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 HIV‑1 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 “F43 pocket” of gp120 in a bridging sheet naïve gp120 conformation of the glycoprotein led to a stable complex. This pose prevents formation of the bridging sheet minidomain, which is required for receptor–coreceptor binding, providing a mechanistic basis for dual-site antagonism of this class of inhibitors. Burial of the peptide triazole at the gp120 inner domain–outer 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)­pyrrolidine] tripartite hydrophobic motif in the peptide is the major contributor of contacts at the gp120–PT interface. Because the model predicts that the peptide Trp side chain hydrogen bonding with gp120 S375 contributes to the stability of the PT–gp120 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 PT–gp120 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 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_YU‑2, 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