Presenilins (PSs) are part of the ␥-secretase complex that produces the amyloid ␤-peptide (A␤) from its precursor [␤-amyloid precursor protein (␤APP)]. Mutations in PS that cause familial Alzheimer's disease (FAD) increase A␤ production and trigger p53-dependent cell death. We demonstrate that PS deficiency, catalytically inactive PS mutants, ␥-secretase inhibitors, and ␤APP or amyloid precursor protein-like protein 2 (APLP2) depletion all reduce the expression and activity of p53 and lower the transactivation of its promoter and mRNA expression. p53 expression also is diminished in the brains of PS-or ␤APP-deficient mice. The ␥-and -secretase-derived amyloid intracellular C-terminal domain (AICD) fragments (AICDC59 and AICDC50, respectively) of ␤APP trigger p53-dependent cell death and increase p53 activity and mRNA. Finally, PS1 mutations enhance p53 activity in human embryonic kidney 293 cells and p53 expression in FAD-affected brains. Thus our study shows that AICDs control p53 at a transcriptional level, in vitro and in vivo, and that FAD mutations increase p53 expression and activity in cells and human brains.

Proprotein convertase subtilisin-like/kexin type 9 (PCSK9) is a key regulator of plasma LDL-cholesterol (LDL-C) and a clinically validated target for the treatment of hypercholesterolemia and coronary artery disease. In this paper, we describe a series of novel cyclic peptides derived from an mRNA display screen which inhibit the protein–protein interaction between PCSK9 and LDLR. Using a structure-based drug design approach, we were able to modify our original screening lead 2 to optimize the potency and metabolic stability and minimize the molecular weight to provide novel bicyclic next-generation PCSK9 inhibitor peptides such as 78. These next-generation peptides serve as a critical foundation for continued exploration of potential oral, once-a-day PCSK9 therapeutics for the treatment of cardiovascular disease.

The mechanism by which hypoxia induces gene transcription is now well established. Hypoxia reduces activity of prolyl hydroxylases (PHD) that hydroxylate specific proline residues in the oxygen-dependent degradation domain (ODD) of hypoxia-inducible factor-1␣ (HIF-1␣). As a consequence, HIF-1␣ accumulates and promotes hypoxic tolerance by activating gene transcription. This paper identifies the three forms of PHDs in rats and shows that a period of hypoxia selectively increases expression of PHD-2 mRNAs levels. We developed assays for PHD activity that used (i) the peptidespecific conversion of labeled 2-oxoglutarate into succinate and (ii) the binding of the von Hippel-Lindau protein to a glutathione S-transferase-ODD fusion protein. The two assays indicated a low enzymatic activity in normoxic and hypoxic cells and a rapid increase during reoxygenation. We also developed hydroxyprolinespecific antibodies that recognized hydroxylated forms of a fusion protein (ODD-green fluorescent protein) that combined the ODD domain of HIF-1␣ and the green fluorescent protein. Using this antibody, we demonstrated that reoxygenation induced a rapid hydroxylation of Pro-564, which was followed by a massive degradation of the proteins. The results suggest that a hypoxic upregulation of PHD (presumably PHD-2) acts as a feedback mechanism to stop hypoxic responses in reoxygenated cells. We propose that proline hydroxylation might play a role in hypoxic preconditioning.Cells respond to reduced oxygen tensions by up-regulating the expression of genes involved in angiogenesis (e.g. vascular endothelial growth factor), erythropoiesis (e.g. erythropoietin), and glycolysis. The transcriptional activation of target genes is induced by a common transcription factor, hypoxia inducible factor-1 (HIF-1).1 HIF-1 was first identified as a heterodimeric transactivator that recognizes a specific DNA sequence termed hypoxia-responsive element in the 3Ј-untranslated region of the erythropoietin gene (1). HIF-1 is composed of two subunits, HIF-1␣ and the aryl hydrocarbon nuclear translocator, both of which belong to the large family of basic helix-loop-helix-perarnt-sim transcription factors (2). The mechanism of the hypoxic induction of HIF-1␣ has recently been identified. Under normoxic conditions, specific HIF-1␣ prolyl hydroxylases (PHD) hydroxylate two proline residues (Pro-402 and Pro-564) in the oxygen-dependent degradation (ODD) domain of HIF-1␣ (3, 4). The von Hippel-Lindau protein (vHL) E3 ubiquitin ligase complex associates to hydroxylated proline residues and targets HIF-1␣ to proteasomal degradation. Under hypoxic conditions, oxygen becomes rate-limiting for proline hydroxylation. As a consequence, HIF-1␣ accumulates, migrates to the nucleus, and associates with the aryl hydrocarbon nuclear translocator and the complex interacts with hypoxia-responsive element of target genes (5, 6).Less attention has been given to the situation in which hypoxic cells are reoxygenated. Tissue reoxygenation is not an unusual condition. It is frequently...

The epipolythiodiketopiperazine (ETP) alkaloids are a highly complex class of natural products with potent anticancer activity. Herein, we report the application of a flexible and scalable synthesis, allowing the construction of dozens of ETP derivatives. The evaluation of these compounds against cancer cell lines in culture allows for the first expansive structure–activity relationship (SAR) to be defined for monomeric and dimeric ETP-containing natural products and their synthetic cognates. Many ETP derivatives demonstrate potent anticancer activity across a broad range of cancer cell lines, and kill cancer cellsviainduction of apoptosis. Several traits thatbode well for the translational potential of the ETP class of natural products includeconcise and efficient synthetic access, potent induction of apoptotic cell death, activity against a wide range of cancer types, and a broad tolerance for modifications at multiple sitesthat should facilitate small-molecule drug development, mechanistic studies, and evaluation in vivo.

The first total synthesis of (+)-gliocladin B is described. Our concise and enantioselective synthesis takes advantage of a new regioselective Friedel–Crafts-based strategy to provide an efficient multigram-scale access to the C3-(3′-indolyl)hexahydropyrroloindole substructure, a molecular foundation present in a significant subset of epipolythiodiketopiperazine natural alkaloids. Our first-generation solution to (+)-gliocladin B involved the stereoselective formation of (+)-12-deoxybionectin A, a plausible biosynthetic precursor. Our synthesis clarified the C15 stereochemistry of (+)-gliocladin B and allowed its full structure confirmation. Further studies of a versatile dihydroxylated diketopiperazine provided a concise and efficient synthesis of (+)-gliocladin B as well as access to (+)-gliocladin C.

Macrocyclic peptides open new opportunities to target intracellular protein–protein interactions (PPIs) that are often considered nondruggable by traditional small molecules. However, engineering sufficient membrane permeability into these molecules is a central challenge for identifying clinical candidates. Currently, there is a lack of high-throughput assays to assess peptide permeability, which limits our capacity to engineer this property into macrocyclic peptides for advancement through drug discovery pipelines. Accordingly, we developed a high throughput and target-agnostic cell permeability assay that measures the relative cumulative cytosolic exposure of a peptide in a concentration-dependent manner. The assay was named NanoClick as it combines in-cell Click chemistry with an intracellular NanoBRET signal. We validated the approach using known cell penetrating peptides and further demonstrated a correlation to cellular activity using a p53/MDM2 model system. With minimal change to the peptide sequence, NanoClick enables the ability to measure uptake of molecules that enter the cell via different mechanisms such as endocytosis, membrane translocation, or passive permeability. Overall, the NanoClick assay can serve as a screening tool to uncover predictive design rules to guide structure–activity–permeability relationships in the optimization of functionally active molecules.

In Lycopersicon esculentum, two cDNAs encoding glutathione peroxidase (GPX)-like proteins were isolated, sequenced and characterized as putative phospholipid hydroperoxide :glutathione peroxidases. After mechanical stimulation (rubbing) of the plant, each GPX tomato gene was found to behave differently in terms of inducibility and tissue distribution. The accumulation of GPX transcripts suggests that reactive oxygen species are involved in the plant response to a mechanical stress.Keywords : Lycopersicon esculentum; free-radical scavenger; gene expression ; glutathione peroxidase ; mechanical stimulation.The consumption of oxygen in normal respiratory processes ing are expressed in tomato. Two GPX-like cDNAs were cloned and the tissue distribution and accumulation of the correspondand the production of oxygen during photosynthesis results in the generation of toxic reactive oxygen species (ROS) [1,2]. ing mRNAs were investigated. Biotic and abiotic stresses can give rise to further increases in the ROS level [3,4] ; this phenomenon is described as oxidative burst. The primary constituents of protective mechanisms MATERIALS AND METHODS against ROS include enzymes, such as superoxide dismutase, Plant material, culture conditions and stimulation. Culcatalase and peroxidase, and free-radical scavengers, such as ca-ture conditions. Tomato (Lycopersicon esculentulum Mill. cv. rotenoid, tocopherol, ascorbate and glutathione [5]. VFN8) plants were raised from seeds in moist vermiculite in a Glutathione peroxidases (GPX) belong to a family of iso-controlled environment room: 16 h daylight at 60 µmol · m Ϫ2 · zymes that catalyses the reduction of H 2 O 2 , organic hydroper-s Ϫ1 , photosynthetically active radiation provided by 40-W white oxides and lipid hydroperoxides using GSH as a reducing agent daylight tubes (Mazda LDL, TF 40), 23Ϯ1°C (day) and and thus help to protect the cells against oxidative damage [6].19Ϯ1°C (night), 60Ϯ 10% relative humidity. At the cotyledon In animals, where these enzymes have been studied extensively, stage, plants were transferred to a mineral solution [13]. both selenium-dependent and selenium-independent GPX have Mechanical stimulation. Mechanical stimulus was applied to been described [7,8]. Much less is known about these enzymes 3-week-old plants, with six developed internodes. The young in plants. Recent reports have shown increases of the steady-growing internode [14] was held between the thumb and forefinstate level of mRNA or protein corresponding to GPX, and ger and rubbed back and forth. stress conditions, such as pathogenic attack, salt treatment and RNA and DNA isolation, northern blotting and Southern osmotic stress [9Ϫ11], seem to be involved. blot hybridizations. Total RNA was extracted from different The present paper reports that rubbing of tomato plants initi-organs (roots, internodes, mature and young leaves) of 3-weekates an oxidative response. Previous data in Bryonia dioica have old tomato plants by the borate method [15]. Extractions were suggested that the mechanica...

Abstract. Rubbing young internodes of Eryonia dioica results in a reduced elongation and an increased diameter of the internodes. In the present study activities of some enzymes involved in the lignification process and levels of lignification were compared in rubbed and non-rubbed internodes. Rubbing caused an increase in the activities of phenylalanine ammonia-lyase and soluble and ionically-and covalently-bound cell wall peroxidases. Sensitivity of the covalently-bound walJ peroxidase assay was markedly increased if syringaldazine was used as a substrate. Mechanical perturbation induced an increase in lignin, lignin monomer (sinapylic, coniferylic and p-coumarylic alcohols) content and the number of lignifying vessels. Conversely, rubbing resulted in a decrease in cellulose content. The hypothetical interpretation of the thigmomorphogenetic response through cell wall lignifrcation and hence rigidification is consistent with all the presented results. A comparison is possible between this accelerated lignification and induced lignification as a mechanism of disease resistance. The thigmomorphogenetic response in Bryonia dioica can be considered as a mechanism of resistance in order to withstand further environmental mechanical perturbation.