scholarly journals Cellular ATP Levels Determine the Stability of a Nucleotide Kinase

2021 ◽  
Vol 8 ◽  
Author(s):  
Oliver Brylski ◽  
Puja Shrestha ◽  
Patricia Gnutt ◽  
David Gnutt ◽  
Jonathan Wolf Mueller ◽  
...  
Keyword(s):  
Protein Stability ◽  
Bound States ◽  
Atp Depletion ◽  
Aps Kinase ◽  
Cellular Processes ◽  
Atp Levels ◽  
Stability Change ◽  
Cell Stability ◽  
The Stability ◽  
Nucleotide Kinase

The energy currency of the cell ATP, is used by kinases to drive key cellular processes. However, the connection of cellular ATP abundance and protein stability is still under investigation. Using Fast Relaxation Imaging paired with alanine scanning and ATP depletion experiments, we study the nucleotide kinase (APSK) domain of 3′-phosphoadenosine-5′-phosphosulfate (PAPS) synthase, a marginally stable protein. Here, we show that the in-cell stability of the APSK is determined by ligand binding and directly connected to cellular ATP levels. The observed protein stability change for different ligand-bound states or under ATP-depleted conditions ranges from ΔGf0 = -10.7 to +13.8 kJ/mol, which is remarkable since it exceeds changes measured previously, for example upon osmotic pressure, cellular stress or differentiation. The results have implications for protein stability during the catalytic cycle of APS kinase and suggest that the cellular ATP level functions as a global regulator of kinase activity.

Applicability of Instability Index for In vitro Protein Stability Prediction

2019 ◽  
Vol 26 (5) ◽  
pp. 339-347 ◽  
Author(s):  
Dilani G. Gamage ◽  
Ajith Gunaratne ◽  
Gopal R. Periyannan ◽  
Timothy G. Russell
Keyword(s):  
Protein Stability ◽  
Caulobacter Crescentus ◽  
Effect Of Temperature ◽  
Instability Index ◽  
Dipeptide Composition ◽  
Experimental Conditions ◽  
The Stability ◽  
Vitro Protein

Background: The dipeptide composition-based Instability Index (II) is one of the protein primary structure-dependent methods available for in vivo protein stability predictions. As per this method, proteins with II value below 40 are stable proteins. Intracellular protein stability principles guided the original development of the II method. However, the use of the II method for in vitro protein stability predictions raises questions about the validity of applying the II method under experimental conditions that are different from the in vivo setting. Objective: The aim of this study is to experimentally test the validity of the use of II as an in vitro protein stability predictor. Methods: A representative protein CCM (CCM - Caulobacter crescentus metalloprotein) that rapidly degrades under in vitro conditions was used to probe the dipeptide sequence-dependent degradation properties of CCM by generating CCM mutants to represent stable and unstable II values. A comparative degradation analysis was carried out under in vitro conditions using wildtype CCM, CCM mutants and two other candidate proteins: metallo-β-lactamase L1 and α -S1- casein representing stable, borderline stable/unstable, and unstable proteins as per the II predictions. The effect of temperature and a protein stabilizing agent on CCM degradation was also tested. Results: Data support the dipeptide composition-dependent protein stability/instability in wt-CCM and mutants as predicted by the II method under in vitro conditions. However, the II failed to accurately represent the stability of other tested proteins. Data indicate the influence of protein environmental factors on the autoproteolysis of proteins. Conclusion: Broader application of the II method for the prediction of protein stability under in vitro conditions is questionable as the stability of the protein may be dependent not only on the intrinsic nature of the protein but also on the conditions of the protein milieu.

Poly(ADP-ribose) polymerase-1 gene ablation protects mice from ischemic renal injury

2005 ◽  
Vol 288 (2) ◽  
pp. F387-F398 ◽  
Author(s):  
Jianfeng Zheng ◽  
Kishor Devalaraja-Narashimha ◽  
Kurinji Singaravelu ◽  
Babu J. Padanilam
Keyword(s):  
Dna Damage ◽  
Cell Death ◽  
Mouse Model ◽  
Renal Injury ◽  
Atp Depletion ◽  
Inflammatory Cascade ◽  
Renal Functions ◽  
Atp Levels ◽  
Ischemic Renal Injury ◽  
Parp 1

Increased generation of reactive oxygen species (ROS) and the subsequent DNA damage and excessive activation of poly(ADP-ribose) polymerase-1 (PARP-1) have been implicated in the pathogenesis of ischemic injury. We previously demonstrated that pharmacological inhibition of PARP protects against ischemic renal injury (IRI) in rats (Martin DR, Lewington AJ, Hammerman MR, and Padanilam BJ. Am J Physiol Regul Integr Comp Physiol 279: R1834–R1840, 2000). To further define the role of PARP-1 in IRI, we tested whether genetic ablation of PARP-1 attenuates tissue injury after renal ischemia. Twenty-four hours after reperfusion following 37 min of bilateral renal pedicle occlusion, the effects of the injury on renal functions in PARP−/− and PARP+/+ mice were assessed by determining glomerular filtration rate (GFR) and the plasma levels of creatinine. The levels of plasma creatinine were decreased and GFR was augmented in PARP−/− mice. Morphological evaluation of the kidney tissues showed that the extent of damage due to the injury in PARP−/− mice was less compared with their wild-type counterparts. The levels of ROS and DNA damage were comparable in the injured kidneys of PARP+/+ and PARP−/− mice. PARP activity was induced in ischemic kidneys of PARP+/+ mice at 6–24 h postinjury. At 6, 12, and 24 h after injury, ATP levels in the PARP+/+ mice kidney declined to 28, 26, and 43%, respectively, whereas it was preserved close to normal levels in PARP−/− mice. The inflammatory cascade was attenuated in PARP−/− mice as evidenced by decreased neutrophil infiltration and attenuated expression of inflammatory molecules such as TNF-α, IL-1β, and intercellular adhesion molecule-1. At 12 h postinjury, no apoptotic cell death was observed in PARP−/− mice kidneys. However, by 24 h postinjury, a comparable number of cells underwent apoptosis in both PARP−/− and PARP+/+ mice kidneys. Thus activation of PARP post-IRI contributes to cell death most likely by ATP depletion and augmentation of the inflammatory cascade in the mouse model. PARP ablation preserved ATP levels, renal functions, and attenuated inflammatory response in the setting of IRI in the mouse model. PARP inhibition may have clinical efficacy in preventing the progression of acute renal failure complications.

scholarly journals Agonist-induced regulation of mitochondrial and endoplasmic reticulum motility

2005 ◽  
Vol 392 (2) ◽  
pp. 291-297 ◽  
Author(s):  
David Brough ◽  
Michael J. Schell ◽  
Robin F. Irvine
Keyword(s):  
Endoplasmic Reticulum ◽  
Confocal Microscopy ◽  
Atp Depletion ◽  
Metabolic Inhibitors ◽  
Muscarinic Agonist ◽  
Organelle Movement ◽  
Atp Levels ◽  
Two Parameters

Using fluorescently tagged markers for organelles in conjunction with confocal microscopy, we have studied the effects of agonist-induced Ca2+ signals on the motility of mitochondria and the ER (endoplasmic reticulum). We observed that the muscarinic agonist carbachol produced a rapid, simultaneous and reversible cessation of the movements of both organelles, which was dependent on a rise in cytosolic Ca2+. This rise in Ca2+ was shown to cause a fall in cellular ATP levels, and the effect of carbachol on organelle movement could be mimicked by depleting ATP with metabolic inhibitors in the absence of any such rise in Ca2+. However, a Ca2+-sensing process independent of ATP appears also to be involved, because we identified conditions where the ATP depletion was blocked (by inhibitors of the Ca2+ pumps), but the organelle movements still ceased following a rise in cytosolic Ca2+. We conclude that the co-ordinated cessation of mitochondria and ER motility is a process regulated by the cytosolic concentration of both Ca2+ and ATP, and that these two parameters are likely to synergize to regulate the localization of the two organelles, and to facilitate the transfer of Ca2+ between them.

scholarly journals Effects of Atovaquone and Diospyrin-Based Drugs on the Cellular ATP of Pneumocystis carinii f. sp.carinii

2000 ◽  
Vol 44 (3) ◽  
pp. 713-719 ◽  
Author(s):  
Melanie T. Cushion ◽  
Margaret Collins ◽  
Banasri Hazra ◽  
Edna S. Kaneshiro
Keyword(s):  
Electron Transport ◽  
Drug Target ◽  
Coenzyme Q ◽  
Pneumocystis Carinii ◽  
Atp Depletion ◽  
Malarial Parasite ◽  
Dihydroorotate Dehydrogenase ◽  
Atp Levels ◽  
Transport Chain ◽  
Kinetics Of

ABSTRACT Atovaquone (also called Mepron, or 566C80) is a napthoquinone used for the treatment of infections caused by pathogens such asPlasmodium spp. and Pneumocystis carinii. The mechanism of action against the malarial parasite is the inhibition of dihydroorotate dehydrogenase (DHOD), a consequence of blocking electron transport by the drug. As an analog of ubiquinone (coenzyme Q [CoQ]), atovaquone irreversibly binds to the mitochondrial cytochrome bc 1 complex; thus, electrons are not able to pass from dehydrogenase enzymes via CoQ to cytochrome c. Since DHOD is a critical enzyme in pyrimidine biosynthesis, and because the parasite cannot scavenge host pyrimidines, the drug is lethal to the organism. Oxygen consumption inP. carinii is inhibited by the drug; thus, electron transport has also been identified as the drug target in P. carinii. However, unlike Plasmodium DHOD, P. carinii DHOD is inhibited only at high atovaquone concentrations, suggesting that the organism may salvage host pyrimidines and that atovaquone exerts its primary effects on ATP biosynthesis. In the present study, the effect of atovaquone on ATP levels in P. carinii was measured directly from 1 to 6 h and then after 24, 48, and 72 h of exposure. The average 50% inhibitory concentration after 24 to 72 h of exposure was 1.5 μg/ml (4.2 μM). The kinetics of ATP depletion were in contrast to those of another family of naphthoquinone compounds, diospyrin and two of its derivatives. Whereas atovaquone reduced ATP levels within 1 h of exposure, the diospyrins required at least 48 h. After 72 h, the diospyrins were able to decrease ATP levels of P. carinii at nanomolar concentrations. These data indicate that although naphthoquinones inhibit the electron transport chain, the molecular targets in a given organism are likely to be distinct among members of this class of compounds.

scholarly journals A glycine-specific N-degron pathway mediates the quality control of protein N-myristoylation

Science ◽  
2019 ◽  
Vol 365 (6448) ◽  
pp. eaaw4912 ◽  
Author(s):  
Richard T. Timms ◽  
Zhiqian Zhang ◽  
David Y. Rhee ◽  
J. Wade Harper ◽  
Itay Koren ◽  
...  
Keyword(s):  
Quality Control ◽  
Protein Stability ◽  
Protein Degradation ◽  
E3 Ligase ◽  
Cleavage Sites ◽  
Caspase Cleavage ◽  
E3 Ligases ◽  
Ring E3 Ligase ◽  
The Stability ◽  
Ring E3

The N-terminal residue influences protein stability through N-degron pathways. We used stability profiling of the human N-terminome to uncover multiple additional features of N-degron pathways. In addition to uncovering extended specificities of UBR E3 ligases, we characterized two related Cullin-RING E3 ligase complexes, Cul2ZYG11B and Cul2ZER1, that act redundantly to target N-terminal glycine. N-terminal glycine degrons are depleted at native N-termini but strongly enriched at caspase cleavage sites, suggesting roles for the substrate adaptors ZYG11B and ZER1 in protein degradation during apoptosis. Furthermore, ZYG11B and ZER1 were found to participate in the quality control of N-myristoylated proteins, in which N-terminal glycine degrons are conditionally exposed after a failure of N-myristoylation. Thus, an additional N-degron pathway specific for glycine regulates the stability of metazoan proteomes.

scholarly journals Spin(p + 1, p + 1) COVARIANT Dp-BRANE BOUND STATES

2001 ◽  
Vol 16 (17) ◽  
pp. 3025-3040 ◽  
Author(s):  
P. SUNDELL
Keyword(s):  
Field Theory ◽  
Ground State ◽  
Bound States ◽  
Type Ii ◽  
Duality Group ◽  
Zero Force ◽  
Force Condition ◽  
The Stability

We construct Spin (p + 1, p + 1) covariant D p-brane bound states by using the fact that the potentials in the RR sector of toroidically compactified type II supergravity transform as a chiral spinor of the T duality group. As an application, we show the invariance of the zero-force condition for a probe D-brane under noncommutative deformations of the background, which gives a holographic proof of the stability of the corresponding field theory ground state under noncommutative deformations. We also identify the Spin (p + 1, p + 1) transformation laws by examining the covariance of the D-brane Lagrangians.

Slow ATP loss and the defense of ion homeostasis in the anoxic frog brain

2001 ◽  
Vol 204 (20) ◽  
pp. 3547-3551
Author(s):  
Debra L. Knickerbocker ◽  
Peter L. Lutz
Keyword(s):  
Initial Phase ◽  
Ion Homeostasis ◽  
Atp Depletion ◽  
Neuronal Membrane ◽  
Anoxia Tolerance ◽  
Frog Brain ◽  
Atp Levels ◽  
Anoxic Depolarization ◽  
Rate Of Increase ◽  
The Brain

SUMMARY For most vertebrates, cutting off the oxygen supply to the brain results in a rapid (within minutes) loss of ATP, the failure of ATP-dependent ion-transport process, subsequent anoxic depolarization of neuronal membrane potential and consequential neuronal death. The few species that survive brain anoxia for days or months, such as the freshwater turtle Trachemys scripta, avoid anoxic depolarization and maintain brain ATP levels through a coordinated downregulation of brain energy demand processes. The frog Rana pipiens represents an intermediate in anoxia-tolerance, being able to survive brain anoxia for hours. However, the anoxic frog brain does not defend its energy stores. Instead, anoxia-tolerance appears to be related to a retarded rate of ATP depletion. To investigate the relationship between this slow ATP depletion and the loss of ionic homeostasis, cerebral extracellular K+ concentrations were monitored and ATP levels measured during anoxia, during the initial phase of anoxic depolarization and during complete anoxic depolarization. Extracellular K+ levels were maintained at normoxic levels for at least 3 h of anoxia, while ATP content decreased by 35 %. When ATP levels reached 0.33±0.06 mmol l–1 (mean ± s.e.m., N=5), extracellular K+ levels slowly started to increase. This value is thought to represent a critical ATP concentration for the maintenance of ion homeostasis. When extracellular [K+] reached an inflection value of 4.77±0.84 mmol l–1 (mean ± s.e.m., N=5), approximately 1 h later, the brain quickly depolarized. Part of the reduction in ATP demand was attributable to an approximately 50 % decrease in the rate of K+ efflux from the anoxic frog brain, which would also contribute to the retarded rate of increase in extracellular [K+] during the initial phase of anoxic depolarization. However, unlike the anoxia-tolerant turtle brain, adenosine did not appear to be involved in the downregulation of K+ leakage in the frog brain. The increased anoxia-tolerance of the frog brain is thought to be a matter more of slow death than of enhanced protective mechanisms.

The Distribution and Stability of Flow in a Rotating Channel

1968 ◽  
Vol 90 (3) ◽  
pp. 229-235 ◽  
Author(s):  
H. S. Fowler
Keyword(s):  
Reynolds Number ◽  
Flow Field ◽  
Centrifugal Compressor ◽  
Channel Wall ◽  
Flow Distribution ◽  
Stability Change ◽  
The Stability ◽  
Downstream Flow ◽  
Adjacent Channels ◽  
Rotating Channel

The importance of flow distribution and stability leaving the impeller of a centrifugal compressor is discussed. Experiments on the flow in converging, parallel, and diffusing channels, representative of sections of impeller channels, are reported and discussed. The convergent or divergent character of the channel appears to have less influence on the flow pattern than on the stability. Change of Reynolds number appears to change the degree of nonuniformity of distribution in the channel, but change of rotation number appears to affect the distribution of the nonuniformity more. It appears that the influence of adjacent channels, and of the downstream flow-field in general, has a large effect on the flow within the rotating channel, particularly as regards detachment from the suction channel wall. “No man is an island unto himself,” and no element in an aerodynamic system can be divorced from its upstream or downstream neighbors.

In Silico Analysis of Non Synonymous Single Nucleotide Polymorphisms (nsSNPs) of SMPX Gene in Hearing Impairment

2018 ◽  
Author(s):  
Md. Arifuzzaman ◽  
Sarmistha Mitra ◽  
Amir Hamza ◽  
Raju Das ◽  
Nurul Absar ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphisms ◽  
Protein Stability ◽  
In Silico Analysis ◽  
Normal Activity ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Binding Motifs ◽  
Stability Change ◽  
And Function ◽  
Dbsnp Database

ABSTRACTBackgroundMutations in SMPX gene can disrupt the normal activity of the SMPX protein which is involved in hearing process.ObjectiveIn this study, deleterious non-synonymous single nucleotide polymorphisms were isolated from the neutral variants by using several bioinformatics tools.MethodFirstly, dbSNP database hosted by NCBI was used to retrieve the SNPs of SMPX gene, secondly, SIFT was used primarily to screen the damaging SNPs. Further, for validation PROVEAN, PredictSNP and PolyPhen 2 were used. I-Mutant 3 was utilized to analyze the protein stability change and MutPred predicted the molecular mechanism of protein stability change. Finally evolutionary conservation was done to study their conservancy by using ConSurf server.ResultsA total of 26 missense (0.6517%) and 3 nonsense variants (0.075%) were retrieved and among them 4 mutations were found deleterious by all the tools of this experiment and are also highly conserved according to ConSurf server. rs772775896, rs759552778, rs200892029 and rs1016314772 are the reference IDs of deleterious mutations where the substitutions are S71L, N19D, A29T and K54N. Loss of Ubiquitination, loss of methylation, loss of glycosylation, and loss of MoRF binding motifs are the root causes of protein stability change.ConclusionThis is the first study regarding nsSNPs of SMPX gene where the most damaging SNPs were screened that are associated with the SMPX gene and can be used for further research to study their effect on protein structure and function, their dynamic behavior and how they actually affect protein’s flexibility.

Sign in / Sign up

Export Citation Format

Share Document