Phosphorylation of adenosine monophosphate in the mitochondrial matrix.
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ABSTRACT:
SUBMITTER: Krebs HA
PROVIDER: S-EPMC1185910 | biostudies-other | 1978 Jul
REPOSITORIES: biostudies-other
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Project description:Ischemia and reperfusion injury following severe trauma or cardiac arrest are major causes of organ damage in intensive care patients. The brain is particularly vulnerable because hypoxia rapidly damages neurons due to their heavy reliance on oxidative phosphorylation. Therapeutic hypothermia can reduce ischemia-induced brain damage, but cooling procedures are slow and technically difficult to perform in critical care settings. It has been previously reported that injection of naturally occurring adenosine 5'-monophosphate (AMP) can rapidly induce hypothermia in mice. We studied the underlying mechanisms and found that AMP transiently reduces the heart rate, respiratory rate, body temperature, and the consciousness of adult male and female C57BL/6J mice. Adding AMP to mouse or human neuronal cell cultures dose-dependently reduced the membrane potential (ΔΨm) and Ca signaling of mitochondria in these cells. AMP treatment increased intracellular AMP levels and activated AMP-activated protein kinase, which resulted in the inhibition of mammalian target of rapamycin complex 1 (mTORC1) and of mitochondrial and cytosolic Ca signaling in resting and stimulated neurons. Pretreatment with an intraperitoneal injection of AMP almost doubled the survival time of mice under hypoxic (6% O2) or anoxic (<1% O2) conditions when compared to untreated mice. These findings suggest that AMP induces a hypometabolic state that slows mitochondrial respiration, reduces oxygen demand, and delays the processes that damage mitochondria in the brain and other organs following hypoxia and reperfusion. Further examination of these mechanisms may lead to new treatments that preserve organ function in critical care patients.
| S-EPMC7044067 | biostudies-literature
Project description:OBJECTIVE:Our goal was to determine the mechanism by which mitochondrial oxidative stress impairs collateral growth in the heart. APPROACH AND RESULTS:Rats were treated with rotenone (mitochondrial complex I inhibitor that increases reactive oxygen species production) or sham-treated with vehicle and subjected to repetitive ischemia protocol for 10 days to induce coronary collateral growth. In control rats, repetitive ischemia increased flow to the collateral-dependent zone; however, rotenone treatment prevented this increase suggesting that mitochondrial oxidative stress compromises coronary collateral growth. In addition, rotenone also attenuated mitochondrial complex I activity and led to excessive mitochondrial aggregation. To further understand the mechanistic pathway(s) involved, human coronary artery endothelial cells were treated with 50 ng/mL vascular endothelial growth factor, 1 µmol/L rotenone, and rotenone/vascular endothelial growth factor for 48 hours. Vascular endothelial growth factor induced robust tube formation; however, rotenone completely inhibited this effect (P<0.05 rotenone versus vascular endothelial growth factor treatment). Inhibition of tube formation by rotenone was also associated with significant increase in mitochondrial superoxide generation. Immunoblot analyses of human coronary artery endothelial cells with rotenone treatment showed significant activation of adenosine monophosphate activated kinase (AMPK)-? and inhibition of mammalian target of rapamycin and p70 ribosomal S6 kinase. Activation of AMPK-? suggested impairments in energy production, which was reflected by decrease in O2 consumption and bioenergetic reserve capacity of cultured cells. Knockdown of AMPK-? (siRNA) also preserved tube formation during rotenone, suggesting the negative effects were mediated by the activation of AMPK-?. Conversely, expression of a constitutively active AMPK-? blocked tube formation. CONCLUSIONS:We conclude that activation of AMPK-? during mitochondrial oxidative stress inhibits mammalian target of rapamycin signaling, which impairs phenotypic switching necessary for the growth of blood vessels.
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Project description:It has become appreciated over the last several years that protein phosphorylation within the cardiac mitochondrial matrix and respiratory complexes is extensive. Given the importance of oxidative phosphorylation and the balance of energy metabolism in the heart, the potential regulatory effect of these classical signaling events on mitochondrial function is of interest. However, the functional impact of protein phosphorylation and the kinase/phosphatase system responsible for it are relatively unknown. Exceptions include the well-characterized pyruvate dehydrogenase and branched chain ?-ketoacid dehydrogenase regulatory system. The first task of this review is to update the current status of protein phosphorylation detection primarily in the matrix and evaluate evidence linking these events with enzymatic function or protein processing. To manage the scope of this effort, we have focused on the pathways involved in energy metabolism. The high sensitivity of modern methods of detecting protein phosphorylation and the low specificity of many kinases suggests that detection of protein phosphorylation sites without information on the mole fraction of phosphorylation is difficult to interpret, especially in metabolic enzymes, and is likely irrelevant to function. However, several systems including protein translocation, adenine nucleotide translocase, cytochrome c, and complex IV protein phosphorylation have been well correlated with enzymatic function along with the classical dehydrogenase systems. The second task is to review the current understanding of the kinase/phosphatase system within the matrix. Though it is clear that protein phosphorylation occurs within the matrix, based on (32)P incorporation and quantitative mass spectrometry measures, the kinase/phosphatase system responsible for this process is ill-defined. An argument is presented that remnants of the much more labile bacterial protein phosphoryl transfer system may be present in the matrix and that the evaluation of this possibility will require the application of approaches developed for bacterial cell signaling to the mitochondria.
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Project description:Apoptosis is coupled with recruitment of macrophages for engulfment of dead cells, and with compensatory proliferation of neighboring cells. Yet, this death process is silent, and it does not cause inflammation. The molecular mechanisms underlying anti-inflammatory nature of the apoptotic process remains poorly understood. In this study, we found that the culture supernatant of apoptotic cells activated the macrophages to express anti-inflammatory genes such as Nr4a and Thbs1. A high level of AMP accumulated in the apoptotic cell supernatant in a Pannexin1-dependent manner. A nucleotidase inhibitor and A2a adenosine receptor antagonist inhibited the apoptotic supernatant-induced gene expression, suggesting AMP was metabolized to adenosine by an ecto-5'-nucleotidase expressed on macrophages, to activate the macrophage A2a adenosine receptor. Intraperitoneal injection of zymosan into Adora2a- or Panx1-deficient mice produced high, sustained levels of inflammatory mediators in the peritoneal lavage. These results indicated that AMP from apoptotic cells suppresses inflammation as a 'calm down' signal. DOI: http://dx.doi.org/10.7554/eLife.02172.001.
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Project description:The plant mirid bug Apolygus lucorum is an omnivorous pest that can cause considerable economic damage. The steroid hormone 20-hydroxyecdysone (20E) is mainly responsible for molting and metamorphosis. The adenosine monophosphate-activated protein kinase (AMPK) is an intracellular energy sensor regulated by 20E, and its activity is regulated allosterically through phosphorylation. It is unknown whether the 20E-regulated insect's molting and gene expression depends on the AMPK phosphorylation. Herein, we cloned the full-length cDNA of the AlAMPK gene in A. lucorum. AlAMPK mRNA was detected at all developmental stages, whereas the dominant expression was in the midgut and, to a lesser extent, in the epidermis and fat body. Treatment with 20E and AMPK activator 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AlCAR) or only AlCAR resulted in activation of AlAMPK phosphorylation levels in the fat body, probed with an antibody directed against AMPK phosphorylated at Thr172, enhancing AlAMPK expression, whereas no phosphorylation occurred with compound C. Compared to compound C, 20E and/or AlCAR increased the molting rate, the fifth instar nymphal weight and shortened the development time of A. lucorum in vitro by inducing the expression of EcR-A, EcR-B, USP, and E75-A. Similarly, the knockdown of AlAMPK by RNAi reduced the molting rate of nymphs, the weight of fifth-instar nymphs and blocked the developmental time and the expression of 20E-related genes. Moreover, as observed by TEM, the thickness of the epidermis of the mirid was significantly increased in 20E and/or AlCAR treatments, molting spaces began to form between the cuticle and epidermal cells, and the molting progress of the mirid was significantly improved. These composite data indicated that AlAMPK, as a phosphorylated form in the 20E pathway, plays an important role in hormonal signaling and, in short, regulating insect molting and metamorphosis by switching its phosphorylation status.
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Project description:Chronic hepatitis B virus (HBV) infection is a major cause of chronic liver disease and cancer worldwide. The mechanisms of viral genome sensing and the evasion of innate immune responses by HBV infection are still poorly understood. Recently, the cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) was identified as a DNA sensor. In this study, we investigated the functional role of cGAS in sensing HBV infection and elucidate the mechanisms of viral evasion. We performed functional studies including loss-of-function and gain-of-function experiments combined with cGAS effector gene expression profiling in an infectious cell culture model, primary human hepatocytes, and HBV-infected human liver chimeric mice. Here, we show that cGAS is expressed in the human liver, primary human hepatocytes, and human liver chimeric mice. While naked relaxed-circular HBV DNA is sensed in a cGAS-dependent manner in hepatoma cell lines and primary human hepatocytes, host cell recognition of viral nucleic acids is abolished during HBV infection, suggesting escape from sensing, likely during packaging of the genome into the viral capsid. While the hepatocyte cGAS pathway is functionally active, as shown by reduction of viral covalently closed circular DNA levels in gain-of-function studies, HBV infection suppressed cGAS expression and function in cell culture models and humanized mice. Conclusion: HBV exploits multiple strategies to evade sensing and antiviral activity of cGAS and its effector pathways.
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Project description:The vascular endothelium responds to damage through activation of multiple signaling events that restore cell-cell adhesion and vascular integrity. However, the molecular mechanisms that integrate these events are not clearly defined. Herein, we identify a previously unexpected role for adenosine monophosphate-activated protein kinase (AMPK) in pulmonary microvascular endothelial cell (PMVEC) repair. PMVECs selectively express the AMPK?1 catalytic subunit, pharmacological and short hairpin RNA-mediated inhibition of which attenuates Ca(2+) entry in these cells induced by the inflammatory Ca(2+)-signaling mimetic thapsigargin. We find that AMPK?1 activity is required for the formation of PMVEC cell-cell networks in a prorepair environment and for monolayer resealing after wounding. Decreasing AMPK?1 expression reduces barrier resistance in PMVEC monolayers, results consistent with a role for AMPK?1 in cell-cell adhesion. AMPK?1 colocalizes and coimmunoprecipitates with the adherens junction protein N-cadherin and cofractionates with proteins selectively expressed in caveolar membranes. Assessment of permeability, by measuring the filtration coefficient (K(f)) in isolated perfused lungs, confirmed that AMPK activation contributes to barrier repair in vivo. Our findings thus provide novel evidence for AMPK?1 in Ca(2+) influx-mediated signaling and wound repair in the endothelium.
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Project description:Mitophagy is an important quality-control mechanism in eukaryotic cells, and defects in mitophagy correlate with aging phenomena and neurodegenerative disorders. It is known that different mitochondrial matrix proteins undergo mitophagy with very different rates but, to date, the mechanism underlying this selectivity at the individual protein level has remained obscure. We now present evidence indicating that protein phosphorylation within the mitochondrial matrix plays a mechanistic role in regulating selective mitophagic degradation in yeast via involvement of the Aup1 mitochondrial protein phosphatase, as well as 2 known matrix-localized protein kinases, Pkp1 and Pkp2. By focusing on a specific matrix phosphoprotein reporter, we also demonstrate that phospho-mimetic and nonphosphorylatable point mutations at known phosphosites in the reporter increased or decreased its tendency to undergo mitophagy. Finally, we show that phosphorylation of the reporter protein is dynamically regulated during mitophagy in an Aup1-dependent manner. Our results indicate that structural determinants on a mitochondrial matrix protein can govern its mitophagic fate, and that protein phosphorylation regulates these determinants.
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Project description:BackgroundWhile intracellular buffers are widely used to study calcium signaling, no such tool exists for the other major second messenger, cyclic AMP (cAMP).Methods/principal findingsHere we describe a genetically encoded buffer for cAMP based on the high-affinity cAMP-binding carboxy-terminus of the regulatory subunit RIbeta of protein kinase A (PKA). Addition of targeting sequences permitted localization of this fragment to the extra-nuclear compartment, while tagging with mCherry allowed quantification of its expression at the single cell level. This construct (named "cAMP sponge") was shown to selectively bind cAMP in vitro. Its expression significantly suppressed agonist-induced cAMP signals and the downstream activation of PKA within the cytosol as measured by FRET-based sensors in single living cells. Point mutations in the cAMP-binding domains of the construct rendered the chimera unable to bind cAMP in vitro or in situ. Cyclic AMP sponge was fruitfully applied to examine feedback regulation of gap junction-mediated transfer of cAMP in epithelial cell couplets.ConclusionsThis newest member of the cAMP toolbox has the potential to reveal unique biological functions of cAMP, including insight into the functional significance of compartmentalized signaling events.
| S-EPMC2766031 | biostudies-literature
Project description:Previously, we reported the identification of a thiazolidinedione-based adenosine monophosphate activated protein kinase (AMPK) activator, compound 1 (N-[4-({3-[(1-methylcyclohexyl)methyl]-2,4-dioxothiazolidin-5-ylidene}methyl)phenyl]-4-nitro-3-(trifluoromethyl)benzenesulfonamide), which provided a proof of concept to delineate the intricate role of AMPK in regulating oncogenic signaling pathways associated with cell proliferation and epithelial-mesenchymal transition (EMT) in cancer cells. In this study, we used 1 as a scaffold to conduct lead optimization, which generated a series of derivatives. Analysis of the antiproliferative and AMPK-activating activities of individual derivatives revealed a distinct structure-activity relationship and identified 59 (N-(3-nitrophenyl)-N'-{4-[(3-{[3,5-bis(trifluoromethyl)phenyl]methyl}-2,4-dioxothiazolidin-5-ylidene)methyl]phenyl}urea) as the optimal agent. Relative to 1, compound 59 exhibits multifold higher potency in upregulating AMPK phosphorylation in various cell lines irrespective of their liver kinase B1 (LKB1) functional status, accompanied by parallel changes in the phosphorylation/expression levels of p70S6K, Akt, Foxo3a, and EMT-associated markers. Consistent with its predicted activity against tumors with activated Akt status, orally administered 59 was efficacious in suppressing the growth of phosphatase and tensin homologue (PTEN)-null PC-3 xenograft tumors in nude mice. Together, these findings suggest that 59 has clinical value in therapeutic strategies for PTEN-negative cancer and warrants continued investigation in this regard.
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