Rate Of Biosynthesis Research Articles

The biological significance of lipogenesis in Nasonia vitripennis.

Parasitic wasps have long been thought to be unable to synthesize fatty acids de novo, but recent 13C-labelling studies have challenged this view. It remained unclear, however, whether the reported biosynthesis rates are of biological relevance. Here, we show in Nasonia vitripennis that ageing females with partly depleted lipid reserves produce biologically relevant amounts of fatty acids de novo. Females with varying oviposition history (0-48 h) prior to feeding 20% 13C-labelled glucose solution showed 13C-incorporation rates of (mean ± SEM) 30 ± 2%, 50 ± 2%, 49 ± 3% and 21 ± 2% in palmitic, stearic, oleic and linoleic acid, respectively. The absolute amounts of fatty acids synthesized de novo across treatments corresponded to 28 ± 3 egg lipid equivalents. Females incorporated de novo synthesized fatty acids into their eggs, and glucose-fed females laid more eggs than water-fed control females. The number of eggs laid prior to glucose feeding did not correlate with the degree of lipogenesis, but the amounts of de novo synthesized fatty acids correlated with constitutive (not synthesized de novo) fatty acids. Hence, glucose feeding has a twofold effect on the fatty acid status of N. vitripennis females by decelerating the catabolism of existing fat reserves and partially replenishing ebbing fat reserves by lipogenesis.

CAGEs are Golgi-localized GT31 enzymes involved in cellulose biosynthesis in Arabidopsis.

SUMMARYCellulose is the main structural component in the plant cell walls. We show that two glycosyltransferase family 31 (GT31) enzymes of Arabidopsis thaliana, here named cellulose synthesis associated glycosyltransferases 1 and 2 (CAGE1 and 2), influence both primary and secondary cell wall cellulose biosynthesis. cage1cage2 mutants show primary cell wall defects manifesting as impaired growth and cell expansion in seedlings and etiolated hypocotyls, along with secondary cell wall defects, apparent as collapsed xylem vessels and reduced xylem wall thickness in the inflorescence stem. Single and double cage mutants also show increased sensitivity to the cellulose biosynthesis inhibitor isoxaben. The cage1cage2 phenotypes were associated with an approximately 30% reduction in cellulose content, an approximately 50% reduction in secondary cell wall CELLULOSE SYNTHASE (CESA) protein levels in stems and reduced cellulose biosynthesis rate in seedlings. CESA transcript levels were not significantly altered in cage1cage2 mutants, suggesting that the reduction in CESA levels was caused by a post‐transcriptional mechanism. Both CAGE1 and 2 localize to the Golgi apparatus and are predicted to synthesize β‐1,3‐galactans on arabinogalactan proteins. In line with this, the cage1cage2 mutants exhibit reduced levels of β‐Yariv binding to arabinogalactan protein linked β‐1,3‐galactan. This leads us to hypothesize that defects in arabinogalactan biosynthesis underlie the cellulose deficiency of the mutants.

Physiological and Molecular Changes in Cherry Red Tobacco in Response to Iron Deficiency Stress.

The genotype CR60 is a spontaneous Cherry Red variant (containing granular red dapples on flue-cured leaves) of the Yunyan 87 (Y87) tobacco; it accumulates higher concentration of iron (Fe) in leaves than Y87, but the physiological differences between them remain largely unknown. We investigated the physiological and molecular mechanisms of CR60 in response to Fe deficiency under hydroponic conditions. Our results showed no significant phenotypic difference between Y87 and CR60 at optimal (40 μM) and high Fe (160 and 320 μM) concentrations. By contrast, CR60 exhibited higher tolerance to Fe deficiency (0 μM) than Y87, as shown by higher concentrations of chlorophyll in CR60 leaves after 21-day Fe-deficiency stress. Transcriptome profiling coupled with RT-PCR analyses found that the expression of IRT1 and several genes associated with chlorophyll biosynthesis and photosynthesis (e.g., PRO, GSA, FD1, PsbO, and PC) was higher in CR60 than Y87. These results indicated that CR60 maintains sufficient Fe uptake, chlorophyll biosynthesis and photosynthetic rate when subjected to Fe starvation.

Lack of xyloglucan in the cell walls of the Arabidopsis xxt1/xxt2 mutant results in specific increases in homogalacturonan and glucomannan.

SUMMARYMutations in both ArabidopsisXyloglucan Xylosyltransferase1 (XXT1) and Xyloglucan Xylosyltransferase2 (XXT2) result in the absence of the major hemicellulose, xyloglucan (XyG). The growth physiology, tensile strength, and cell wall compositions of etiolated hypocotyls of xxt1/xxt2 double and xxt1/xxt2/xxt5 triple mutants were characterized and compared with those of mur2 and mur3 mutations that alter the side group composition of XyG. Wild type and mur2 demonstrated normal elongation growth, whereas the xxt double and triple mutants and mur3 showed reduced elongation and cell swelling at the base of the hypocotyl at the later stages of elongation. Whereas mur3 hypocotyls had reduced tensile strength, the xxt double and triple mutants had similar tensile strengths as wild type, but reached breaking points at lower strains. Loss of XyG in hypocotyls and rosette leaf cell walls was associated with specific increases in the relative abundance of homogalacturonan and the hemicellulose glucomannan, but not arabinoxylan. The increased homogalacturonan and glucomannan abundance did not result from enhanced expression of their polymer synthases. An analysis of published transcriptomic data indicated that genes encoding some members of the expansin, xyloglucan endotransglucosylase/hydrolase, arabinogalactan‐protein, and structural protein families were upregulated in the xxt mutants. We hypothesize that an as yet unknown mechanism exists by which pectin and hemicellulose abundance can be altered other than by increased rates of biosynthesis to maintain a structurally sound cell wall.

Physiological maturity and wound-based orchard practices influence the antioxidant content and metabolic activity of two species of açai fruit at harvest and during storage

Pre-harvest treatments like wound-based orchard management practices and early harvest were applied to açai plants (Euterpe oleracea Mart., Euterpe precatoria Mart.) to yield higher levels of antioxidants. Orchard practices like 50% shoot suppression and 50% cluster thinning when applied 87 d before harvest (187 days DAA) were similar to control fruits at harvest and during storage (20 °C). However, lesions in the stipe applied 187 DAA altered the acid, carbohydrate, phenolic content and the ethylene biosynthesis compared to control fruits, showing enhanced fruit antioxidant activity. Early harvest of fruit including 120 and 150 DAA, showed higher acid, lower sugars, higher phenolic content and higher ethylene biosynthesis and respiration rate compared to control fruits, showing the highest levels of fruit antioxidant activity. The selected strategies studied may achieve higher yields of phenolic antioxdants from açai fruit and target high value health markets including functional foods and dietary supplements.

Enhancing tryptophan production by balancing precursors in Escherichia coli.

Tryptophan, an essential aromatic amino acid, is widely used in animal feed, food additives, and pharmaceuticals. Although sustainable and environmentally friendly, microbial tryptophan production from renewable feedstocks is limited by low biosynthesis and transport rates. Here, an Escherichia coli strain capable of efficient tryptophan production was generated by improving and balancing the supply of precursors and by engineering membrane transporters. Tryptophan biosynthesis was increased by eliminating negative regulatory factors, blocking competing pathways, and preventing tryptophan degradation. Promoter engineering balanced the supply of the precursors erythrose-4-phosphate and phosphoenolpyruvate, as well as the availability of serine. Finally, the engineering of tryptophan transporters prevented feedback inhibition and growth toxicity. Fed-batch fermentation of the final strain (TRP12) in a 5 L bioreactor produced 52.1 g·L-1 of tryptophan, with a yield of 0.171 g·g-1 glucose and productivity of 1.45 g·L-1 ·h-1 . The metabolic engineering strategy described here paves the way for high-performance microbial cell factories aimed at the production of tryptophan as well as other valuable chemicals.

Recombinant Production of Biliverdin IXβ and δ Isomers in the T7 Promoter Compatible Escherichia coli Nissle.

The ability to obtain purified biliverdin IX (BVIX) isomers other than the commercially available BVIXα is limited due to the low yields obtained by the chemical coupled oxidation of heme. Chemical oxidation requires toxic chemicals, has very poor BVIX yields (<0.05%), and is not conducive to scalable production. Alternative approaches utilizing recombinant E. coli BL21 expressing a cyanobacterial heme oxygenase have been employed for the production BVIXα, but yields are limited by the rate of endogenous heme biosynthesis. Furthermore, the emerging roles of BVIXβ and BVIXδ in biology and their lack of commercial availability has led to a need for an efficient and scalable method with the flexibility to produce all three physiologically relevant BVIX isomers. Herein, we have taken advantage of an optimized non-pathogenic E. coli Nissle (EcN(T7)) strain that encodes an endogenous heme transporter and an integrated T7 polymerase gene. Protein production of the Pseudomonas aeruginosa BVIXβ and BVIXδ selective heme oxygenase (HemO) or its BVIXα producing mutant (HemOα) in the EcN(T7) strain provides a scalable method to obtain all three isomers, that is not limited by the rate of endogenous heme biosynthesis, due to the natural ability of EcN(T7) to transport extracellular heme. Additionally, we have optimized our previous LC-MS/MS protocol for semi-preparative separation and validation of the BVIX isomers. Utilizing this new methodology for scalable production and separation we have increased the yields of the BVIXβ and -δ isomers >300-fold when compared to the chemical oxidation of heme.

Characterization of Sequence-Specific Binding of LARP6 to the 5′ Stem-Loop of Type I Collagen mRNAs and Implications for Rational Design of Antifibrotic Drugs

Excessive synthesis of type I collagen is a hallmark of fibrotic diseases. Binding of La-related protein 6 (LARP6) to the 5′ stem-loop (5′SL) of collagen mRNAs regulates their translation leading to an unnaturally elevated rate of collagen biosynthesis in fibrosis. Previous work suggested that LARP6 needs two domains to form stable complex with 5′SL RNA, the La domain and the juxtaposed RNA recognition motif (RRM), jointly called the La-module. Here we describe that La domain of LARP6 is necessary and sufficient for recognition of 5′SL in RNA sequence specific manner. A three-amino-acid motif located in the flexible loop connecting the second α-helix to the β-sheet of the La domain, called the RNK-motif, is critical for binding. Mutation of any of these three amino acids abolishes the binding of the La domain to 5′SL. The major site of crosslinking of LARP6 to 5′SL RNA was mapped to this motif, as well. The RNK-motif is not found in other LARPs, which cannot bind 5′SL. Presence of RRM increases the stability of complex between La domain and 5′SL RNA and RRM domain does not make extensive contacts with 5′SL RNA. We propose a model in which the initial recognition of 5′SL by LARP6 is mediated by the RNK epitope and further stabilized by the RRM domain. This discovery suggests that the interaction between LARP6 and collagen mRNAs can be blocked by small molecules that target the RNK epitope and will help rational design of the LARP6 binding inhibitors as specific antifibrotic drugs.

Mechanisms Affecting the Biosynthesis and Incorporation Rate of Selenocysteine.

Selenocysteine (Sec) is the 21st non-standard proteinogenic amino acid. Due to the particularity of the codon encoding Sec, the selenoprotein synthesis needs to be completed by unique mechanisms in specific biological systems. In this paper, the underlying mechanisms for the biosynthesis and incorporation of Sec into selenoprotein were comprehensively reviewed on five aspects: (i) the specific biosynthesis mechanism of Sec and the role of its internal influencing factors (SelA, SelB, SelC, SelD, SPS2 and PSTK); (ii) the elements (SECIS, PSL, SPUR and RF) on mRNA and their functional mechanisms; (iii) the specificity (either translation termination or translation into Sec) of UGA; (iv) the structure–activity relationship and action mechanism of SelA, SelB, SelC and SelD; and (v) the operating mechanism of two key enzyme systems for inorganic selenium source flow before Sec synthesis. Lastly, the size of the translation initiation interval, other action modes of SECIS and effects of REPS (Repetitive Extragenic Palindromic Sequences) that affect the incorporation efficiency of Sec was also discussed to provide scientific basis for the large-scale industrial fermentation for the production of selenoprotein.

Super Rice With High Sink Activities Has Superior Adaptability to Low Filling Stage Temperature.

To investigate the differential responses of super rice grain filling to low filling stage temperature (LT) and the regulative effect of nitrogen panicle fertilizer (NPF), physiological and molecular experiments were conducted with two super rice varieties (Nanjing 7th: N7 and Nanjing 9108th: N9108) on two different filling stage temperature treatments implemented by applying two sowing dates [Normal filling stage temperature (CK): Sowed on May 30, Tmean = 24.7°C and low filling stage temperature (LT): Sowed on July 1, Tmean = 20.3°C], and two NPF levels (0 and 150 kg N ha−1). In this study, LT, NPF, and simultaneous LT and NPF treatments suppressed the grain filling in all varieties with different levels. Under LT or NPF treatments, the reduction of grain weight, seed setting rate, and filling rate were closely associated with suppressed starch biosynthesis rate in inferior seeds, suggesting that reduced starch biosynthesis rate, expression, and activities of enzymes encoded by related genes, Floury endosperm-4 (FLO4), Starch branching enzyme-I (SBE1), and Starch phosphorylase-L (PHO-l), were responsible for the grain filling reduction. Under LT or NPF treatments, significantly higher grain filling rates and lower variance were found in N9108 compared to that in N7, which were closely related to their higher starch biosynthesis ability, related gene expression, and enzymes activities. One of the probable explanations of the grain filling difference was the variation in the relative amount of key regulative hormones, Abscisic acid (ABA) and 1-aminocyclopropane-1-carboxylic acid (ACC). These results raise a possibility that super rice with higher sink activities has superior adaptability to LT and NPF due to their higher sink activities.

Cellular Models for Primary CoQ Deficiency Pathogenesis Study

Primary coenzyme Q10 (CoQ) deficiency includes a heterogeneous group of mitochondrial diseases characterized by low mitochondrial levels of CoQ due to decreased endogenous biosynthesis rate. These diseases respond to CoQ treatment mainly at the early stages of the disease. The advances in the next generation sequencing (NGS) as whole-exome sequencing (WES) and whole-genome sequencing (WGS) have increased the discoveries of mutations in either gene already described to participate in CoQ biosynthesis or new genes also involved in this pathway. However, these technologies usually provide many mutations in genes whose pathogenic effect must be validated. To functionally validate the impact of gene variations in the disease’s onset and progression, different cell models are commonly used. We review here the use of yeast strains for functional complementation of human genes, dermal skin fibroblasts from patients as an excellent tool to demonstrate the biochemical and genetic mechanisms of these diseases and the development of human-induced pluripotent stem cells (hiPSCs) and iPSC-derived organoids for the study of the pathogenesis and treatment approaches.

Systems Biology Applied to the Study of Papaya Fruit Ripening: The Influence of Ethylene on Pulp Softening.

Papaya is a fleshy fruit that undergoes fast ethylene-induced modifications. The fruit becomes edible, but the fast pulp softening is the main factor that limits the post-harvest period. Papaya fast pulp softening occurs due to cell wall disassembling coordinated by ethylene triggering that massively expresses pectinases. In this work, RNA-seq analysis of ethylene-treated and non-treated papayas enabled a wide transcriptome overview that indicated the role of ethylene during ripening at the gene expression level. Several families of transcription factors (AP2/ERF, NAC, and MADS-box) were differentially expressed. ACO, ACS, and SAM-Mtase genes were upregulated, indicating a high rate of ethylene biosynthesis after ethylene treatment. The correlation among gene expression and physiological data demonstrated ethylene treatment can indeed simulate ripening, and regulation of changes in fruit color, aroma, and flavor could be attributed to the coordinated expression of several related genes. Especially about pulp firmness, the identification of 157 expressed genes related to cell wall metabolism demonstrated that pulp softening is accomplished by a coordinated action of several different cell wall-related enzymes. The mechanism is different from other commercially important fruits, such as strawberry, tomato, kiwifruit, and apple. The observed behavior of this new transcriptomic data confirms ethylene triggering is the main event that elicits fast pulp softening in papayas.

Ins and Outs of the TCA Cycle: The Central Role of Anaplerosis.

The reactions of the tricarboxylic acid (TCA) cycle allow the controlled combustion of fat and carbohydrate. In principle, TCA cycle intermediates are regenerated on every turn and can facilitate the oxidation of an infinite number of nutrient molecules. However, TCA cycle intermediates can be lost to cataplerotic pathways that provide precursors for biosynthesis, and they must be replaced by anaplerotic pathways that regenerate these intermediates. Together, anaplerosis and cataplerosis help regulate rates of biosynthesis by dictating precursor supply, and they play underappreciated roles in catabolism and cellular energy status. They facilitate recycling pathways and nitrogen trafficking necessary for catabolism, and they influence redox state and oxidative capacity by altering TCA cycle intermediate concentrations. These functions vary widely by tissue and play emerging roles in disease. This article reviews the roles of anaplerosis and cataplerosis in various tissues and discusses how they alter carbon transitions, and highlights their contribution to mechanisms of disease.

Standard Thermodynamic Properties, Biosynthesis Rates, and the Driving Force of Growth of Five Agricultural Plants.

Elemental composition of Gossypium hirsutum L. (cotton), Oryza sativa L. (Asian rice), Phaseolus vulgaris L. (common bean), Saccharum spp. L. (sugarcane), and Zea mays L. (corn) was used to calculate their empirical formulas (unit carbon formulas) and growth stoichiometry. The empirical formulas were used to find standard enthalpy of formation, standard molar entropy, standard Gibbs energy of formation, and standard molar heat capacity. A comparison was made between thermodynamic properties of live matter of the analyzed plants and other unicellular and multicellular organisms. Moreover, the growth process was analyzed through standard enthalpy, entropy, and Gibbs energy of biosynthesis. The average standard Gibbs energy of biosynthesis was found to be +463.0 kJ/C-mol. Thus, photosynthesis provides energy and carbon for plant growth. The average intercepted photosynthetic energy was found to be 15.5 MJ/C-mol for the analyzed plants. However, due to inefficiency, a great fraction of the intercepted photosynthetic energy cannot be used by plants. The average usable photosynthetic energy was found to be –2.3 MJ/C-mol. The average thermodynamic driving force for growth is –1.9 MJ/C-mol. Driving forces of growth of C3 and C4 plants were compared. It was found that C4 plants have a greater driving force of growth than C3 plants, which reflects the greater efficiency of C4 photosynthesis. The relationship between the driving force and growth rates was analyzed by determining phenomenological L coefficients. The determined phenomenological coefficients span two orders of magnitude, depending on plant species and environmental conditions. The L coefficient of P. vulgaris was found to be lower than that of other plants, due to additional energy requirements of nitrogen fixation.

Is dietary phosphatidylcholine essential for juvenile slipper lobster (Thenus australiensis)?

Phospholipids, especially phosphatidylcholine (PC), are considered essential nutrients for larval and juvenile stages of many decapod crustacean species due to low rates of endogenous biosynthesis and potential nutritional benefits in assisting lipid digestion and transport. This study was conducted to test the hypothesis that dietary PC is essential and would subsequently have beneficial effects on lipid utilisation and digestibility of fish oil triacylglycerol (TAG) in the slipper lobster, Thenus australiensis. Juvenile slipper lobsters were supplied six formulated experimental feeds with actual PC contents of 0.05, 0.48, 0.65, 0.98, 1.15 and 1.43% (dry matter) for 12 weeks. Manipulating dietary PC content had no effects on survival, growth, chemical composition, whole body or tissue lipid distribution, or apparent digestibility (AD) of gross energy (GE), total lipid (TL) or PC. Juvenile slipper lobsters appeared to possess fully developed and efficient mechanisms for GE, TAG and PC digestion (>95% AD). In vivo lipid class mass balance calculations demonstrated net intake of TL and TAG was considerably greater than was accumulated in the whole-body during growth. Thus, a significant proportion of dietary TL and TAG was utilised for energy and other metabolic processes. In contrast, particularly for low PL dietary treatments (≤ 0.5%), net gain of PC was greater than net intake, indicating a reliance on biosynthesis of PC. Thus, it is concluded that there is no or a very low requirement for dietary PC and thus may not be essential for the examined size class of juvenile T. australiensis when supplied adequate dietary PC biosynthetic precursors and other potentially related dietary nutrients. Further targeted research is recommended to verify if these findings are applicable to larval and early juvenile stages, and to enhance overall understanding of lipid nutritional requirements for T. australiensis.

N1-acetylspermidine is a determinant of hair follicle stem cell fate.

ABSTRACTStem cell differentiation is accompanied by increased mRNA translation. The rate of protein biosynthesis is influenced by the polyamines putrescine, spermidine and spermine, which are essential for cell growth and stem cell maintenance. However, the role of polyamines as endogenous effectors of stem cell fate and whether they act through translational control remains obscure. Here, we investigate the function of polyamines in stem cell fate decisions using hair follicle stem cell (HFSC) organoids. Compared to progenitor cells, HFSCs showed lower translation rates, correlating with reduced polyamine levels. Surprisingly, overall polyamine depletion decreased translation but did not affect cell fate. In contrast, specific depletion of natural polyamines mediated by spermidine/spermine N1-acetyltransferase (SSAT; also known as SAT1) activation did not reduce translation but enhanced stemness. These results suggest a translation-independent role of polyamines in cell fate regulation. Indeed, we identified N1-acetylspermidine as a determinant of cell fate that acted through increasing self-renewal, and observed elevated N1-acetylspermidine levels upon depilation-mediated HFSC proliferation and differentiation in vivo. Overall, this study delineates the diverse routes of polyamine metabolism-mediated regulation of stem cell fate decisions.This article has an associated First Person interview with the first author of the paper.

The complexity of sound quantification of specialized metabolite biosynthesis: The stress related impact on the alkaloid content of Catharanthus roseus

Medicinal plants grown under stress conditions reveal higher concentrations of relevant specialized metabolites than well-watered plants, putatively due to an enhanced biosynthesis. Yet, stress also reduced the biomass gain. Accordingly, the concentration increase in comparison to control plants could also be due to lesser biomass employed as the reference value, whereas the rate of biosynthesis may remain unchanged. For an unequivocal proof that stress indeed enhances the biosynthesis, the total amount of the substances per plant has to be determined. In this study, we investigated the stress-induced impact on the alkaloids accumulated in Catharanthus roseus and quantified both, the changes in concentration and in the entire amount of alkaloids.At any time, all Catharanthus roseus plants grown under drought stress exhibited a markedly higher alkaloid concentration compared to the well-watered controls. However, by calculating the entire alkaloid content per plant, a corresponding increment occurred only within the first two weeks of drought stress. Thereafter, no significant differences among drought treatments and control were detected. Finally, within the last week, the alkaloid content per plant decreased markedly, although there was a meaningfully higher concentration of alkaloids in the drought-stressed plants. In contrast, when plants had been exposed to high salt concentrations, the alkaloid concentrations were quite the same in stressed and control plants. The related total contents were significantly lower in plants exposed to salt stress.These results display that both phenomena, an increased rate of biosynthesis and lesser reference values, i.e., the biomass, contribute to the stress-related increase in the concentration of natural product. Moreover, it has to be considered that the enhancement of biosynthesis could be due to either an “active” up-regulation of biosynthetic capacity or a “passive” shift caused by the over-reduced status as a result of the stress-induced stomatal closure.

Optimization of Synthetic Media Composition for Kluyveromyces marxianus Fed-Batch Cultivation

The Kluyveromyces marxianus yeast recently has gained considerable attention due to its applicability in high-value-added product manufacturing. In order to intensify the biosynthesis rate of a target product, reaching high biomass concentrations in the reaction medium is mandatory. Fed-batch processes are an attractive and efficient way how to achieve high cell densities. However, depending on the physiology of the particular microbial strain, an optimal media composition should be used to avoid by-product synthesis and, subsequently, a decrease in overall process effi-ciency. Thus, the aim of the present study was to optimise the synthetic growth medium and feeding solution compositions (in terms of carbon, nitrogen, phosphorous, magnesium, and calcium concentrations) for high cell density K. marxianus fed‑batch cultivations. Additionally, the biomass yields from the vitamin mixture and other macro/microelements were identified. A model predictive control algorithm was successfully applied for a fed-batch cultivation control. Biomass growth and substrate consumption kinetics were compared with the mathematical model predictions. Finally, 2‑phenylethanol biosynthesis was induced and its productivity was estimated. The determined optimal macronutrient ratio for K. marxianus biomass growth was identified as C:N:P = 1:0.07:0.011. The maximal attained yeast biomass concentration was close to 70 g·L-1 and the 2-PE biosynthesis rate was 0.372 g·L−1·h−1, with a yield of 74% from 2-phenylalanine.

Labelling and Visualization of Mitochondrial Genome Expression Products in Baker's Yeast Saccharomyces cerevisiae.

Mitochondria are essential organelles of eukaryotic cells capable of aerobic respiration. They contain circular genome and gene expression apparatus. A mitochondrial genome of baker's yeast encodes eight proteins: three subunits of the cytochrome c oxidase (Cox1p, Cox2p, and Cox3p), three subunits of the ATP synthase (Atp6p, Atp8p, and Atp9p), a subunit of the ubiquinol-cytochrome c oxidoreductase enzyme, cytochrome b (Cytb), and mitochondrial ribosomal protein Var1p. The purpose of the method described here is to specifically label these proteins with 35S methionine, separate them by electrophoresis and visualize the signals as discrete bands on the screen. The procedure involves several steps. First, yeast cells are cultured in a galactose-containing medium until they reach the late logarithmic growth stage. Next, cycloheximide treatment blocks cytoplasmic translation and allows 35S methionine incorporation only in mitochondrial translation products. Then, all proteins are extracted from yeast cells and separated by polyacrylamide gel electrophoresis. Finally, the gel is dried and incubated with the storage phosphor screen. The screen is scanned on a phosphorimager revealing the bands. The method can be applied to compare the biosynthesis rate of a single polypeptide in the mitochondria of a mutant yeast strain versus the wild type, which is useful for studying mitochondrial gene expression defects. This protocol gives valuable information about the translation rate of all yeast mitochondrial mRNAs. However, it requires several controls and additional experiments to make proper conclusions.

Collagen metabolism as a regulator of proline dehydrogenase/proline oxidase-dependent apoptosis/autophagy

Recent studies on the regulatory role of amino acids in cell metabolism have focused on the functional significance of proline degradation. The process is catalysed by proline dehydrogenase/proline oxidase (PRODH/POX), a mitochondrial flavin-dependent enzyme converting proline into ∆1-pyrroline-5-carboxylate (P5C). During this process, electrons are transferred to electron transport chain producing ATP for survival or they directly reduce oxygen, producing reactive oxygen species (ROS) inducing apoptosis/autophagy. However, the mechanism for switching survival/apoptosis mode is unknown. Although PRODH/POX activity and energetic metabolism were suggested as an underlying mechanism for the survival/apoptosis switch, proline availability for this enzyme is also important. Proline availability is regulated by prolidase (proline supporting enzyme), collagen biosynthesis (proline utilizing process) and proline synthesis from glutamine, glutamate, α-ketoglutarate (α-KG) and ornithine. Proline availability is dependent on the rate of glycolysis, TCA and urea cycles, proline metabolism, collagen biosynthesis and its degradation. It is well established that proline synthesis enzymes, P5C synthetase and P5C reductase as well as collagen prolyl hydroxylases are up-regulated in most of cancer types and control rates of collagen biosynthesis. Up-regulation of collagen prolyl hydroxylase and its exhaustion of ascorbate and α-KG may compete with DNA and histone demethylases (that require the same cofactors) to influence metabolic epigenetics. This knowledge led us to hypothesize that up-regulation of prolidase and PRODH/POX with inhibition of collagen biosynthesis may represent potential pharmacotherapeutic approach to induce apoptosis or autophagic death in cancer cells. These aspects of proline metabolism are discussed in the review as an approach to understand complex regulatory mechanisms driving PRODH/POX-dependent apoptosis/survival.