Short telomeres in yeast are highly recombinogenic

We report that recombination rates specifically increase by up to 10(3) near shortened telomeres in K. lactis cells. This occurs in cells lacking telomerase that undergo growth senescence as well as in cells with stably shortened telomeres that cause little effect on cell growth. The high rates of gene conversion allowed a subtelomeric marker, initially present at a single telomere, to efficiently spread to most or all other telomeres in the cell. We propose that short telomeres in K. lactis are not fully competent at capping chromosome ends and hence are occasionally processed by proteins that normally act to repair broken DNA ends through recombination. This helps explain how recombination can be frequent enough to permit maintenance of telomeres in yeast cells lacking telomerase.     

The tetraspan protein epithelial membrane protein-2 interacts with beta1 integrins and regulates adhesion

The growth arrest-specific-3 (GAS3)/PMP22 proteins are members of the four-transmembrane (tetraspan) superfamily. Although the function of these proteins is poorly understood, GAS3/PMP22 proteins have been implicated in the control of growth and progression of certain cancers. Epithelial membrane protein-2 (EMP2), a GAS3/PMP22 family member, was recently identified as a putative tumor suppressor gene. Here, we addressed the normal function of EMP2 by testing the prediction that it influences integrin-related cell functions. We observed that EMP2 associates with the beta(1) integrin subunit. Co-immunoprecipitation and immunodepletion experiments indicated that approximately 60% of beta(1) integrins and EMP2 can be isolated in common protein complexes. Whereas this association between EMP2 and beta(1) integrin may be direct or indirect, it has features of integrin heterodimer selectivity. Thus, by laser confocal microscopy, EMP2 colocalized with alpha(6)beta(1) but not alpha(5)beta(1) integrin. Increased expression of EMP2 also influenced the integrin heterodimer repertoire present on the plasma membrane. EMP2 specifically increased the surface expression of the alpha(6)beta(1) integrin while decreasing that of the alpha(5)beta(1) protein. Reciprocally, reduction in EMP2 expression using a specific ribozyme decreased surface expression of alpha(6)beta(1) integrin. Accordingly, these EMP2-mediated changes resulted in a dramatic alteration in cellular adhesion to extracellular matrix proteins. This study demonstrates for the first time the interaction of a GAS3/PMP22 family member with an integrin protein and suggests that such interactions and their functional consequences are a physiologic role of GAS3/PMP22 proteins.     

Long CTG.CAG repeats from myotonic dystrophy are preferred sites for intermolecular recombination

Homologous recombination was shown to enable the expansion of CTG.CAG repeat sequences. Other prior investigations revealed the involvement of replication and DNA repair in these genetic instabilities. Here we used a genetic assay to measure the frequency of homologous intermolecular recombination between two CTG.CAG tracts. When compared with non-repeating sequences of similar lengths, long (CTG.CAG)(n) repeats apparently recombine with an approximately 60-fold higher frequency. Sequence polymorphisms that interrupt the homogeneity of the CTG.CAG repeat tracts reduce the apparent recombination frequency as compared with the pure uninterrupted repeats. The orientation of the repeats relative to the origin of replication strongly influenced the apparent frequency of recombination. This suggests the involvement of DNA replication in the recombination process of triplet repeats. We propose that DNA polymerases stall within the CTG.CAG repeat tracts causing nicks or double-strand breaks that stimulate homologous recombination. The recombination process is RecA-dependent.     

Zinc protoporphyrin IX binds heme crystals to inhibit the process of crystallization in Plasmodium falciparum

The intraerythrocytic Plasmodium falciparum parasite converts most of host hemoglobin heme into a nontoxic heme crystal. Erythrocyte zinc protoporphyrin IX, normally present at 0.5 microM, which is a ratio of 1:40,000 hemes, can elevate 10-fold in some of the anemias associated with malaria disease protection. This work examines a binding mechanism for zinc protoporphyrin IX inhibition of heme crystallization similar to the antimalarial quinolines. Zinc protoporphyrin IX neither forms crystals alone nor extends on preformed heme crystals. Inhibition of both seed heme crystal formation and crystal extension occurs with an inhibitory concentration (IC)50 of 5 microM. Field emission in-lens scanning electron microscopy depicts the transition and inhibition of heme monomer aggregates to heme crystals with and without seeding of preformed hemozoin templates. In vitro zinc protoporphyrin IX, like the quinolines, binds to heme crystals in a saturable, specific, pH, and time-dependent manner. The ratio at saturation is approximately 1 zinc protoporphyrin IX per 250 hemes of the crystal. Unlike the quinolines, zinc protoporphyrin IX binds measurably in the absence of heme. Isolated ring and trophozoite stage parasites have an elevated zinc protoporphyrin IX to heme ratio 6 to 10 times that in the erythrocyte cytosol, which also corresponds to elevated ratios found in heme crystals purified from Plasmodium parasites. This work implicates protection from malaria by a mechanism where elevated zinc protoporphyrin IX in anemic erythrocytes binds to heme crystals to inhibit further crystallization. In endemic malaria areas, severe iron deficiency anemia should be treated with antimalarials along with iron replenishment.     

Selective activation of antitumor activity of macrophages by the delivery of muramyl dipeptide using a novel polynucleotide-based carrier recognized by scavenger receptors

We have shown that muramyl dipeptide (MDP) conjugated to a 10-mer polyguanylic acid (PolyG) is specifically internalized by macrophages through scavenger receptor (SCR)-mediated endocytosis. Macrophages activated by PolyG-MDP displayed about 20-fold higher cytotoxic activity against nonmacrophage tumor cells compared to that elicited by free MDP. The PolyG-MDP was found to trigger the secretion of higher levels of interleukin-6, interleukin-1alpha, TNF-alpha, and nitric oxide in comparison to free MDP. Addition of antibodies directed against IL-6 and TNF-alpha to macrophage culture completely abrogated the tumoricidal response of PolyG-MDP, indicating that these two cytokines are primarily responsible for bioefficacy. This general approach of PolyG as a vehicle may find wide application in the delivery of genes and antisense oligonucleotides to macrophages.     

Molecular basis for the polyamine-ompF porin interactions: inhibitor and mutant studies

By testing the sensitivity of Escherichia coli OmpF porin to various natural and synthetic polyamines of different lengths, charge and other molecular characteristics, we were able to identify the molecular properties required for compounds to act as inhibitors of OmpF in the nanomolar range. Inhibitors require at least two amine groups to be effective. For diamines, the optimum length of the hydrocarbon spacer was found to be of eight to ten methylene groups. Triamine molecules based on a 12-carbon motif were found to be more effective that spermidine, an eight-carbon trivalent derivative. But differences in inhibition efficiencies were also found for trivalent compounds depending on the relative position of the internal secondary amine group with respect to the terminal groups. Finally, quaternary ammonium derivatives had no effect, suggesting that the nature of the terminal amine is important for the interaction. From these observations, we deduce that inhibition efficiency in the nanomolar range requires a 12-carbon chain triamine with terminal primary amine groups and replacement of the eighth methylene by a secondary amine. The need for this type of molecular architecture suggests that inhibition is governed by interactions between specific amine groups and protein residues, and that this is not simply due to the accumulation of charges into the pore. Together with previous observations from site-directed mutagenesis studies and inspection of the crystal structure of OmpF, these results allowed us to propose three residues (D113, D121 and Y294) as putative sites of interaction between the channel and spermine. Alanine substitution at each of these three residues resulted in a loss of inhibition by spermine, while mutations of only D113 and D121 affected inhibition by spermidine. Based on these observations, we suggest a model for the molecular determinants involved in the porin-polyamine interaction.     

Breaking the Hydrophobicity of the MscL Pore: Insights into a Charge-Induced Gating Mechanism

The mechanosensitive channel of large conductance (MscL) is a protein that responds to membrane tension by opening a transient pore during osmotic downshock. Due to its large pore size and functional reconstitution into lipid membranes, MscL has been proposed as a promising artificial nanovalve suitable for biotechnological applications. For example, site-specific mutations and tailored chemical modifications have shown how MscL channel gating can be triggered in the absence of tension by introducing charged residues at the hydrophobic pore level. Recently, engineered MscL proteins responsive to stimuli like pH or light have been reported. Inspired by experiments, we present a thorough computational study aiming at describing, with atomistic detail, the artificial gating mechanism and the molecular transport properties of a light-actuated bacterial MscL channel, in which a charge-induced gating mechanism has been enabled through the selective cleavage of photo-sensitive alkylating agents. Properties such as structural transitions, pore dimension, ion flux and selectivity have been carefully analyzed. Besides, the effects of charge on alternative sites of the channel with respect to those already reported have been addressed. Overall, our results provide useful molecular insights into the structural events accompanying the engineered MscL channel gating and the interplay of electrostatic effects, channel opening and permeation properties. In addition, we describe how the experimentally observed ionic current in a single-subunit charged MscL mutant is obtained through a hydrophobicity breaking mechanism involving an asymmetric inter-subunit motion.     

Design and activity of AP endonuclease-1 inhibitors

Apurinic/apyrimidinic endonuclease-1/redox effector factor-1 (APE-1) is a critical component of base excision repair that excises abasic lesions created enzymatically by the action of DNA glycosylases on modified bases and non-enzymatically by hydrolytic depurination/depyrimidination of nucleobases. Many anticancer drugs generate DNA adducts that are processed by base excision repair, and tumor resistance is frequently associated with enhanced APE-1 expression. Accordingly, APE-1 is a potential therapeutic target to treat cancer. Using computational approaches and the high resolution structure of APE-1, we developed a 5-point pharmacophore model for APE-1 small molecule inhibitors. One of the nM APE-1 inhibitors (AJAY-4) that was identified based on this model exhibited an overall median growth inhibition (GI₅₀) of 4.19 μM in the NCI-60 cell line panel. The mechanism of action is shown to be related to the buildup of abasic sites that cause PARP activation and PARP cleavage, and the activation of caspase-3 and caspase-7, which is consistent with cell death by apoptosis. In a drug combination growth inhibition screen conducted in 10 randomly selected NCI-60 cell lines and with 20 clinically used non-genotoxic anticancer drugs, a synergy was flagged in the SK-MEL-5 melanoma cell line exposed to combinations of vemurafenib, which targets melanoma cells with V600E mutated BRAF, and AJAY-4, our most potent APE-1 inhibitor. The synergy between AJAY-4 and vemurafenib was not observed in cell lines expressing wild-type B-Raf protein. This synergistic combination may provide a solution to the resistance that develops in tumors treated with B-Raf-targeting drugs.

Electronic supplementary material

The online version of this article (doi:10.1007/s12154-015-0131-7) contains supplementary material, which is available to authorized users.