The oncoproteins MDM2 and MDMX negatively regulate the activity and stability of the tumor suppressor protein p53-a cellular process initiated by MDM2 and/or MDMX binding to the Nterminal transactivation domain of p53. MDM2 and MDMX in many tumors confer p53 inactivation and tumor survival, and are important molecular targets for anticancer therapy. We screened a duodecimal peptide phage library against site-specifically biotinylated p53-binding domains of human MDM2 and MDMX chemically synthesized via native chemical ligation, and identified several peptide inhibitors of the p53-MDM2/MDMX interactions. The most potent inhibitor (TSFAEYWNLLSP), termed PMI, bound to MDM2 and MDMX at low nanomolar affinities-approximately 2 orders of magnitude stronger than the wild-type p53 peptide of the same length (ETFSDLWKLLPE). We solved the crystal structures of synthetic MDM2 and MDMX, both in complex with PMI, at 1.6 Å resolution. Comparative structural analysis identified an extensive, tightened intramolecular H-bonding network in bound PMI that contributed to its conformational stability, thus enhanced binding to the 2 oncogenic proteins. Importantly, the C-terminal residue Pro of PMI induced formation of a hydrophobic cleft in MDMX previously unseen in the structures of p53-bound MDM2 or MDMX. Our findings deciphered the structural basis for highaffinity peptide inhibition of p53 interactions with MDM2 and MDMX, shedding new light on structure-based rational design of different classes of p53 activators for potential therapeutic use.

Reactive oxygen species (ROS) have been proposed to participate in the induction of cardiac preconditioning. However, their source and mechanism of induction are unclear. We tested whether brief hypoxia induces preconditioning by augmenting mitochondrial generation of ROS in chick cardiomyocytes. Cells were preconditioned with 10 min of hypoxia, followed by 1 h of simulated ischemia and 3 h of reperfusion. Preconditioning decreased cell death from 47 ؎ 3% to 14 ؎ 2%. Return of contraction was observed in 3/3 preconditioned versus 0/6 non-preconditioned experiments. During induction, ROS oxidation of the probe dichlorofluorescin (sensitive to H 2 O 2 ) increased ϳ2.5-fold. As a substitute for hypoxia, the addition of H 2 O 2 (15 mol/liter) during normoxia also induced preconditioning-like protection. Conversely, the ROS signal during hypoxia was attenuated with the thiol reductant 2-mercaptopropionyl glycine, the cytosolic Cu,Zn-superoxide dismutase inhibitor diethyldithiocarbamic acid, and the anion channel inhibitor 4,4-diisothiocyanato-stilbene-2,2-disulfonate, all of which also abrogated protection. ROS generation during hypoxia was attenuated by myxothiazol, but not by diphenyleneiodonium or the nitric-oxide synthase inhibitor L-nitroarginine. We conclude that hypoxia increases mitochondrial superoxide generation which initiates preconditioning protection. Furthermore, mitochondrial anion channels and cytosolic dismutation to H 2 O 2 may be important steps for oxidant induction of hypoxic preconditioning.Myocardial preconditioning was initially described as an adaptive response of the heart to brief episodes of ischemia that decreased necrosis during subsequent prolonged ischemia (1). Reactive oxygen species (ROS 1 ; e.g. superoxide, H 2 O 2 , hydroxyl radicals) generated from brief ischemia/reperfusion have been recognized as possible "triggers" in the initiation of preconditioning (2). Evidence for this role includes intact heart studies where exposure to superoxide or H 2 O 2 caused preconditioninglike protection (2, 3), and other studies demonstrating that antioxidants abolished the induction of preconditioning (4, 5). Few studies have directly measured ROS generation during brief hypoxia or ischemia induction (6). Such direct measures are needed to clarify important questions that remain regarding the role of ROS as inducing agents, including their source, where they are metabolized, and the relative contributions of different oxidant species to the induction of preconditioning protection.Within the intact heart, possible sources of ROS include the cardiomyocytes, endothelial cells, neutrophils, or the auto-oxidation of catecholamines (7,8). Within cardiomyocytes, sources of ROS could include superoxide generation from NAD(P)H or other oxidases such as cytochrome P450 (9 -11), the mitochondrial electron transport chain (12), or even nitric-oxide synthase under conditions where arginine is depleted (13-15). Although it is likely that superoxide is the initial oxidant generated from these systems, the rel...

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Noble metal (Pt, Ru, and Ir)-based electrocatalysts are currently considered the most active materials for the hydrogen evolution reaction (HER). Although they have been associated with high cost, easy agglomeration, and poor stability during the HER reaction, recent efforts to intentionally tailor noble-metal-based catalysts have led to promising improvements, with lower cost and superior activity, which are critical to achieving large-scale production of pure hydrogen. In this mini-review, we focus on the recent advances in noble-metal-based HER electrocatalysts. In particular, the synthesis strategies to enhance cost-effectiveness and the catalytic activity for HER are highlighted.

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Inhibition of the interaction between the tumor suppressor protein p53 and its negative regulators MDM2 and MDMX is of great interest in cancer biology and drug design. We previously reported a potent duodecimal peptide inhibitor, termed PMI (TSFAEYWNLLSP), of the p53-MDM2 and -MDMX interactions. PMI competes with p53 for MDM2 and MDMX binding at an affinity roughly two orders of magnitude higher than that of 17–28p53 (ETFSDLWKLLPE) of the same length; both peptides adopt nearly identical α-helical conformations in the complexes, where the three highlighted hydrophobic residues Phe, Trp and Leu dominate PMI or 17–28p53 binding to MDM2 and MDMX. To elucidate the molecular determinants for PMI activity and specificity, we performed a systematic Ala scanning mutational analysis of PMI and 17–28p53. The binding affinities for MDM2 and MDMX of a total of 35 peptides including 10 truncation analogs were quantified, affording a complete dissection of energetic contributions of individual residues of PMI and 17–28p53 to MDM2 and MDMX association. Importantly, the N8A mutation turned PMI into the most potent dual specific antagonist of MDM2 and MDMX reported to date, registering respective Kd values of 490 pM and 2.4 nM. The co-crystal structure of N8A-PMI-25–109MDM2 was determined at 1.95 Å, affirming that high-affinity peptide binding to MDM2/MDMX necessitates, in addition to optimized inter-molecular interactions, enhanced helix stability or propensity contributed by non-contact residues. The powerful empirical binding data and crystal structures present a unique opportunity for computational studies of peptide inhibition of the p53-MDM2/MDMX interactions.

The oncoproteins MDM2 and MDMX negatively regulate the activity and stability of the tumor suppressor protein p53, conferring tumor development and survival. Antagonists targeting the p53-binding domains of MDM2 and MDMX kill tumor cells both in vitro and in vivo by reactivating the p53 pathway, promising a class of antitumor agents for cancer therapy. Aided by native chemical ligation and mirror image phage display, we recently identified a D-peptide inhibitor of the p53-MDM2 interaction termed The tumor suppressor protein p53 is a transcription factor that transactivates, in response to cellular stresses, the expression of various target genes that mediate cell cycle arrest, senescence, or apoptosis (1). Dubbed the "guardian of the genome" (2), p53 is critical for maintaining genetic stability and preventing tumor development (3). Not surprisingly, loss of p53 activity resulting from point mutations in the TP53 gene is responsible for approximately 50% of human tumors. Although p53 retains WT status in many other tumors, its tumor suppressor activity and in vivo stability are abrogated by regulatory molecules such as the E3 ubiquitin ligase MDM2 and its homologue MDMX (4, 5). Amplified or over-expressed in a significant fraction of cancers without concomitant TP53 mutation, MDM2 and MDMX directly contribute to p53 inactivation and tumor survival.MDM2 itself is transcriptionally inducible by p53 in a negative feedback loop (6). MDM2 binds the N-terminal transactivation domain of p53 with high affinity to block p53 regulating responsive gene expression (7). More importantly, MDM2 controls p53 stability by targeting the tumor suppressor protein for ubiquitinmediated constitutive degradation (8-10). Although MDMX lacks E3 ubiquitin ligase activity, the MDM2 homologue acts as an effective transcriptional antagonist of p53, and nonredundantly impedes p53-induced growth inhibitory and apoptotic responses (4, 5). In addition, MDMX forms heterodimers with MDM2 through their C-terminal RING finger domains, stimulating MDM2-mediated ubiquitination and degradation of p53 and MDMX itself (11-13). The interplay between MDM2 and MDMX confers a robust p53 inactivation in tumorigenesis (14).Recent studies show that restoring endogenous p53 activity can halt the growth of cancerous tumors in mice through cell typedependent multiple mechanisms, including apoptosis, senescence, and senescence-triggered innate inflammatory responses (15-17). Thus, antagonists of MDM2 and MDMX that activate the p53 pathway can potentially be developed into a class of therapeutic agents for cancer treatment (14). Much of the current efforts have been focused on combinatorial library search for and structurebased rational design of low molecular weight antagonists of MDM2 (18). Successful examples include a cis-imidazoline analogue, termed nutlin-3, and, a spiro-oxindole-derived compound, termed 20). For optimal efficacy, however, dual specific inhibitors may be needed to target both MDM2 and MDMX (14).We previously reported the synthesis of the p53...

a b s t r a c tDefensins constitute a major class of cationic antimicrobial peptides in mammals and vertebrates, acting as effectors of innate immunity against infectious microorganisms. It is generally accepted that defensins are bactericidal by disrupting the anionic microbial membrane. Here, we provide evidence that membrane activity of human a-defensins does not correlate with antibacterial killing. We further show that the a-defensin human neutrophil peptide-1 (HNP1) binds to the cell wall precursor lipid II and that reduction of lipid II levels in the bacterial membrane significantly reduces bacterial killing. The interaction between defensins and lipid II suggests the inhibition of cell wall synthesis as a novel antibacterial mechanism of this important class of host defense peptides.Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.