14 research outputs found
Legislative Documents
Also, variously referred to as: House bills; House documents; House legislative documents; legislative documents; General Court documents
C-Terminal Tetrapeptides Inhibit Aβ42-Induced Neurotoxicity Primarily through Specific Interaction at the N-Terminus of Aβ42
Inhibition of amyloid β-protein (Aβ)-induced
toxicity
is a promising therapeutic strategy for Alzheimer’s disease
(AD). Previously, we reported that the C-terminal tetrapeptide Aβ(39–42)
is a potent inhibitor of neurotoxicity caused by Aβ42, the form
of Aβ most closely associated with AD. Here, initial structure–activity
relationship studies identified key structural requirements, including
chirality, side-chain structure, and a free N-terminus, which control
Aβ(39–42) inhibitory activity. To elucidate the binding
siteÂ(s) of Aβ(39–42) on Aβ42, we used intrinsic
tyrosine (Y) fluorescence and solution-state NMR. The data suggest
that Aβ(39–42) binds at several sites, of which the predominant
one is located in the N-terminus of Aβ42, in agreement with
recent modeling predictions. Thus, despite the small size of Aβ(39–42)
and the hydrophobic, aliphatic nature of all four side-chains, the
interaction of Aβ(39–42) with Aβ42 is controlled
by specific intermolecular contacts requiring a combination of hydrophobic
and electrostatic interactions and a particular stereochemistry
Innovative Insight into O<sub>2</sub>/N<sub>2</sub> Permeation Behavior through an Ionomer Film in Cathode Catalyst Layers of Polymer Electrolyte Membrane Fuel Cells
It is crucial to clarify the permeation behavior of O2 through the ionomer film for enhancing local O2 transport
in cathodes of fuel cells. However, all existing studies mainly deal
with pure O2 rather than air. Herein, the permeation behavior
of the O2/N2 mixture through the ionomer film
has been well explored in view of molecular bond length variations
by molecular dynamics simulations. The bond lengths for O2 and N2 are shortened under a low hydration level when
permeating through a dense layer with small free voids while no obvious
change occurs at higher hydration. In the bulk ionomer region, O2 molecules residing in water domains are energetically unstable
because the bond lengths deviate far from the equilibrium length;
thus, O2 diffuses through the interfacial or hydrophobic
regions. N2 molecules show similar properties with O2. This study provides a novel perspective on the permeation
behavior of O2 and N2 through the ionomer film,
which definitely benefits enhancing local O2 transport
Peptide Triazole Inactivators of HIV‑1 Utilize a Conserved Two-Cavity Binding Site at the Junction of the Inner and Outer Domains of Env gp120
We
used coordinated mutagenesis, synthetic design, and flexible
docking to investigate the structural mechanism of Env gp120 encounter
by peptide triazole (PT) inactivators of HIV-1. Prior results demonstrated
that the PT class of inhibitors suppresses binding at both CD4 and
coreceptor sites on Env and triggers gp120 shedding, leading to cell-independent
irreversible virus inactivation. Despite these enticing anti-HIV-1
phenotypes, structural understanding of the PT–gp120 binding
mechanism has been incomplete. Here we found that PT engages two inhibitor
ring moieties at the junction between the inner and outer domains
of the gp120 protein. The results demonstrate how combined occupancy
of two gp120 cavities can coordinately suppress both receptor and
coreceptor binding and conformationally entrap the protein in a destabilized
state. The two-cavity model has common features with small molecule
gp120 inhibitor binding sites and provides a guide for further design
of peptidomimetic HIV-1 inactivators based on the PT pharmacophore
Covalent Conjugation of a Peptide Triazole to HIV‑1 gp120 Enables Intramolecular Binding Site Occupancy
The HIV-1 gp120 glycoprotein is the
main viral surface protein
responsible for initiation of the entry process and, as such, can
be targeted for the development of entry inhibitors. We previously
identified a class of broadly active peptide triazole (PT) dual antagonists
that inhibit gp120 interactions at both its target receptor and coreceptor
binding sites, induce shedding of gp120 from virus particles prior
to host–cell encounter, and consequently can prevent viral
entry and infection. However, our understanding of the conformational
alterations in gp120 by which PT elicits its dual receptor antagonism
and virus inactivation functions is limited. Here, we used a recently
developed computational model of the PT–gp120 complex as a
blueprint to design a covalently conjugated PT–gp120 recombinant
protein. Initially, a single-cysteine gp120 mutant, E275C<sub>YU‑2</sub>, was expressed and characterized. This variant retains excellent
binding affinity for peptide triazoles, for sCD4 and other CD4 binding
site (CD4bs) ligands, and for a CD4-induced (CD4i) ligand that binds
the coreceptor recognition site. In parallel, we synthesized a PEGylated
and biotinylated peptide triazole variant that retained gp120 binding
activity. An N-terminally maleimido variant of this PEGylated PT,
denoted AE21, was conjugated to E275C gp120 to produce the AE21–E275C
covalent conjugate. Surface plasmon resonance interaction analysis
revealed that the PT–gp120 conjugate exhibited suppressed binding
of sCD4 and 17b to gp120, signatures of a PT-bound state of envelope
protein. Similar to the noncovalent PT–gp120 complex, the covalent
conjugate was able to bind the conformationally dependent mAb 2G12.
The results argue that the PT–gp120 conjugate is structurally
organized, with an intramolecular interaction between the PT and gp120
domains, and that this structured state embodies a conformationally
entrapped gp120 with an altered bridging sheet but intact 2G12 epitope.
The similarities of the PT–gp120 conjugate to the noncovalent
PT–gp120 complex support the orientation of binding of PT to
gp120 predicted in the molecular dynamics simulation model of the
PT–gp120 noncovalent complex. The conformationally stabilized
covalent conjugate can be used to expand the structural definition
of the PT-induced “off” state of gp120, for example,
by high-resolution structural analysis. Such structures could provide
a guide for improving the subsequent structure-based design of inhibitors
with the peptide triazole mode of action
Disulfide Sensitivity in the Env Protein Underlies Lytic Inactivation of HIV‑1 by Peptide Triazole Thiols
We investigated the mode of action
underlying lytic inactivation
of HIV-1 virions by peptide triazole thiol (PTT), in particular the
relationship between gp120 disulfides and the C-terminal cysteine-SH
required for virolysis. Obligate PTT dimer obtained by PTT SH cross-linking
and PTTs with serially truncated linkers between pharmacophore isoleucine–ferrocenyltriazole-proline–tryptophan
and cysteine-SH were synthesized. PTT variants showed loss of lytic
activity but not binding and infection inhibition upon SH blockade.
A disproportionate loss of lysis activity vs binding and infection
inhibition was observed upon linker truncation. Molecular docking
of PTT onto gp120 argued that, with sufficient linker length, the
peptide SH could approach and disrupt several alternative gp120 disulfides.
Inhibition of lysis by gp120 mAb 2G12, which binds at the base of
the V3 loop, as well as disulfide mutational effects, argued that
PTT-induced disruption of the gp120 disulfide cluster at the base
of the V3 loop is an important step in lytic inactivation of HIV-1.
Further, PTT-induced lysis was enhanced after treating virus with
reducing agents dithiothreitol and tris (2-carboxyethyl)Âphosphine.
Overall, the results are consistent with the view that the binding
of PTT positions the peptide SH group to interfere with conserved
disulfides clustered proximal to the CD4 binding site in gp120, leading
to disulfide exchange in gp120 and possibly gp41, rearrangement of
the Env spike, and ultimately disruption of the viral membrane. The
dependence of lysis activity on thiol–disulfide interaction
may be related to intrinsic disulfide exchange susceptibility in gp120
that has been reported previously to play a role in HIV-1 cell infection
Design of Cell-Permeable Stapled Peptides as HIV‑1 Integrase Inhibitors
HIV-1 integrase (IN) catalyzes the
integration of viral DNA into
the host genome, involving several interactions with the viral and
cellular proteins. We have previously identified peptide IN inhibitors
derived from the α-helical
regions along the dimeric interface of HIV-1 IN. Herein, we show that
appropriate hydrocarbon stapling of these peptides to stabilize their
helical structure remarkably improves the cell permeability, thus
allowing inhibition of the HIV-1 replication in cell culture. Furthermore,
the stabilized peptides inhibit the interaction of IN with the cellular
cofactor LEDGF/p75. Cellular uptake of the stapled peptide was confirmed
in four different cell lines using a fluorescein-labeled analogue.
Given their enhanced potency and cell permeability, these stapled
peptides can serve as not only lead IN inhibitors but also prototypical
biochemical probes or “nanoneedles” for the elucidation
of HIV-1 IN dimerization and host cofactor interactions within their
native cellular environment
Additional file 3: of Targeted genome engineering in human induced pluripotent stem cells from patients with hemophilia B using the CRISPR-Cas9 system
Figure S1. showing sequencing results of parental and inserted iPSCs. a Parental iPSCs have the known F9 gene mutation c.676C > T, p.Arg226Trp. b Inserted iPSCs (colony 5) have a heterozygous mutation of c.676C > T, p.Arg226Trp. (DOCX 393 kb
Additional file 7: of Targeted genome engineering in human induced pluripotent stem cells from patients with hemophilia B using the CRISPR-Cas9 system
Figure S4. showing characterization of hepatocytic functions. Differentiated cells had functions of glycogen storage (a) and ICG uptake (b), and also expressed LDL-receptor (c) and had ability for LDL uptake (d). All scale bars represent 100 ĂŽĹşm. (DOCX 1747 kb
Additional file 1: of Targeted genome engineering in human induced pluripotent stem cells from patients with hemophilia B using the CRISPR-Cas9 system
Table S1. presenting antibodies used for immunofluorescence staining and flow cytometry analysis. (DOCX 14 kb