The anti-apoptotic proteins NAF-1 and iASPP interact to drive apoptosis in cancer cells.

Suppression of apoptosis is a key Hallmark of cancer cells, and reactivation of apoptosis is a major avenue for cancer therapy. We reveal an interaction between the two anti-apoptotic proteins iASPP and NAF-1, which are overexpressed in many types of cancer cells and tumors. iASPP is an inhibitory member of the ASPP protein family, whereas NAF-1 belongs to the NEET 2Fe-2S protein family. We show that the two proteins are stimulated to interact in cells during apoptosis. Using peptide array screening and computational methods we mapped the interaction interfaces of both proteins to residues 764-778 of iASPP that bind to a surface groove of NAF-1. A peptide corresponding to the iASPP 764-780 sequence stabilized the NAF-1 cluster, inhibited NAF-1 interaction with iASPP, and inhibited staurosporine-induced apoptosis activation in human breast cancer, as well as in PC-3 prostate cancer cells in which p53 is inactive. The iASPP 764-780 IC50 value for inhibition of cell death in breast cancer cells was 13 ± 1 µM. The level of cell death inhibition by iASPP 764-780 was altered in breast cancer cells expressing different levels and/or variants of NAF-1, indicating that the peptide activity is associated with NAF-1 function. We propose that the interaction between iASPP and NAF-1 is required for apoptosis activation in cancer cells. This interaction uncovers a new layer in the highly complex regulation of cell death in cancer cells and opens new avenues of exploration into the development of novel anticancer drugs that reactivate apoptosis in malignant tumors.

Covalent Inhibition of HIV-1 Integrase by N-Succinimidyl Peptides.

We present a new approach for the covalent inhibition of HIV-1 integrase (IN) by an LEDGF/p75-derived peptide modified with an N-terminal succinimide group. The covalent inhibition is mediated by direct binding of the succinimide to the amine group of a lysine residue in IN. The peptide serves as a specific recognition sequence for the target protein, while the succinimide serves as the binding moiety. The combination of a readily synthesizable peptide precursor with easy and efficient binding to the target protein makes this approach a promising new strategy for designing lead compounds.

An Intrinsically Disordered Region in the Proapoptotic ASPP2 Protein Binds to the Helicobacter pylori Oncoprotein CagA.

The leading risk factor for gastric cancer in humans is infection by Helicobacter pylori strains that express and translocate the oncoprotein CagA into host epithelial cells. Once inside host cells, CagA interacts with ASPP2, which specifically stimulates p53-mediated apoptosis and reverses its pro-apoptotic function to promote ASPP2-dependent degradation of p53. The X-ray crystal structure of a complex between the N-terminal domain of CagA and a 56-residue fragment of ASPP2, of which 22 residues were resolved, was recently described. Here, we present biochemical and biophysical analyses of the interaction between the additional regions of CagA and ASPP2 potentially involved in this interaction. Using size exclusion chromatography-multiangle laser light scattering, circular dichroism, and nuclear magnetic resonance analyses, we observed that the ASPP2 region spanning residues 331-692, which was not part of the ASPP2 fragment used for crystallization, is intrinsically disordered in its unbound state. By surface plasmon resonance analysis and isothermal titration calorimetry, we found that a portion of this disordered region in ASPP2, residues 448-692, binds to the N-terminal domain of CagA. We also measured the affinity of the complex between the ASPP2 fragment composed of residues 693-918 and inclusive of the fragment used for crystallization and CagA. Additionally, we mapped the binding regions between ASPP2 and CagA using peptide arrays, demonstrating interactions between CagA and numerous peptides distributed throughout the ASPP2 protein sequence. Our results identify previously uncharacterized regions distributed throughout the protein sequence of ASPP2 as determinants of CagA binding, providing mechanistic insight into apoptosis reprogramming by CagA and potential new drug targets for H. pylori-mediated gastric cancer.

Identifying protein-protein interaction sites using peptide arrays.

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may result in disease, making protein interactions important drug targets. It is thus highly important to understand these interactions at the molecular level. Protein interactions are studied using a variety of techniques ranging from cellular and biochemical assays to quantitative biophysical assays, and these may be performed either with full-length proteins, with protein domains or with peptides. Peptides serve as excellent tools to study protein interactions since peptides can be easily synthesized and allow the focusing on specific interaction sites. Peptide arrays enable the identification of the interaction sites between two proteins as well as screening for peptides that bind the target protein for therapeutic purposes. They also allow high throughput SAR studies. For identification of binding sites, a typical peptide array usually contains partly overlapping 10-20 residues peptides derived from the full sequences of one or more partner proteins of the desired target protein. Screening the array for binding the target protein reveals the binding peptides, corresponding to the binding sites in the partner proteins, in an easy and fast method using only small amount of protein. In this article we describe a protocol for screening peptide arrays for mapping the interaction sites between a target protein and its partners. The peptide array is designed based on the sequences of the partner proteins taking into account their secondary structures. The arrays used in this protocol were Celluspots arrays prepared by INTAVIS Bioanalytical Instruments. The array is blocked to prevent unspecific binding and then incubated with the studied protein. Detection using an antibody reveals the binding peptides corresponding to the specific interaction sites between the proteins.

Characterization of the translocation-competent complex between the Helicobacter pylori oncogenic protein CagA and the accessory protein CagF.

CagA is a virulence factor that Helicobacter pylori inject into gastric epithelial cells through a type IV secretion system where it can cause gastric adenocarcinoma. Translocation is dependent on the presence of secretion signals found in both the N- and C-terminal domains of CagA and an interaction with the accessory protein CagF. However, the molecular basis of this essential protein-protein interaction is not fully understood. Herein we report, using isothermal titration calorimetry, that CagA forms a 1:1 complex with a monomer of CagF with nM affinity. Peptide arrays and isothermal titration calorimetry both show that CagF binds to all five domains of CagA, each with µM affinity. More specifically, a coiled coil domain and a C-terminal helix within CagF contacts domains II-III and domain IV of CagA, respectively. In vivo complementation assays of H. pylori with a double mutant, L36A/I39A, in the coiled coil region of CagF showed a severe weakening of the CagA-CagF interaction to such an extent that it was nearly undetectable. However, it had no apparent effect on CagA translocation. Deletion of the C-terminal helix of CagF also weakened the interaction with CagA but likewise had no effect on translocation. These results indicate that the CagA-CagF interface is distributed broadly across the molecular surfaces of these two proteins to provide maximal protection of the highly labile effector protein CagA.