Loss of CLPP alleviates mitochondrial cardiomyopathy without affecting the mammalian UPRmt

The mitochondrial matrix protease CLPP plays a central role in the activation of the mitochondrial unfolded protein response (UPR^mt) in Caenorhabditis elegans. Far less is known about mammalian UPR^mt signaling, although similar roles were assumed for central players, including CLPP. To better understand the mammalian UPR^mt signaling, we deleted CLPP in hearts of DARS2‐deficient animals that show robust induction of UPR^mt due to strong dysregulation of mitochondrial translation. Remarkably, our results clearly show that mammalian CLPP is neither required for, nor it regulates the UPR^mt in mammals. Surprisingly, we demonstrate that a strong mitochondrial cardiomyopathy and diminished respiration due to DARS2 deficiency can be alleviated by the loss of CLPP, leading to an increased de novo synthesis of individual OXPHOS subunits. These results question our current understanding of the UPR^mt signaling in mammals, while introducing CLPP as a possible novel target for therapeutic intervention in mitochondrial diseases.     

GnRH-II analogs for selective activation and inhibition of non-mammalian and type-II mammalian GnRH receptors

Recently, we identified three types of non-mammalian gonadotropin-releasing hormone receptors (GnRHR) in the bullfrog (designated bfGnRHR-1-3), and a mammalian type-II GnRHR in green monkey cell lines (denoted gmGnRHR-2). All these receptors responded better to GnRH-II than GnRH-I, while mammalian type-I GnRHR showed greater sensitivity to GnRH-I than GnRH-II. In the present study, we designed new GnRH-II analogs and examined whether they activated or inhibited non-mammalian and mammalian type-II GnRHRs. [D-Ala6]GnRH-II, with D-Ala substituted for Gly6 in GnRH-II, increased inositol phosphate (IP) production in cells stably expressing non-mammalian GnRHRs more effectively than native GnRH-II. However, it exhibited lower activity for mammalian type-I GnRHR than GnRH-I itself. Trptorelix-1, a GnRH-II antagonist, inhibited GnRH-induced IP production in cells expressing non-mammalian GnRHRs more effectively than Cetrorelix, a GnRH-I antagonist. Trptorelix-1, however, had lower potency for mammalian type-I GnRHR than Cetrorelix. Ligand-receptor binding assays revealed that [D-Ala6]GnRH-II and Trptorelix-1 have higher affinities for non-mammalian GnRHRs but lower affinities for mammalian type-I GnRHR than GnRH-II and Cetrorelix, respectively. Moreover, [D-Ala6]GnRH-II and Trptorelix-1 had a higher affinity for gmGnRHR-2 than GnRH-II and Cetrorelix, respectively. These results indicate that [D-Ala6]GnRH-II and Trptorelix-1 are highly effective agonist and antagonist, respectively, for non-mammalian and type-II mammalian GnRHRs.     

Hoechst 33258 binds to G-quadruplex in the promoter region of human c-myc

In vitro binding of Hoechst 33258 to the promoter region of human c-myc, d(GG GGAGGG TGG GGA GGG TGG GGA AGG TGG GG) which forms G-quadruplex, both in vitro and in vivo in the presence of metal ions, was investigated by equilibrium absorption, fluorescence, and kinetic surface plasmon resonance methods. Hypochromic effect in UV absorption spectra and blue shift in fluorescence emission maxima of Hoechst in the presence of quadruplex revealed that Hoechst binds to the quadruplex. Analysis of UV and fluorescence titration data revealed that Hoechst binds to quadruplex with binding affinity of the order of 10(6). Anisotropy measurements and higher lifetime obtained from time-resolved decay experiments revealed that quadruplex-bound Hoechst is rotationally restricted in a less polar environment than the bulk buffer medium. From surface plasmon resonance studies, we obtained kinetic association (k(a)) and dissociation (k(d)) of 1.23+/-0.04 x 10(5)M(-1)s(-1) and 0.686+/-0.009 s(-1), respectively. As Hoechst is known to bind A-T-rich region of duplex DNA, here we propose the likelihood of Hoechst interacting with the AAGGT loop of the quadruplex.     

Effect of green tea polyphenols on angiogenesis induced by an angiogenin-like protein

Angiogenesis is a fundamental process by which new blood vessels are formed. The angiogenesis process is induced by several growth factors. Among them angiogenin is the most potent blood vessel inducer known. In this paper, we have investigated the effect of green tea polyphenols, mainly the catechins, on an angiogenin-like protein induced angiogenesis process. The angiogenin-like protein was isolated from goat serum and the effect of green tea components was tested by the chicken chorioallantoic membrane (CAM) assay. The results show that green tea components are capable of reducing the vascularization on CAM that is induced by the angiogenin-like protein.     

RPE65 operates in the vertebrate visual cycle by stereospecifically binding all-trans-retinyl esters

RPE65 is a major protein of unknown function found associated with the retinyl pigment epithelial (RPE) membranes [Hamel, C. P., Tsilou, E., Pfeffer, B. A., Hooks, J. J., Detrick, B., and Redmond, T. M. (1993) J. Biol. Chem. 268, 15751-15757; Bavik, C. O., Levy, F., Hellman, U., Wernstedt, C., and Eriksson, U. (1993) J. Biol. Chem. 268, 20540-20546]. RPE65 knockouts fail to synthesize 11-cis-retinal, the chromophore of rhodopsin, and accumulate all-trans-retinyl esters in the RPE. Previous studies have also shown that RPE65 is specifically labeled with all-trans-retinyl ester based affinity labeling agents, suggesting a retinyl ester binding role for the protein. In the present work, we show that purified RPE65 binds all-trans-retinyl palmitate (tRP) with a K(D) = 20 pM. These quantitative experiments are performed by measuring the quenching of RPE65 fluorescence by added tRP. The binding for tRP is highly specific because 11-cis-retinyl palmitate binds with a K(D) = 14 nM, 11-cis-retinol binds with a K(D) = 3.8 nM, and all-trans-retinol (vitamin A) binds with a K(D) = 10.8 nM. This stereospecificity for tRP is to be compared to the binding of retinoids to BSA, where virtually no discrimination is found in the binding of the same retinoids. This work provides further evidence that RPE65 functions by binding to and mobilizing the highly hydrophobic all-trans-retinyl esters, allowing them to enter the visual cycle.     

The amyloid beta peptide (Abeta(1-40)) is thermodynamically soluble at physiological concentrations

Precipitation of the 39-43-residue amyloid beta peptide (Abeta) is a crucial factor in Alzheimer's disease (AD). In normal as well as in AD-afflicted brain, the Abeta concentration is estimated to be a few nanomolar. Here we show that Abeta(1-40) precipitates in vitro only if the dissolved concentration is >14 microM. Using fluorescence correlation spectroscopy, we further show that the precipitation is complete in 1 day, after which the size distribution of Abeta monomer/oligomers in the solution phase becomes stationary in time and independent of the starting Abeta concentration. Mass spectra confirm that both the solution phase and the coexisting precipitate contain chemically identical Abeta molecules. Incubation at 68 degrees C for 1 h reduces the solubility by <12%. Together, these results show that the thermodynamic saturation concentration (C(sat)) of Abeta(1-40) in phosphate-buffered saline (PBS) at pH 7.4 has a well-defined lower limit of 15.5 +/- 1 microM. Divalent metal ions (believed to play a role in AD) at near-saturation concentrations in PBS reduce C(sat) only marginally (2 mM Mg(2+) by 6%, 2.5 microM Ca(2+) by 7%, and 4 microM Zn(2+) by 11%). Given that no precipitation is possible at concentrations below C(sat), we infer that coprecipitant(s), and not properties of Abeta(1-40) alone, are key factors in the in vivo aggregation of Abeta.     

Influence of DNA structures on the conversion of sanguinarine alkanolamine form to iminium form

Sanguinarine exhibits pH dependent structural equilibrium between iminium form (structure I) and alkanolamine form (structure II) with a pKa of 7.4 as revealed from spectrophotometric titration. The titration data show that the compound exists almost exclusively as structure I and structure II in the pH range 1 to 6 and 8.5 to 11, respectively. The interaction of structure I and structure II to several B-form natural and synthetic double and single stranded DNAs has been studied by spectrophotometric, spectrofluorimetric and circular dichroic measurements in buffers of pH 5.2 and pH 10.4 where the physicochemical properties of DNA remain in B-form structure. The results show that structure I bind strongly to all B-form DNA structures showing typical hypochromism and bathochromism of the alkaloid's absorption maximum, quenching of steady-state fluorescence intensity and perturbations in circular dichroic spectrum. The structure II does not bind to DNA, but in presence of large amount of DNA significant population of structure I is generated, which binds to DNA and forms a structure I-DNA intercalated complex. The nature and magnitude of the spectral pattern are very much dependent on the structure as well as base composition of each DNA. The generation of the structure I from structure II is significantly affected by increasing ionic strength of the medium. The conversion of structure II to structure I in presence of high concentration of DNA in solution is explained through formation of a binding equilibrium process between structure II and structure I-DNA intercalated complex.