodies target the same site, composed of residues from both gH and gL, distinct from two other neutralizing epitopes identified by negative-stain electron microscopy and mutational analysis. Inhibition of gB/gHgL-mediated membrane fusion and structural comparisons with herpesvirus homologs suggest that the IgG-RC/94 epitope is in proximity to the site on VZV gHgL that activates gB. Immunization studies proved that the anti-gHgL IgG-RC/94 epitope is a critical target for antibodies that neutralize VZV. Thus, the gHgL/Fab structures delineate a site of herpesvirus vulnerability targeted by natural immunity.

Download Full-text

The Use of Negative Stain Electron Microscopy for the Examination of Pharmaceutical Preparations

Microscopy and Microanalysis ◽

10.1017/s1431927605500114 ◽

2005 ◽

Vol 11 (S02) ◽

Author(s):

R Hamilton ◽

M Walker

Keyword(s):

Electron Microscopy ◽

Pharmaceutical Preparations ◽

Negative Stain ◽

Negative Stain Electron Microscopy

Download Full-text

Isolation of the yeast nuclear pore complex.

The Journal of Cell Biology ◽

10.1083/jcb.123.4.771 ◽

1993 ◽

Vol 123 (4) ◽

pp. 771-783 ◽

Cited By ~ 169

Author(s):

M P Rout ◽

G Blobel

Keyword(s):

Electron Microscopy ◽

Image Reconstruction ◽

Nuclear Pore Complex ◽

Sedimentation Coefficient ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Negative Stain ◽

Negative Stain Electron Microscopy ◽

Pore Complexes

Nuclear pore complexes (NPCs) have been isolated from the yeast Saccharomyces. Negative stain electron microscopy of the isolated NPCs and subsequent image reconstruction revealed the octagonal symmetry and many of the ultrastructural features characteristic of vertebrate NPCs. The overall dimensions of the yeast NPC, both in its isolated form as well as in situ, are smaller than its vertebrate counterpart. However, the diameter of the central structures are similar. The isolated yeast NPC has a sedimentation coefficient of approximately 310 S and an M(r) of approximately 66 MD. It retains all but one of the eight known NPC proteins. In addition it contains as many as 80 uncharacterized proteins that are candidate NPC proteins.

Download Full-text

Postentry Neutralization of Adenovirus Type 5 by an Antihexon Antibody

Journal of Virology ◽

10.1128/jvi.78.22.12320-12332.2004 ◽

2004 ◽

Vol 78 (22) ◽

pp. 12320-12332 ◽

Cited By ~ 61

Author(s):

Robin Varghese ◽

Yeshi Mikyas ◽

Phoebe L. Stewart ◽

Robert Ralston

Keyword(s):

Virus Particle ◽

Intracellular Transport ◽

Nuclear Periphery ◽

Adenovirus Type ◽

Nuclear Entry ◽

Viral Attachment ◽

Virus Particles ◽

Negative Stain ◽

Virus Complex ◽

Negative Stain Electron Microscopy

ABSTRACT Antibodies against hexon, the major coat protein of adenovirus (Ad), are an important component of the neutralizing activity in serum from naturally infected humans and experimentally infected animals. The mechanisms by which antihexon antibodies neutralize the virus have not been defined. As a model system, murine monoclonal antibodies raised against Ad type 5 (Ad5) were screened for antihexon binding and neutralization activity; one monoclonal antibody, designated 9C12, was selected for further characterization. The minimum ratio of 9C12 to Ad5 required for neutralization was 240 antibody molecules per virus particle, or 1 antibody per hexon trimer. Analysis of antibody-virus complexes by dynamic light scattering and negative-stain electron microscopy (EM) showed that the virus particles were coated with electron-dense material but not aggregated at neutralizing ratios. Cryo-EM image reconstruction of the antibody-virus complex showed that the surface of the virus particle was covered by a meshwork of 9C12 antibody density, consistent with bivalent binding at multiple sites. Confocal analysis revealed that viral attachment, cell entry, and intracellular transport to the nuclear periphery still occur in the presence of neutralizing levels of 9C12. A model is presented for neutralization of Ad by an antihexon antibody in which the hexon capsid is cross-linked by antibodies, thus preventing virus uncoating and nuclear entry of viral DNA.

Download Full-text