Elevated Levels of FMR1 mRNA in Granulosa Cells Are Associated with Low Ovarian Reserve in FMR1 Premutation Carriers

Aim

To assess the role of mRNA accumulation in granulosa cells as the cause of low ovarian response among FMR1 premutation carriers undergoing pre-implantation genetic diagnosis (PGD).

Design

Case control study in an academic IVF unit. Twenty-one consecutive FMR1 premutation carriers and 15 control women were included. After oocyte retrieval the granulosa cells mRNA levels of FMR1 was measured using RT-PCR.

Results

In FMR1 premutation carriers, there was a significant non-linear association between the number of CGG repeats and the number of retrieved oocytes (p<0.0001) and a trend to granulosa cells FMR1 mRNA levels (p = 0.07). The lowest number of retrieved oocytes and the highest level of mRNA were seen in women with mid-size CGG repeats (80–120). A significant negative linear correlation was observed between the granulosa cells FMR1 mRNA levels and the number of retrieved oocytes (R² linear = 0.231, P = 0.02).

Conclusion

We suggest that there is a no-linear association between the number of CGG repeats and ovarian function, resulting from an increased granulosa cells FMR1 mRNA accumulation in FMR1 carriers in the mid-range (80–120 repeats).     

Inhibition of HIV-1 entry before gp41 folds into its fusion-active conformation

HIV-1 entry into its host cell is modulated by its transmembrane envelope glycoprotein (gp41). The core of the activated conformation of gp41 consists of a trimer of heterodimers comprising a leucine/isoleucine zipper sequence (represented here by the synthetic peptide N36 or by the longer N51 peptide) and a C-terminal highly conserved region (represented here by C34). A correlation was found between the action of DP178, which is a potent inhibitor of HIV-1 entry into its host cell, and its ability to interact with the leucine/isoleucine zipper sequence. This correlation was further tested and confirmed by circular dichroism spectroscopy. We found that whereas DP178 perturbs the partial alpha-helix nature of peptides corresponding to the leucine/isoleucine zipper sequence (N36 or N51), it cannot perturb the trimer of heterodimers conformation, modeled by the complex of N36 or N51 with C34. Therefore, we suggest that the already formed trimer of heterodimers is not the target of inhibition by DP178. Our results are consistent with a model in which DP178 acquires its inhibitory activity by binding to an earlier intermediate of gp41, in which the N and C peptide regions are not yet associated, thus allowing DP178 to bind to the leucine/isoleucine zipper sequence and consequently to inhibit transition to the fusion-active conformation.     

Modulation of the Voltage-Dependent Anion Channel (VDAC) by Glutamate1

The voltage-dependent anion channel (VDAC), also known as mitochondrial porin, is a large channel permeable to anions, cations, ATP, and other metabolites. VDAC was purified from sheep brain synaptosomes or rat liver mitochondria using a reactive red-agarose column, in addition to the hydroxyapatitate column. The red-agarose column allowed further purification (over 98%), concentration of the protein over ten-fold, decreasing Triton X-100 concentration, and/or replacing Triton X-100 with other detergents, such as Nonidet P-40 or octylglucoside. This purified VDAC reconstituted into planar-lipid bilayer, had a unitary maximal conductance of 3.7 ± 0.1 nS in 1 M NaCl, at 10 mV and was permeable to both large cations and anions. In the maximal conducting state, the permeability ratios for Na⁺, acetylcholine⁺, dopamine,⁺ and glutamate^–, relative to Cl^–, were estimated to be 0.73, 0.6, 0.44, and 0.4, respectively. In contrast, in the subconducting state, glutamate^– was impermeable, while the relative permeability to acetylcholine⁺ increased and to dopamine⁺ remained unchanged. At the high concentrations (0.1–0.5 M) used in the permeability experiments, glutamate eliminated the bell shape of the voltage dependence of VDAC channel conductance. Glutamate at concentrations of 1 to 20 mM, in the presence of 1 M NaCl, was found to modulate the VDAC channel activity. In single-channel experiments, at low voltages (±10 mV), glutamate induced rapid fluctuations of the channel between the fully open state and long-lived low-conducting states or short-lived closed state. Glutamate modification of the channel activity, at low voltages, is dependent on voltage, requiring short-time (20–60 sec) exposure of the channel to high membrane potentials. The effect of glutamate is specific, since it was observed in the presence of 1 M NaCl and it was not obtained with aspartate or GABA. These results suggest that VDAC possesses a specific glutamate-binding site that modulates its activity.     

Epizootic Hemorrhagic Disease Virus Induces and Benefits from Cell Stress,Autophagy, and Apoptosis

The mode and timing of virally induced cell death hold the potential of regulating viral yield, viral transmission, and the severity of virally induced disease. Orbiviruses such as the epizootic hemorrhagic disease virus (EHDV) are nonenveloped and cytolytic. To date, the death of cells infected with EHDV, the signal transduction pathways involved in this process, and the consequence of their inhibition have yet to be characterized. Here, we report that the Ibaraki strain of EHDV2 (EHDV2-IBA) induces apoptosis, autophagy, a decrease in cellular protein synthesis, the activation of c-Jun N-terminal kinase (JNK), and the phosphorylation of the JNK substrate c-Jun. The production of infectious virions decreased upon inhibition of apoptosis with the pan-caspase inhibitor Q-VD-OPH (quinolyl-valyl-O-methylaspartyl-[-2,6-difluorophenoxy]-methyl ketone), upon inhibition of autophagy with 3-methyladenine or via the knockout of the autophagy regulator Atg5, or upon treatment of infected cells with the JNK inhibitor SP600125 or the cyclin-dependent kinase (CDK) inhibitor roscovitine, which also inhibited c-Jun phosphorylation. Moreover, Q-VD-OPH, SP600125, and roscovitine partially reduced EHDV2-IBA-induced cell death, and roscovitine diminished the induction of autophagy by EHDV2-IBA. Taken together, our results imply that EHDV induces and benefits from the activation of signaling pathways involved in cell stress and death.     

A novel substrate mimetic inhibitor of PKB/Akt inhibits prostate cancer tumor growth in mice by blocking the PKB pathway

We describe a novel, potent peptide substrate mimetic inhibitor of protein kinase B (PKB/Akt). The compound selectively kills prostate cancer cells, in which PKB is highly activated, but not normal cells, or cancer cells in which PKB is not activated. The inhibitor induces apoptosis and inhibits the phosphorylation of PKB substrates in prostate cancer cell lines and significantly increases the efficacy of chemotherapy agents to induce prostate cancer cell death, when given in combination. In vivo, the inhibitor exhibits a strong antitumor effect in two prostate cancer mouse models. Moreover, treated animals develop significantly less lung metastases compared to untreated ones, and the effect is accompanied by a significant decrease in blood PSA [prostate-specific antigen] levels in treated animals. This compound and its potential analogues may be developed into novel, potent, and safe anticancer agents, both as stand-alone treatment and in combination with other chemotherapy agents.     

Secondary structure and gating rearrangements of transmembrane segments in rat P2X4 receptor channels

P2X receptors are cation selective channels that are activated by extracellular nucleotides. These channels are likely formed by three identical or related subunits, each having two transmembrane segments (TM1 and TM2). To identify regions that undergo rearrangement during gating and to probe their secondary structure, we performed tryptophan scanning mutagenesis on the two putative TMs of the rat P2X4 receptor channel. Mutant channels were expressed in Xenopus oocytes, concentration-response relationships constructed for ATP, and the EC50 estimated by fitting the Hill equation to the data. Of the 22 mutations in TM1 and 24 in TM2, all but one in TM1 and seven in TM2 result in functional channels. Interestingly, the majority of the functional mutants display an increased sensitivity to ATP, and in general these perturbations are more pronounced for TM2 when compared with TM1. For TM1 and for the outer half of TM2, the perturbations are consistent with these regions adopting alpha-helical secondary structures. In addition, the greatest perturbations in the gating equilibrium occur for mutations near the outer ends of both TM1 and TM2. Surface biotinylation experiments reveal that all the nonfunctional mutants traffic to the surface membrane at levels comparable to the WT channel, suggesting that these mutations likely disrupt ion conduction or gating. Taken together, these results suggest that the outer parts of TM1 and TM2 are helical and that they move during activation. The observation that the majority of nonconducting mutations are clustered toward the inner end of TM2 suggests a critical functional role for this region.     

Identification of new protein substrates for the chloroplast ATP-dependent Clp protease supports its constitutive role in Arabidopsis

The ATP-dependent Clp protease in plant chloroplasts consists of a heterogeneous proteolytic core containing multiple ClpP and ClpR paralogues. In this study, we have examined in detail the only viable knockout mutant to date of one of these subunits in Arabidopsis thaliana, ClpR1. Loss of ClpR1 caused a slow-growth phenotype, with chlorotic leaves during early development that later partially recovered upon maturity. Analysis of the Clp proteolytic core in the clpR1 mutant (clpR1-1) revealed approx. 10% of the wild-type levels remaining, probably due to a relative increase in the closely related ClpR3 protein and its partial substitution of ClpR1 in the core complex. A proteomic approach using an in organello proteolytic assay revealed 19 new potential substrates for the chloroplast Clp protease. Many of these substrates were constitutive enzymes involved in different metabolic pathways, including photosynthetic carbon fixation, nitrogen metabolism and chlorophyll/haem biosynthesis, whereas others function in housekeeping roles such as RNA maturation, protein synthesis and maturation, and recycling processes. In contrast, degradation of the stress-related chloroplast proteins Hsp21 (heat-shock protein 21) and lipoxygenase 2 was unaffected in the clpR1-1 line and thus not facilitated by the Clp protease. Overall, we show that the chloroplast Clp protease is principally a constitutive enzyme that degrades numerous stromal proteins, a feature that almost certainly underlies its vital importance for chloroplast function and plant viability.     

Insertion and organization within membranes of the delta-endotoxin pore-forming domain, helix 4-loop-helix 5, and inhibition of its activity by a mutant helix 4 peptide

The pore-forming domain of Bacillus thuringiensis Cry1Ac insecticidal protein comprises of a seven alpha-helix bundle (alpha1-alpha7). According to the "umbrella model," alpha4 and alpha5 helices form a hairpin structure thought to be inserted into the membrane upon binding. Here, we have synthesized and characterized the hairpin domain, alpha4-loop-alpha5, its alpha4 and alpha5 helices, as well as mutant alpha4 peptides based on mutations that increased or decreased toxin toxicity. Membrane permeation studies revealed that the alpha4-loop-alpha5 hairpin is extremely active compared with the isolated helices or their mixtures, indicating the complementary role of the two helices and the need for the loop for efficient insertion into membranes. Together with spectrofluorometric studies, we provide direct evidence for the role of alpha4-loop-alpha5 as the membrane-inserted pore-forming hairpin in which alpha4 and alpha5 line the lumen of the channel and alpha5 also participates in the oligomerization of the toxin. Strikingly, the addition of the active alpha4 mutant peptide completely inhibits alpha4-loop-alpha5 pore formation, thus providing, to our knowledge, the first example that a mutated helix within a pore can function as an "immunity protein" by directly interacting with the segments that form the pore. This presents a potential means of interfering with the assembly and function of other membrane proteins as well.