scholarly journals Cooperative subunit dynamics modulate p97 function

2018 ◽  
Vol 116 (1) ◽  
pp. 158-167 ◽  
Author(s):  
Rui Huang ◽  
Zev A. Ripstein ◽  
John L. Rubinstein ◽  
Lewis E. Kay
Keyword(s):  
Bound State ◽  
Nmr Studies ◽  
Biologically Relevant ◽  
Aaa Atpase ◽  
Cellular Processes ◽  
Allosteric Communication ◽  
Functional Dynamics ◽  
Nmr Study ◽  
Wide Range

p97 is an essential hexameric AAA+ ATPase involved in a wide range of cellular processes. Mutations in the enzyme are implicated in the etiology of an autosomal dominant neurological disease in which patients are heterozygous with respect to p97 alleles, containing one copy each of WT and disease-causing mutant genes, so that, in vivo, p97 molecules can be heterogeneous in subunit composition. Studies of p97 have, however, focused on homohexameric constructs, where protomers are either entirely WT or contain a disease-causing mutation, showing that for WT p97, the N-terminal domain (NTD) of each subunit can exist in either a down (ADP) or up (ATP) conformation. NMR studies establish that, in the ADP-bound state, the up/down NTD equilibrium shifts progressively toward the up conformation as a function of disease mutant severity. To understand NTD functional dynamics in biologically relevant p97 heterohexamers comprising both WT and disease-causing mutant subunits, we performed a methyl-transverse relaxation optimized spectroscopy (TROSY) NMR study on a series of constructs in which only one of the protomer types is NMR-labeled. Our results show positive cooperativity of NTD up/down equilibria between neighboring protomers, allowing us to define interprotomer pathways that mediate the allosteric communication between subunits. Notably, the perturbed up/down NTD equilibrium in mutant subunits is partially restored by neighboring WT protomers, as is the two-pronged binding of the UBXD1 adaptor that is affected in disease. This work highlights the plasticity of p97 and how subtle perturbations to its free-energy landscape lead to significant changes in NTD conformation and adaptor binding.

scholarly journals The AAA+ ATPase p97, a cellular multitool

2017 ◽  
Vol 474 (17) ◽  
pp. 2953-2976 ◽  
Author(s):  
Lasse Stach ◽  
Paul S. Freemont
Keyword(s):  
Atp Hydrolysis ◽  
Current Status ◽  
Cellular Functions ◽  
Aaa Atpase ◽  
Cellular Processes ◽  
Proteotoxic Stress ◽  
Binding Domains ◽  
Wide Range ◽  
Aaa Atpases ◽  
Substrate Proteins

The AAA+ (ATPases associated with diverse cellular activities) ATPase p97 is essential to a wide range of cellular functions, including endoplasmic reticulum-associated degradation, membrane fusion, NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) activation and chromatin-associated processes, which are regulated by ubiquitination. p97 acts downstream from ubiquitin signaling events and utilizes the energy from ATP hydrolysis to extract its substrate proteins from cellular structures or multiprotein complexes. A multitude of p97 cofactors have evolved which are essential to p97 function. Ubiquitin-interacting domains and p97-binding domains combine to form bi-functional cofactors, whose complexes with p97 enable the enzyme to interact with a wide range of ubiquitinated substrates. A set of mutations in p97 have been shown to cause the multisystem proteinopathy inclusion body myopathy associated with Paget's disease of bone and frontotemporal dementia. In addition, p97 inhibition has been identified as a promising approach to provoke proteotoxic stress in tumors. In this review, we will describe the cellular processes governed by p97, how the cofactors interact with both p97 and its ubiquitinated substrates, p97 enzymology and the current status in developing p97 inhibitors for cancer therapy.

NMR studies of enzymatic rates in vitro and in vivo by magnetization transfer

1984 ◽  
Vol 17 (1) ◽  
pp. 83-124 ◽  
Author(s):  
J. R. Alger ◽  
R. G. Shulman
Keyword(s):  
Creatine Kinase ◽  
Chemical Reactions ◽  
Magnetization Transfer ◽  
Living Systems ◽  
Nmr Studies ◽  
Wide Range ◽  
Potential Applications

Magnetization transfer techniques are specialized NMR experiments which can measure the rate of chemical reactions while concentrations of products and reactants are maintained constant. These techniques are being used to measure the rates of enzyme catalysed reactions in a variety of living systems and in vitro. The magnetization transfer measurements in vivo of the ATP synthetase and the creatine kinase reactions have been particularly useful in describing rates of major energy transducing reactions involving ATP and phosphocreatine. As a result, a wide range of biomedicai scientists are becoming aware of the potentials of these techniques. The purpose of this review is thus threefold: first, to present a concise, conceptual review of the underlying principles for these non-specialists; secondly, to review the important biochemical applications of the method which have appeared, and thirdly, to discuss potential applications and limitations of the method.

CAN COMPLEX CELLULAR PROCESSES BE GOVERNED BY SIMPLE LINEAR RULES?

2009 ◽  
Vol 07 (01) ◽  
pp. 243-268 ◽  
Author(s):  
KUMAR SELVARAJOO ◽  
MASARU TOMITA ◽  
MASA TSUCHIYA
Keyword(s):  
Biological Networks ◽  
Biological Network ◽  
Living Systems ◽  
Regulatory Motifs ◽  
Cellular Processes ◽  
Self Organized ◽  
Wide Range ◽  
Activation Response ◽  
Physical Reasons

Complex living systems have shown remarkably well-orchestrated, self-organized, robust, and stable behavior under a wide range of perturbations. However, despite the recent generation of high-throughput experimental datasets, basic cellular processes such as division, differentiation, and apoptosis still remain elusive. One of the key reasons is the lack of understanding of the governing principles of complex living systems. Here, we have reviewed the success of perturbation–response approaches, where without the requirement of detailed in vivo physiological parameters, the analysis of temporal concentration or activation response unravels biological network features such as causal relationships of reactant species, regulatory motifs, etc. Our review shows that simple linear rules govern the response behavior of biological networks in an ensemble of cells. It is daunting to know why such simplicity could hold in a complex heterogeneous environment. Provided physical reasons can be explained for these phenomena, major advancement in the understanding of basic cellular processes could be achieved.

scholarly journals Mitochondrial division, fusion and degradation

2019 ◽  
Vol 167 (3) ◽  
pp. 233-241 ◽  
Author(s):  
Daisuke Murata ◽  
Kenta Arai ◽  
Miho Iijima ◽  
Hiromi Sesaki
Keyword(s):  
Super Resolution ◽  
Healthy Population ◽  
Lipid Biochemistry ◽  
Mitochondrial Division ◽  
Cellular Processes ◽  
Size Number ◽  
Wide Range ◽  
Autophagic Degradation ◽  
And Function

Abstract The mitochondrion is an essential organelle for a wide range of cellular processes, including energy production, metabolism, signal transduction and cell death. To execute these functions, mitochondria regulate their size, number, morphology and distribution in cells via mitochondrial division and fusion. In addition, mitochondrial division and fusion control the autophagic degradation of dysfunctional mitochondria to maintain a healthy population. Defects in these dynamic membrane processes are linked to many human diseases that include metabolic syndrome, myopathy and neurodegenerative disorders. In the last several years, our fundamental understanding of mitochondrial fusion, division and degradation has been significantly advanced by high resolution structural analyses, protein-lipid biochemistry, super resolution microscopy and in vivo analyses using animal models. Here, we summarize and discuss this exciting recent progress in the mechanism and function of mitochondrial division and fusion.

scholarly journals Interlinked GTPase cascades provide a motif for both robust switches and oscillators

2019 ◽  
Vol 16 (157) ◽  
pp. 20190198 ◽  
Author(s):  
Andreas Ehrmann ◽  
Basile Nguyen ◽  
Udo Seifert
Keyword(s):  
Single Species ◽  
Protein Translation ◽  
Bound State ◽  
Gtp Hydrolysis ◽  
Cellular Processes ◽  
Guanosine Diphosphate ◽  
Negative Feedback Loops ◽  
Consistent Model ◽  
Wide Range ◽  
Positive And Negative Feedback

GTPases regulate a wide range of cellular processes, such as intracellular vesicular transport, signal transduction and protein translation. These hydrolase enzymes operate as biochemical switches by toggling between an active guanosine triphosphate (GTP)-bound state and an inactive guanosine diphosphate (GDP)-bound state. We compare two network motifs, a single-species switch and an interlinked cascade that consists of two species coupled through positive and negative feedback loops. We find that interlinked cascades are closer to the ideal all-or-none switch and are more robust against fluctuating signals. While the single-species switch can only achieve bistability, interlinked cascades can be converted into oscillators by tuning the cofactor concentrations, which catalyse the activity of the cascade. These regimes can only be achieved with sufficient chemical driving provided by GTP hydrolysis. In this study, we present a thermodynamically consistent model that can achieve bistability and oscillations with the same feedback motif.

scholarly journals Identification of Z nucleotides as an ancient signal for two-component system activation in bacteria

2020 ◽  
Vol 117 (52) ◽  
pp. 33530-33539
Author(s):  
Oscar J. Vázquez-Ciros ◽  
Adrián F. Alvarez ◽  
Dimitris Georgellis
Keyword(s):  
De Novo ◽  
Response Regulator ◽  
Phosphoryl Group ◽  
Response Regulators ◽  
Carbamoyl Phosphate ◽  
Cellular Processes ◽  
Wide Range ◽  
Two Component ◽  
Two Component Systems

Two-component systems (TCSs) in bacteria are molecular circuits that allow the perception of and response to diverse stimuli. These signaling circuits rely on phosphoryl-group transfers between transmitter and receiver domains of sensor kinase and response regulator proteins, and regulate several cellular processes in response to internal or external cues. Phosphorylation, and thereby activation, of response regulators has been demonstrated to occur by their cognate histidine kinases but also by low molecular weight phosphodonors such as acetyl phosphate and carbamoyl phosphate. Here, we present data indicating that the intermediates of the de novo syntheses of purines and histidine, 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranosyl 5′-monophosphate (ZMP) and/or 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranosyl 5′-triphosphate (ZTP), activate the response regulator UvrY, by promoting its autophosphorylation at the conserved aspartate at position 54. Moreover, these Z nucleotides are shown to also activate the nonrelated response regulators ArcA, CpxR, RcsB, and PhoQ. We propose that ZMP and/or ZTP act as alarmones for a wide range of response regulators in vivo, providing a novel mechanism by which they could impact gene expression in response to metabolic cues.

scholarly journals Microbiota-Mediated Modulation of Organophosphate Insecticide Toxicity by Species-Dependent Interactions with Lactobacilli in a Drosophila melanogaster Insect Model

2018 ◽  
Vol 84 (9) ◽  
Author(s):  
Brendan A. Daisley ◽  
Mark Trinder ◽  
Tim W. McDowell ◽  
Stephanie L. Collins ◽  
Mark W. Sumarah ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Gut Microbiota ◽  
Environmental Sustainability ◽  
Lactobacillus Rhamnosus ◽  
Organophosphate Insecticide ◽  
Content Type ◽  
Biologically Relevant ◽  
Wide Range ◽  
Probiotic Lactobacilli

ABSTRACT Despite the benefits to the global food supply and agricultural economies, pesticides are believed to pose a threat to the health of both humans and wildlife. Chlorpyrifos (CP), a commonly used organophosphate insecticide, has poor target specificity and causes acute neurotoxicity in a wide range of species via the suppression of acetylcholinesterase. This effect is exacerbated 10- to 100-fold by chlorpyrifos oxon (CPO), a principal metabolite of CP. Since many animal-associated symbiont microorganisms are known to hydrolyze CP into CPO, we used a Drosophila melanogaster insect model to investigate the hypothesis that indigenous and probiotic bacteria could affect CP metabolism and toxicity. Antibiotic-treated and germfree D. melanogaster insects lived significantly longer than their conventionally reared counterparts when exposed to 10 μM CP. Drosophila melanogaster gut-derived Lactobacillus plantarum , but not Acetobacter indonesiensis , was shown to metabolize CP. Liquid chromatography tandem-mass spectrometry confirmed that the L. plantarum isolate preferentially metabolized CP into CPO when grown in CP-spiked culture medium. Further experiments showed that monoassociating germfree D. melanogaster with the L. plantarum isolate could reestablish a conventional-like sensitivity to CP. Interestingly, supplementation with the human probiotic Lactobacillus rhamnosus GG (a strain that binds but does not metabolize CP) significantly increased the survival of the CP-exposed germfree D. melanogaster . This suggests strain-specific differences in CP metabolism may exist among lactobacilli and emphasizes the need for further investigation. In summary, these results suggest that (i) CPO formation by the gut microbiota can have biologically relevant consequences for the host, and (ii) probiotic lactobacilli may be beneficial in reducing in vivo CP toxicity. IMPORTANCE An understudied area of research is how the microbiota (microorganisms living in/on an animal) affects the metabolism and toxic outcomes of environmental pollutants such as pesticides. This study focused specifically on how the microbial biotransformation of chlorpyrifos (CP; a common organophosphate insecticide) affected host exposure and toxicity parameters in a Drosophila melanogaster insect model. Our results demonstrate that the biotransformation of CP by the gut microbiota had biologically relevant and toxic consequences on host health and that certain probiotic lactobacilli may be beneficial in reducing CP toxicity. Since inadvertent pesticide exposure is suspected to negatively impact the health of off-target species, these findings may provide useful information for wildlife conservation and environmental sustainability planning. Furthermore, the results highlight the need to consider microbiota composition differences between beneficial and pest insects in future insecticide designs. More broadly, this study supports the use of beneficial microorganisms to modulate the microbiota-mediated biotransformation of xenobiotics.

scholarly journals What macromolecular crystallogenesis tells us – what is needed in the future

IUCrJ ◽  
2017 ◽  
Vol 4 (4) ◽  
pp. 340-349 ◽  
Author(s):  
Richard Giegé
Keyword(s):  
Self Assembly ◽  
Inorganic Materials ◽  
Biological Processes ◽  
X Ray ◽  
Biologically Relevant ◽  
Cellular Processes ◽  
The Future

Crystallogenesis is a longstanding topic that has transformed into a discipline that is mainly focused on the preparation of crystals for practising crystallographers. Although the idiosyncratic features of proteins have to be taken into account, the crystallization of proteins is governed by the same physics as the crystallization of inorganic materials. At present, a diversified panel of crystallization methods adapted to proteins has been validated, and although only a few methods are in current practice, the success rate of crystallization has increased constantly, leading to the determination of ∼105X-ray structures. These structures reveal a huge repertoire of protein folds, but they only cover a restricted part of macromolecular diversity across the tree of life. In the future, crystals representative of missing structures or that will better document the structural dynamics and functional steps underlying biological processes need to be grown. For the pertinent choice of biologically relevant targets, computer-guided analysis of structural databases is needed. From another perspective, crystallization is a self-assembly process that can occur in the bulk of crowded fluids, with crystals being supramolecular assemblies. Life also uses self-assembly and supramolecular processes leading to transient, or less often stable, complexes. An integrated view of supramolecularity implies that proteins crystallizing eitherin vitroorin vivoor participating in cellular processes share common attributes, notably determinants and antideterminants that favour or disfavour their correct or incorrect associations. As a result, underin vivoconditions proteins show a balance between features that favour or disfavour association. If this balance is broken, disorders/diseases occur. Understanding crystallization underin vivoconditions is a challenge for the future. In this quest, the analysis of packing contacts and contacts within oligomers will be crucial in order to decipher the rules governing protein self-assembly and will guide the engineering of novel biomaterials. In a wider perspective, understanding such contacts will open the route towards supramolecular biology and generalized crystallogenesis.

scholarly journals Whole-Plant Measure of Temperature-Induced Changes in the Cytosolic pH of Potato Plants Using Genetically Encoded Fluorescent Sensor Pt-GFP

Agriculture ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 1131
Author(s):  
Anna Pecherina ◽  
Marina Grinberg ◽  
Maria Ageyeva ◽  
Tatiana Zdobnova ◽  
Maria Ladeynova ◽  
...  
Keyword(s):  
Fluorescent Sensor ◽  
Signal Transmission ◽  
Ph Sensor ◽  
Threshold Temperature ◽  
Cytosolic Ph ◽  
Cellular Processes ◽  
Wide Range ◽  
Open Ground ◽  
Work Model

Cytosolic pH (pHcyt) regulates a wide range of cellular processes in plants. Changes in pHcyt occurring under the effect of different stressors can participate in signal transmission. The dynamics of pHcyt under the action of external factors, including significant factors for open ground crops such as temperature, remains poorly understood, which is largely due to the difficulty of intracellular pH registration using standard methods. In this work, model plants of potato (one of the essential crops) expressing a fluorescent ratiometric pH sensor Pt-GFP were created. The calibration obtained in vivo allowed for the determination of the pHcyt values of the cells of the leaves, which is 7.03 ± 0.03 pH. Cooling of the whole leaf caused depolarization and rapid acidification of the cytosol, the amplitude of which depended on the cooling strength, amounting to about 0.2 pH units when cooled by 15 °C. When the temperature rises to 35–40 °C, the cytosol was alkalized by 0.2 pH units. Heating above the threshold temperature caused the acidification of cytosol and generation of variation potential. The observed rapid changes in pHcyt can be associated with changes in the activity of H+-ATPases, which was confirmed by inhibitory analysis.

scholarly journals Mapping physiological ADP-ribosylation using Activated Ion Electron Transfer Dissociation (AI-ETD)

2020 ◽  
Author(s):  
Sara C. Buch-Larsen ◽  
Ivo A. Hendriks ◽  
Jean M. Lodge ◽  
Martin Rykær ◽  
Benjamin Furtwängler ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Electron Transfer Dissociation ◽  
Post Translational Modification ◽  
Physiological Regulation ◽  
Physiological Conditions ◽  
Adp Ribosylation ◽  
Cellular Processes ◽  
Wide Range ◽  
Enrichment Strategy

SUMMARYADP-ribosylation (ADPr) is a post-translational modification that plays pivotal roles in a wide range of cellular processes. Mass spectrometry (MS)-based analysis of ADPr under physiological conditions, without relying on genetic or chemical perturbation, has been hindered by technical limitations. Here, we describe the applicability of Activated Ion Electron Transfer Dissociation (AI-ETD) for MS-based proteomics analysis of physiological ADPr using our unbiased Af1521 enrichment strategy. To benchmark AI-ETD, we profiled 9,000 ADPr peptides mapping to >5,000 unique ADPr sites from a limited number of cells exposed to oxidative stress, corresponding to 120% and 28% more ADPr peptides compared to contemporary strategies using ETD and EThcD, respectively. Under physiological conditions AI-ETD identified 450 ADPr sites on low-abundant proteins, including in vivo cysteine auto-modifications on PARP8 and tyrosine auto-modifications on PARP14, hinting at specialist enzymatic functions for these enzymes. Collectively, our data provides new insights into the physiological regulation of ADP-ribosylation.

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