Design and synthesis of β-strand-fixed peptides inhibiting aggregation of amyloid β-protein

Aggregation of 42-residue amyloid β-protein (Aβ42) can be prevented by β-sheet breaker peptides (BSBps) homologous to LVFFA residues, which are included in a β-sheet region of Aβ42 aggregates. To enhance the affinity of BSBps to the Aβ42 aggregates, we designed and synthesized β-strand-fixed peptides (BSFps) whose side chains were cross-linked by ring closing metathesis. Conformation analysis verified that the designed peptides could be fixed in β-strand conformation. Among the synthesized pentapeptides, 1 and 12, whose side chains of 2nd and 4th residues were cross-linked, significantly inhibited the aggregation of Aβ42. This suggested that β-strand-fixation of BSBps could enhance their inhibitory activity against the Aβ42 aggregation. However, pentapeptides 1 and 12 had little effect on morphology of Aβ42 aggregates (fibrils) and neurotoxicity of Aβ42 against SH-SY5Y cells.     

Olive oil compounds inhibit vascular endothelial growth factor receptor-2 phosphorylation

Vascular endothelial growth factor (VEGF) triggers crucial signaling processes that regulate tumor angiogenesis and, therefore, represents an attractive target for the development of novel anticancer therapeutics. Several epidemiological studies have confirmed that abundant consumption of foods from plant origin is associated with reduced risk of developing cancers. In the Mediterranean basin, the consumption of extra virgin olive oil is an important constituent of the diet. Compared to other vegetable oils, the presence of several phenolic antioxidants in olive oil is believed to prevent the occurrence of a variety of pathological processes, such as cancer. While the strong antioxidant potential of these molecules is well characterized, their antiangiogenic activities remain unknown. The aim of this study is to investigate whether tyrosol (Tyr), hydroxytyrosol (HT), taxifolin (Tax), oleuropein (OL) and oleic acid (OA), five compounds contained in extra virgin olive oil, can affect in vitro angiogenesis. We found that HT, Tax and OA were the most potent angiogenesis inhibitors through their inhibitory effect on specific autophosphorylation sites of VEGFR-2 (Tyr951, Tyr1059, Tyr1175 and Tyr1214) leading to the inhibition of endothelial cell (EC) signaling. Inhibition of VEGFR-2 by these olive oil compounds significantly reduced VEGF-induced EC proliferation and migration as well as their morphogenic differentiation into capillary-like tubular structures in Matrigel. Our study demonstrates that HT, Tax and OA are novel and potent inhibitors of the VEGFR-2 signaling pathway. These findings emphasize the chemopreventive properties of olive oil and highlight the importance of nutrition in cancer prevention.     

New Cdc2 Tyr 4 phosphorylation by dsRNA‐activated protein kinase triggers Cdc2 polyubiquitination and G2 arrest under genotoxic stresses

Cell division cycle 2 (Cdc2) protein is an essential subunit of M‐phase kinase (MPK), which has a key role in G2/M transition. Even though the control of MPK activity has been well established with regard to the phosphorylation of Cdc2 at Thr 14 and/or Tyr 15 and Thr 161, little is known about the proteolytic control of Cdc2. In this study, we observed that Cdc2 was downregulated under genotoxic stresses and that double‐stranded RNA‐activated protein kinase (PKR) was involved in the process. The PKR‐mediated Tyr4 phosphorylation triggered Cdc2 ubiquitination. Phospho‐mimic mutations at the Tyr 4 residue (Y4D or Y4E) caused significant ubiquitination of Cdc2 even in the absence of PKR. Our findings demonstrate that (i) PKR, Ser/Thr kinase, phosphorylates its new substrate Cdc2 at the Tyr 4 residue, (ii) PKR‐mediated Tyr 4‐phosphorylation facilitates Cdc2 ubiquitination and proteosomal degradation, (iii) unphosphorylated Tyr 4 prevents Cdc2 ubiquitination, and (iv) downstream from p53, PKR has a crucial role in G2 arrest and triggers Cdc2 downregulation under genotoxic conditions.     

High-Resolution Crystal Structures of Drosophila melanogaster Angiotensin-Converting Enzyme in Complex with Novel Inhibitors and Antihypertensive Drugs

Angiotensin I-converting enzyme (ACE), one of the central components of the renin-angiotensin system, is a key therapeutic target for the treatment of hypertension and cardiovascular disorders. Human somatic ACE (sACE) has two homologous domains (N and C). The N- and C-domain catalytic sites have different activities toward various substrates. Moreover, some of the undesirable side effects of the currently available and widely used ACE inhibitors may arise from their targeting both domains leading to defects in other pathways. In addition, structural studies have shown that although both these domains have much in common at the inhibitor binding site, there are significant differences and these are greater at the peptide binding sites than regions distal to the active site. As a model system, we have used an ACE homologue from Drosophila melanogaster (AnCE, a single domain protein with ACE activity) to study ACE inhibitor binding. In an extensive study, we present high-resolution structures for native AnCE and in complex with six known antihypertensive drugs, a novel C-domain sACE specific inhibitor, lisW-S, and two sACE domain-specific phosphinic peptidyl inhibitors, RXPA380 and RXP407 (i.e., nine structures). These structures show detailed binding features of the inhibitors and highlight subtle changes in the orientation of side chains at different binding pockets in the active site in comparison with the active site of N- and C-domains of sACE. This study provides information about the structure-activity relationships that could be utilized for designing new inhibitors with improved domain selectivity for sACE.     

Dual effects of [Tyr6]‐γ2‐MSH(6–12) on pain perception and in vivo hyperalgesic activity of its analogues

[Tyr⁶]‐γ2‐MSH(6–12) with a short effecting time of about 20 min is one of the most potent rMrgC receptor agonists. To possibly increase its potency and metabolic stability, a series of analogues were prepared by replacing the Tyr⁶ residue with the non‐canonical amino acids 3‐(1‐naphtyl)‐L ‐alanine, 4‐fluoro‐L ‐phenylalanine, 4‐methoxy‐L ‐phenylalanine and 3‐nitro‐L ‐tyrosine. Dose‐dependent nociceptive assays performed in conscious rats by intrathecal injection of the MSH peptides showed [Tyr⁶]‐γ2‐MSH(6–12) hyperalgesic effects at low doses (5–20 nmol) and analgesia at high doses (100–200 nmol). This analgesic activity is fully reversed by the kyotorphin receptor‐specific antagonist Leu–Arg. For the two analogues containing in position 6, 4‐fluoro‐L ‐phenylalanine and 3‐nitro‐L ‐tyrosine, a hyperalgesic activity was not observed, while the 3‐(1‐naphtyl)‐L ‐alanine analogue at 10 nmol dose was found to induce hyperalgesia at a potency very similar to γ2‐MSH(6–12), but with longer duration of the effect. Finally, the 4‐methoxy‐L ‐phenylalanine analogue (0.5 nmol) showed greatly improved hyperalgesic activity and prolonged effects compared to the parent [Tyr⁶]‐γ2‐MSH(6–12) compound. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Syntheses and activities of backbone‐side chain cyclic octapeptide ligands with N‐functionalized phosphotyrosine for the N‐terminal SH2‐domain of the protein tyrosine phosphatase SHP‐1

The protein tyrosine phosphatase SHP‐1 plays an important role in many physiological and pathophysiological processes. This phosphatase is activated through binding of ligands to its SH2‐domains, mainly to the N‐terminal one. Based on a theoretical docking model, backbone‐to‐side chain cyclized octapeptides were designed as ligands. Assembly of such modelled structures required the synthesis of N‐functionalized tyrosine derivatives and their incorporation into the sequence. Because of difficulties encountered in the condensation of N‐protected amino acids to the N‐alkylated tyrosine‐peptide we synthesized and used preformed dipeptide building units. As all attempts to obtain phosphorylated dipeptide units failed, the syntheses had to be performed with a free phenolic function. Use of different N‐alkyl or cycloalkyl residues in the N‐functionalized side chains allowed to investigate the effect of ring size, flexibility and hydrophobicity of formed lactam bridges on stimulatory activity. All tested linear and cyclic octapeptides stimulate the phosphatase activity of SHP‐1. Stimulatory activities of cyclic ligands increase with the chain length of the lactam bridges resulting in increased flexibility and better entropic preformation of the binding conformation. The strong activity of some cyclic octapeptides supports the modelled structure. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

The beta-tubulin genes of two Strongyloides species

The World Health Organization is sponsoring major treatment programs with the aim of controlling helminth infection throughout the tropical world. Prominent among the anthelmintics recommended for use in these programs are drugs in the benzimidazole (BZ) class. Resistance to these drugs has been associated with polymorphisms in the beta-tubulin gene. We have cloned and sequenced the beta-tubulin genes of Strongyloides stercoralis and Strongyloides ratti and have proceeded to develop a protocol for genotyping single worms for polymorphisms in beta-tubulin. Our findings indicate that S. ratti has a single beta-tubulin gene, making DNA sequence analysis of a single larva PCR product a feasible means of studying BZ resistance in these species. Our genotyping test allows the identification of polymorphisms at codons 167, 198, and 200 in the Strongyloides beta-tubulin gene, thus enabling survey for BZ resistant genotypes.     

Abi1/Hssh3bp1 pY213 links Abl kinase signaling to p85 regulatory subunit of PI-3 kinase in regulation of macropinocytosis in LNCaP cells

Macropinocytosis is regulated by Abl kinase via an unknown mechanism. We previously demonstrated that Abl kinase activity is, itself, regulated by Abi1 subsequent to Abl kinase phosphorylation of Abi1 tyrosine 213 (pY213) [1]. Here we show that blocking phosphorylation of Y213 abrogated the ability of Abl to regulate macropinocytosis, implicating Abi1 pY213 as a key regulator of macropinocytosis. Results from screening the human SH2 domain library and mapping the interaction site between Abi1 and the p85 regulatory domain of PI-3 kinase, coupled with data from cells transfected with loss-of-function p85 mutants, support the hypothesis that macropinocytosis is regulated by interactions between Abi1 pY213 and the C-terminal SH2 domain of p85—thereby linking Abl kinase signaling to p85-dependent regulation of macropinocytosis.

Structured summary

MINT-7908602: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to SHIP2 (uniprotkb:O15357) by array technology (MI:0008)MINT-7908362: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Emt (uniprotkb:Q08881) by array technology (MI:0008)MINT-7908235: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Lyn (uniprotkb:P07948) by array technology (MI:0008)MINT-7908075: Abi1 (uniprotkb:Q8IZP0)binds (MI:0407) to Fgr (uniprotkb:P09769) by array technology (MI:0008)MINT-7908330, MINT-7908522: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Vav1 (uniprotkb:P15498) by array technology (MI:0008)MINT-7907962: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Fyn (uniprotkb:P06241) by array technology (MI:0008)MINT-7908203: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Src (uniprotkb:P12931) by array technology (MI:0008)MINT-7908570: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to SHP-2 (uniprotkb:P35235) by array technology (MI:0008)MINT-7908187, MINT-7908586: Abi1(uniprotkb:Q8IZP0) binds (MI:0407) to Gap (uniprotkb:P20936) by array technology (MI:0008)MINT-7907981, MINT-7907995: Abi1 (uniprotkb:Q8IZP0) physically interacts (MI:0915) with p85a (uniprotkb:P26450) by anti tag coimmunoprecipitation (MI:0007)MINT-7908251: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to PLCG1 (uniprotkb:P19174) by array technology (MI:0008)MINT-7908346: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Grb2 (uniprotkb:P62993) by array technology (MI:0008)MINT-7907945: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Abl (uniprotkb:P00519) by array technology (MI:0008)MINT-7908474: Abi1 (uniprotkb:Q8IZP0)binds (MI:0407) to p85b (uniprotkb:O00459) by array technology (MI:0008)MINT-7908107: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Hck (uniprotkb:P08631) by array technology (MI:0008)MINT-7908011: p85a (uniprotkb:P26450) physically interacts (MI:0915) with Abi1 (uniprotkb:Q8IZP0) by pull down (MI:0096)MINT-7908155: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to FynT (uniprotkb:P06241-2) by array technology (MI:0008)MINT-7908283, MINT-7908490: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to p55g (uniprotkb:Q92569) by array technology (MI:0008)MINT-7907929, MINT-7907815, MINT-7907832, MINT-7907865, MINT-7907897, MINT-7907913, MINT-7907881, MINT-7907848: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to p85a (uniprotkb:P27986) by array technology (MI:0008)MINT-7908059: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Frk (uniprotkb:P42685) by array technology (MI:0008)MINT-7908378: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to CblC (uniprotkb:Q9ULV8) by array technology (MI:0008)MINT-7908618: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to CblA (uniprotkb:B5MC15) by array technology (MI:0008)MINT-7908139, MINT-7908538: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Nap4 (uniprotkb:O14512) by array technology (MI:0008)MINT-7908426: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to CblB (uniprotkb:Q13191) by array technology (MI:0008)MINT-7908506: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Crk (uniprotkb:P46108) by array technology (MI:0008)MINT-7908554: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to mAbl (uniprotkb:P00520) by array technology (MI:0008)MINT-7908043, MINT-7908394: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Vav2 (uniprotkb:P52735) by array technology (MI:0008)MINT-7908458: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to mSck/ShcB (uniprotkb:Q8BMC3) by array technology (MI:0008)MINT-7908091: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Yes (uniprotkb:P07947) by array technology (MI:0008)MINT-7908219: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Src (uniprotkb:P00523) by array technology (MI:0008)MINT-7908123: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Fer (uniprotkb:P16591) by array technology (MI:0008)MINT-7908410: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to CrkL (uniprotkb:P46109) by array technology (MI:0008)MINT-7908314, MINT-7908442: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Arg (uniprotkb:P42684) by array technology (MI:0008)MINT-7908299: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to PLCG1 (uniprotkb:P10686) by array technology (MI:0008)MINT-7908171: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Fes (uniprotkb:P07332) by array technology (MI:0008)MINT-7908027: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Lck (uniprotkb:P06239) by array technology (MI:0008)     

The loops facing the active site of prolyl oligopeptidase are crucial components in substrate gating and specificity

Prolyl oligopeptidase (POP) has emerged as a drug target for neurological diseases. A flexible loop structure comprising loop A (res. 189–209) and loop B (res. 577–608) at the domain interface is implicated in substrate entry to the active site. Here we determined kinetic and structural properties of POP with mutations in loop A, loop B, and in two additional flexible loops (the catalytic His loop, propeller Asp/Glu loop). POP lacking loop A proved to be an inefficient enzyme, as did POP with a mutation in loop B (T590C). Both variants displayed an altered substrate preference profile, with reduced ligand binding capacity. Conversely, the T202C mutation increased the flexibility of loop A, enhancing the catalytic efficiency beyond that of the native enzyme. The T590C mutation in loop B increased the preference for shorter peptides, indicating a role in substrate gating. Loop A and the His loop are disordered in the H680A mutant crystal structure, as seen in previous bacterial POP structures, implying coordinated structural dynamics of these loops. Unlike native POP, variants with a malfunctioning loop A were not inhibited by a 17-mer peptide that may bind non-productively to an exosite involving loop A. Biophysical studies suggest a predominantly closed resting state for POP with higher flexibility at the physiological temperature. The flexible loop A, loop B and His loop system at the active site is the main regulator of substrate gating and specificity and represents a new inhibitor target.