The zinc finger proteins Mxr1p and repressor of phosphoenolpyruvate carboxykinase (ROP) have the same DNA binding specificity but regulate Methanol Metabolism antagonistically in pichia pastoris

The methanol-inducible alcohol oxidase I (AOXI) promoter of the methylotrophic yeast, Pichia pastoris, is used widely for the production of recombinant proteins. AOXI transcription is regulated by the zinc finger protein Mxr1p (methanol expression regulator 1). ROP (repressor of phosphoenolpyruvate carboxykinase, PEPCK) is a methanol- and biotin starvation-inducible zinc finger protein that acts as a negative regulator of PEPCK in P. pastoris cultured in biotin-deficient, glucose-ammonium medium. The function of ROP during methanol metabolism is not known. In this study, we demonstrate that ROP represses methanol-inducible expression of AOXI when P. pastoris is cultured in a nutrient-rich medium containing yeast extract, peptone, and methanol (YPM). Deletion of the gene encoding ROP results in enhanced expression of AOXI and growth promotion whereas overexpression of ROP results in repression of AOXI and growth retardation of P. pastoris cultured in YPM medium. Surprisingly, deletion or overexpression of ROP has no effect on AOXI gene expression and growth of P. pastoris cultured in a minimal medium containing yeast nitrogen base and methanol (YNBM). Subcellular localization studies indicate that ROP translocates from cytosol to nucleus of cells cultured in YPM but not YNBM. In vitro DNA binding studies indicate that AOXI promoter sequences containing 5' CYCCNY 3' motifs serve as binding sites for Mxr1p as well as ROP. Thus, Mxr1p and ROP exhibit the same DNA binding specificity but regulate methanol metabolism antagonistically in P. pastoris. This is the first report on the identification of a transcriptional repressor of methanol metabolism in any yeast species.

NMR studies of preimplantation embryo metabolism in human assisted reproductive techniques: a new biomarker for assessment of embryo implantation potential

There has been growing interest in understanding energy metabolism in human embryos generated using assisted reproductive techniques (ART) for improving the overall success rate of the method. Using NMR spectroscopy as a noninvasive tool, we studied human embryo metabolism to identify specific biomarkers to assess the quality of embryos for their implantation potential. The study was based on estimation of pyruvate, lactate and alanine levels in the growth medium, ISM1, used in the culture of embryos. An NMR study involving 127 embryos from 48 couples revealed that embryos transferred on Day 3 (after 72 h in vitro culture) with successful implantation (pregnancy) exhibited significantly (p < 10(-5)) lower pyruvate/alanine ratios compared to those that failed to implant. Lactate levels in media were similar for all embryos. This implies that in addition to lactate production, successfully implanted embryos use pyruvate to produce alanine and other cellular functions. While pyruvate and alanine individually have been used as biomarkers, the present study highlights the potential of combining them to provide a single parameter that correlates strongly with implantation potential. Copyright (C) 2012 John Wiley & Sons, Ltd.

Transcription factor erect wing (EWG) is involved in indirect flight muscle patterning, development and maintenance in Drosophila

Muscle development is a multistep process which includes myoblast diversification, proliferation, migration, fusion, differentiation and growth. A hierarchical exhibition of myogenic factors is important for dexterous execution of progressive events in muscle formation. EWG (erect wing) is a transcription factor known to have a role in indirect flight muscle development (IFM) in Drosophila. We marked out the precise spatio-temporal expression profile of EWG in the myoblasts, and in the developing muscles. Mutant adult flies null for EWG in myoblasts show variable number of IFM, suggesting that EWG is required for patterning of the IFM. The remnant muscle found in the EWG null flies show proper assembly of the structural proteins, which implies that some myoblasts manage to fuse, develop and differentiate normally indicating that EWG is not required for differentiation program per se. However, when EWG expression is extended beyond its expression window in a wild type background, muscle thinning is observed implying EWG function in protein synthesis inhibition. Mis-expression studies in wing disc myoblasts hinted at its role in myoblast proliferation. We thus conclude that EWG is important for regulating fusion events which in turn decides the IFM pattern. Also IFM in EWG null mutants show clumps containing broken fibres and an altered mitochondrial morphology. The vertebrate homolog of EWG is nuclear respiratory factor1 (NRF1) which is known to have a function in mitochondrial biogenesis and protection against oxidative stress. Gene expression for inner mitochondrial membrane protein, Opa1-like was found to be absent in these mutants. Also, these flies were more sensitive to oxidative stress, indicating a compromised mitochondrial functioning. Our results therefore demonstrate that EWG functions in maintaining muscles’ structural integrity by ensuing proper mitochondrial activity.

Cytochrome P450-mediated metabolism in brain: functional roles and their implications

Introduction: Cytochromes P450 (P450) and associated monooxygenases are a family of heme proteins involved in metabolism of endogenous compounds (arachidonic acid, eicosanoids and prostaglandins) as also xenobiotics including drugs and environmental chemicals. Liver is the major organ involved in P450-mediated metabolism and hepatic enzymes have been characterized. Extrahepatic organs, such as lung, kidney and brain have the capability for biotransformation through P450 enzymes. Brain, including human brain, expresses P450 enzymes that metabolize xenobiotics and endogenous compounds. Areas covered: An overview of P450-mediated metabolism in brain is presented focusing on distinct differences seen in expression of P450 enzymes, generation of unique P450 enzymes in brain through alternate splicing and their consequences in terms of metabolism of psychoactive drugs and inflammatory prompts, such as leukotrienes, thus modulating inflammatory response. Expert opinion: The brain possesses unique P450s that metabolize drugs and endogenous compounds through pathways that are markedly different from that seen in liver indicating that extrapolation directly from liver to brain is not appropriate. It is therefore necessary to characterize the unique brain P450s and their ability to metabolize xenobiotics and endogenous compounds to better understand the functions of this important class of enzymes in brain, especially human brain.

Cyclic AMP-dependent protein lysine acylation in mycobacteria regulates fatty acid and propionate metabolism

Acetylation of lysine residues is a posttranslational modification that is used by both eukaryotes and prokaryotes to regulate a variety of biological processes. Here we identify multiple substrates for the cAMP-dependent protein lysine acetyltransferase from Mycobacterium tuberculosis (KATmt). We demonstrate that a catalytically important lysine residue in a number of FadD (fatty acyl CoA synthetase) enzymes is acetylated by KATmt in a cAMP-dependent manner and that acetylation inhibits the activity of FadD enzymes. A sirtuin-like enzyme can deacetylate multiple FadDs, thus completing the regulatory cycle. Using a strain deleted for the KATmt ortholog in Mycobacterium bovis Bacillus Calmette-Guerin (BCG), we show for the first time that acetylation is dependent on intracellular cAMP levels. KATmt can utilize propionyl CoA as a substrate and, therefore, plays a critical role in alleviating propionyl CoA toxicity in mycobacteria by inactivating acyl CoA synthetase (ACS). The precision by which mycobacteria can regulate the metabolism of fatty acids in a cAMP-dependent manner appears to be unparalleled in other biological organisms and is ideally suited to adapt to the complex environment that pathogenic mycobacteria experience in the host.

Effect of myonuclear number and mitochondrial fusion on Drosophila indirect flight muscle organization and size

Mechanisms involved in establishing the organization and numbers of fibres in a muscle are not completely understood. During Drosophila indirect flight muscle (IFM) formation, muscle growth is achieved by both incorporating hundreds of nuclei, and hypertrophy. As a result, IFMs provide a good model with which to understand the mechanisms that govern overall muscle organization and growth. We present a detailed analysis of the organization of dorsal longitudinal muscles (DLMs), a subset of the IFMs. We show that each DLM is similar to a vertebrate fascicle and consists of multiple muscle fibres. However, increased fascicle size does not necessarily change the number of constituent fibres, but does increase the number of myofibrils packed within the fibres. We also find that altering the number of myoblasts available for fusion changes DLM fascicle size and fibres are loosely packed with myofibrils. Additionally, we show that knock down of genes required for mitochondrial fusion causes a severe reduction in the size of DLM fascicles and fibres. Our results establish the organization levels of DLMs and highlight the importance of the appropriate number of nuclei and mitochondrial fusion in determining the overall organization, growth and size of DLMs. (C) 2013 Elsevier Inc. All rights reserved.

Drosophila Erect wing (Ewg) controls mitochondrial fusion during muscle growth and maintenance by regulation of the Opa1-like gene

Mitochondrial biogenesis and morphological changes are associated with tissue-specific functional demand, but the factors and pathways that regulate these processes have not been completely identified. A lack of mitochondrial fusion has been implicated in various developmental and pathological defects. The spatiotemporal regulation of mitochondrial fusion in a tissue such as muscle is not well understood. Here, we show in Drosophila indirect flight muscles (IFMs) that the nuclear-encoded mitochondrial inner membrane fusion gene, Opa1-like, is regulated in a spatiotemporal fashion by the transcription factor/co-activator Erect wing (Ewg). In IFMs null for Ewg, mitochondria undergo mitophagy and/or autophagy accompanied by reduced mitochondrial functioning and muscle degeneration. By following the dynamics of mitochondrial growth and shape in IFMs, we found that mitochondria grow extensively and fuse during late pupal development to form the large tubular mitochondria. Our evidence shows that Ewg expression during early IFM development is sufficient to upregulate Opa1-like, which itself is a requisite for both late pupal mitochondrial fusion and muscle maintenance. Concomitantly, by knocking down Opa1-like during early muscle development, we show that it is important for mitochondrial fusion, muscle differentiation and muscle organization. However, knocking down Opa1-like, after the expression window of Ewg did not cause mitochondrial or muscle defects. This study identifies a mechanism by which mitochondrial fusion is regulated spatiotemporally by Ewg through Opa1-like during IFM differentiation and growth.

Skeletal Muscle Degeneration and Regeneration in Mice and Flies

Many aspects of skeletal muscle biology are remarkably similar between mammals and tiny insects, and experimental models of mice and flies (Drosophila) provide powerful tools to understand factors controlling the growth, maintenance, degeneration (atrophy and necrosis), and regeneration of normal and diseased muscles, with potential applications to the human condition. This review compares the limb muscles of mice and the indirect flight muscles of flies, with respect to the mechanisms of adult myofiber formation, homeostasis, atrophy, hypertrophy, and the response to muscle degeneration, with some comment on myogenic precursor cells and common gene regulatory pathways. There is a striking similarity between the species for events related to muscle atrophy and hypertrophy, without contribution of any myoblast fusion. Since the flight muscles of adult flies lack a population of reserve myogenic cells (equivalent to satellite cells), this indicates that such cells are not required for maintenance of normal muscle function. However, since satellite cells are essential in postnatal mammals for myogenesis and regeneration in response to myofiber necrosis, the extent to which such regeneration might be possible in flight muscles of adult flies remains unclear. Common cellular and molecular pathways for both species are outlined related to neuromuscular disorders and to age-related loss of skeletal muscle mass and function (sarcopenia). The commonality of events related to skeletal muscles in these disparate species (with vast differences in size, growth duration, longevity, and muscle activities) emphasizes the combined value and power of these experimental animal models.