Stereochemistry-Driven Interactions of α,γ-Peptide Ligands with the Neuropeptide Y Y4-Receptor.

The G-protein-coupled Y4-receptor (Y4R) and its endogenous ligand, pancreatic polypeptide (PP), suppress appetite in response to food intake and, thus, are attractive drug targets for body-weight control. The C-terminus of human PP (hPP), T32-R33-P34-R35-Y36-NH2, penetrates deep into the binding pocket with its tyrosine-amide and di-arginine motif. Here, we present two C-terminally amidated α,γ-hexapeptides (1a/b) with sequence Ac-R31-γ-CBAA32-R33-L34-R35-Y36-NH2, where γ-CBAA is the (1R,2S,3R)-configured 2-(aminomethyl)-3-phenylcyclobutanecarboxyl moiety (1a) or its mirror image (1b). Both peptides bind the Y4R (Ki of 1a/b: 0.66/12 nM) and act as partial agonists (intrinsic activity of 1a/b: 50/39%). Their induced-fit binding poses in the Y4R pocket are unique and build ligand-receptor contacts distinct from those of the C-terminus of the endogenous ligand hPP. We conclude that energetically favorable interactions, although they do not match those of the native ligand hPP, still guarantee high binding affinity (with 1a rivaling hPP) but not the maximum receptor activation.

Backbone distortions in lactam-bridged helical peptides.

Side-chain-to-side-chain cyclization is frequently used to stabilize the α-helical conformation of short peptides. In a previous study, we incorporated a lactam bridge between the side chains of Lys-i and Asp-i+4 in the nonapeptide 1Y, cyclo-(2,6)-(Ac-VKRLQDLQY-NH2 ), an artificial ligand of the inhibitor of DNA binding and cell differentiation (ID) protein with antiproliferative activity on cancer cells. Herein, we show that only the cyclized five-residue segment adopts a helical turn whereas the C-terminal residues remain flexible. Moreover, we present nine 1Y analogs arising from different combinations of hydrophobic residues (leucine, isoleucine, norleucine, valine, and tyrosine) at positions 1, 4, 7, and 9. All cyclopeptides except one build a lactam-bridged helical turn; however, residue-4 reveals less helix character than the neighboring Arg-3 and Gln-5, especially with residue-4 being isoleucine, valine, and tyrosine. Surprisingly, only two cyclopeptides exhibit helix propagation until the C-terminus, whereas the others share a remarkable outward tilting of the backbone carbonyl of the lactam-bridged Asp-6 (>40° deviation from the orientation parallel to the helix axis), which prevents the formation of the H-bond between Arg-3 CO and residue-7 NH: As a result, the propagation of the helix beyond the lactam-bridged sequence becomes unfavorable. We conclude that, depending on the amino-acid sequence, the lactam bridge between Lys-i and Asp-i+4 can stabilize a helical turn but deviations from the ideal helix geometry are possible: Indeed, besides the outward tilting of the backbone carbonyls, the residues per turn increased from 3.6 (typical of a regular α-helix) to 4.2, suggesting a partial helix unwinding.

A Conformationally Stable Acyclic ß-Hairpin Scaffold Tolerating the Incorporation of Poorly ß-Sheet-Prone Amino Acids.

The ß-hairpin is a structural element of native proteins, but it is also a useful artificial scaffold for finding lead compounds to convert into peptidomimetics or non-peptide structures for drug discovery. Since linear peptides are synthetically more easily accessible than cyclic ones, but are structurally less well-defined, we propose XWXWXpPXK(/R)X(R) as an acyclic but still rigid ß-hairpin scaffold that is robust enough to accommodate different types of side chains, regardless of the secondary-structure propensity of the X residues. The high conformational stability of the scaffold results from tight contacts between cross-strand cationic and aromatic side chains, combined with the strong tendency of the d-Pro-l-Pro dipeptide to induce a type II' ß-turn. To demonstrate the robustness of the scaffold, we elucidated the NMR structures and performed molecular dynamics (MD) simulations of a series of peptides displaying mainly non-ß-branched, poorly ß-sheet-prone residues at the X positions. Both the NMR and MD data confirm that our acyclic ß-hairpin scaffold is highly versatile as regards the amino-acid composition of the ß-sheet face opposite to the cationic-aromatic one.

Detecting aspartate isomerization and backbone cleavage after aspartate in intact proteins by NMR spectroscopy.

The monitoring of non-enzymatic post-translational modifications (PTMs) in therapeutic proteins is important to ensure drug safety and efficacy. Together with methionine and asparagine, aspartic acid (Asp) is very sensitive to spontaneous alterations. In particular, Asp residues can undergo isomerization and peptide-bond hydrolysis, especially when embedded in sequence motifs that are prone to succinimide formation or when followed by proline (Pro). As Asp and isoAsp have the same mass, and the Asp-Pro peptide-bond cleavage may lead to an unspecific mass difference of + 18 Da under native conditions or in the case of disulfide-bridged cleavage products, it is challenging to directly detect and characterize such modifications by mass spectrometry (MS). Here we propose a 2D NMR-based approach for the unambiguous identification of isoAsp and the products of Asp-Pro peptide-bond cleavage, namely N-terminal Pro and C-terminal Asp, and demonstrate its applicability to proteins including a therapeutic monoclonal antibody (mAb). To choose the ideal pH conditions under which the NMR signals of isoAsp and C-terminal Asp are distinct from other random coil signals, we determined the pKa values of isoAsp and C-terminal Asp in short peptides. The characteristic 1H-13C chemical shift correlations of isoAsp, N-terminal Pro and C-terminal Asp under standardized conditions were used to identify these PTMs in lysozyme and in the therapeutic mAb rituximab (MabThera) upon prolonged storage under acidic conditions (pH 4-5) and 40 °C. The results show that the application of our 2D NMR-based protocol is straightforward and allows detecting chemical changes of proteins that may be otherwise unnoticed with other analytical methods.

Structural and functional studies of Arabidopsis thaliana legumain beta reveal isoform specific mechanisms of activation and substrate recognition.

The vacuolar cysteine protease legumain plays important functions in seed maturation and plant programmed cell death. Because of their dual protease and ligase activity, plant legumains have become of particular biotechnological interest, e.g. for the synthesis of cyclic peptides for drug design or for protein engineering. However, the molecular mechanisms behind their dual protease and ligase activities are still poorly understood, limiting their applications. Here, we present the crystal structure of Arabidopsis thaliana legumain isoform ß (AtLEGß) in its zymogen state. Combining structural and biochemical experiments, we show for the first time that plant legumains encode distinct, isoform-specific activation mechanisms. Whereas the autocatalytic activation of isoform γ (AtLEGγ) is controlled by the latency-conferring dimer state, the activation of the monomeric AtLEGß is concentration independent. Additionally, in AtLEGß the plant-characteristic two-chain intermediate state is stabilized by hydrophobic rather than ionic interactions, as in AtLEGγ, resulting in significantly different pH stability profiles. The crystal structure of AtLEGß revealed unrestricted nonprime substrate binding pockets, consistent with the broad substrate specificity, as determined by degradomic assays. Further to its protease activity, we show that AtLEGß exhibits a true peptide ligase activity. Whereas cleavage-dependent transpeptidase activity has been reported for other plant legumains, AtLEGß is the first example of a plant legumain capable of linking free termini. The discovery of these isoform-specific differences will allow us to identify and rationally design efficient ligases with application in biotechnology and drug development.

A novel FRET peptide assay reveals efficient Helicobacter pylori HtrA inhibition through zinc and copper binding.

Helicobacter pylori (H. pylori) secretes the chaperone and serine protease high temperature requirement A (HtrA) that cleaves gastric epithelial cell surface proteins to disrupt the epithelial integrity and barrier function. First inhibitory lead structures have demonstrated the essential role of HtrA in H. pylori physiology and pathogenesis. Comprehensive drug discovery techniques allowing high-throughput screening are now required to develop effective compounds. Here, we designed a novel fluorescence resonance energy transfer (FRET) peptide derived from a gel-based label-free proteomic approach (direct in-gel profiling of protease specificity) as a valuable substrate for H. pylori HtrA. Since serine proteases are often sensitive to metal ions, we investigated the influence of different divalent ions on the activity of HtrA. We identified Zn++ and Cu++ ions as inhibitors of H. pylori HtrA activity, as monitored by in vitro cleavage experiments using casein or E-cadherin as substrates and in the FRET peptide assay. Putative binding sites for Zn++ and Cu++ were then analyzed in thermal shift and microscale thermophoresis assays. The findings of this study will contribute to the development of novel metal ion-dependent protease inhibitors, which might help to fight bacterial infections.

Unambiguous Identification of Pyroglutamate in Full-Length Biopharmaceutical Monoclonal Antibodies by NMR Spectroscopy.

Biotherapeutic proteins are an indispensable class of pharmaceuticals that present a high degree of structural complexity and are prone to chemical modifications during production, processing, and storage, which have to be tightly controlled. Pyroglutamate (pGlu), a cyclization product of N-terminal Gln or Glu residues, is a widespread post-translational modification in proteins, including monoclonal antibodies (mAbs). The unambiguous identification and quantification of this modification in proteins is challenging, since the mass difference of -17 Da or -18 Da, when formed from Gln or Glu, respectively, is not unique. Moreover, deamidation and dehydration occur not only during cyclization to pGlu, but also during other reactions leading to different types of modifications, like succinimide or isopeptide bond moieties due to cross-linking between Asn or Gln and Lys side chains. Here we report the unambiguous identification and quantification of pGlu in intact mAbs with natural isotope distribution by NMR spectroscopy. The assignment of all 1H, 13C and 15N random coil chemical shifts of pGlu in short reference peptides led to the identification of unique chemical shift pairs that are distinct from the random coil chemical shifts of the natural amino-acid residues. These characteristic correlations are suited for the detection of pGlu in denatured proteins. We achieved complete denaturation of mAbs using a straightforward protocol, and could detect and quantify pGlu, in agreement with available mass spectrometric data. The application to the mAbs rituximab and adalimumab illustrates the potential of our approach for the characterization of biotherapeutics containing isotopes at natural abundance.

Susceptibility of protein therapeutics to spontaneous chemical modifications by oxidation, cyclization, and elimination reactions.

Peptides and proteins are preponderantly emerging in the drug market, as shown by the increasing number of biopharmaceutics already approved or under development. Biomolecules like recombinant monoclonal antibodies have high therapeutic efficacy and offer a valuable alternative to small-molecule drugs. However, due to their complex three-dimensional structure and the presence of many functional groups, the occurrence of spontaneous conformational and chemical changes is much higher for peptides and proteins than for small molecules. The characterization of biotherapeutics with modern and sophisticated analytical methods has revealed the presence of contaminants that mainly arise from oxidation- and elimination-prone amino-acid side chains. This review focuses on protein chemical modifications that may take place during storage due to (1) oxidation (methionine, cysteine, histidine, tyrosine, tryptophan, and phenylalanine), (2) intra- and inter-residue cyclization (aspartic and glutamic acid, asparagine, glutamine, N-terminal dipeptidyl motifs), and (3) ß-elimination (serine, threonine, cysteine, cystine) reactions. It also includes some examples of the impact of such modifications on protein structure and function.

Multiple roles of Bet v 1 ligands in allergen stabilization and modulation of endosomal protease activity.

BACKGROUND: Over 100 million people worldwide suffer from birch pollen allergy. Bet v 1 has been identified as the major birch pollen allergen. However, the molecular mechanisms of birch allergic sensitization, including the roles of Bet v 1 and other components of the birch pollen extract, remain incompletely understood. Here, we examined how known birch pollen-derived molecules influence the endolysosomal processing of Bet v 1, thereby shaping its allergenicity. METHODS: We analyzed the biochemical and immunological interaction of ligands with Bet v 1. We then investigated the proteolytic processing of Bet v 1 by endosomal extracts in the presence and absence of ligands, followed by a detailed kinetic analysis of Bet v 1 processing by individual endolysosomal proteases as well as the T-cell epitope presentation in BMDCs. RESULTS: We identified E1 phytoprostanes as novel Bet v 1 ligands. Pollen-derived ligands enhanced the proteolytic resistance of Bet v 1, affecting degradation kinetics and preferential cleavage sites of the endolysosomal proteases cathepsin S and legumain. E1 phytoprostanes exhibited a dual role by stabilizing Bet v 1 and inhibiting cathepsin protease activity. CONCLUSION: Bet v 1 can serve as a transporter of pollen-derived, bioactive compounds. When carried to the endolysosome, such compounds can modulate the proteolytic activity, including its processing by cysteine cathepsins. We unveil a paradigm shift from an allergen-centered view to a more systemic view that includes the host endolysosomal enzymes.

An explorative study towards the chemical synthesis of the immunoglobulin G1 Fc CH3 domain.

Monoclonal antibodies, fusion proteins including the immunoglobulin fragment c (Ig Fc) CH2-CH3 domains, and engineered antibodies are prominent representatives of an important class of drugs and drug candidates, which are referred to as biotherapeutics or biopharmaceuticals. These recombinant proteins are highly heterogeneous due to their glycosylation pattern. In addition, enzyme-independent reactions, like deamidation, dehydration, and oxidation of sensitive side chains, may contribute to their heterogeneity in a minor amount. To investigate the biological impact of a spontaneous chemical modification, especially if found to be recurrent in a biotherapeutic, it would be necessary to reproduce it in a homogeneous manner. Herein, we undertook an explorative study towards the chemical synthesis of the IgG1 Fc CH3 domain, which has been shown to undergo spontaneous changes like succinimide formation and methionine oxidation. We used Fmoc-solid-phase peptide synthesis (SPPS) and native chemical ligation (NCL) to test the accessibility of large fragments of the IgG1 Fc CH3 domain. In general, the incorporation of pseudoproline dipeptides improved the quality of the crude peptide precursors; however, sequences larger than 44 residues could not be achieved by standard stepwise elongation with Fmoc-SPPS. In contrast, the application of NCL with cysteine residues, which were either native or introduced ad hoc, allowed the assembly of the C-terminal IgG1 Fc CH3 sequence 371 to 450. The syntheses reported here show advantages and limitations of the chemical approaches chosen for the preparation of the synthetic IgG1 Fc CH3 domain and will guide future plans towards the synthesis of both the native and selectively modified full-length domain.

Structural analyses of Arabidopsis thaliana legumain γ reveal differential recognition and processing of proteolysis and ligation substrates.

Legumain is a dual-function protease-peptide ligase whose activities are of great interest to researchers studying plant physiology and to biotechnological applications. However, the molecular mechanisms determining the specificities for proteolysis and ligation are unclear because structural information on the substrate recognition by a fully activated plant legumain is unavailable. Here, we present the X-ray structure of Arabidopsis thaliana legumain isoform γ (AtLEGγ) in complex with the covalent peptidic Ac-YVAD chloromethyl ketone (CMK) inhibitor targeting the catalytic cysteine. Mapping of the specificity pockets preceding the substrate-cleavage site explained the known substrate preference. The comparison of inhibited and free AtLEGγ structures disclosed a substrate-induced disorder-order transition with synergistic rearrangements in the substrate-recognition sites. Docking and in vitro studies with an AtLEGγ ligase substrate, sunflower trypsin inhibitor (SFTI), revealed a canonical, protease substrate-like binding to the active site-binding pockets preceding and following the cleavage site. We found the interaction of the second residue after the scissile bond, P2'-S2', to be critical for deciding on proteolysis versus cyclization. cis-trans-Isomerization of the cyclic peptide product triggered its release from the AtLEGγ active site and prevented inadvertent cleavage. The presented integrative mechanisms of proteolysis and ligation (transpeptidation) explain the interdependence of legumain and its preferred substrates and provide a rational framework for engineering optimized proteases, ligases, and substrates.

The Recombinant Inhibitor of DNA Binding Id2 Forms Multimeric Structures via the Helix-Loop-Helix Domain and the Nuclear Export Signal.

The inhibitor of DNA binding and cell differentiation 2 (Id2) is a helix-loop-helix (HLH) protein that acts as negative dominant regulator of basic-HLH transcription factors during development and in cancer. The structural properties of Id2 have been investigated so far by using synthetic or recombinant fragments reproducing single domains (N-terminus, HLH, C-terminus): the HLH domain tends to dimerize into a four-helix bundle, whereas the flanking regions are flexible. In this work, the intact protein was expressed in E. coli, solubilized from inclusion bodies with urea, purified and dissolved in water at pH~4. Under these conditions, Id2 was obtained with both cysteine residues disulfide-bonded to ß-mercaptoethanol that was present during the solubilization process. Moreover, it existed in a self-assembled state, in which the N-terminus remained highly flexible, while the HLH domain and, surprisingly, part of the C-terminus, which corresponds to the nuclear export signal (NES), both were involved in slowly tumbling, rigid structures. The protein oligomers also formed twisted fibrils that were several micrometers long and up to 80 nm thick. These results show that self-assembly decreases the backbone flexibility of those two protein regions (HLH and NES) that are important for interaction with basic-HLH transcription factors or for nucleocytoplasmic shuttling.

Crystal Structure of Plant Legumain Reveals a Unique Two-Chain State with pH-Dependent Activity Regulation.

The vacuolar cysteine protease legumain can cleave and selectively rebuild peptide bonds, thereby vastly expanding the sequential repertoire of biomolecules. In this context, plant legumains have recently attracted particular interest. Furthermore, legumains have important roles in many physiological processes, including programmed cell death. Their efficient peptide bond ligase activity has gained tremendous interest in the design of cyclic peptides for drug design. However, the mechanistic understanding of these dual activities is incomplete and partly conflicting. Here, we present the crystal structure of a plant legumain, Arabidopsis thaliana isoform-γ (AtLEGγ). Employing a conserved legumain fold, the plant legumain AtLEGγ revealed unique mechanisms of autoactivation, including a plant-specific two-chain activation state, which remains conformationally stable at neutral pH, which is a prerequisite for full ligase activity and survival in different cell compartments. The charge distribution around the α6-helix mediates the pH-dependent dimerization and serves as a gatekeeper for the active site, thus regulating its protease and ligase activity.

Reduction of cancer cell viability by synergistic combination of photodynamic treatment with the inhibition of the Id protein family.

The inhibitor of DNA binding and cell differentiation (Id) proteins are dominant negative regulators of the helix-loop-helix transcription factor family and play a key role during development as well as in vascular disorders and cancer. In fact, impairing the Id-protein activity in cancer cells reduces cell growth and even chemoresistance. Recently, we have shown that a synthetic Id-protein ligand (1Y) consisting of a cyclic nonapeptide can reduce the viability of the two breast cancer cell lines MCF-7 and T47D and of the bladder cancer cells T24 to about 50% at concentrations ≥100µM. Moreover, the cyclopeptide displays both proapoptotic and antiproliferative effects on MCF-7 cells. Herein, we show that the cyclopeptide does not induce cell death at the dose of 5µΜ, but it still inhibits MCF-7 and T24 cell proliferation, which correlates with an increased protein level of the cell-cycle regulator p27Kip1. Furthermore, 1Y-pretreated MCF-7, T47D, and T24 cells are more susceptible than untreated cells to the phototoxic effects of the three photosensitizers meta-tetra(hydroxyphenyl)chlorin, porfimer sodium, and hypericin, which are applied in photodynamic therapy (PDT). The combination of the Id-protein ligand with each of the light-activated photosensitizers shows synergistic effects on the reduction of cell viability. In conclusion, an Id-protein ligand with moderate cancer cell killing activity at concentrations ≥100µM can be applied at a 20-fold lower and barely toxic dose to raise the sensitivity of cancer cells towards phototoxicity associated with photodynamic treatment. This suggests the potential benefit of targeting the Id proteins in combined drug approaches for cancer therapy.

Complete NMR Assignment of Succinimide and Its Detection and Quantification in Peptides and Intact Proteins.

Detecting and quantifying post-translational modifications (PTMs) in full-length proteins is a challenge, especially in the case of spontaneously occurring, nonenzymatic PTMs. Such a PTM is the formation of succinimide (Snn) in a protein that occurs spontaneously in prone primary sequences and leads typically to an equilibrium between Snn and its hydrolysis products isoaspartate (isoAsp) and aspartate. In order to detect these modifications in proteins by NMR spectroscopy, chemical shift assignments of reference compounds are required. We used peptide synthesis and 2D NMR spectroscopy to assign all 1H and 13C chemical shifts of Snn and isoAsp and found characteristic chemical shift correlations. To provide chemical shift reference data suitable for comparison with data of denatured proteins, we repeated the assignment in 7 M urea (pH 2.3) and in DMSO. Most characteristic of Snn are the two downfield shifted carbonyl chemical shifts, the chemical shift correlations of Cß-Hß of Snn and Cα-Hα of the succeeding residue which are clearly distinct from random coil chemical shift correlations. The characteristic 2D NMR fingerprints of Snn were used to detect and quantify this PTM in the model protein lysozyme, the biotherapeutic filgrastim, and the Fc part of immunoglobulin G1. Mass spectrometry (MS) was applied as an additional independent method. The orthogonality of the NMR and MS techniques allows cross-validation, which is especially important to search for subtle PTMs in proteins. Studying PTMs by NMR spectroscopy is a promising method to analyze proteins and peptides from natural sources, recombinant expression, or chemical synthesis.

Targeting of a Helix-Loop-Helix Transcriptional Regulator by a Short Helical Peptide.

The Id proteins (Id1-4) are cell-cycle regulators that play a key role during development, in cancer and vascular disorders. They contain a conserved helix-loop-helix (HLH) domain that folds into a parallel four-helix bundle upon self- or hetero-association with basic-HLH transcription factors. By using such protein-protein interactions, the Id proteins inhibit cell differentiation and promote cell-cycle progression. Accordingly, their supporting role in cancer has been convincingly demonstrated, which makes these proteins interesting therapeutic targets. Herein we present a short peptide containing an (i,i+4)-lactam bridge and a hydrophobic (Φ) three-residue motif Φ(i)-Φ(i+3)-Φ(i+6), which adopts a helical conformation in water, shows Id protein binding in the low-micromolar range, penetrates into breast (MCF-7 and T47D) and bladder (T24) cancer cells, accumulates in the nucleus, and decreases cell viability to ∼50 %. Thus, this cyclopeptide is a promising scaffold for the development of Id protein binders that impair cancer cell viability.

Visible-light photoredox-catalyzed desulfurization of thiol- and disulfide-containing amino acids and small peptides.

A scalable protocol for the desulfurization of cysteine by using visible light, the photocatalyst Ir(dF(CF3 )ppy)2 (dtb-bpy)PF6 and triethylphosphite under biphasic reaction conditions has been developed. The loading of the catalyst can be as low as 0.01 mol%, which can be efficiently removed during the workup (≤0.3 ppm), giving rise to the corresponding desulfurized product in high yields. This method has been applied also to cystine, penicillamine, and reduced and oxidized glutathione. The desulfurization has been found to be pH sensitive, with an optimal pH value of 6.5 and 7.0 for the cysteine derivatives and glutathione, respectively. In addition, during the desulfurization of a decapeptide containing cysteine and methionine, concurrent oxidation of the two sulfur-containing residues to disulfide and sulfoxide has been observed. Therefore, whereas the presented protocol allows a straightforward visible light-mediated desulfurization of simple thiols by using very low catalyst loading and a cost-effective trialkylphosphite as thiyl radical trapping agent, its application to complex substrates needs to be carefully validated. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Impact of the amino acid sequence on the conformation of side chain lactam-bridged octapeptides.

Synthetic helical peptides are valuable scaffolds for the development of modulators of protein-protein interactions involving helical motifs. Backbone-to-side chain or side chain-to-side chain constraints have been and still are intensively exploited to stabilize short α-helices. Very often, these constraints have been combined with backbone modifications induced by Cα-tetrasubstituted, ß-, or γ-amino acids, which facilitate the α-peptide or α/ß/γ-peptide adopting an α-helical conformation. In this work, we investigated the helical character of octapeptides that were cyclized by a Lys-Asp-(i,i + 4)-lactam bridge. We started with two sequences extracted from the helix-loop-helix region of the Id proteins, which are inhibitors of cell differentiation during development and in cancer. Nineteen analogs containing the lactam bridge at different positions and displaying different amino acid core triads (i + 1,2,3) as well as outer residues were prepared by solid-phase methodology. Their conformation in water and water/2,2,2-trifluoroethanol mixtures was investigated by circular dichroism (CD) spectroscopy. The cyclopeptides could be grouped in helix-prone and non-helix-prone structures. Both the amino acid core triad (i + 1,2,3) and the pendant residues positively or negatively affected the formation of a helical structure. Computational studies based on the NMR-derived helical structure of a cyclopeptide containing Aib at position (i + 2) of the triad were generally in agreement with the secondary structure propensity of the cyclopeptides observed by CD spectroscopy. In conclusion, the Lys-Asp-(i,i + 4)-lactam bridge may succeed or fail in the stabilization of short helices, depending on the primary structure. Moreover, computational methods may be valuable tools to discriminate helix-prone from non-helix-prone peptide-based macrolactams. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Inhibition of delta-secretase improves cognitive functions in mouse models of Alzheimer's disease.

δ-secretase, also known as asparagine endopeptidase (AEP) or legumain, is a lysosomal cysteine protease that cleaves both amyloid precursor protein (APP) and tau, mediating the amyloid-ß and tau pathology in Alzheimer's disease (AD). Here we report the therapeutic effect of an orally bioactive and brain permeable δ-secretase inhibitor in mouse models of AD. We performed a high-throughput screen and identified a non-toxic and selective δ-secretase inhibitor, termed compound 11, that specifically blocks δ-secretase but not other related cysteine proteases. Co-crystal structure analysis revealed a dual active site-directed and allosteric inhibition mode of this compound class. Chronic treatment of tau P301S and 5XFAD transgenic mice with this inhibitor reduces tau and APP cleavage, ameliorates synapse loss and augments long-term potentiation, resulting in protection of memory. Therefore, these findings demonstrate that this δ-secretase inhibitor may be an effective clinical therapeutic agent towards AD.

The Id-protein family in developmental and cancer-associated pathways.

Inhibitors of DNA binding and cell differentiation (Id) proteins are members of the large family of the helix-loop-helix (HLH) transcription factors, but they lack any DNA-binding motif. During development, the Id proteins play a key role in the regulation of cell-cycle progression and cell differentiation by modulating different cell-cycle regulators both by direct and indirect mechanisms. Several Id-protein interacting partners have been identified thus far, which belong to structurally and functionally unrelated families, including, among others, the class I and II bHLH transcription factors, the retinoblastoma protein and related pocket proteins, the paired-box transcription factors, and the S5a subunit of the 26 S proteasome. Although the HLH domain of the Id proteins is involved in most of their protein-protein interaction events, additional motifs located in their N-terminal and C-terminal regions are required for the recognition of diverse protein partners. The ability of the Id proteins to interact with structurally different proteins is likely to arise from their conformational flexibility: indeed, these proteins contain intrinsically disordered regions that, in the case of the HLH region, undergo folding upon self- or heteroassociation. Besides their crucial role for cell-fate determination and cell-cycle progression during development, other important cellular events have been related to the Id-protein expression in a number of pathologies. Dysregulated Id-protein expression has been associated with tumor growth, vascularization, invasiveness, metastasis, chemoresistance and stemness, as well as with various developmental defects and diseases. Herein we provide an overview on the structural properties, mode of action, biological function and therapeutic potential of these regulatory proteins.