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...
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