Kinase-independent inhibition of cyclophosphamide-induced pathways protects the ovarian reserve and prolongs fertility.

Premature ovarian failure and infertility are adverse effects of cancer therapies. The mechanism underlying chemotherapy-mediated depletion of the ovarian reserve remains unclear. Here, we aim to identify the signaling pathways involved in the loss of the ovarian reserve to prevent the damaging effects of chemotherapy. We evaluated the effects of cyclophosphamide, one of the most damaging chemotherapeutic drugs, against follicle reserve. In vivo studies showed that the cyclophosphamide-induced loss of ovarian reserve occurred through a sequential mechanism. Cyclophosphamide exposure induced the activation of both DNAPK-γH2AX-checkpoint kinase 2 (CHK2)-p53/TAp63α isoform and protein kinase B (AKT)-forkhead box O3 (FOXO3a) signaling axes in the nucleus of oocytes. Concomitant administration of an allosteric ABL inhibitor and cyclophosphamide modulated both pathways while protecting the ovarian reserve from chemotherapy assaults. As a consequence, the fertility of the treated mice was prolonged. On the contrary, the administration of an allosteric ABL activator enhanced the lethal effects of cyclophosphamide while shortening mouse fertility. Therefore, kinase-independent inhibition may serve as an effective ovarian-protective strategy in women under chemotherapy.

N-myristoyltransferases inhibitory activity of ellagitannins from Terminalia bentzoë (L.) L. f. subsp. bentzoë.

N-myristoylation (Myr) is an eukaryotic N-terminal co- or post-translational protein modification in which the enzyme N-myristoyltransferase (NMT) transfers a fatty acid (C14:0) to the N-terminal glycine residues of several cellular key proteins. Depending on the cellular context, NMT may serve as a molecular target in anticancer or anti-infectious therapy, and drugs that inhibit this enzyme may be useful in the treatment of cancer or infectious diseases. As part of an on-going project to identify natural Homo sapiens N-myristoyltransferase 1 inhibitors (HsNMT1), two ellagitannins, punicalagin (1) and isoterchebulin (2), along with eschweilenol C (3) and ellagic acid (4) were isolated from the bark of Terminalia bentzoë (L.) L. f. subsp. bentzoë. Their structures were determined by means of spectroscopic analyses and comparison with literature data. Punicalagin (1) and isoterchebulin (2) showed significant inhibitory activity towards HsNMT1, and also against Plasmodium falciparum NMT (PfNMT) both in vitro and in cellulo, opening alternative paths for new NMT inhibitors development. This is the first report identifying natural products from a botanical source as inhibitors of HsNMT and PfNMT.

Structural and genomic decoding of human and plant myristoylomes reveals a definitive recognition pattern.

An organism's entire protein modification repertoire has yet to be comprehensively mapped. N-myristoylation (MYR) is a crucial eukaryotic N-terminal protein modification. Here we mapped complete Homo sapiens and Arabidopsis thaliana myristoylomes. The crystal structures of human modifier NMT1 complexed with reactive and nonreactive target-mimicking peptide ligands revealed unexpected binding clefts and a modifier recognition pattern. This information allowed integrated mapping of myristoylomes using peptide macroarrays, dedicated prediction algorithms, and in vivo mass spectrometry. Global MYR profiling at the genomic scale identified over a thousand novel, heterogeneous targets in both organisms. Surprisingly, MYR involved a non-negligible set of overlapping targets with N-acetylation, and the sequence signature marks for a third proximal acylation-S-palmitoylation-were genomically imprinted, allowing recognition of sequences exhibiting both acylations. Together, the data extend the N-end rule concept for Gly-starting proteins to subcellular compartmentalization and reveal the main neighbors influencing protein modification profiles and consequent cell fate.

P53 and Sirt1: routes of metabolism and genome stability.

The tumor suppressor p53 is a transcription factor that regulates key processes. But, the outcomes of the p53 response go beyond its role as a nuclear transcription factor. Sirtuin (SIRT1) regulates p53 functions as transcription factor. At the same time, SIRT1 protects the genome under stress conditions. The link between p53 and SIRT1 responses is unique. Both regulate metabolism, stress signaling, cell survival, cell cycle control and genome stability. Recent studies have proposed cancer as a metabolic disease. This is due to the switch from aerobic to anaerobic metabolism during tumor development. Yet, the complex molecular circuits (in and out of the nucleus) of tumor progression remain elusive. In this review, we will focus on the interplay between p53 and SIRT1. We will discuss their roles as nodes for possible therapeutic intervention.

Design of a novel LOX-1 receptor antagonist mimicking the natural substrate.

The lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), the major receptor for oxidized low-density lipoprotein (ox-LDL) in endothelial cells, is overexpressed in atherosclerotic lesions. LOX-1 specific inhibitors, urgently necessary to reduce the rate of atherosclerotic and inflammation processes, are not yet available. We have designed and synthesized a new modified oxidized phospholipid, named PLAzPC, which plays to small scale the ligand-receptor recognition scheme. Molecular docking simulations confirm that PLAzPC disables the hydrophobic component of the ox-LDL recognition domain and allows the interaction of the l-lysine backbone charged groups with the solvent and with the charged/polar residues located around the edges of the LOX-1 hydrophobic tunnel. Binding assays, in a cell model system expressing human LOX-1 receptors, confirm that PLAzPC markedly inhibits ox-LDL binding to LOX-1 with higher efficacy compared to previously identified inhibitors.

Parkinson's disease: a complex interplay of mitochondrial DNA alterations and oxidative stress.

Parkinson's disease (PD) is one of the most common age-related neurodegenerative diseases. This pathology causes a significant loss of dopaminergic neurons in the Substantia Nigra. Several reports have claimed a role of defective nuclear and mitochondrial DNA repair pathways in PD etiology, in particular, of the Base Excision Repair (BER) system. In addition, recent findings, related to PD progression, indicate that oxidative stress pathways involving c-Abl and GST could also be implicated in this pathology. This review focuses on recently described networks most likely involved in an integrated manner in the course of PD.

Cholesterol-lowering drugs inhibit lectin-like oxidized low-density lipoprotein-1 receptor function by membrane raft disruption.

Lectin-like oxidized low-density lipoprotein (LOX-1), the primary receptor for oxidized low-density lipoprotein (ox-LDL) in endothelial cells, is up-regulated in atherosclerotic lesions. Statins are the principal therapeutic agents for cardiovascular diseases and are known to down-regulate LOX-1 expression. Whether the effect on the LOX-1 receptor is related to statin-mediated cholesterol-lowering activity is unknown. We investigate the requirement of cholesterol for LOX-1-mediated lipid particle internalization, trafficking, and processing and the role of statins as inhibitors of LOX-1 function. Disruption of cholesterol-rich membrane microdomains by acute exposure of cells to methyl-ß-cyclodextrin or chronic exposure to different statins (lovastatin and atorvastatin) led to a spatial disorganization of LOX-1 in plasma membranes and a marked loss of specific LOX-1 function in terms of ox-LDL binding and internalization. Subcellular fractionation and immunochemical studies indicate that LOX-1 is naturally present in caveolae-enriched lipid rafts and, by cholesterol reduction, the amount of LOX-1 in this fraction is highly decreased (≥60%). In contrast, isoprenylation inhibition had no effect on the distribution and function of LOX-1 receptors. Furthermore, in primary cultures from atherosclerotic human aorta lesions, we confirm the presence of LOX-1 in caveolae-enriched lipid rafts and demonstrate that lovastatin treatment led to down-regulation of LOX-1 in lipid rafts and rescue of the ox-LDL-induced apoptotic phenotype. Taken together, our data reveal a previously unrecognized essential role of membrane cholesterol for LOX-1 receptor activity and suggest that statins protect vascular endothelium against the adverse effect of ox-LDL by disruption of membrane rafts and impairment of LOX-1 receptor function.

Monomer-dimer equilibrium in glutathione transferases: a critical re-examination.

Glutathione transferases (GSTs) are dimeric enzymes involved in cell detoxification versus many endogenous toxic compounds and xenobiotics. In addition, single monomers of GSTs appear to be involved in particular protein-protein interactions as in the case of the pi class GST that regulates the apoptotic process by means of a GST-c-Jun N-terminal kinase complex. Thus, the dimer-monomer transition of GSTs may have important physiological relevance, but many studies reached contrasting conclusions both about the modality and extension of this event and about the catalytic competence of a single subunit. This paper re-examines the monomer-dimer question in light of novel experiments and old observations. Recent papers claimed the existence of a predominant monomeric and active species among pi, alpha, and mu class GSTs at 20-40 nM dilution levels, reporting dissociation constants (K(d)) for dimeric GST of 5.1, 0.34, and 0.16 microM, respectively. However, we demonstrate here that only traces of monomers could be found at these concentrations since all these enzymes display K(d) values of <<1 nM, values thousands of times lower than those reported previously. Time-resolved and steady-state fluorescence anisotropy experiments, two-photon fluorescence correlation spectroscopy, kinetic studies, and docking simulations have been used to reach such conclusions. Our results also indicate that there is no clear evidence of the existence of a fully active monomer. Conversely, many data strongly support the idea that the monomeric form is scarcely active or fully inactive.

The anti-cancer drug chlorambucil as a substrate for the human polymorphic enzyme glutathione transferase P1-1: kinetic properties and crystallographic characterisation of allelic variants.

The commonly used anti-cancer drug chlorambucil is the primary treatment for patients with chronic lymphocytic leukaemia. Chlorambucil has been shown to be detoxified by human glutathione transferase Pi (GST P1-1), an enzyme that is often found over-expressed in cancer tissues. The allelic variants of GST P1-1 are associated with differing susceptibilities to leukaemia and differ markedly in their efficiency in catalysing glutathione (GSH) conjugation reactions. Here, we perform detailed kinetic studies of the allelic variants with the aid of three representative co-substrates. We show that the differing catalytic properties of the variants are highly substrate-dependent. We show also that all variants exhibit the same temperature stability in the range 10 degrees C to 45 degrees C. We have determined the crystal structures of GST P1-1 in complex with chlorambucil and its GSH conjugate for two of these allelic variants that have different residues at positions 104 and 113. Chlorambucil is found to bind in a non-productive mode to the substrate-binding site (H-site) in the absence of GSH. This result suggests that under certain stress conditions where GSH levels are low, GST P1-1 can inactivate the drug by sequestering it from the surrounding medium. However, in the presence of GSH, chlorambucil binds in the H-site in a productive mode and undergoes a conjugation reaction with GSH present in the crystal. The crystal structure of the GSH-chlorambucil complex bound to the *C variant is identical with the *A variant ruling out the hypothesis that primary structure differences between the variants cause structural changes at the active site. Finally, we show that chlorambucil is a very poor inhibitor of the enzyme in contrast to ethacrynic acid, which binds to the enzyme in a similar fashion but can act as both substrate and inhibitor.