A peptide strategy for inhibiting different protein aggregation pathways.

Protein aggregation correlates with many human diseases. Protein aggregates differ in structure and shape. Strategies to develop effective aggregation inhibitors that reach the clinic failed so far. Here, we developed a family of peptides targeting early aggregation stages for both amorphous and fibrillar aggregates of proteins unrelated in sequence and structure. They act on dynamic precursors before mechanistic differentiation takes place. Using peptide arrays, we first identified peptides inhibiting the amorphous aggregation of a molten globular, aggregation-prone mutant of the Axin tumor suppressor. Optimization revealed that the peptides activity did not depend on their sequences but rather on their molecular determinants: a composition of 20-30% flexible, 30-40% aliphatic and 20-30% aromatic residues, a hydrophobicity/hydrophilicity ratio close to 1, and an even distribution of residues of different nature throughout the sequence. The peptides also suppressed fibrillation of Tau, a disordered protein that forms amyloids in Alzheimer's disease, and slowed down that of Huntingtin Exon1, an amyloidogenic protein in Huntington's disease, both entirely unrelated to Axin. Our compounds thus target early aggregation stages of different aggregation mechanisms, inhibiting both amorphous and amyloid aggregation. Such cross-mechanistic, multi-targeting aggregation inhibitors may be lead compounds for developing drug candidates against various protein aggregation diseases.

Targeting Protein Interaction Hotspots Using Structured and Disordered Chimeric Peptide Inhibitors.

The main challenge in inhibiting protein-protein interactions (PPI) for therapeutic purposes is designing molecules that bind specifically to the interaction hotspots. Adding to the complexity, such hotspots can be within both structured and disordered interaction interfaces. To address this, we present a strategy for inhibiting the structured and disordered hotspots of interactions using chimeric peptides that contain both structured and disordered parts. The chimeric peptides we developed are comprised of a cyclic structured part and a disordered part, which target both disordered and structured hotspots. We demonstrate our approach by developing peptide inhibitors for the interactions of the antiapoptotic iASPP protein. First, we developed a structured, α-helical stapled peptide inhibitor, derived from the N-terminal domain of MDM2. The peptide bound two hotspots on iASPP at the low micromolar range and had a cytotoxic effect on A2780 cancer cells with a half-maximal inhibitory concentration (IC50) value of 10 ± 1 µM. We then developed chimeric peptides comprising the structured stapled helical peptide and the disordered p53-derived LinkTer peptide that we previously showed to inhibit iASPP by targeting its disordered RT loop. The chimeric peptide targeted both structured and disordered domains in iASPP with higher affinity compared to the individual structured and disordered peptides and caused cancer cell death. Our strategy overcomes the inherent difficulty in inhibiting the interactions of proteins that possess structured and disordered regions. It does so by using chimeric peptides derived from different interaction partners that together target a much wider interface covering both the structured and disordered domains. This paves the way for developing such inhibitors for therapeutic purposes.

Targeting an Interaction Between Two Disordered Domains by Using a Designed Peptide.

Invited for the cover of this issue is the group of Assaf Friedler at the Hebrew University of Jerusalem. The image depicts the protein-protein interactions reported in this work. Read the full text of the article at 10.1002/chem.202000465.

Multiphosphorylated peptides: importance, synthetic strategies, and applications for studying biological mechanisms.

Unraveling the role of post-translational modification (PTM) patterns is one of the most urgent and unresolved issues facing the scientific community. Attempts to crack the phosphorylation bio-barcode led to significant findings, which suggest that many proteins cannot be regarded as a single entity but exist as several forms which differ in their phosphorylation patterns and their functions. While protein regions that do not contain PTMs can be rather simply mimicked using peptide libraries, heavily phosphorylated regions are much harder to study using the same tools. The differences between the syntheses of simple mono-, di- and tri-phosphopeptides and the synthesis of multiphosphopeptides are dramatic. While simple phosphopeptides can be synthesized using almost standard SPPS strategies, the synthesis of multiphosphopeptides is to date a major synthetic challenge. Synthesis of multiphosphopeptides requires the insertion of several phosphate groups simultaneously or sequentially into various positions on the peptide in the presence of many other potential modification sites. These groups are bulky, unstable and cannot be easily introduced when in close proximity. Moreover, since the same protein region can possess many alternative multiphosphorylation patterns, libraries comprising a large number of peptides with different degrees and positions of phosphorylation are essential. Many strategies have been developed to provide routes to enable the preparation of multiphosphopeptides. These methods are essentially different from the methods used for the preparation of simple phosphopeptides. In this review, we specifically emphasize the challenges and importance of synthesizing multiphosphopeptides and their libraries. The historical perspective and state of the art strategies are described. We demonstrate here how the different synthetic approaches attempt to address the special problems associated with the synthesis of multiphosphopeptides. The advantages and disadvantages of each strategy are discussed in order to provide a roadmap for the synthesis of such libraries. An overview of the existing strategies and some comments regarding future directions are provided. Applications of multiphosphopeptide libraries as tools to study the effect of phosphorylation patterns on the biological function of proteins are also described.

Targeting an Interaction Between Two Disordered Domains by Using a Designed Peptide.

Intrinsically disordered regions in proteins (IDRs) mediate many disease-related protein-protein interactions. However, the unfolded character and continuous conformational changes of IDRs make them difficult to target for therapeutic purposes. Here, we show that a designed peptide based on the disordered p53 linker domain can be used to target a partner IDR from the anti-apoptotic iASPP protein, promoting apoptosis of cancer cells. The p53 linker forms a hairpin-like structure with its two termini in close proximity. We designed a peptide derived from the disordered termini without the hairpin, designated as p53 LinkTer. The LinkTer peptide binds the disordered RT loop of iASPP with the same affinity as the parent p53 linker peptide, and inhibits the p53-iASPP interaction in vitro. The LinkTer peptide shows increased stability to proteolysis, penetrates cancer cells, causes nuclei shrinkage, and compromises the viability of cells. We conclude that a designed peptide comprising only the IDR from a peptide sequence can serve as an improved inhibitor since it binds its target protein without the need for pre-folding, paving the way for therapeutic targeting of IDRs.

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.

Highly homologous proteins exert opposite biological activities by using different interaction interfaces.

We present a possible molecular basis for the opposite activity of two homologues proteins that bind similar ligands and show that this is achieved by fine-tuning of the interaction interface. The highly homologous ASPP proteins have opposite roles in regulating apoptosis: ASPP2 induces apoptosis while iASPP inhibits it. The ASPP proteins are regulated by an autoinhibitory interaction between their Ank-SH3 and Pro domains. We performed a detailed biophysical and molecular study of the Pro - Ank-SH3 interaction in iASPP and compared it to the interaction in ASPP2. We found that iASPP Pro is disordered and that the interaction sites are entirely different: iASPP Ank-SH3 binds iASPP Pro via its fourth Ank repeat and RT loop while ASPP2 Ank-SH3 binds ASPP2 Pro via its first Ank repeat and the n-src loop. It is possible that by using different moieties in the same interface, the proteins can have distinct and specific interactions resulting in differential regulation and ultimately different biological activities.

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.

Azoospermia as a new feature of Fabry disease.

OBJECTIVE: To describe two cases of azoospermia in men with Fabry disease. DESIGN: Case report. SETTING: Centre hospitalier universitaire, maternité Pellegrin, Bordeaux, France. PATIENT(S): Two infertile men with azoospermia and with Fabry disease. INTERVENTION: Testicular biopsies. MAIN OUTCOME MEASURE: Histological and electron microscopy analysis of testicular biopsies. RESULT(S): Testicular biopsies revealed characteristic aspects of trihexosid ceramid deposits in Leydig cells by optical and electronic microscopic analysis. Using testicular sperm extraction and intracytoplasmic sperm injection, sperm retrieval led to pregnancies and deliveries of healthy children. CONCLUSION(S): Azoospermia should be considered as a possible complication of Fabry disease. We recommend a routine sperm analysis in the follow-up of young patients with Fabry disease. Azoospermia was still present after 4 years of agalsidase-beta therapy. Because we do not know the efficacy of agalsidase therapy on the genital involvement in Fabry disease, sperm cryopreservation is recommended.

Chlamydia trachomatis in subfertile couples undergoing an in vitro fertilization program: a prospective study.

OBJECTIVES: The objectives were to estimate the prevalence of Chlamydia trachomatis infection in subfertile couples and to study the relationship between markers of C. trachomatis infection and male infertility as well as pregnancy rates after in vitro fertilization (IVF). STUDY DESIGN: All consecutive couples consulting for infertility and IVF in Pellegrin Hospital were screened for C. trachomatis by direct (PCR test) and serological methods. RESULTS: Two hundred and seventy-seven couples were included in the study (mean age in years: 35 for men, 32 for women; mean duration of infertility: 4 years). The most frequent indication for IVF was tubal factor in 33%, endometriosis in 6%, dysovarian function in 12%, male infertility in 36% and others in 13%. C. trachomatis PCR was positive in 1.2% of men, 95% confidence interval (CI95%): (0.2%; 3.3%) and in 2.7% of women, CI95%: (1.1%; 5.5%). When combining all chlamydial markers, 17.3% of men, CI95%: (12.7%; 22.8%) and 20.4% of women, CI95%: (15.6%; 25.9%) had at least one positive marker. The presence of positive markers was not associated with altered semen characteristics. Couples with positive markers had a pregnancy rate of 23.1% (12 out of 52) compared with 20.2% (24 out of 119) among those with negative markers. CONCLUSION: In this population, the presence of past or current C. trachomatis infection was associated with neither semen characteristics nor outcome of IVF in subfertile couples.