The oncoproteins MDM2 and MDMX negatively regulate the activity and stability of the tumor suppressor protein p53--a cellular process initiated by MDM2 and/or MDMX binding to the N-terminal ...transactivation domain of p53. MDM2 and MDMX in many tumors confer p53 inactivation and tumor survival, and are important molecular targets for anticancer therapy. We screened a duodecimal peptide phage library against site-specifically biotinylated p53-binding domains of human MDM2 and MDMX chemically synthesized via native chemical ligation, and identified several peptide inhibitors of the p53-MDM2/MDMX interactions. The most potent inhibitor (TSFAEYWNLLSP), termed PMI, bound to MDM2 and MDMX at low nanomolar affinities--approximately 2 orders of magnitude stronger than the wild-type p53 peptide of the same length (ETFSDLWKLLPE). We solved the crystal structures of synthetic MDM2 and MDMX, both in complex with PMI, at 1.6 Å resolution. Comparative structural analysis identified an extensive, tightened intramolecular H-bonding network in bound PMI that contributed to its conformational stability, thus enhanced binding to the 2 oncogenic proteins. Importantly, the C-terminal residue Pro of PMI induced formation of a hydrophobic cleft in MDMX previously unseen in the structures of p53-bound MDM2 or MDMX. Our findings deciphered the structural basis for high-affinity peptide inhibition of p53 interactions with MDM2 and MDMX, shedding new light on structure-based rational design of different classes of p53 activators for potential therapeutic use.
The oncoproteins MDM2 and MDMX negatively regulate the activity and stability of the tumor suppressor protein p53, conferring tumor development and survival. Antagonists targeting the p53-binding ...domains of MDM2 and MDMX kill tumor cells both in vitro and in vivo by reactivating the p53 pathway, promising a class of antitumor agents for cancer therapy. Aided by native chemical ligation and mirror image phage display, we recently identified a D-peptide inhibitor of the p53-MDM2 interaction termed D PMI-α (TNWYANLEKLLR) that competes with p53 for MDM2 binding at an affinity of 219 nM. Increased selection stringency resulted in a distinct D-peptide inhibitor termed D PMI-γ (DWWPLAFEALLR) that binds MDM2 at an affinity of 53 nM. Structural studies coupled with mutational analysis verified the mode of action of these D-peptides as MDM2-dependent p53 activators. Despite being resistant to proteolysis, both d PMI-α and d PMI-γ failed to actively traverse the cell membrane and, when conjugated to a cationic cell-penetrating peptide, were indiscriminately cytotoxic independently of p53 status. When encapsulated in liposomes decorated with an integrin-targeting cyclic-RGD peptide, however, d PMI-α exerted potent p53-dependent growth inhibitory activity against human glioblastoma in cell cultures and nude mouse xenograft models. Our findings validate D-peptide antagonists of MDM2 as a class of p53 activators for targeted molecular therapy of malignant neoplasms harboring WT p53 and elevated levels of MDM2.
The negative strand of HIV-1 encodes a highly hydrophobic antisense protein (ASP) with no known homologs. The presence of humoral and cellular immune responses to ASP in HIV-1 patients indicates that ...ASP is expressed
, but its role in HIV-1 replication remains unknown. We investigated ASP expression in multiple chronically infected myeloid and lymphoid cell lines using an anti-ASP monoclonal antibody (324.6) in combination with flow cytometry and microscopy approaches. At baseline and in the absence of stimuli, ASP shows polarized subnuclear distribution, preferentially in areas with low content of suppressive epigenetic marks. However, following treatment with phorbol 12-myristate 13-acetate (PMA), ASP translocates to the cytoplasm and is detectable on the cell surface, even in the absence of membrane permeabilization, indicating that 324.6 recognizes an ASP epitope that is exposed extracellularly. Further, surface staining with 324.6 and anti-gp120 antibodies showed that ASP and gp120 colocalize, suggesting that ASP might become incorporated in the membranes of budding virions. Indeed, fluorescence correlation spectroscopy studies showed binding of 324.6 to cell-free HIV-1 particles. Moreover, 324.6 was able to capture and retain HIV-1 virions with efficiency similar to that of the anti-gp120 antibody VRC01. Our studies indicate that ASP is an integral protein of the plasma membranes of chronically infected cells stimulated with PMA, and upon viral budding, ASP becomes a structural protein of the HIV-1 envelope. These results may provide leads to investigate the possible role of ASP in the virus replication cycle and suggest that ASP may represent a new therapeutic or vaccine target.
The HIV-1 genome contains a gene expressed in the opposite, or antisense, direction to all other genes. The protein product of this antisense gene, called ASP, is poorly characterized, and its role in viral replication remains unknown. We provide evidence that the antisense protein, ASP, of HIV-1 is found within the cell nucleus in unstimulated cells. In addition, we show that after PMA treatment, ASP exits the nucleus and localizes on the cell membrane. Moreover, we demonstrate that ASP is present on the surfaces of viral particles. Altogether, our studies identify ASP as a new structural component of HIV-1 and show that ASP is an accessory protein that promotes viral replication. The presence of ASP on the surfaces of both infected cells and viral particles might be exploited therapeutically.
HIV-1 conceals epitopes of its envelope glycoproteins (Env) recognized by antibody (Ab)-dependent cellular cytotoxicity (ADCC)-mediating antibodies. These Abs, including anti-coreceptor binding site ...(CoRBS) and anti-cluster A antibodies, preferentially recognize Env in its "open" conformation. The binding of anti-CoRBS Abs has been shown to induce conformational changes that further open Env, allowing interaction of anti-cluster A antibodies. We explored the possibility that CoRBS Abs synergize with anti-cluster A Abs to engage Fc-gamma receptors to mediate ADCC. We found that binding of anti-CoRBS and anti-cluster A Abs to the same gp120 is required for interaction with soluble dimeric FcγRIIIa in enzyme-linked immunosorbent assays (ELISAs). We also found that Fc regions of both Abs are required to optimally engage FcγRIIIa and mediate robust ADCC. Taken together, our results indicate that these two families of Abs act together in a sequential and synergistic fashion to promote FcγRIIIa engagement and ADCC.
The "open" CD4-bound conformation of HIV-1 envelope glycoproteins is the primary target of antibody-dependent cellular cytotoxicity (ADCC)-mediating antibodies present in HIV-positive (HIV
) sera, such as anti-coreceptor binding site and anti-cluster A antibodies. Here we report that the binding of these two families of antibodies is required to engage FcγRIIIa and mediate ADCC.
To minimize immune responses against infected cells, HIV-1 limits the surface expression of its envelope glycoprotein (Env). Here, we demonstrate that this mechanism is specific for the Env ...conformation and affects the efficiency of antibody-dependent cellular cytotoxicity (ADCC). Using flow cytometry and confocal microscopy, we show that broadly neutralizing antibodies (bNAbs) targeting the "closed" conformation of Env induce its internalization from the surface. In contrast, non-neutralizing antibodies (nNAbs) are displayed on the cell surface for prolonged period of times. The bNAb-induced Env internalization can be decreased by blocking dynamin function, which translates into higher susceptibilities of infected cells to ADCC. Our results suggest that antibody-mediated Env internalization is a mechanism used by HIV-1 to evade immune responses against the "closed" conformation of Env expressed on HIV-1-infected cells.
HIV-1 has evolved to acquire several strategies to limit the exposure of its envelope glycoproteins (Env) on the surface of infected cells. In this study, we show that antibody-induced Env internalization is conformation specific and reduces the susceptibility of infected cells to antibody-dependent cellular cytotoxicity (ADCC). Thus, a better understanding of this mechanism might help develop antibodies with improved capacities to mediate ADCC.
The HIV-1 envelope glycoprotein (Env) undergoes conformational transitions consequent to CD4 binding and coreceptor engagement during viral entry. The physical steps in this process are becoming ...defined, but less is known about their significance as targets of antibodies potentially protective against HIV-1 infection. Here we probe the functional significance of transitional epitope exposure by characterizing 41 human mAbs specific for epitopes exposed on trimeric Env after CD4 engagement. These mAbs recognize three epitope clusters: cluster A, the gp120 face occluded by gp41 in trimeric Env; cluster B, a region proximal to the coreceptor-binding site (CoRBS) and involving the V1/V2 domain; and cluster C, the coreceptor-binding site. The mAbs were evaluated functionally by antibody-dependent, cell-mediated cytotoxicity (ADCC) and for neutralization of Tiers 1 and 2 pseudoviruses. All three clusters included mAbs mediating ADCC. However, there was a strong potency bias for cluster A, which harbors at least three potent ADCC epitopes whose cognate mAbs have electropositive paratopes. Cluster A epitopes are functional ADCC targets during viral entry in an assay format using virion-sensitized target cells. In contrast, only cluster C contained epitopes that were recognized by neutralizing mAbs. There was significant diversity in breadth and potency that correlated with epitope fine specificity. In contrast, ADCC potency had no relationship with neutralization potency or breadth for any epitope cluster. Thus, Fc-mediated effector function and neutralization coselect with specificity in anti-Env antibody responses, but the nature of selection is distinct for these two antiviral activities.
Inhibition of the interaction between the tumor suppressor protein p53 and its negative regulators MDM2 and MDMX is of great interest in cancer biology and drug design. We previously reported a ...potent duodecimal peptide inhibitor, termed PMI (TS
FAEY
WNL
LSP), of the p53–MDM2 and –MDMX interactions. PMI competes with p53 for MDM2 and MDMX binding at an affinity roughly 2 orders of magnitude higher than that of
17–28p53 (ET
FSDL
WKL
LPE) of the same length; both peptides adopt nearly identical α-helical conformations in the complexes, where the three highlighted hydrophobic residues Phe, Trp, and Leu dominate PMI or
17–28p53 binding to MDM2 and MDMX. To elucidate the molecular determinants for PMI activity and specificity, we performed a systematic Ala scanning mutational analysis of PMI and
17–28p53. The binding affinities for MDM2 and MDMX of a total of 35 peptides including 10 truncation analogs were quantified, affording a complete dissection of energetic contributions of individual residues of PMI and
17–28p53 to MDM2 and MDMX association. Importantly, the N8A mutation turned PMI into the most potent dual-specific antagonist of MDM2 and MDMX reported to date, registering respective
K
d values of 490 pM and 2.4 nM. The co-crystal structure of N8A–PMI–
25–109MDM2 was determined at 1.95 Å, affirming that high-affinity peptide binding to MDM2/MDMX necessitates, in addition to optimized intermolecular interactions, enhanced helix stability or propensity contributed by non-contact residues. The powerful empirical binding data and crystal structures present a unique opportunity for computational studies of peptide inhibition of the p53–MDM2/MDMX interactions.
CD4 mimetics sensitize HIV-1-infected cells to ADCC Richard, Jonathan; Veillette, Maxime; Brassard, Nathalie ...
Proceedings of the National Academy of Sciences - PNAS,
05/2015, Letnik:
112, Številka:
20
Journal Article
Recenzirano
Odprti dostop
Significance The prevention of HIV-1 transmission and progression likely requires approaches that can specifically eliminate HIV-1-infected cells. Rationally designed CD4-mimetic compounds (CD4mc) ...have been shown to efficiently inhibit viral entry and sensitize HIV-1 particles to neutralization by otherwise nonneutralizing CD4-induced antibodies. Here we found that CD4mc can also sensitize HIV-1-infected cells to antibody-dependent cell-mediated cytotoxicity (ADCC). Indeed, CD4mc induced the CD4-bound conformation of HIV-1 envelope glycoproteins, exposing CD4-induced epitopes recognized by easy-to-elicit antibodies present in sera, cervicovaginal lavages, and breast milk from HIV-1-infected individuals. Importantly, we provide evidence that CD4mc can efficiently sensitize primary CD4 T cells from HIV-1-infected individuals to ADCC mediated by autologous sera and effector cells. Therefore, CD4mc might represent an attractive approach to prevent and control HIV-1 infection.
HIV-1-infected cells presenting envelope glycoproteins (Env) in the CD4-bound conformation on their surface are preferentially targeted by antibody-dependent cell-mediated cytotoxicity (ADCC). HIV-1 has evolved a sophisticated mechanism to avoid exposure of ADCC-mediating Env epitopes by down-regulating CD4 and by limiting the overall amount of Env at the cell surface. Here we report that small-molecule CD4-mimetic compounds induce the CD4-bound conformation of Env, and thereby sensitize cells infected with primary HIV-1 isolates to ADCC mediated by antibodies present in sera, cervicovaginal lavages, and breast milk from HIV-1-infected individuals. Importantly, we identified one CD4 mimetic with the capacity to sensitize endogenously infected ex vivo-amplified primary CD4 T cells to ADCC killing mediated by autologous sera and effector cells. Thus, CD4 mimetics hold the promise of therapeutic utility in preventing and controlling HIV-1 infection.
Throwing tumors a left‐hook punch: The oncoprotein MDM2 negatively regulates the activity and stability of the tumor suppressor protein p53, and is an important molecular target for anticancer ...therapy. Mirror‐image phage display identified a high‐affinity D‐peptide ligand of MDM2 (see structure) that could be developed into a potent and protease‐resistant p53 activator with potential antitumor activity.
Previous studies have shown that sera from HIV-1-infected individuals contain antibodies able to mediate antibody-dependent cellular cytotoxicity (ADCC). These antibodies preferentially recognize ...envelope glycoprotein (Env) epitopes induced upon CD4 binding. Here, we show that a highly conserved tryptophan at position 69 of the gp120 inner domain is important for ADCC mediated by anti-cluster A antibodies and sera from HIV-1-infected individuals.
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