13 research outputs found
Computational and Experimental Investigation of the Structure of Peptide Monolayers on Gold Nanoparticles
The
self-assembly and self-organization of small molecules on the
surface of nanoparticles constitute a potential route toward the preparation
of advanced proteinlike nanosystems. However, their structural characterization,
critical to the design of bionanomaterials with well-defined biophysical
and biochemical properties, remains highly challenging. Here, a computational
model for peptide-capped gold nanoparticles (GNPs) is developed using
experimentally characterized Cys-Ala-Leu-Asn-Asn (CALNN)- and Cys-Phe-Gly-Ala-Ile-Leu-Ser-Ser
(CFGAILSS)-capped GNPs as a benchmark. The structure of CALNN and
CFGAILSS monolayers is investigated using both structural biology
techniques and molecular dynamics simulations. The calculations reproduce
the experimentally observed dependence of the monolayer secondary
structure on the peptide capping density and on the nanoparticle size,
thus giving us confidence in the model. Furthermore, the computational
results reveal a number of new features of peptide-capped monolayers,
including the importance of sulfur movement for the formation of secondary
structure motifs, the presence of water close to the gold surface
even in tightly packed peptide monolayers, and the existence of extended
2D parallel β-sheet domains in CFGAILSS monolayers. The model
developed here provides a predictive tool that may assist in the design
of further bionanomaterials
Galectin‑3 Binding to α<sub>5</sub>β<sub>1</sub> Integrin in Pore Suspended Biomembranes
Galectin-3 (Gal3)
is a β-galactoside binding lectin that
mediates many physiological functions, including the binding of cells
to the extracellular matrix for which the glycoprotein α5β1 integrin is of critical importance. The
mechanisms by which Gal3 interacts with membranes have not been widely
explored to date due to the complexity of cell membranes and the difficulty
of integrin reconstitution within model membranes. Herein, to study
their interaction, Gal3 and α5β1 were purified, and the latter reconstituted into pore-suspended
lipid bilayers comprised eggPC:eggPA. Using electrochemical impedance
and fluorescence lifetime correlation spectroscopy, we found that
on incubation with low nanomolar concentrations of wild-type Gal3,
the membrane’s admittance and fluidity, as well as integrin’s
lateral diffusivity, were enhanced. These effects were diminished
in the following conditions: (i) absence of integrin, (ii) presence
of lactose as a competitive inhibitor of glycan–Gal3 interaction,
and (iii) use of a Gal3 mutant that lacked the N-terminal oligomerization
domain (Gal3ΔNter). These findings indicated that WTGal3 oligomerized
on α5β1 integrin in a glycan-dependent
manner and that the N-terminal domain interacted directly with membranes
in a way that is yet to be fully understood. At concentrations above
10 nM of WTGal3, membrane capacitance started to decrease and very
slowly diffusing molecular species appeared, which indicated the formation
of protein clusters made from WTGal3−α5β1 integrin assemblies. Overall, our study demonstrates the
capacity of WTGal3 to oligomerize in a cargo protein-dependent manner
at low nanomolar concentrations. Of note, these WTGal3 oligomers appeared
to have membrane active properties that could only be revealed using
our sensitive methods. At slightly higher WTGal3 concentrations, the
capacity to generate lateral assemblies between cargo proteins was
observed. In cells, this could lead to the construction of tubular
endocytic pits according to the glycolipid–lectin (GL–Lect)
hypothesis or to the formation of galectin lattices, depending on
cargo glycoprotein stability at the membrane, the local Gal3 concentration,
or plasma membrane intrinsic parameters. The study also demonstrates
the utility of microcavity array-suspended lipid bilayers to address
the biophysics of transmembrane proteins
Silencing of the insulin receptor isoform A favors formation of type 1 insulin-like growth factor receptor (IGF-IR) homodimers and enhances ligand-induced IGF-IR activation and viability of human colon carcinoma cells
Insulin receptor (IR) overexpression is common in cancers, with expression of the A isoform (IR-A, exon 11–) predominating over the B isoform. The IR-A signals a proliferative, antiapoptotic response to IGF-II, which itself can be secreted by tumors to establish an autocrine proliferative loop. Therefore, IGF-II signaling via the IR-A could mediate resistance to type 1 IGF receptor (IGF-IR) inhibitory drugs that are currently in development. This study addressed the role of the IR-A, using a small interfering RNA-based approach in SW480 human colon adenocarcinoma cells that coexpress the IGF-IR. Clonogenic survival was inhibited by depletion of the IGF-IR but not the IR-A, and dual receptor depletion had no greater effect than IGF-IR knockdown alone, suggesting that the IR-A could not compensate for IGF-IR loss. IGF-IR knockdown also resulted in a decrease in viability, whereas IR-A depletion resulted in increased viability. Consistent with this, upon IR-A depletion, we found a concomitant enhancement of IGF-IR activation by IGF-I and IGF-II, reduced formation of IGF-IR:IR-A hybrid receptors and increased IGF-IR homodimer formation. Together, these results suggest that IGF bioactivity is mediated more effectively by the IGF-IR than by the IR-A or receptor hybrids and that signaling via the IGF-IR is dominant to the IR-A in colon cancer cells that express both receptors.G.V. Brierley, S.L. Macaulay, B.E. Forbes, J.C. Wallace, L.J. Cosgrove and V.M. Macaula