Validation and characterisation of a novel peptide that binds monomeric and aggregated beta-amyloid and inhibits the formation of neurotoxic oligomers

Although the formation of β-amyloid (Aβ) deposits in the brain is a hallmark of Alzheimer disease (AD), the soluble oligomers rather than the mature amyloid fibrils most likely contribute to Aβ toxicity and neurodegeneration. Thus, the discovery of agents targeting soluble Aβ oligomers is highly desirable for early diagnosis prior to the manifestation of a clinical AD phenotype and also more effective therapies. We have previously reported that a novel 15-amino acid peptide (15-mer), isolated via phage display screening, targeted Aβ and attenuated its neurotoxicity (Taddei, K., Laws, S. M., Verdile, G., Munns, S., D'Costa, K., Harvey, A. R., Martins, I. J., Hill, F., Levy, E., Shaw, J. E., and Martins, R. N. (2010) Neurobiol. Aging 31, 203–214). The aim of the current study was to generate and biochemically characterize analogues of this peptide with improved stability and therapeutic potential. We demonstrated that a stable analogue of the 15-amino acid peptide (15M S.A.) retained the activity and potency of the parent peptide and demonstrated improved proteolytic resistance in vitro (stable to t = 300 min, c.f. t = 30 min for the parent peptide). This candidate reduced the formation of soluble Aβ42 oligomers, with the concurrent generation of non-toxic, insoluble aggregates measuring up to 25–30 nm diameter as determined by atomic force microscopy. The 15M S.A. candidate directly interacted with oligomeric Aβ42, as shown by coimmunoprecipitation and surface plasmon resonance/Biacore analysis, with an affinity in the low micromolar range. Furthermore, this peptide bound fibrillar Aβ42 and also stained plaques ex vivo in brain tissue from AD model mice. Given its multifaceted ability to target monomeric and aggregated Aβ42 species, this candidate holds promise for novel preclinical AD imaging and therapeutic strategies

The Use of Phage-Displayed Peptide Libraries to Develop Tumor-Targeting Drugs

Monoclonal antibodies have been successfully utilized as cancer-targeting therapeutics and diagnostics, but the efficacies of these treatments are limited in part by the size of the molecules and non-specific uptake by the reticuloendothelial system. Peptides are much smaller molecules that can specifically target cancer cells and as such may alleviate complications with antibody therapy. Although many endogenous and exogenous peptides have been developed into clinical therapeutics, only a subset of these consists of cancer-targeting peptides. Combinatorial biological libraries such as bacteriophage-displayed peptide libraries are a resource of potential ligands for various cancer-related molecular targets. Target-binding peptides can be affinity selected from complex mixtures of billions of displayed peptides on phage and further enriched through the biopanning process. Various cancer-specific ligands have been isolated by in vitro, in vivo, and ex vivo screening methods. As several peptides derived from phage-displayed peptide library screenings have been developed into therapeutics in current clinical trials, which validates peptide-targeting potential, the use of phage display to identify cancer-targeting therapeutics should be further exploited

Structure-Based Design of Non-Natural Amino Acid Inhibitors of Amyloid Fibrillation

Many globular and natively disordered proteins can convert into amyloid fibers. These fibers are associated with numerous pathologies1 as well as with normal cellular functions2,3, and frequently form during protein denaturation4,5. Inhibitors of pathological amyloid fibers could serve as leads for therapeutics, provided the inhibitors were specific enough to avoid interfering with normal processes. Here we show that computer-aided, structure-based design can yield highly specific peptide inhibitors of amyloid formation. Using known atomic structures of segments of amyloid fibers as templates, we have designed and characterized an all D-amino acid inhibitor of fibrillation of the tau protein found in Alzheimer’s disease, and a non-natural L-amino acid inhibitor of an amyloid fiber that enhances sexual transmission of HIV. Our results indicate that peptides from structure-based designs can disrupt the fibrillation of full-length proteins, including those like tau that lack fully ordered native structures.We thank M.I. Ivanova, J. Corn, T. Kortemme, D. Anderson, M.R. Sawaya, M. Phillips, S. Sambashivan, J. Park, M. Landau, Q. Zhang, R. Clubb, F. Guo, T. Yeates, J. Nowick, J. Zheng, and M.J. Thompson for discussions, HHMI, NIH, NSF, the GATES foundation, and the Joint Center for Translational Medicine for support, R. Peterson for help with NMR experiments, E. Mandelkow for providing tau constructs, R. Riek for providing amyloid beta, J. Stroud for amyloid beta preparation. Support for JK was from the Damon Runyon Cancer Research Foundation, for HWC by the Ruth L. Kirschstein National Research Service Award, for JM from the programme for junior-professors by the ministry of science, Baden-Württemberg, and for SAS by a UCLA-IGERT bioinformatics traineeship

Insights into Human Lck SH3 Domain Binding Specificity: Different Binding Modes of Artificial and Native Ligands

We analyzed the ligand binding specificity of the lymphocyte specific kinase (Lck) SH3 domain. We identified artificial Lck SH3 ligands using phage display. In addition, we analyzed Lck SH3 binding sites within known natural Lck SH3 binding proteins using an Lck specific binding assay on membrane-immobilized synthetic peptides. On one hand, from the phage-selected peptides, representing mostly special class I' ligands, a well-defined consensus sequence was obtained. Interestingly, a histidine outside the central polyproline motif contributes significantly to Lck SH3 binding affinity and specificity. On the other hand, we confirmed previously mapped Lck SH3 binding sites in ADAM15, HS1, SLP76, and NS5A, and identified putative Lck SH3 binding sites of Sam68, FasL, c-Cbl, and Cbl-b. Without exception, the comparatively diverse Lck SH3 binding sites of all analyzed natural Lck SH3 binding proteins emerged as class II proteins. Possible explanations for the observed variations between artificial and native ligands-which are not due to significant K(D) value differences as shown by calculating Lck SH3 affinities of artificial peptide PD1-Y(-3)R as well as for peptides comprising putative Lck SH3 binding sites of NS5A, Sos, and Sam68-are discussed. Our data suggest that phage display, a popular tool for determining SH3 binding specificity, must-at least in the case of Lck-not irrevocably mirror physiologically relevant protein-ligand interactions

Identification of calreticulin as ligand of GABARAP by phage display screening of a peptide library

4-Aminobutyrate type A (GABA(A)) receptor-associated protein (GABARAP) is a ubiquitin-like modifier implicated in the intracellular trafficking of GABA(A) receptors, and belongs to a family of proteins involved in intracellular vesicular transport processes, such as autophagy and intra-Golgi transport. In this article, it is demonstrated that calreticulin is a high affinity ligand of GABARAP. Calreticulin, although best known for its functions as a Ca(2+) -dependent chaperone and a Ca(2+) -buffering protein in the endoplasmic reticulum, is also localized to the cytosol and exerts a variety of extra-endoplasmic reticulum functions. By phage display screening of a randomized peptide library, peptides that specifically bind GABARAP were identified. Their amino acid sequences allowed us to identify calreticulin as a potential GABARAP binding protein. GABARAP binding to calreticulin was confirmed by pull-down experiments with brain lysate and colocalization studies in N2a cells. Calreticulin and GABARAP interact with a dissociation constant K(d) = 64 nm and a mean lifetime of the complex of 20 min. Thus, the interaction between GABARAP and calreticulin is the strongest so far reported for each protein

Purification of recombinantly expressed and cytotoxic human amyloid-beta peptide

The amyloid cascade hypothesis assigns the amyloid-beta peptide (Abeta) a central role in the pathogenesis of Alzheimer's disease (AD). Although there are strong efforts to biophysically characterize formation of Abeta aggregates and fibrils, as well as their prevention, progress is still severly hampered by the availability of tens of milligrams of recombinant Abeta(1-42). Here, we describe a reliable and easy procedure to recombinantly express and purify Abeta(1-42), which is fully cytotoxic and able to form fibrils without any further refolding steps. The yield of the procedure is 5-8 mg of tag-less peptide per liter culture volume

Expression, purification and membrane reconstitution of a CD4 fragment comprising the transmembrane and cyctoplasmic domains of the receptor

The transmembrane glycoprotein CD4 plays a prominent role in the adaptive immune response. CD4 is displayed primarily on the surface of T helper cells, but also on subsets of memory and regulatory T lymphocytes, macrophages, and dendritic cells. Binding of the lymphocyte specific tyrosine kinase p56(lck) to the cytoplasmic domain of CD4 is crucial for antigen receptor-mediated signal transduction. The human immunodeficiency virus (HIV) utilizes CD4 as the main receptor for T cell invasion. The virus has developed multiple strategies for down-regulation of CD4 in infected cells. Physical interactions of viral proteins VpU and Nef with the cytoplasmic tail of CD4 initiate a cascade of events leading to degradation of CD4. Here we report heterologous expression and purification of a CD4 fragment comprising the transmembrane and cytoplasmic domains of human CD4. A synthetic gene encoding CD4 amino acid residues 372-433 and a protease cleavage site was cloned into the pTKK19xb/ub plasmid. The CD4 fragment was expressed in Escherichia coli C43(DE3) cells as a ubiquitin fusion with an N-terminal His-tag, isolated, released by PreScission proteolytic cleavage, and purified to homogeneity. Incorporation of the recombinant CD4 fragment in lipid membranes and physical interaction with the cytoplasmic domain of VpU was demonstrated by centrifugation assays followed by reversed phase chromatographic analysis of the composition of the proteoliposomes. A high resolution NMR spectrum of uniformly (15)N-labeled CD4 peptide in membrane simulating micelles proves the possibility of solution NMR studies of this CD4 fragment and of its molecular complexes