Integrative Analysis of the Mitochondrial Proteome in Yeast

In this study yeast mitochondria were used as a model system to apply, evaluate, and integrate different genomic approaches to define the proteins of an organelle. Liquid chromatography mass spectrometry applied to purified mitochondria identified 546 proteins. By expression analysis and comparison to other proteome studies, we demonstrate that the proteomic approach identifies primarily highly abundant proteins. By expanding our evaluation to other types of genomic approaches, including systematic deletion phenotype screening, expression profiling, subcellular localization studies, protein interaction analyses, and computational predictions, we show that an integration of approaches moves beyond the limitations of any single approach. We report the success of each approach by benchmarking it against a reference set of known mitochondrial proteins, and predict approximately 700 proteins associated with the mitochondrial organelle from the integration of 22 datasets. We show that a combination of complementary approaches like deletion phenotype screening and mass spectrometry can identify over 75% of the known mitochondrial proteome. These findings have implications for choosing optimal genome-wide approaches for the study of other cellular systems, including organelles and pathways in various species. Furthermore, our systematic identification of genes involved in mitochondrial function and biogenesis in yeast expands the candidate genes available for mapping Mendelian and complex mitochondrial disorders in humans

Etude par spectrométrie de masse de la vaporisation par laser du graphite et du dioxyde d'uranium jusqu'à 4000k

Une nouvelle méthode de spectrométrie de masse (TOF MS) à haute température a été développée. La surface de l échantillon y est chauffée par laser pendant environ 20 ms, et température et spectres de masse sont mesurés en fonction du temps. Chaque expérience couvre tout un intervalle de température. Cette méthode a été appliquée au graphite pyrolytique et au dioxyde d uranium.L étude du graphite a clairement montré que la sublimation est de type Langmuir (ou surface libre), malgré les très hautes températures et pressions. Les pressions partielles relatives de C1, C2, C3, C4 et C5 ont été mesurées jusqu à 4100 K, ainsi que les enthalpies de sublimation des trois espèces principales de la vapeur. Les coefficients d évaporation relatifs de C1-C5 ont été estimés par comparaison des pressions partielles obtenues ici à 4000 K avec celles à l équilibre thermodynamique de la littérature.La courbe de pression de vapeur de UO2 au-dessus du dioxyde d uranium a été mesurée entre 2800 et 3400 K. Des enthalpies de sublimation et de vaporisation sont proposées pour UO2, ainsi qu une première valeur expérimentale de l enthalpie de vaporisation de UO3. Les rapports de pressions partielles p(UO2)/p(UO), p(UO2)/p(UO3) et p(UO2+)/p(UO+) ont été mesurés aux alentours de 3300 K et indiquent que l évaporation se fait dans des conditions proches de l équilibre thermodynamique.La méthode développée ici est adaptée à l étude par spectrométrie de masse jusqu à de très hautes températures de la vaporisation de matériaux réfractaires, et pourrait être utilisée pour l étude de matériaux chimiquement instables comme le dioxyde d uranium hyperstœchiométrique ou des carbures et nitrures.A new method of high-temperature mass spectrometry (TOF MS) was developed, where the specimen surface is heated by a laser pulse of approx. 20 ms. During it, time-resolved measurements of mass spectra and of the temperature are performed. Each experiment covers an entire temperature interval. The method was applied to pyrolytic graphite and uranium dioxide.In graphite study, it was clearly shown that the sublimation occurs in a Langmuir-like mode (free surface vaporisation), despite the very high temperatures and thus pressures. Relative partial pressures of C1, C2, C3, C4 and C5 were measured up to 4100 K. Obtained sublimation enthalpies of the main three vapour species are in a good agreement with literature values. Relative vaporisation coefficients of C1-C5 were estimated by comparison of the present partial pressures at 4000 K with equilibrium ones given in the literature.The vapour pressure curve of UO2 over stoichiometric uranium dioxide was measured between 2800 and 3400 K. Obtained sublimation and vaporisation enthalpies are in agreement with the literature. The vaporisation enthalpy of UO3 was measured for the first time. Relative partial pressure ratios p(UO2)/p(UO), p(UO2)/p(UO3) and p(UO2+)/p(UO+) were measured at around 3300 K and indicate that the vaporisation occurs in a regime close to thermodynamic equilibrium.This method is suitable for the fast and time-resolved mass spectrometric measurements of refractory materials up to very high temperatures, and could now be applied to the study of chemically unstable materials such as hyperstoichiometric urania and some carbides and nitrides.Key words: pyrolytic graphite, HOPG, uranium dioxide, laser vaporisation, TOF MS, vaporisation coefficients, Langmuir evaporation.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Ultrasonics and sonochemistry: Editors’ perspective

Ultrasonic waves can induce physical and chemical changes in liquid media via acoustic cavitation. Various applications have benefitted from utilizing these effects, including but not limited to the synthesis of functional materials, emulsification, cleaning, and processing. Several books and review articles in the public domain cover both fundamental and applied aspects of ultrasonics and sonochemistry. The Editors of the Ultrasonics Sonochemistry journal possess diverse expertise in this field, from theoretical and experimental aspects of acoustic cavitation to materials synthesis, environmental remediation, and sonoprocessing. This article provides Editors' perspectives on various aspects of ultrasonics and sonochemistry that may benefit students and early career researchers

Ataxin-2 regulates RGS8 translation in a new BAC-SCA2 transgenic mouse model.

Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominant disorder with progressive degeneration of cerebellar Purkinje cells (PCs) and other neurons caused by expansion of a glutamine (Q) tract in the ATXN2 protein. We generated BAC transgenic lines in which the full-length human ATXN2 gene was transcribed using its endogenous regulatory machinery. Mice with the ATXN2 BAC transgene with an expanded CAG repeat (BAC-Q72) developed a progressive cellular and motor phenotype, whereas BAC mice expressing wild-type human ATXN2 (BAC-Q22) were indistinguishable from control mice. Expression analysis of laser-capture microdissected (LCM) fractions and regional expression confirmed that the BAC transgene was expressed in PCs and in other neuronal groups such as granule cells (GCs) and neurons in deep cerebellar nuclei as well as in spinal cord. Transcriptome analysis by deep RNA-sequencing revealed that BAC-Q72 mice had progressive changes in steady-state levels of specific mRNAs including Rgs8, one of the earliest down-regulated transcripts in the Pcp2-ATXN2[Q127] mouse line. Consistent with LCM analysis, transcriptome changes analyzed by deep RNA-sequencing were not restricted to PCs, but were also seen in transcripts enriched in GCs such as Neurod1. BAC-Q72, but not BAC-Q22 mice had reduced Rgs8 mRNA levels and even more severely reduced steady-state protein levels. Using RNA immunoprecipitation we showed that ATXN2 interacted selectively with RGS8 mRNA. This interaction was impaired when ATXN2 harbored an expanded polyglutamine. Mutant ATXN2 also reduced RGS8 expression in an in vitro coupled translation assay when compared with equal expression of wild-type ATXN2-Q22. Reduced abundance of Rgs8 in Pcp2-ATXN2[Q127] and BAC-Q72 mice supports our observations of a hyper-excitable mGluR1-ITPR1 signaling axis in SCA2, as RGS proteins are linked to attenuating mGluR1 signaling

Cell type-dependent escape of capsid inhibitors by simian immunodeficiency virus SIVcpz

Pandemic human immunodeficiency virus type 1 (HIV-1) is the result of the zoonotic transmission of simian immunodeficiency virus (SIV) from the chimpanzee subspecies Pan troglodytes troglodytes (SIVcpzPtt). The related subspecies Pan troglodytes schweinfurthii is the host of a similar virus, SIVcpzPts, which did not spread to humans. We tested these viruses with small-molecule capsid inhibitors (PF57, PF74, and GS-CA1) that interact with a binding groove in the capsid that is also used by CPSF6. While HIV-1 was sensitive to capsid inhibitors in cell lines, human macrophages, and peripheral blood mononuclear cells (PBMCs), SIVcpzPtt was resistant in rhesus FRhL-2 cells and human PBMCs but was sensitive to PF74 in human HOS and HeLa cells. SIVcpzPts was insensitive to PF74 in FRhL-2 cells, HeLa cells, PBMCs, and macrophages but was inhibited by PF74 in HOS cells. A truncated version of CPSF6 (CPSF6-358) inhibited SIVcpzPtt and HIV-1, while in contrast, SIVcpzPts was resistant to CPSF6-358. Homology modeling of HIV-1, SIVcpzPtt, and SIVcpzPts capsids and binding energy estimates suggest that these three viruses bind similarly to the host proteins cyclophilin A (CYPA) and CPSF6 as well as the capsid inhibitor PF74. Cyclosporine treatment, mutation of the CYPA-binding loop in the capsid, or CYPA knockout eliminated the resistance of SIVcpzPts to PF74 in HeLa cells. These experiments revealed that the antiviral capacity of PF74 is controlled by CYPA in a virus- and cell type-specific manner. Our data indicate that SIVcpz viruses can use infection pathways that escape the antiviral activity of PF74. We further suggest that the antiviral activity of PF74 capsid inhibitors depends on cellular cofactors.IMPORTANCE HIV-1 originated from SIVcpzPtt but not from the related virus SIVcpzPts, and thus, it is important to describe molecular infection by SIVcpzPts in human cells to understand the zoonosis of SIVs. Pharmacological HIV-1 capsid inhibitors (e.g., PF74) bind a capsid groove that is also a binding site for the cellular protein CPSF6. SIVcpzPts was resistant to PF74 in HeLa cells but sensitive in HOS cells, thus indicating cell line-specific resistance. Both SIVcpz viruses showed resistance to PF74 in human PBMCs. Modulating the presence of cyclophilin A or its binding to capsid in HeLa cells overcame SIVcpzPts resistance to PF74. These results indicate that early cytoplasmic infection events of SIVcpzPts may differ between cell types and affect, in an unknown manner, the antiviral activity of capsid inhibitors. Thus, capsid inhibitors depend on the activity or interaction of currently uncharacterized cellular factors

In Vitro Assessment of the Genotoxic Hazard of Novel Hydroxamic Acid- and Benzamide-Type Histone Deacetylase Inhibitors (HDACi)

Histone deacetylase inhibitors (HDACi) are already approved for the therapy of leukemias. Since they are also emerging candidate compounds for the treatment of non-malignant diseases, HDACi with a wide therapeutic window and low hazard potential are desirable. Here, we investigated a panel of 12 novel hydroxamic acid- and benzamide-type HDACi employing non-malignant V79 hamster cells as toxicology guideline-conform in vitro model. HDACi causing a ≥10-fold preferential cytotoxicity in malignant neuroblastoma over non-malignant V79 cells were selected for further genotoxic hazard analysis, including vorinostat and entinostat for control. All HDACi selected, (i.e., KSK64, TOK77, DDK137 and MPK77) were clastogenic and evoked DNA strand breaks in non-malignant V79 cells as demonstrated by micronucleus and comet assays, histone H2AX foci formation analyses (γH2AX), DNA damage response (DDR) assays as well as employing DNA double-strand break (DSB) repair-defective VC8 hamster cells. Genetic instability induced by hydroxamic acid-type HDACi seems to be independent of bulky DNA adduct formation as concluded from the analysis of nucleotide excision repair (NER) deficient mutants. Summarizing, KSK64 revealed the highest genotoxic hazard and DDR stimulating potential, while TOK77 and MPK77 showed the lowest DNA damaging capacity. Therefore, these compounds are suggested as the most promising novel candidate HDACi for subsequent pre-clinical in vivo studies

Class I histone deacetylase inhibitor MS-275 attenuates vasoconstriction and inflammation in angiotensin II-induced hypertension.

OBJECTIVE:Non-selective histone deacetylase (HDAC) inhibitors are known to improve hypertension. Here, we investigated the therapeutic effect and regulatory mechanism of the class I HDAC selective inhibitors, MS-275 and RGFP966, in angiotensin (Ang) II-induced hypertensive mice. METHODS AND RESULTS:MS-275 inhibited the activity of HDAC1, HDAC2, and HDAC3, while RGFP966 weakly inhibited that of HDAC3 in a cell-free system. MS-275 and RGFP966 treatment reduced systolic blood pressure and thickness of the aorta wall in Ang II-induced hypertensive mice. MS-275 treatment reduced aorta collagen deposition, as determined by Masson's trichrome staining. MS-275 decreased the components of the renin angiotensin system and increased vascular relaxation of rat aortic rings via the nitric oxide (NO) pathway. NO levels reduced by Ang II were restored by MS-275 treatment in vascular smooth muscle cells (VSMCs). However, MS-275 dose (3 mg·kg-1·day-1) was not enough to induce NO production in vivo. In addition, MS-275 did not prevent endothelial nitric oxide synthase (eNOS) uncoupling in the aorta of Ang II-induced mice. Treatment with MS-275 failed to inhibit Ang II-induced expression of NADPH oxidase (Nox)1, Nox2, and p47phox. MS-275 treatment reduced proinflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and monocyte chemoattractant protein (MCP)-1, as well as adhesion molecules. Histological analysis showed that Ang II-induced macrophage infiltration was reduced by MS-275 and RGFP966 administration. CONCLUSIONS:Our results indicate that class I HDAC selective inhibitors may be good therapeutic agents for the treatment of hypertension through the regulation of vascular remodeling and vasoconstriction, as well as inflammation

Decreased steady-state levels of <i>Rgs8</i> message and protein in BAC-Q72 mice.

<p><b>(A)</b> qRT-PCR analyses of cerebellar RNAs from wild-type and BAC-Q22 mice show unchanged <i>Rgs8</i> levels, whereas BAC-Q72 mice show significant and progressive reduction of <i>Rgs8</i> mRNA levels starting at 5 weeks of age. n: number of animals in each group. The data are means ± SD, **p<0.01, ***p<0.001. <b>(B)</b> Western blot analyses indicate reduction of Rgs8 steady-state levels in cerebella of BAC-Q72 mice, but no change in BAC-Q22 mice when compared with wild-type mice. The blot is a representative Western blot of 3 independently performed experiments with 2 animals each per BAC line. <b>(C)</b> SCA2 patient-derived LB cells demonstrate decreased <i>RGS8</i> transcripts. Total RNAs were isolated from LB cell lines derived from two normal control individuals and two SCA2 patients and subjected to RT-PCR analysis using primers specifically amplifying the human <i>ATXN2</i> CAG repeat. RT-PCR analyses indicate the expression of ATXN2 with expanded CAG repeats (46 or 52) (left panel). qRT-PCR analyses of synthesized cDNAs from LB cells show significant reduction of <i>RGS8</i> in both SCA2-LB cell lines. The data represent mean ± SD, **p<0.01 (right panel).</p

Motor phenotype of <i>ATXN2</i> BAC transgenic mice on the accelerating rotarod.

<p><b>(A)</b> BAC-Q22 mice performed as well as wild-type mice at all ages. <b>(B)</b> BAC-Q72 mice performed significantly worse than wild-type littermates on the rotarod starting at 16 weeks of age. Data represent mean ± SEM of three trials on the test day (day 3). Number of animals tested are shown within the bars. Significance was determined using repeated measures ANOVA with post-hoc test correction. *p<0.05 and ***p<0.001.</p