10 research outputs found
A Molecular Dynamics Simulation of Peptide-Triazole HIV Entry Inhibitor Binding to gp120 Hydrophobic Core
Changes in the Transcriptome of Human Astrocytes Accompanying Oxidative Stress-Induced Senescence
Aging is a major risk factor for many neurodegenerative disorders. A key feature of aging biology that may underlie these diseases is cellular senescence. Senescent cells accumulate in tissues with age, undergo widespread changes in gene expression, and typically demonstrate altered, pro-inflammatory profiles. Astrocyte senescence has been implicated in neurodegenerative disease, and to better understand senescence-associated changes in astrocytes, we investigated changes in their transcriptome using RNA sequencing. Senescence was induced in human fetal astrocytes by transient oxidative stress. Brain-expressed genes, including those involved in neuronal development and differentiation, were downregulated in senescent astrocytes. Remarkably, several genes indicative of astrocytic responses to injury were also downregulated, including glial fibrillary acidic protein and genes involved in the processing and presentation of antigens by major histocompatiblity complex class II proteins, while pro-inflammatory genes were upregulated. Overall, our findings suggest that senescence-related changes in the function of astrocytes may impact the pathogenesis of age-related brain disorders
A Model of Peptide Triazole Entry Inhibitor Binding to HIVâ1 gp120 and the Mechanism of Bridging Sheet Disruption
Peptide
triazole (PT) entry inhibitors prevent HIV-1 infection by blocking
the binding of viral gp120 to both the HIV-1 receptor and the coreceptor
on target cells. Here, we used all-atom explicit solvent molecular
dynamics (MD) to propose a model for the encounter complex of the
peptide triazoles with gp120. Saturation transfer difference nuclear
magnetic resonance (STD NMR) and single-site mutagenesis experiments
were performed to test the simulation results. We found that docking
of the peptide to a conserved patch of residues lining the âF43
pocketâ of gp120 in a bridging sheet naiÌve gp120 conformation
of the glycoprotein led to a stable complex. This pose prevents formation
of the bridging sheet minidomain, which is required for receptorâcoreceptor
binding, providing a mechanistic basis for dual-site antagonism of
this class of inhibitors. Burial of the peptide triazole at the gp120
inner domainâouter domain interface significantly contributed
to complex stability and rationalizes the significant contribution
of hydrophobic triazole groups to peptide potency. Both the simulation
model and STD NMR experiments suggest that the I-X-W [where X is (2<i>S</i>,4<i>S</i>)-4-(4-phenyl-1<i>H</i>-1,2,3-triazol-1-yl)Âpyrrolidine]
tripartite hydrophobic motif in the peptide is the major contributor
of contacts at the gp120âPT interface. Because the model predicts
that the peptide Trp side chain hydrogen bonding with gp120 S375 contributes
to the stability of the PTâgp120 complex, we tested this prediction
through analysis of peptide binding to gp120 mutant S375A. The results
showed that a peptide triazole KR21 inhibits S375A with 20-fold less
potency than WT, consistent with predictions of the model. Overall,
the PTâgp120 model provides a starting point for both the rational
design of higher-affinity peptide triazoles and the development of
structure-minimized entry inhibitors that can trap gp120 into an inactive
conformation and prevent infection
Covalent Conjugation of a Peptide Triazole to HIVâ1 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 hostâcell 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 PTâgp120 complex as a
blueprint to design a covalently conjugated PTâgp120 recombinant
protein. Initially, a single-cysteine gp120 mutant, E275C<sub>YUâ2</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 AE21âE275C
covalent conjugate. Surface plasmon resonance interaction analysis
revealed that the PTâgp120 conjugate exhibited suppressed binding
of sCD4 and 17b to gp120, signatures of a PT-bound state of envelope
protein. Similar to the noncovalent PTâgp120 complex, the covalent
conjugate was able to bind the conformationally dependent mAb 2G12.
The results argue that the PTâgp120 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 PTâgp120 conjugate to the noncovalent
PTâgp120 complex support the orientation of binding of PT to
gp120 predicted in the molecular dynamics simulation model of the
PTâgp120 noncovalent complex. The conformationally stabilized
covalent conjugate can be used to expand the structural definition
of the PT-induced âoffâ 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