In utero pulse injection of isotopic amino acids quantifies protein turnover rates during murine fetal development.

Protein translational control is critical for ensuring that the fetus develops correctly and that necessary organs and tissues are formed and functional. We developed an in utero method to quantify tissue-specific protein dynamics by monitoring amino acid incorporation into the proteome after pulse injection. Fetuses of pregnant mice were injected with isotopically labeled lysine and arginine via the vitelline vein at various embyonic days, and organs and tissues were harvested. By analyzing the nascent proteome, unique signatures of each tissue were identified by hierarchical clustering. In addition, the quantified proteome-wide turnover rates were calculated between 3.81E-5 and 0.424 h-1. We observed similar protein turnover profiles for analyzed organs (e.g., liver vs. brain); however, their distributions of turnover rates vary significantly. The translational kinetic profiles of developing organs displayed differentially expressed protein pathways and synthesis rates, which correlated with known physiological changes during mouse development.

In utero pulse injection of isotopic amino acids quantifies protein turnover rates during murine fetal development.

Protein translational control is highly regulated step in the gene expression program during mammalian development that is critical for ensuring that the fetus develops correctly and that all of the necessary organs and tissues are formed and functional. Defects in protein expression during fetal development can lead to severe developmental abnormalities or premature death. Currently, quantitative techniques to monitor protein synthesis rates in a developing fetus (in utero) are limited. Here, we developed a novel in utero stable isotope labeling approach to quantify tissue-specific protein dynamics of the nascent proteome during mouse fetal development. Fetuses of pregnant C57BL/6J mice were injected with isotopically labeled lysine (Lys8) and arginine (Arg10) via the vitelline vein at various gestational days. After treatment, fetal organs/tissues including brain, liver, lung, and heart were harvested for sample preparation and proteomic analysis. We show that the mean incorporation rate for injected amino acids into all organs was 17.50 ± 0.6%. By analyzing the nascent proteome, unique signatures of each tissue were identified by hierarchical clustering. In addition, the quantified proteome-wide turnover rates (kobs) were calculated between 3.81E-5 and 0.424 hour-1. We observed similar protein turnover profiles for analyzed organs (e.g., liver versus brain), however, their distributions of turnover rates vary significantly. The translational kinetic profiles of developing organs displayed differentially expressed protein pathways and synthesis rates which correlated with known physiological changes during mouse development.

Arginine Methylation of the PGC-1α C-Terminus Is Temperature-Dependent.

We set out to determine whether the C-terminus (amino acids 481-798) of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α, UniProt Q9UBK2), a regulatory metabolic protein involved in mitochondrial biogenesis, and respiration, is an arginine methyltransferase substrate. Arginine methylation by protein arginine methyltransferases (PRMTs) alters protein function and thus contributes to various cellular processes. In addition to confirming methylation of the C-terminus by PRMT1 as described in the literature, we have identified methylation by another member of the PRMT family, PRMT7. We performed in vitro methylation reactions using recombinant mammalian PRMT7 and PRMT1 at 37, 30, 21, 18, and 4 °C. Various fragments of PGC-1α corresponding to the C-terminus were used as substrates, and the methylation reactions were analyzed by fluorography and mass spectrometry to determine the extent of methylation throughout the substrates, the location of the methylated PGC-1α arginine residues, and finally, whether temperature affects the deposition of methyl groups. We also employed two prediction programs, PRmePRed and MePred-RF, to search for putative methyltransferase sites. Methylation reactions show that arginine residues R548 and R753 in PGC-1α are methylated at or below 30 °C by PRMT7, while methylation by PRMT1 was detected at these same residues at 30 °C. Computational approaches yielded additional putative methylarginine sites, indicating that since PGC-1α is an intrinsically disordered protein, additional methylated arginine residues have yet to be experimentally verified. We conclude that temperature affects the extent of arginine methylation, with more methylation by PRMT7 occurring below physiological temperature, uncovering an additional control point for PGC-1α.

Targeting acetyl-CoA metabolism attenuates the formation of fear memories through reduced activity-dependent histone acetylation.

Histone acetylation is a key component in the consolidation of long-term fear memories. Histone acetylation is fueled by acetyl-coenzyme A (acetyl-CoA), and recently, nuclear-localized metabolic enzymes that produce this metabolite have emerged as direct and local regulators of chromatin. In particular, acetyl-CoA synthetase 2 (ACSS2) mediates histone acetylation in the mouse hippocampus. However, whether ACSS2 regulates long-term fear memory remains to be determined. Here, we show that Acss2 knockout is well tolerated in mice, yet the Acss2-null mouse exhibits reduced acquisition of long-term fear memory. Loss of Acss2 leads to reductions in both histone acetylation and expression of critical learning and memory-related genes in the dorsal hippocampus, specifically following fear conditioning. Furthermore, systemic administration of blood-brain barrier-permeable Acss2 inhibitors during the consolidation window reduces fear-memory formation in mice and rats and reduces anxiety in a predator-scent stress paradigm. Our findings suggest that nuclear acetyl-CoA metabolism via ACSS2 plays a critical, previously unappreciated, role in the formation of fear memories.

Enzymatic transfer of acetate on histones from lysine reservoir sites to lysine activating sites.

Histone acetylation is governed by nuclear acetyl-CoA pools generated, in part, from local acetate by metabolic enzyme acetyl-CoA synthetase 2 (ACSS2). We hypothesize that during gene activation, a local transfer of intact acetate occurs via sequential action of epigenetic and metabolic enzymes. Using stable isotope labeling, we detect transfer between histone acetylation sites both in vitro using purified mammalian enzymes and in vivo using quiescence exit in Saccharomyces cerevisiae as a change-of-state model. We show that Acs2, the yeast ortholog of ACSS2, is recruited to chromatin during quiescence exit and observe dynamic histone acetylation changes proximal to Acs2 peaks. We find that Acs2 is preferentially associated with the most up-regulated genes, suggesting that acetyl group transfer plays an important role in gene activation. Overall, our data reveal direct transfer of acetate between histone lysine residues to facilitate rapid transcriptional induction, an exchange that may be critical during changes in nutrient availability.

A Dynamic and Combinatorial Histone Code Drives Malaria Parasite Asexual and Sexual Development.

Histone posttranslational modifications (PTMs) frequently co-occur on the same chromatin domains or even in the same molecule. It is now established that these "histone codes" are the result of cross talk between enzymes that catalyze multiple PTMs with univocal readout as compared with these PTMs in isolation. Here, we performed a comprehensive identification and quantification of histone codes of the malaria parasite, Plasmodium falciparum. We used advanced quantitative middle-down proteomics to identify combinations of PTMs in both the proliferative, asexual stages and transmissible, sexual gametocyte stages of P. falciparum. We provide an updated, high-resolution compendium of 77 PTMs on H3 and H3.3, of which 34 are newly identified in P. falciparum. Coexisting PTMs with unique stage distinctions were identified, indicating that many of these combinatorial PTMs are associated with specific stages of the parasite life cycle. We focused on the code H3R17me2K18acK23ac for its unique presence in mature gametocytes; chromatin proteomics identified a gametocyte-specific SAGA-like effector complex including the transcription factor AP2-G2, which we tied to this specific histone code, as involved in regulating gene expression in mature gametocytes. Ultimately, this study unveils previously undiscovered histone PTMs and their functional relationship with coexisting partners. These results highlight that investigating chromatin regulation in the parasite using single histone PTM assays might overlook higher-order gene regulation for distinct proliferation and differentiation processes.

p53 mediates target gene association with nuclear speckles for amplified RNA expression.

Nuclear speckles are prominent nuclear bodies that contain proteins and RNA involved in gene expression. Although links between nuclear speckles and gene activation are emerging, the mechanisms regulating association of genes with speckles are unclear. We find that speckle association of p53 target genes is driven by the p53 transcription factor. Focusing on p21, a key p53 target, we demonstrate that speckle association boosts expression by elevating nascent RNA amounts. p53-regulated speckle association did not depend on p53 transactivation functions but required an intact proline-rich domain and direct DNA binding, providing mechanisms within p53 for regulating gene-speckle association. Beyond p21, a substantial subset of p53 targets have p53-regulated speckle association. Strikingly, speckle-associating p53 targets are more robustly activated and occupy a distinct niche of p53 biology compared with non-speckle-associating p53 targets. Together, our findings illuminate regulated speckle association as a mechanism used by a transcription factor to boost gene expression.

Phosphoserine inhibits neighboring arginine methylation in the RKS motif of histone H3.

The effects of phosphorylation of histone H3 at serine 10 have been studied in the context of other posttranslational modifications such as lysine methylation. We set out to investigate the impact of phosphoserine-10 on arginine-8 methylation. We performed methylation reactions using peptides based on histone H3 that contain a phosphorylated serine and compared the extent of arginine methylation with unmodified peptides. Results obtained via fluorography indicate that peptides containing a phosphorylated serine-10 inhibit deposition of methyl groups to arginine-8 residues. To further explore the effects of phosphoserine on neighboring arginine residues, we physically characterized the non-covalent interactions between histone H3 phosphoserine-10 and arginine-8 using 31P NMR spectroscopy. A salt bridge was detected between the negatively charged phosphoserine-10 and the positively charged unmodified arginine-8 residue. This salt bridge was not detected when arginine-8 was symmetrically dimethylated. Finally, molecular simulations not only confirm the presence of a salt bridge but also identify a subset of electrostatic interactions present when arginine is replaced with alanine. Taken together, our work suggests that the negatively charged phosphoserine maximizes its interactions. By limiting its exposure and creating new contacts with neighboring residues, it will inhibit deposition of neighboring methyl groups, not through steric hindrance, but by forming intrapeptide interactions that may mask substrate recognition. Our work provides a mechanistic framework for understanding the role of phosphoserine on nearby amino acid residues and arginine methylation.

Bullet points to evaluate the performance of the middle-down proteomics workflow for histone modification analysis.

Middle-down proteomics has emerged as the method of choice to study combinatorial histone post translational modifications (PTMs). In the common bottom-up workflow, histones are digested into relatively short peptides (4-20 aa), separated using reversed-phase chromatography and analyzed using typical proteomics methods in mass spectrometry. In middle-down, histones are cleaved into longer polypeptides (50-60 aa) mostly corresponding to their N-terminal tails, resolved using weak cation exchange-hydrophilic interaction liquid chromatography (WCX-HILIC) and analyzed with less conventional mass spectrometry, i.e. using Electron Transfer Dissociation (ETD) for analyte fragmentation. Middle-down is not nearly as utilized as bottom-up for PTM analysis, partially due to its limited reproducibility and robustness. This has also limited the establishment of rigorous benchmarks to discriminate good vs poor quality experiments. Here, we describe critical aspects of the middle-down workflow to assist the user in evaluating the presence of biased and misleading results. Specifically, we tested the use of porous graphitic carbon (PGC) during the desalting step, demonstrating that desalting using only C18 material leads to sample loss. We also tested different salts in the WCX-HILIC buffers for their effect on retention, selectivity, and reproducibility of analysis of variants of histone tail fragments, in particular replacing ammonium ion with ethylenediammonium ion in buffer A. These substitutions had marked effects on selectivity and retention. Our results provide a streamlined way to evaluate middle-down performance to identify and quantify combinatorial histone PTMs.

Reactive oxygen species regulate lovastatin biosynthesis in Aspergillus terreus during submerged and solid-state fermentations.

In a previous work we detected an important increase in reactive oxygen species (ROS) concentrations during idiophase in lovastatin fermentations. Hence, the objective of the present work was to determine if ROS contributes to the regulation of lovastatin biosynthesis. Exogenous antioxidants were used to reduce ROS accumulation. The addition of N-Acetyl-L-cysteine (NAC) decreased ROS accumulation and concurrent lovastatin production. In solid-state fermentation (SSF), the addition of 100 mM of NAC lowered ROS accumulation by 53%, together with a 79% decrease in lovastatin biosynthesis. A similarly, situation was observed in submerged fermentation (SmF). Decreased lovastatin production was due to a lower expression of the regulatory gene lovE, and gene lovF. Moreover, the addition of H2O2 to the culture caused precocious gene expression and lovastatin biosynthesis. These results indicate that ROS accumulation in idiophase contributes to the regulation of the biosynthetic genes. It was considered that Yap1 (Atyap1) could be a transcription factor linking ROS with lovastatin biosynthesis. In a Northern analysis, Aspergillus terreus yap1 gene (Atyap1) was highly expressed during trophophase but down regulated during idiophase. Conversely, expression pattern of srrA gene, suggested that SrrA could positively control lovastatin biosynthesis, and also explaining the characteristics of the biosynthesis in SSF.

Embarazo ectopico complicado por aborto tubarico / Ectopic pregnancy complicated by tubal abortion

El embarazo ectópico es una complicación de la primera mitad del embarazo, cuya evolución clinica puede concluir de forma adversa, si es que no se adoptan las medidas oportunas y certeras dirigidas a salvar la vida de la mujer afectada en primera instancia, y la interidad del aparato genital en la segunda instancia. Si bien la presentación y evaluación clinica del cuadro guardas cierta caracteristica que nos permiten su disgnóstico, existe la posiblidad de una signo-sintomatologia casi ausente que confunde y nos puede conducir a una sub-valoración y concluisiones erradas. El caso en discusión , de desmarca en sintomatología y concluye en una sobredistención lubárica, sin solución de continuidad de la misma y aborto final hacia cavidad pélvica de un proceso gestacional que sobrepasa el primer trimestre (13 a 14 semanas). con invación placentaria en luz tubárica. Si bien su solución final es quirúrgica, el tipo de manifestación clínica puso en riesgo la vida de la mujer afectada.