Efficiency Of Phosphorylation Research Articles

Persistently Altered Brain Mitochondrial Bioenergetics After Apparently Successful Resuscitation From Cardiac Arrest.

BackgroundAlthough advances in cardiopulmonary resuscitation have improved survival from cardiac arrest (CA), neurologic injury persists and impaired mitochondrial bioenergetics may be critical for targeted neuroresuscitation. The authors sought to determine if excellent cardiopulmonary resuscitation and postresuscitation care and good traditional survival rates result in persistently disordered cerebral mitochondrial bioenergetics in a porcine pediatric model of asphyxia-associated ventricular fibrillation CA.Methods and ResultsAfter 7 minutes of asphyxia, followed by ventricular fibrillation, 5 female 1-month-old swine (4 sham) received blood pressure–targeted care: titration of compression depth to systolic blood pressure of 90 mm Hg and vasopressor administration to a coronary perfusion pressure >20 mm Hg. All animals received protocol-based vasopressor support after return of spontaneous circulation for 4 hours before they were killed. The primary outcome was integrated mitochondrial electron transport system (ETS) function. CA animals displayed significantly decreased maximal, coupled oxidative phosphorylating respiration (OXPHOSCI+CII) in cortex (P<0.02) and hippocampus (P<0.02), as well as decreased phosphorylation and coupling efficiency (cortex, P<0.05; hippocampus, P<0.05). Complex I– and complex II–driven respiration were both significantly decreased after CA (cortex: OXPHOSCI P<0.01, ETSCII P<0.05; hippocampus: OXPHOSCI P<0.03, ETSCII P<0.01). In the hippocampus, there was a significant decrease in maximal uncoupled, nonphosphorylating respiration (ETSCI+CII), as well as a 30% reduction in citrate synthase activity (P<0.04).ConclusionsMitochondria in both the cortex and hippocampus displayed significant alterations in respiratory function after CA despite excellent cardiopulmonary resuscitation and postresuscitation care in asphyxia-associated ventricular fibrillation CA. Analysis of integrated ETS function identifies mitochondrial bioenergetic failure as a target for goal-directed neuroresuscitation after CA. IACUC Protocol: IAC 13-001023.

Sequential phosphorylation analysis using dye-tethered peptides and microfluidic isoelectric focusing electrophoresis

We report a simple method for analyzing sequential phosphorylation by protein kinases using fluorescent peptide substrates and microfluidic isoelectric focusing (μIEF) electrophoresis. When a dye-labeled peptide substrate was sequentially phosphorylated by two consecutive protein kinases (mitogen-activated protein kinase (MAPK) and glycogen synthase kinase 3 (GSK3)), its differently phosphorylated forms were easily separated and visualized by fluorescent focusing zones in the μIEF channel based on a change in the isoelectric point (pI) by phosphorylation. As a result, ratiometric and quantitative analysis of the fluorescent focusing regions shifted by phosphorylation enabled the analysis of phosphorylation efficiency and the relevant inhibition of protein kinases (MAPK and GSK3) with high simplicity and selectivity. Furthermore, the GSK3 activity in the cell lysates was elucidated by μIEF electrophoresis in combination with immunoprecipitation. Our results suggest that this method has great potential for analyzing the sequential phosphorylation of multiple protein kinases that are implicated in cellular signaling pathways.

Influence of Antioxidant SkQ1 on Accumulation of Mitochondrial DNA Deletions in the Hippocampus of Senescence-Accelerated OXYS Rats.

Reduction of efficiency of oxidative phosphorylation associated with aging and the development of neurodegenerative diseases including Alzheimer's disease is thought to be linked to the accumulation of deletions in mitochondrial DNA (ΔmtDNA), which are seen as a marker of oxidative damage. Recently, we have shown that mitochondria-targeted antioxidant SkQ1 (10-(6'-plastoquinonyl)decyltriphenylphosphonium) can slow the development of signs of Alzheimer's disease in senescence-accelerated OXYS rats. The purpose of this study was to explore the relationship between the development of neurodegenerative changes in the brain of OXYS rats and changes in the amount of mtDNA and the 4834-bp mitochondrial DNA deletion (ΔmtDNA4834) as well as the effect of SkQ1. We studied the relative amount of mtDNA and ΔmtDNA4834 in the hippocampus of OXYS and Wistar (control) rats at ages of 1, 2, 6, 10, and 20 days and 3, 6, and 24 months. During the period crucial for manifestation of the signs of accelerated aging of OXYS rats (from 1.5 to 3 months of age), we evaluated the effects of administration of SkQ1 (250nmol/kg) and vitamin E (670mmol/kg, reference treatment) on the amount of mtDNA and ΔmtDNA4834 and on the formation of the behavioral feature of accelerated senescence in OXYS rats - passive type of behavior in the open field test. In OXYS rats, the level of ΔmtDNA4834 in the hippocampus is increased compared to the Wistar rats, especially at the stage of completion of brain development in the postnatal period. This level remains elevated not only at the stages preceding the manifestation of the signs of accelerated brain aging and the development of pathological changes linked to Alzheimer's disease, but also during their progression. However, at age of 24 months, there were no detectable differences between the two strains. SkQ1 treatment reduced the level of ΔmtDNA4834 in the hippocampus of Wistar and OXYS rats and slowed the formation of passive behavior in OXYS rats. These results support the possible use of SkQ1 for prophylaxis of brain aging.

Mitochondrial uncoupling protein-2 is not involved in palmitate-induced impairment of glucose-stimulated insulin secretion in INS-1E insulinoma cells and is not needed for the amplification of insulin release

We have recently shown that overnight exposure of INS-1E insulinoma cells to palmitate in the presence of high glucose causes defects in both mitochondrial energy metabolism and glucose-stimulated insulin secretion (GSIS). Here we report experiments designed to test the involvement of mitochondrial uncoupling protein-2 (UCP2) in these glucolipotoxic effects. Measuring real-time oxygen consumption in siRNA-transfected INS-1E cells, we show that deleterious effects of palmitate on the glucose sensitivity of mitochondrial respiration and on the coupling efficiency of oxidative phosphorylation are independent of UCP2. Consistently, palmitate impairs GSIS to the same extent in cells with and without UCP2. Furthermore, we knocked down UCP2 in spheroid INS-1E cell clusters (pseudoislets) to test whether or not UCP2 regulates insulin secretion during prolonged glucose exposure. We demonstrate that there are no differences in temporal GSIS kinetics between perifused pseudoislets with and without UCP2. We conclude that UCP2 is not involved in palmitate-induced impairment of GSIS in INS-1E insulinoma cells and is not needed for the amplification of insulin release. These conclusions inform ongoing debate on the disputed biochemical and physiological functions of the beta cell UCP2.

Anti-aging dilemma: to restore the hardware or to reinstall the software?

Sinceitsearlydays,thevalueofdecipheringthehumanDNAhasbeenseenprimarilyinextractingthe set of messages that run the cells that constitute the body. In common understanding, thesemessages are encoded in DNA and transcribed as cell-specific sets of RNAs, some of which aretranslatedtoproteins,thenmodifiedwithvariouspost-translationaladd-onsmadeofsugars,lipidsand other moieties. This complex chain of events is further complicated by multi-layered possi-bilities for the modifications allowed at every step–epigenetics for DNA, editing for RNAs andtherecentlydiscoveredphenomenonofnon-templatepolypeptideextensionallowedbyribosomes(Shen et al., 2015). It seems that when looked at as a whole, the DNA, and all the messages associ-ated with DNA, do not look like a blueprint, or even a clear set of instructions, but rather a messydraft or a stack of notes that are scribbled all over and full of ambiguities.However, let us hack through the majority of the “omics” and look upon the set of smallmolecules known as metabolites, and the budding discipline of Metabolomics that researches thetrueunderpinningsoftheabundantlycomplexmechanicsofthelivingcell.Itisworthwhiletonotethat,toasomewhatdefineddegree,thecellwilltoleratethelossofageneorchangesinthelevelsofRNAs or even the most important of proteins, while even slight deregulation of the levels of someof the smallest metabolites leads to immediate and catastrophic consequences. The potassium ionandATPmaybeusedastheprimaryexamplesofsmallestmoleculescapableofelicitingasystemicresponse.Accordingtoourcalculation,amere0.5%increaseinthetotalcontentofpotassiumchlo-ride, one of the most common electrolytes in the human body, leads to immediate cardiac arrest.TheconsequencesofthedepletionofATPmaymanifestasavarietyofailments,withtheirdurationinversely proportional to the severity of the defect. Aging, in particular, is associated with a declinein the efficiency of oxidative phosphorylation and an increase in the risk of resulting pathologies.Of course, there are other small molecules, possibly not as well-known as ATP, but still indispens-able and irreplaceable. In particular, the metabolites derived from the amino acid tryptophan havethe capacity for similar dramatic alteration of system-wide function. Most pertinent to the topicof this discussion, the changes in metabolic profiles are considered as drivers for the pathogene-sis of age-associated disorders, including Alzheimer’s disease (Tacutu et al., 2010; Demetrius andDriver, 2013; Jia et al., 2014; Obre and Rossignol, 2015). It also is of note that metabolites are notasabundantascommonlystudiedspeciesofproteinsandRNAs.Hence,theworldofmetabolitesisimmensely easier to comprehend than the overly complicated world of other famous “omics.” Thelatter point is extremely important, as it provides a possibility for the use of a powerful reduction-ist approach without falling into ill-fitting or over-fitting of the underlying model, a well-knownperpetual source of entrapment.Letuscomparetheliving(andaging)cell,withitsendless“omics”-scalelayersofinterconnectedcomponents, to modern computers. Computer hardware is a collection of interconnected physicaldevices used in or with your machine. One of these parts may wear itself out and die; however,

PKA-dependent phosphorylation of ribosomal protein S6 does not correlate with translation efficiency in striatonigral and striatopallidal medium-sized spiny neurons.

Ribosomal protein S6 (rpS6), a component of the 40S ribosomal subunit, is phosphorylated on several residues in response to numerous stimuli. Although commonly used as a marker for neuronal activity, its upstream mechanisms of regulation are poorly studied and its role in protein synthesis remains largely debated. Here, we demonstrate that the psychostimulant d-amphetamine (d-amph) markedly increases rpS6 phosphorylation at Ser235/236 sites in both crude and synaptoneurosomal preparations of the mouse striatum. This effect occurs selectively in D1R-expressing medium-sized spiny neurons (MSNs) and requires the cAMP/PKA/DARPP-32/PP-1 cascade, whereas it is independent of mTORC1/p70S6K, PKC, and ERK signaling. By developing a novel assay to label nascent peptidic chains, we show that the rpS6 phosphorylation induced in striatonigral MSNs by d-amph, as well as in striatopallidal MSNs by the antipsychotic haloperidol or in both subtypes by papaverine, is not correlated with the translation of global or 5' terminal oligopyrimidine tract mRNAs. Together, these results provide novel mechanistic insights into the in vivo regulation of the post-translational modification of rpS6 in the striatum and point out the lack of a relationship between PKA-dependent rpS6 phosphorylation and translation efficiency.

Temperature controls oxidative phosphorylation and reactive oxygen species production through uncoupling in rat skeletal muscle mitochondria

Mitochondrial respiratory and phosphorylation activities, mitochondrial uncoupling, and hydrogen peroxide formation were studied in isolated rat skeletal muscle mitochondria during experimentally induced hypothermia (25°C) and hyperthermia (42°C) compared to the physiological temperature of resting muscle (35°C). For nonphosphorylating mitochondria, increasing the temperature from 25 to 42°C led to a decrease in membrane potential, hydrogen peroxide production, and quinone reduction levels. For phosphorylating mitochondria, no temperature-dependent changes in these mitochondrial functions were observed. However, the efficiency of oxidative phosphorylation decreased, whereas the oxidation and phosphorylation rates and oxidative capacities of the mitochondria increased, with increasing assay temperature. An increase in proton leak, including uncoupling protein-mediated proton leak, was observed with increasing assay temperature, which could explain the reduced oxidative phosphorylation efficiency and reactive oxygen species production.

Acute ketamine impairs mitochondrial function and promotes superoxide dismutase activity in the rat brain.

Ketamine is often associated with altered mitochondrial function and oxidative stress. Nevertheless, limited data are still available regarding the in vivo action of ketamine in mitochondrial bioenergetics and redox state. Accumulating evidence supports a role for nitric oxide (NO) as a possible modulator of ketamine's side effects. In the present study, we investigated the role of NO modulation on ketamine anesthesia at the level of brain mitochondrial function and redox status. Adult male rats received a single dose of ketamine (50, 100, or 150 mg/kg IP) or a combination of ketamine and N-nitro-L-arginine (3 mg/kg IP). Animals were killed 6 hours after treatment. Brain and blood samples were collected for plasma NO determination and mitochondria isolation. Several variables of brain mitochondrial function were evaluated. Ketamine interfered with complex I function, revealing increased oxygen consumption in state 4, impaired oxidative phosphorylation efficiency of glutamate-malate substrate, and decreased NADH-ubiquinone oxidoreductase activity. In addition, mitochondrial NO synthase (mtNOS) activity and NO plasma levels were increased for the 50 and 100 mg/kg doses. Ketamine administration increased hydrogen peroxide generation and triggered superoxide dismutase activity. All these effects could totally or partially be prevented by mtNOS inhibition through N-nitro-L-arginine. Acute ketamine administration impaired the function of mitochondrial complex I leading to increased mtNOS activity, increased generation of hydrogen peroxide and NO, resulting in superoxide dismutase triggering, and improved antioxidant activity. The present findings clarify the role of NO modulation in ketamine anesthesia, providing new data on a relevant clinical mechanism.

Temperature acclimation of mitochondria function from the hearts of a temperate wrasse (Notolabrus celidotus)

Understanding how mitochondrial function alters with acclimation may provide insight to the limits these organelles place on temperate fish hearts facing seasonal temperature fluctuations. This investigation determined if compromised cardiac mitochondrial function contributed to heart failure (HF) in the New Zealand wrasse Notolabrus celidotus acclimated at their mean summer and winter ocean temperatures. To test this hypothesis, fish were acclimated to cold (CA, 15°C) and warm (WA, 21°C) temperatures. The temperature of HF was determined by Doppler sonography and mitochondrial function in permeabilised cardiac fibres was tested using high resolution respirometry. Heat stress mediated HF occurred at a THF of 26.7±0.4°C for CA fish, and at 28.2±0.6°C for WA fish. Biochemical analyses also revealed that WA fish had elevated resting plasma lactate indicating an increased dependence on anaerobic pathways. When cardiac fibres were tested with increasing temperatures, apparent breakpoints in the respiratory control ratio (RCR-I) with substrates supporting complex I (CI) oxygen flux occurred below the THF for both acclimated groups. While WA cardiac mitochondria were less sensitive to increasing temperature for respirational flux supported by CI, Complex II, and chemically uncoupled flux, CA fish maintained higher RCRs at higher temperatures. We conclude that while acclimation to summer temperatures does alter cardiac mitochondrial function in N. celidotus, these changes need not be beneficial in terms of oxidative phosphorylation efficiency and may come at an energetic cost, which would be detrimental in the face of further habitat warming.

Biogenesis of mitochondria in cauliflower (Brassica oleracea var. botrytis) curds subjected to temperature stress and recovery involves regulation of the complexome, respiratory chain activity, organellar translation and ultrastructure

The biogenesis of the cauliflower curd mitochondrial proteome was investigated under cold, heat and the recovery. For the first time, two dimensional fluorescence difference gel electrophoresis was used to study the plant mitochondrial complexome in heat and heat recovery. Particularly, changes in the complex I and complex III subunits and import proteins, and the partial disintegration of matrix complexes were observed. The presence of unassembled subunits of ATP synthase was accompanied by impairment in mitochondrial translation of its subunit. In cold and heat, the transcription profiles of mitochondrial genes were uncorrelated. The in-gel activities of respiratory complexes were particularly affected after stress recovery. Despite a general stability of respiratory chain complexes in heat, functional studies showed that their activity and the ATP synthesis yield were affected. Contrary to cold stress, heat stress resulted in a reduced efficiency of oxidative phosphorylation likely due to changes in alternative oxidase (AOX) activity. Stress and stress recovery differently modulated the protein level and activity of AOX. Heat stress induced an increase in AOX activity and protein level, and AOX1a and AOX1d transcript level, while heat recovery reversed the AOX protein and activity changes. Conversely, cold stress led to a decrease in AOX activity (and protein level), which was reversed after cold recovery. Thus, cauliflower AOX is only induced by heat stress. In heat, contrary to the AOX activity, the activity of rotenone-insensitive internal NADH dehydrogenase was diminished. The relevance of various steps of plant mitochondrial biogenesis to temperature stress response and recovery is discussed.

Defects in Mitochondrial Efficiency and H2O2 Emissions in Obese Women Are Restored to a Lean Phenotype With Aerobic Exercise Training

The notion that mitochondria contribute to obesity-induced insulin resistance is highly debated. Therefore, we determined whether obese (BMI 33 kg/m2), insulin-resistant women with polycystic ovary syndrome had aberrant skeletal muscle mitochondrial physiology compared with lean, insulin-sensitive women (BMI 23 kg/m2). Maximal whole-body and mitochondrial oxygen consumption were not different between obese and lean women. However, obese women exhibited lower mitochondrial coupling and phosphorylation efficiency and elevated mitochondrial H2O2 (mtH2O2) emissions compared with lean women. We further evaluated the impact of 12 weeks of aerobic exercise on obesity-related impairments in insulin sensitivity and mitochondrial energetics in the fasted state and after a high-fat mixed meal. Exercise training reversed obesity-related mitochondrial derangements as evidenced by enhanced mitochondrial bioenergetics efficiency and decreased mtH2O2 production. A concomitant increase in catalase antioxidant activity and decreased DNA oxidative damage indicate improved cellular redox status and a potential mechanism contributing to improved insulin sensitivity. mtH2O2 emissions were refractory to a high-fat meal at baseline, but after exercise, mtH2O2 emissions increased after the meal, which resembles previous findings in lean individuals. We demonstrate that obese women exhibit impaired mitochondrial bioenergetics in the form of decreased efficiency and impaired mtH2O2 emissions, while exercise effectively restores mitochondrial physiology toward that of lean, insulin-sensitive individuals.

Mechanisms of ATM Activation.

The ataxia-telangiectasia mutated (ATM) protein kinase is a master regulator of the DNA damage response, and it coordinates checkpoint activation, DNA repair, and metabolic changes in eukaryotic cells in response to DNA double-strand breaks and oxidative stress. Loss of ATM activity in humans results in the pleiotropic neurodegeneration disorder ataxia-telangiectasia. ATM exists in an inactive state in resting cells but can be activated by the Mre11-Rad50-Nbs1 (MRN) complex and other factors at sites of DNA breaks. In addition, oxidation of ATM activates the kinase independently of the MRN complex. This review discusses these mechanisms of activation, as well as the posttranslational modifications that affect this process and the cellular factors that affect the efficiency and specificity of ATM activation and substrate phosphorylation. I highlight functional similarities between the activation mechanisms of ATM, phosphatidylinositol 3-kinases (PI3Ks), and the other PI3K-like kinases, as well as recent structural insights into their regulation.

Skeletal muscle phenotype affects fasting-induced mitochondrial oxidative phosphorylation flexibility in cold-acclimated ducklings.

Starvation is particularly challenging for endotherms that remain active in cold environments or during winter. The aim of this study was to determine whether fasting-induced mitochondrial coupling flexibility depends upon the phenotype of skeletal muscles. The rates of oxidative phosphorylation and mitochondrial efficiency were measured in pectoralis (glycolytic) and gastrocnemius (oxidative) muscles from cold-acclimated ducklings (Cairina moschata). Pyruvate and palmitoyl-l-carnitine were used in the presence of malate as respiratory substrates. Plasma metabolites, skeletal muscle concentrations of triglycerides, glycogen and total protein and mitochondrial levels of oxidative phosphorylation complexes were also quantified. Results from ad libitum fed ducklings were compared with those from ducklings that were fasted for 4 days. During the 4 days of nutritional treatment, birds remained in the cold, at 4°C. The 4 days of starvation preferentially affected the pectoralis muscles, inducing an up-regulation of mitochondrial efficiency, which was associated with a reduction of both total muscle and mitochondrial oxidative phosphorylation protein, and with an increase of intramuscular lipid concentration. By contrast, fasting decreased the activity of oxidative phosphorylation but did not alter the coupling efficiency and protein expression of mitochondria isolated from the gastrocnemius muscles. Hence, the adjustment of mitochondrial efficiency to fasting depends upon the muscle phenotype of cold-acclimated birds. Furthermore, these results suggest that the reduced cost of mitochondrial ATP production in pectoralis muscles may trigger lipid storage within this tissue and help to sustain an important metabolic homeostatic function of skeletal muscles, which is to maintain levels of amino acids in the circulation during the fast.

Mitochondrial Uncoupling Protein 2 Is Present in Human Platelets and Regulates Platelet Activation

It has long been recognized that platelets contain functional mitochondria and accumulating data suggest that several aspects of mitochondrial function, including reactive oxygen species production, modulate platelet thrombotic function. We have recently shown that platelet mitochondria isolated from patients with Sickle Cell Disease generate significantly greater concentrations of reactive oxygen species than healthy African American subjects and that this mitochondrial oxidant generation contributes to higher levels of basal platelet activation in these patients (Blood. 2014 May 1;123(18):2864-72). Based on these data, we have now investigated the factors that regulate platelet mitochondrial superoxide generation in healthy and Sickle Cell Disease platelets. Here we demonstrate that human platelets express mitochondrial uncoupling protein-2 (UCP2), a protein that is known to decrease the efficiency of oxidative phosphorylation and oxidant generation in other cell types but has previously not been identified in platelets. In this study we show that UCP2 protein is expressed in healthy human platelets and is fully functional as it facilitates proton leak across the inner mitochondrial membrane, leading to decreased mitochondrial membrane potential. Further, we demonstrate that the expression of this protein attenuates platelet mitochondrial superoxide generation, as treatment of platelets with Genipin (2-10µM), a pharmacological inhibitor of UCPs, concentration-dependently increases mitochondrial membrane potential and reactive oxygen species production. Further, an approximately 70% inhibition of UCP activity results in platelet activation demonstrated by increased membrane p-selectin expression (65±7% versus 6±3% in untreated controls) and augmented glycoprotein IIb/IIIa activation (57±9% versus 9±4% in untreated controls). The use of the mitochondrial-targeted antioxidant mitoTEMPO (10µM) decreases genipin-induced superoxide generation and significantly attenuates platelet activation. Notably, preliminary data presented here also suggest that UCP expression is decreased in platelets isolated from Sickle Cell Disease patients (3-fold) compared to healthy African American subjects. Additionally, ongoing studies are investigating platelet function in mice deficient in UCP2. Taken together, these data demonstrate a novel mechanism of regulation of platelet thrombotic function whose physiological relevance is apparent in the context of Sickle Cell Disease. More broadly this study advances the understanding of the role of the mitochondrion in platelet biology and thrombotic disease. DisclosuresNo relevant conflicts of interest to declare.

Evolution and Functional Implications of the Tricarboxylic Acid Cycle as Revealed by Phylogenetic Analysis

The tricarboxylic acid (TCA) cycle, a crucial component of respiratory metabolism, is composed of a set of eight enzymes present in the mitochondrial matrix. However, most of the TCA cycle enzymes are encoded in the nucleus in higher eukaryotes. In addition, evidence has accumulated demonstrating that nuclear genes were acquired from the mitochondrial genome during the course of evolution. For this reason, we here analyzed the evolutionary history of all TCA cycle enzymes in attempt to better understand the origin of these nuclear-encoded proteins. Our results indicate that prior to endosymbiotic events the TCA cycle seemed to operate only as isolated steps in both the host (eubacterial cell) and mitochondria (alphaproteobacteria). The origin of isoforms present in different cell compartments might be associated either with gene-transfer events which did not result in proper targeting of the protein to mitochondrion or with duplication events. Further in silico analyses allow us to suggest new insights into the possible roles of TCA cycle enzymes in different tissues. Finally, we performed coexpression analysis using mitochondrial TCA cycle genes revealing close connections among these genes most likely related to the higher efficiency of oxidative phosphorylation in this specialized organelle. Moreover, these analyses allowed us to identify further candidate genes which might be used for metabolic engineering purposes given the importance of the TCA cycle during development and/or stress situations.

Recruiting a silent partner for activation of the protein kinase SRPK1.

The SRPK family of protein kinases regulates mRNA splicing by phosphorylating an essential group of factors known as SR proteins, so named for a C-terminal domain enriched in arginine–serine dipeptide repeats (RS domains). SRPKs phosphorylate RS domains at numerous sites altering SR protein subcellular localization and splicing function. The RS domains in these splicing factors differ considerably in overall length and dipeptide layout. Despite their importance, little is known about how these diverse RS domains interact with SRPKs and regulate SR protein phosphorylation. We now show that sequences distal to the SRPK1 consensus region in the RS domain of the prototype SR protein SRSF1 are not passive as originally thought but rather play active roles in accelerating phosphorylation rates. Located in the C-terminal end of the RS domain, this nonconsensus region up-regulates rate-limiting ADP release through the nucleotide release factor, a structural module in SRPK1 composed of two noncontiguous sequence elements outside the kinase core domain. The data show that the RS domain in SRSF1 is multifunctional and that sequences once thought to be catalytically silent can be recruited to enhance the efficiency of SR protein phosphorylation.

Effect of maternal protein restriction on liver metabolism in rat offspring.

Consequences of gestational protein restriction (GPR) on liver metabolism in rat offspring were investigated. Pregnant dams were divided into groups: normal (NP, 17% casein) or low-protein diet (LP, 6% casein). Livers were collected from 30-day-old offspring (d30) for analysis or isolation of mitochondria. At d30, hepatic and muscle glycogen was increased in LP group. Mitochondrial swelling and oxygen uptake (recorded with a Clark-type electrode) were significantly reduced in NP female and LP pups. Thiobarbituric acid reactive substances production was lower in females (NP or LP), suggesting significant inhibition of lipid peroxidation. Measurement of mitochondrial respiration (states 3 and 4 stimulated by succinate) showed a higher ADP/O ratio in LP pups, particularly females, suggesting higher phosphorylation efficiency. In the 1st month of life, under our experimental conditions, GPR protects liver mitochondria against oxidative stress and females seem to be more resistant or more suitable for survival.

Could thermal sensitivity of mitochondria determine species distribution in a changing climate?

For many aquatic species, the upper thermal limit (Tmax) and the heart failure temperature (THF) are only a few degrees away from the species' current environmental temperatures. While the mechanisms mediating temperature-induced heart failure (HF) remain unresolved, energy flow and/or oxygen supply disruptions to cardiac mitochondria may be impacted by heat stress. Recent work using a New Zealand wrasse (Notolabrus celidotus) found that ATP synthesis capacity of cardiac mitochondria collapses prior to T(HF). However, whether this effect is limited to one species from one thermal habitat remains unknown. The present study confirmed that cardiac mitochondrial dysfunction contributes to heat stress-induced HF in two additional wrasses that occupy cold temperate (Notolabrus fucicola) and tropical (Thalassoma lunare) habitats. With exposure to heat stress, T. lunare had the least scope to maintain heart function with increasing temperature. Heat-exposed fish of all species showed elevated plasma succinate, and the heart mitochondria from the cold temperate N. fucicola showed decreased phosphorylation efficiencies (depressed respiratory control ratio, RCR), cytochrome c oxidase (CCO) flux and electron transport system (ETS) flux. In situ assays conducted across a range of temperatures using naive tissues showed depressed complex II (CII) and CCO capacity, limited ETS reserve capacities and lowered efficiencies of pyruvate uptake in T. lunare and N. celidotus. Notably, alterations of mitochondrial function were detectable at saturating oxygen levels, indicating that cardiac mitochondrial insufficiency can occur prior to HF without oxygen limitation. Our data support the view that species distribution may be related to the thermal limits of mitochondrial stability and function, which will be important as oceans continue to warm.

Ob/ob mouse livers show decreased oxidative phosphorylation efficiencies and anaerobic capacities after cold ischemia.

BackgroundHepatic steatosis is a major risk factor for graft failure in liver transplantation. Hepatic steatosis shows a greater negative influence on graft function following prolonged cold ischaemia. As the impact of steatosis on hepatocyte metabolism during extended cold ischaemia is not well-described, we compared markers of metabolic capacity and mitochondrial function in steatotic and lean livers following clinically relevant durations of cold preservation.MethodsLivers from 10-week old leptin-deficient obese (ob/ob, n = 9) and lean C57 mice (n = 9) were preserved in ice-cold University of Wisconsin solution. Liver mitochondrial function was then assessed using high resolution respirometry after 1.5, 3, 5, 8, 12, 16 and 24 hours of storage. Metabolic marker enzymes for anaerobiosis and mitochondrial mass were also measured in conjunction with non-bicarbonate tissue pH buffering capacity.Results Ob/ob and lean mice livers showed severe (>60%) macrovesicular and mild (<30%) microvesicular steatosis on Oil Red O staining, respectively. Ob/ob livers had lower baseline enzymatic complex I activity but similar adenosine triphosphate (ATP) levels compared to lean livers. During cold storage, the respiratory control ratio and complex I-fueled phosphorylation deteriorated approximately twice as fast in ob/ob livers compared to lean livers. Ob/ob livers also demonstrated decreased ATP production capacities at all time-points analyzed compared to lean livers. Ob/ob liver baseline lactate dehydrogenase activities and intrinsic non-bicarbonate buffering capacities were depressed by 60% and 40%, respectively compared to lean livers.ConclusionsSteatotic livers have impaired baseline aerobic and anaerobic capacities compared to lean livers, and mitochondrial function indices decrease particularly from after 5 hours of cold preservation. These data provide a mechanistic basis for the clinical recommendation of shorter cold storage durations in steatotic donor livers.

Cardiac, skeletal, and smooth muscle mitochondrial respiration: are all mitochondria created equal?

Unlike cardiac and skeletal muscle, little is known about vascular smooth muscle mitochondrial respiration. Therefore, the present study examined mitochondrial respiratory rates in smooth muscle of healthy human feed arteries and compared with that of healthy cardiac and skeletal muscles. Cardiac, skeletal, and smooth muscles were harvested from a total of 22 subjects (53 ± 6 yr), and mitochondrial respiration was assessed in permeabilized fibers. Complex I + II, state 3 respiration, an index of oxidative phosphorylation capacity, fell progressively from cardiac to skeletal to smooth muscles (54 ± 1, 39 ± 4, and 15 ± 1 pmol·s(-1)·mg(-1), P < 0.05, respectively). Citrate synthase (CS) activity, an index of mitochondrial density, also fell progressively from cardiac to skeletal to smooth muscles (222 ± 13, 115 ± 2, and 48 ± 2 μmol·g(-1)·min(-1), P < 0.05, respectively). Thus, when respiration rates were normalized by CS (respiration per mitochondrial content), oxidative phosphorylation capacity was no longer different between the three muscle types. Interestingly, complex I state 2 normalized for CS activity, an index of nonphosphorylating respiration per mitochondrial content, increased progressively from cardiac to skeletal to smooth muscles, such that the respiratory control ratio, state 3/state 2 respiration, fell progressively from cardiac to skeletal to smooth muscles (5.3 ± 0.7, 3.2 ± 0.4, and 1.6 ± 0.3 pmol·s(-1)·mg(-1), P < 0.05, respectively). Thus, although oxidative phosphorylation capacity per mitochondrial content in cardiac, skeletal, and smooth muscles suggest all mitochondria are created equal, the contrasting respiratory control ratio and nonphosphorylating respiration highlight the existence of intrinsic functional differences between these muscle mitochondria. This likely influences the efficiency of oxidative phosphorylation and could potentially alter ROS production.