Direct Binding Interaction Research Articles

V-ATPase Membrane Sector Associates with Synaptobrevin to Modulate Neurotransmitter Release

Acidification of synaptic vesicles by the vacuolar proton ATPase is essential for loading with neurotransmitter. Debated findings have suggested that V-ATPase membrane domain (V0) also contributes to Ca(2+)-dependent transmitter release via a direct role in vesicle membrane fusion, but the underlying mechanisms remain obscure. We now report a direct interaction between V0 c-subunit and the v-SNARE synaptobrevin, constituting a molecular link between the V-ATPase and SNARE-mediated fusion. Interaction domains were mapped to the membrane-proximal domain of VAMP2 and the cytosolic 3.4 loop of c-subunit. Acute perturbation of this interaction with c-subunit 3.4 loop peptides did not affect synaptic vesicle proton pump activity, but induced a substantial decrease in neurotransmitter release probability, inhibiting glutamatergic as well as cholinergic transmission in cortical slices and cultured sympathetic neurons, respectively. Thus, V-ATPase may ensure two independent functions: proton transport by a fully assembled V-ATPase and a role in SNARE-dependent exocytosis by the V0 sector.

Identification of a new mechanism for targeting myosin II heavy chain phosphorylation by Dictyostelium myosin heavy chain kinase B

BackgroundHeavy chain phosphorylation plays a central role in regulating myosin II bipolar filament assembly in Dictyostelium, as well as in higher eukaryotic nonmuscle cells. Our previous work has demonstrated that the WD-repeat domain of Dictyostelium myosin II heavy chain kinase B (MHCK-B), unlike its counterpart in MHCK-A, is not absolutely required for targeting of the kinase to phosphorylate MHC. Thus, we tested the hypothesis that an asparagine-rich and structurally disordered region that is unique to MHCK-B can by itself function in substrate targeting.FindingsBiochemical assays comparing the activities of full-length MHCK-B, a truncation lacking only the WD-repeat domain (B-Δ-WD), and a truncation lacking both the N-rich region and the WD-repeat domain (B-Δ-N-WD) revealed that the N-rich region targets MHCK-B to phosphorylate MHC in a manner that leads to bipolar filament disassembly. This targeting is physiologically relevant since cellular over-expression of the B-Δ-WD truncation, but not the B-Δ-N-WD truncation, leads to dramatically reduced levels of myosin II filament assembly and associated defects in cytokinesis and multicellular development.ConclusionsThe results presented here demonstrate that an intrinsically unstructured, and asparagine-rich, region of a MHCK-B can mediate specific targeting of the kinase to phosphorylate myosin II heavy chain. This targeting involves a direct binding interaction with myosin II filaments. In terms of regulating myosin bipolar filament assembly, our results suggest that factors affecting the activity of this unique region of MHCK-B could allow for regulation of MHCK-B in a manner that is distinct from the other MHCKs in Dictyostelium.

Binding of a potent small-molecule inhibitor of six-helix bundle formation requires interactions with both heptad-repeats of the RSV fusion protein

Six-helix bundle (6HB) formation is an essential step for many viruses that rely on a class I fusion protein to enter a target cell and initiate replication. Because the binding modes of small molecule inhibitors of 6HB formation are largely unknown, precisely how they disrupt 6HB formation remains unclear, and structure-based design of improved inhibitors is thus seriously hampered. Here we present the high resolution crystal structure of TMC353121, a potent inhibitor of respiratory syncytial virus (RSV), bound at a hydrophobic pocket of the 6HB formed by amino acid residues from both HR1 and HR2 heptad-repeats. Binding of TMC353121 stabilizes the interaction of HR1 and HR2 in an alternate conformation of the 6HB, in which direct binding interactions are formed between TMC353121 and both HR1 and HR2. Rather than completely preventing 6HB formation, our data indicate that TMC353121 inhibits fusion by causing a local disturbance of the natural 6HB conformation.

Abstract C174: Therapeutic reactivation of p53 through dual targeting of HDM2 and HDMX with a stapled p53 peptide

Abstract p53 is a transcription factor that induces cell cycle arrest or apoptosis in response to DNA damage and cellular stress, and thereby plays a critical role in protecting cells from malignant transformation. The E3 ubiquitin ligase HDM2 controls p53 levels through a direct binding interaction that neutralizes the transactivation activity of p53 and targets it for degradation via the ubiquitylation-proteasomal pathway. Whereas the HDM2-homologue HDMX lacks ubiquitin ligase function, it participates in regulating the p53 axis by heterodimerizing with HDM2 to form an active E3 ligase and/or by binding to p53 to prevent it from functioning as a transcriptional regulator. Loss of p53 activity, either by deletion, mutation, or HDM2/HDMX overexpression, is the most common defect in human cancer. Tumors expressing wild type p53 are rendered vulnerable by pharmacologic approaches that stabilize and upregulate p53. In this context, HDM2 and HDMX have emerged as independent therapeutic targets for restoring p53 activity and resensitizing cancer cells to apoptosis in vitro and in vivo. The small molecule nutlin-3 is an effective antagonist of the p53-HDM2 interaction. However, several studies have demonstrated the inability of nutlin-3 to disrupt the p53-HDMX complex, rendering tumor cells that overexpress HDMX nutlin-3-resistant. We have previously described the synthesis and characterization of a hydrocarbon-stapled α-helical p53 peptide (SAH-p53-8) that binds HDM2 with low nanomolar affinity, targets HDM2 in situ, and reactivates the p53 tumor suppressor pathway in HDM2overexpressing osteosarcoma cells. We now report that SAH-p53-8 binds HDMX with even greater affinity, co-immunoprecipitates with endogenous HDMX, and induces p53-dependent apoptosis in nutlin-resistant cancer cells that overexpress HDMX. Thus, by inserting a chemical staple into a peptide fragment of the p53 transactivation domain, we have generated the first bifunctional HDM2/HDMX inhibitor to investigate and modulate both targets in cancer cells. We further report a novel mechanism to maximize pharmacological activity through combined use of HDM2-specific and bifunctional HDM2-HDMX antagonists. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C174.

Abstract 4919: Contradictive Estrogen Effects on BMP Signaling in Pulmonary Artery Depend on Oxygen Concentration

Epidemiologic studies have shown that the female predominance in the morbidity of Idiopathic PAH (pulmonary arterial hypertension) in the world, which suggests a role of female sex hormones in the pathogenesis of PAH. Our previous study demonstrated the contradictory effects of estrogen on pulmonary arterial endothelial cells (PAEC) via BMP (Bone Morphogenetic Protein) signaling depend on cellular oxygen environment. This study is to investigate the mechanism of the contradictory effect of β -estradiol (E2) upon BMP signaling in PAEC. <MATERIALS and METHODS> Human and rat PAEC were cultured and the expression of BMPR2, Smad1/5/8, and Id1 were examined under 21%O 2 (normoxic; N) or 1%O 2 (hypoxic; H) condition in the presence of E2. We investigated the effect of estrogen receptor antagonist (ICI 182.780.) and searched for an evidence of binding interaction between estrogen receptor (ER) and Smad. In the presence of E2 (10 −7 M), the expression of BMPR2, phosphorylated Smad (p-Smad)1/5/8 proteins, Id1 mRNA was augmented under N and suppressed under H. We confirmed same phenomena by using Id1 promoter assay. These alterations of BMPR signal expression were inhibited by ICI182.780. An Immunoprecipitation experiment demonstrated a direct binding interaction between ER and p-Smad1/5/8 proteins only under H, whereas estrogen response activity measured by luciferase assay was increased by E2 only under N. In addition, HIF-1 α augmentation by cobalt chloride suppressed BMP signaling, whereas HIF-1 α inhibition by YC-1 augmented those expression. By inhibiting de novo synthesis by administration of cycloheximide, p-Smad1/5/8 were suppressed under N, but could not change under H. We demonstrated that the alteration of BMP signaling in PAEC under different O 2 condition was associated with HIF-1 α expression and the presence of E2. This alteration may also relate to an interaction between BMP signaling and receptor-mediated estrogen pathway under hypoxia, and to de novo synthesis under normoxia. Our observations provide the new mechanism how sex hormone affects on BMP signaling, a key signal pathway for PAH, which can offer novel therapeutic targets in the treatment of PAH.

Cell Adhesion to Tropoelastin Is Mediated via the C-terminal GRKRK Motif and Integrin αVβ3

Elastin fibers are predominantly composed of the secreted monomer tropoelastin. This protein assembly confers elasticity to all vertebrate elastic tissues including arteries, lung, skin, vocal folds, and elastic cartilage. In this study we examined the mechanism of cell interactions with recombinant human tropoelastin. Cell adhesion to human tropoelastin was divalent cation-dependent, and the inhibitory anti-integrin alpha(V)beta(3) antibody LM609 inhibited cell spreading on tropoelastin, identifying integrin alpha(V)beta(3) as the major fibroblast cell surface receptor for human tropoelastin. Cell adhesion was unaffected by lactose and heparin sulfate, indicating that the elastin-binding protein and cell surface glycosaminoglycans are not involved. The C-terminal GRKRK motif of tropoelastin can bind to cells in a divalent cation-dependent manner, identifying this as an integrin binding motif required for cell adhesion.

A Stapled p53 Helix Targets HDMX to Overcome Nutlin-3 Resistance and Reactivate the p53 Tumor Suppressor Pathway in Cancer

p53 is a transcription factor that induces cell cycle arrest or apoptosis in response to DNA damage and cellular stress, and thereby plays a critical role in protecting cells from malignant transformation. The E3 ubiquitin ligase HDM2 controls p53 levels through a direct binding interaction that neutralizes the transactivation activity of p53 and targets it for degradation via the ubiquitylation-proteasomal pathway. Whereas the HDM2-homologue HDMX lacks ubiquitin ligase function, it participates in regulating the p53 axis by heterodimerizing with HDM2 and sequestering p53 through protein interaction. Loss of p53 activity, either by deletion, mutation, or HDM2/HDMX overexpression, is the most common defect in human cancer. Tumors expressing wild type p53 are rendered vulnerable by pharmacologic approaches that stabilize and upregulate p53. In this context, HDM2 and HDMX have emerged as independent therapeutic targets for restoring p53 activity and resensitizing cancer cells to apoptosis in vitro and in vivo. The small molecule nutlin-3 is an effective antagonist of the p53-HDM2 interaction. However, several studies have demonstrated the inability of nutlin-3 to disrupt the p53-HDMX complex, rendering tumor cells that overexpress HDMX nutlin-3-resistant. We have previously described the synthesis and characterization of a hydrocarbon-stapled alpha-helical p53 peptide (SAH-p53-8) that binds HDM2 with low nanomolar affinity, targets HDM2 in situ, and reactivates the p53 tumor suppressor pathway in HDM2-overexpressing osteosarcoma cells. We now report that SAH-p53-8 binds HDMX with even higher affinity, co-immunoprecipitates with endogenous HDMX, and induces apoptosis and cell cycle arrest in nutlin-3-resistant cancer cells that overexpress HDMX. Thus, by inserting a chemical staple into a peptide fragment of the p53 transactivation domain, we have generated the first bifunctional inhibitor of HDM2 and HDMX, enabling the investigation and pharmacologic modulation of both targets in human cancer.

Integration of CREB and bHLH transcriptional signaling pathways through direct heterodimerization of the proteins: Role in muscle and testis development

The cAMP response element binding protein/activating transcription factor (CREB/ATF) family of transcription factors is hormone responsive and critical for nearly all mammalian cell types. The basic helix-loop-helix (bHLH) family of transcription factors is important during the development and differentiation of a wide variety of cell types. Independent studies of the role of the bHLH protein scleraxis in testicular Sertoli cells and paraxis in muscle development using yeast-2-hybrid screens provided the novel observation that bHLH proteins can directly interact with ATF/CREB family members. Analysis of the interactions demonstrated the helix-loop-helix domain of bHLH proteins directly interacts with the leucine zipper (ZIP) region of CREB2/ATF4 to form heterodimers. The direct bHLH-CREB2 binding interactions were supported using co-immunoprecipitation of recombinant proteins. Structural analysis of bHLH and ATF4 heterodimer using previous crystal structures demonstrated the heterodimer likely involves the HLH and Zip domains and has the potential capacity to bind DNA. Transfection assays demonstrated CREB2/ATF4 over-expression blocked stimulatory actions of scleraxis or paraxis. CREB1 inhibited MyoD induced myogenic conversion of C3H10T1/2 cells. CREB2/ATF4 and scleraxis are expressed throughout embryonic and postnatal testis development, with scleraxis specifically expressed in Sertoli cells. ATF4 and scleraxis null mutant mice both had similar adult testis phenotypes of reduced spermatogenic capacity. In summary, bHLH and CREB family members were found to directly heterodimerize and inhibit the actions of bHLH dimers on Sertoli cells and myogenic precursor cells. The observations suggest a mechanism for direct cross-talk between cAMP induced and bHLH controlled cellular differentiation.

Intra and Inter-Molecular Interactions Dictate the Aggregation State of Irinotecan Co-Encapsulated with Floxuridine Inside Liposomes

The inter/intramolecular interactions between drugs (floxuridine, irinotecan) and excipients (copper gluconate, triethanolamine) in the dual-drug liposomal formulation CPX-1 were elucidated in order to identify the physicochemical properties that allow coordinated release of irinotecan and floxuridine and maintenance of the two agents at a fixed, synergistic 1:1 molar ratio. Release of irinotecan and floxuridine from the liposomes was assessed using an in vitro-release assay. Fluorescence, Nuclear Magnetic Resonance spectroscopy (NMR) and UV-Vis were used to characterize the aggregation state of the drugs within the liposomes. Coordinated release of the drugs from liposomes was disrupted by removing copper gluconate. Approximately 45% of the total irinotecan was detectable in the copper-containing CPX-1 formulation by NMR, which decreased to 19% without copper present in the liposomal interior. Formation of higher order, NMR-silent aggregates was associated with slower and uncoordinated irinotecan release relative to floxuridine and loss of the synergistic drug/drug ratio. Solution spectroscopy and calorimetry revealed that while all formulation components were required to achieve the highest solubility of irinotecan, direct drug-excipient binding interactions were absent. Long-range interactions between irinotecan, floxuridine and excipients modulate the aggregation state of irinotecan, allowing for simultaneous release of both drugs from the liposomes.

Breast Cancer Cell Proliferation Is Inhibited by BAD: REGULATION OF CYCLIN D1

Recent investigations suggest that functions of the proapoptotic BCL2 family members, including BAD, are not limited to regulation of apoptosis. Here we demonstrate that BAD inhibits G(1) to S phase transition in MCF7 breast cancer cells independent of apoptosis. BAD overexpression inhibited G(1) transit and cell growth as well as cyclin D1 expression. Inhibition of cyclin D1 expression was mediated through inhibition of transcription activated by AP1. Chromatin immunoprecipitation assays indicated that BAD is localized at the 12-O-tetradecanoylphorbol-13-acetate-response element (TRE) and cAMP-response element (CRE) in the cyclin D1 promoter. This was shown to reflect direct binding interactions of BAD with c-Jun, and this interaction inhibited the activity of AP1 complexes at TRE. BAD did not interact with phosphorylated forms of c-Jun. Our data suggest that inhibitory TRE/CRE-c-Jun-BAD complexes are present at the cyclin D1 promoter in quiescent cells. Estrogen stimulation displaced BAD from TRE/CRE elements in MCF7 cells, whereas BAD overexpression inhibited estrogen-induced cyclin D1 synthesis and cell proliferation. Inhibition of endogenous BAD in MCF7 cells markedly increased the proliferative fraction and DNA synthesis, activated Cdks, and increased cyclin D1 protein levels. This action of BAD required serine residues Ser(75) and Ser(99). Both phosphorylated and unphosphorylated forms of BAD localized to the nuclei of human breast epithelial cells. Thus, we demonstrate a novel role for BAD in cell cycle regulation dependent upon its phosphorylation state and independent of the BAD/BCL2 interaction and apoptosis.

In Vitro Activity of Human β-Defensin 2 againstPseudomonas aeruginosain the Presence of Tear Fluid

Pseudomonas aeruginosa causes vision-threatening keratitis and is difficult to treat due to emerging resistance. Human beta-defensin 2 (hBD-2) is an antimicrobial peptide expressed by ocular surface epithelia with broad-spectrum activity against various pathogens, including P. aeruginosa. The activity of hBD-2 against P. aeruginosa in the presence of human tears or NaCl was studied. In some experiments, tears were heat-inactivated, filtered, and separated into cationic/anionic fractions or mucin MUC5AC was removed by immunoprecipitation before use. Immunoprecipitation was performed to study the interaction between hBD-2 and MUC5AC. hBD-2 activity was reduced by 40 to 90% in the presence of 17.5 to 70% (vol/vol) tears. NaCl reduced hBD-2 activity, but at most it could account for only 36% of the inhibitory effect of tears. Heat inactivation and filtration attenuated the ability of tears to inhibit hBD-2 activity by 65 and 68%, respectively. Anionic tear fractions significantly reduced (86%) the activity of hBD-2, whereas only a 22% reduction was observed with the cationic fractions. In the absence of MUC5AC, the activity of hBD-2 was restored by 64%. Immunoprecipitation studies suggested that the loss of hBD-2 activity in tears is due to a direct binding interaction with MUC5AC. Our data showed that the antimicrobial activity of hBD-2 is sensitive to the presence of human tears and that this is partly due to the salt content and also the presence of MUC5AC. These data cast doubt on the effectiveness of hBD-2 as an antimicrobial peptide, and additional studies are required to conclusively elucidate its role in innate immunity at the ocular surface in vivo.

Assessment of the Roles of Serines 5.43(239) and 5.46(242) for Binding and Potency of Agonist Ligands at the Human Serotonin 5-HT2AReceptor

We assessed the relative importance of two serine residues located near the top of transmembrane helix 5 of the human 5-HT(2A) receptor, comparing the wild type with S5.43(239)A or S5.46(242)A mutations. Using the ergoline lysergic acid diethylamide (LSD), and a series of substituted tryptamine and phenethylamine 5-HT(2A) receptor agonists, we found that Ser5.43(239) is more critical for agonist binding and function than Ser5.46(242). Ser5.43(239) seems to engage oxygen substituents at either the 4- or 5-position of tryptamine ligands and the 5-position of phenylalkylamine ligands. Even when a direct binding interaction cannot occur, our data suggest that Ser5.43(239) is still important for receptor activation. Polar ring-substituted tryptamine ligands also seem to engage Ser5.46(242), but tryptamines lacking such a substituent may adopt an alternate binding orientation that does not engage this residue. Our results are consistent with the role of Ser5.43(239) as a hydrogen bond donor, whereas Ser5.46(242) seems to serve as a hydrogen bond acceptor. These results are consistent with the functional topography and utility of our in silico-activated homology model of the h5-HT(2A) receptor. In addition, being more distal from the absolutely conserved Pro5.50, a strong interaction with Ser5.43(239) may be more effective in straightening the kink in helix 5, a feature that is possibly common to all type A GPCRs that have polar residues at position 5.43.

Ler and H-NS, Regulators Controlling Expression of the Long Polar Fimbriae of Escherichia coli O157:H7

Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 colonizes the human intestine and is responsible for diarrheal outbreaks worldwide. Previously we showed that EHEC produces long polar fimbriae (LPF) and that maximum expression is observed during the exponential phase of growth at 37 degrees C and pH 6.5. In this study, we analyzed the roles of several regulators in the expression of LPF using the beta-galactosidase reporter system, and we found that H-NS functions as a transcriptional silencer while Ler functions as an antisilencer of LPF expression. Interestingly, deletion of the hns and ler genes in EHEC caused constitutive expression of the fusion reporter protein. Semiquantitative reverse transcription (RT)-PCR was also used to analyze LPF expression in the EHEC ler or hns mutant strain. The hns mutant exhibited an increase in lpf mRNA expression, while expression in the ler mutant was decreased, compared to that in the wild-type strain. Using primer extension analysis, we identified two potential transcriptional start sites within the regulatory region of lpf and located consensus hexamers of -10 (CAAGAT) and -35 (TTCAAA), which are commonly found in sigma(70)-dependent promoters. Further, we determined whether H-NS and Ler interact directly with the lpf promoter region by using purified His-tagged proteins and electrophoretic mobility shift assays. Our data are the first to show direct binding interactions between the H-NS and Ler proteins within the regulatory sequence of the lpf operon. Based on the electrophoretic mobility shift assay, RT-PCR, primer extension, and beta-galactosidase assay results, we concluded that the E. coli O157:H7 lpf operon possesses a promoter dependent on sigma(70), that H-NS binds to the regulatory sequence of lpfA and "silences" the transcription of lpf, and that Ler binds to the regulatory sequence and inhibits the action of the H-NS protein.

Regulation of the Pro-apoptotic Scaffolding Protein POSH by Akt

POSH (Plenty of SH3 domains) binds to activated Rac and promotes apoptosis by acting as a scaffold to assemble a signal transduction pathway leading from Rac to JNK activation. Overexpression of POSH induces apoptosis in a variety of cell types, but apoptosis can be prevented by co-expressing the pro-survival protein kinase Akt. We report here that POSH is a direct substrate for phosphorylation by Akt in vivo and in vitro, and we identify a major site of Akt phosphorylation as serine 304 of POSH, which lies within the Rac-binding domain. We further show that phosphorylation of POSH results in a decreased ability to bind activated Rac, as does phosphomimetic S304D and S304E mutation of POSH. S304D mutant POSH also shows a strongly reduced ability to induce apoptosis. These findings identify a novel mechanism by which Akt promotes cell survival.

The Autoregulatory and Transactivating Functions of the Human Cytomegalovirus IE86 Protein Use Independent Mechanisms for Promoter Binding

The functions of the human cytomegalovirus (HCMV) IE86 protein are paradoxical, as it can both activate and repress viral gene expression through interaction with the promoter region. Although the mechanism for these functions is not clearly defined, it appears that a combination of direct DNA binding and protein-protein interactions is involved. Multiple sequence alignment of several HCMV IE86 homologs reveals that the amino acids (534)LPIYE(538) are conserved between all primate and nonprimate CMVs. In the context of a bacterial artificial chromosome (BAC), mutation of both P535 and Y537 to alanines (P535A/Y537A) results in a nonviable BAC. The defective HCMV BAC does not undergo DNA replication, although the P535A/Y537A mutant IE86 protein appears to be stably expressed. The P535A/Y537A mutant IE86 protein is able to negatively autoregulate transcription from the major immediate-early (MIE) promoter and was recruited to the MIE promoter in a chromatin immunoprecipitation (ChIP) assay. However, the P535A/Y537A mutant IE86 protein was unable to transactivate early viral genes and was not recruited to the early viral UL4 and UL112 promoters in a ChIP assay. From these data, we conclude that the transactivation and repressive functions of the HCMV IE86 protein can be separated and must occur through independent mechanisms.

Identification of cyclin D3 as a new interaction partner of lamin A/C

Lamin A/C is a major component of the nuclear lamina. An intact nuclear lamina has been proposed to be necessary for muscle differentiation. Cyclin D3 is known to be upregulated in differentiated muscle cells and to form insoluble complexes with cell-cycle regulatory factors in these cells. We have examined the possibility of direct binding interactions between lamin A/C and cyclin D3 by in vitro binding assays and co-immunoprecipitation studies with muscle cells. Our results indicate that cyclin D3 binds specifically to amino acid residues 383–474 of lamin A/C and associates with lamin A/C in muscle cells. The identification of cyclin D3 as a novel binding partner of lamin A/C has important implications for a role for lamin A/C in muscle differentiation.

Selective Targeting of the p53-hDM2 Interaction Using Hydrocarbon-Stapled p53 Peptides.

p53 is a transcription factor that induces cell cycle arrest and apoptosis in response to DNA damage and cellular stress, and thereby plays a critical role in protecting cells from malignant transformation. The E3 ubiquitin ligase hDM2 controls p53 levels through a direct binding interaction that neutralizes p53 transactivation activity, exports nuclear p53, and targets it for degradation via the ubiquitination-proteasomal pathway. Loss of p53 activity, either by deletion, mutation, or hDM2 overexpression, is the most common defect in human cancer. Tumors with preserved expression of wild type p53 are rendered vulnerable to pharmacologic approaches that stabilize and upregulate p53. In this context, hDM2 inhibition has emerged as a validated approach to restore p53 activity and resensitize cancer cells to apoptosis in vitro and in vivo. The inhibition of the p53-hDM2 interaction has been studied intensively with the goal of developing novel therapeutics for cancer. Here we describe the synthesis and evaluation of hydrocarbon-stapled α-helical peptides based on the transactivation domain of the p53 tumor suppressor protein. Select p53 stapled peptides exhibit subnanomolar binding to hDM2, displaying the highest affinity hDM2 binders reported to date. We find that these structurally-stabilized peptides are resistant to proteolysis and exhibit cell permeability as documented by flow cytometry and confocal microscopy analyses. In vivo binding of p53 stapled peptides to nuclear hDM2 is highly specific as demonstrated by co-immunoprecipitation experiments. In response to stapled peptide treatment, we observe upregulation of p53 and its transcriptional targets, including p21 and Bax. As a consequence, the peptides are capable of activating apoptosis in hDM2-overexpressing tumor cells through the p53 tumor suppressor signaling pathway. Thus, we find that hydrocarbon-stapled p53 peptides can trigger apoptosis in cancer cells by effectively modulating the p53-hDM2 protein interaction.

A Stapled BID BH3 Helix Directly Binds and Activates BAX

BAX is a multidomain proapoptotic BCL-2 family protein that resides in the cytosol until activated by an incompletely understood trigger mechanism, which facilitates BAX translocation to mitochondria and downstream death events. Whether BAX is activated by direct contact with select BH3-only members of the BCL-2 family is highly debated. Here we detect and quantify a direct binding interaction between BAX and a hydrocarbon-stapled BID BH3 domain, which triggers the functional activation of BAX at nanomolar doses in vitro. Chemical reinforcement of BID BH3 alpha helicity was required to reveal the direct BID BH3-BAX association. We confirm the specificity of this BH3 interaction by characterizing a stapled BAD BH3 peptide that interacts with antiapoptotic BCL-X(L) but does not bind or activate BAX. We further demonstrate that membrane targeting of stapled BID BH3 optimizes its ability to activate BAX, supporting a model in which BID directly engages BAX to trigger mitochondrial apoptosis.

Characterization of ExsC and ExsD Self-Association and Heterocomplex Formation

Expression of the Pseudomonas aeruginosa type III secretion system (T3SS) is induced by calcium depletion and is positively regulated by the ExsA transcriptional activator and negatively regulated by the ExsD antiactivator. Under conditions permissive for expression of the T3SS, the negative regulatory activity of ExsD is antagonized by a direct binding interaction with ExsC. In the present study, the ExsC-ExsD binding interaction was characterized. Individually, both ExsC and ExsD form self-associated complexes, as judged by bacterial monohybrid and gel filtration experiments. A mixture of purified ExsC and ExsD readily formed a complex that elutes from gel filtration medium as a single included peak. The calculated molecular weight of the ExsC-ExsD complex is consistent with a complex containing multiple copies of ExsC and ExsD. Isothermic titration calorimetry experiments found formation of the ExsC-ExsD complex to be thermodynamically favorable, with a Kd of approximately 18 nM and a likely binding ratio of 1:1. To identify amino acid residues important for the regulatory activities of ExsC and ExsD, self-association, and complex formation, charged-cluster mutagenesis was performed. Two of the resulting ExsD charged-cluster mutants (DM2 and DM3) demonstrated a hyperrepressive phenotype for expression of the T3SS. By two-hybrid and copurification assays, the DM3 mutant was found to be impaired in its interaction with ExsC. This finding demonstrates that the binding of ExsC to ExsD is required for transcriptional induction of the T3SS under calcium-limiting growth conditions.

P63 mediates survival in squamous cell carcinoma by suppression of p73-dependent apoptosis

We demonstrate that deltaNp63alpha is an essential survival factor in head and neck squamous cell carcinoma (HNSCC) through its ability to suppress p73-dependent apoptosis. Inhibition of endogenous p63 expression by RNAi induces apoptosis selectively in HNSCC cells that overexpress deltaNp63alpha. Knockdown of p63 induces the proapoptotic bcl-2 family members Puma and Noxa, and both their induction and subsequent cell death are p53 independent but require transactivating isoforms of p73. Inhibition of p73-dependent transcription by deltaNp63alpha involves both direct promoter binding and physical interaction with p73. In HNSCC cells lacking endogenous deltaNp63alpha expression, bcl-2 is instead upregulated and can suppress p73-mediated death. Together, these data define a pathway whereby deltaNp63alpha promotes survival in squamous epithelial malignancy by repressing a p73-dependent proapoptotic transcriptional program.