scholarly journals Ensemble cryo-EM structures demonstrate human IMPDH2 filament assembly tunes allosteric regulation

2019 ◽  
Author(s):  
Matthew C. Johnson ◽  
Justin M. Kollman
Keyword(s):  
T Cell Activation ◽  
Cellular Proliferation ◽  
Feedback Inhibition ◽  
Adenine Nucleotide ◽  
Allosteric Regulation ◽  
Regulatory Control ◽  
Guanine Nucleotide ◽  
Inosine Monophosphate Dehydrogenase ◽  
Filament Assembly ◽  
Nucleotide Pools

SummaryInosine monophosphate dehydrogenase (IMPDH) mediates the first committed step in guanine nucleotide biosynthesis and plays important roles in cellular proliferation and the immune response. The enzyme is heavily regulated to maintain balance between guanine and adenine nucleotide pools. IMPDH reversibly polymerizes in cells and tissues in response to changes in metabolic demand, providing an additional layer of regulatory control associated with increased flux through the guanine synthesis pathway. Here, we report a series of human IMPDH2 cryo-EM structures in active and inactive conformations, and show that the filament resists inhibition by guanine nucleotides. The structures define the mechanism of filament assembly, and reveal how assembly interactions tune the response to guanine inhibition. Filament-dependent allosteric regulation of IMPDH2 makes the enzyme less sensitive to feedback inhibition, explaining why assembly occurs under physiological conditions, like stem cell proliferation and T-cell activation, that require expansion of guanine nucleotide pools.

scholarly journals Cryo-EM structures demonstrate human IMPDH2 filament assembly tunes allosteric regulation

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Matthew C Johnson ◽  
Justin M Kollman
Keyword(s):  
Self Assembly ◽  
Cellular Proliferation ◽  
Feedback Inhibition ◽  
Allosteric Regulation ◽  
General Mechanism ◽  
Guanine Nucleotide ◽  
Inosine Monophosphate Dehydrogenase ◽  
Metabolic Demand ◽  
Nucleotide Biosynthesis ◽  
Filament Assembly

Inosine monophosphate dehydrogenase (IMPDH) mediates the first committed step in guanine nucleotide biosynthesis and plays important roles in cellular proliferation and the immune response. IMPDH reversibly polymerizes in cells and tissues in response to changes in metabolic demand. Self-assembly of metabolic enzymes is increasingly recognized as a general mechanism for regulating activity, typically by stabilizing specific conformations of an enzyme, but the regulatory role of IMPDH filaments has remained unclear. Here, we report a series of human IMPDH2 cryo-EM structures in both active and inactive conformations. The structures define the mechanism of filament assembly, and reveal how filament-dependent allosteric regulation of IMPDH2 makes the enzyme less sensitive to feedback inhibition, explaining why assembly occurs under physiological conditions that require expansion of guanine nucleotide pools. Tuning sensitivity to an allosteric inhibitor distinguishes IMPDH from other metabolic filaments, and highlights the diversity of regulatory outcomes that can emerge from self-assembly.

Mycophenolate mofetil treatment following renal transplantation decreases GTP concentrations in mononuclear leucocytes

2004 ◽  
Vol 107 (1) ◽  
pp. 69-74 ◽  
Author(s):  
Piotr JAGODZINSKI ◽  
Slawomir LIZAKOWSKI ◽  
Ryszard T. SMOLENSKI ◽  
Ewa M. SLOMINSKA ◽  
David GOLDSMITH ◽  
...  
Keyword(s):  
Renal Failure ◽  
Mycophenolate Mofetil ◽  
Renal Transplantation ◽  
Adenine Nucleotide ◽  
Target Cells ◽  
Renal Transplant Recipients ◽  
Guanine Nucleotide ◽  
Inosine Monophosphate Dehydrogenase ◽  
Pyrimidine Nucleotides ◽  
Mononuclear Leucocytes

MMF (mycophenolate mofetil) has been proven to provide an effective immunosuppression by non-competitive selective reversible inhibition of IMPDH (inosine monophosphate dehydrogenase), the enzyme playing a crucial role in GTP biosynthesis. However, the exact metabolic changes induced by inhibition of IMPDH in target cells of the immune system have been the subject of recent debate. The aim of the present study was to evaluate whether MMF treatment produced sustained changes in the guanosine nucleotide pool of MNLs (mononuclear leucocytes) in vivo. Sixty-two renal failure patients were divided into three groups: chronic renal failure patients undergoing haemodialysis (CRF-HD; n=20) and two groups of patients after renal transplantation, the first on AZA (azathioprine; TN-AZA; n=23) and the second treated with MMF (TN-MMF; n=19). In addition, MNLs from 25 healthy subjects were analysed as controls. Anion-exchange HPLC was used to quantify purine and pyrimidine nucleotides in MNLs. We report a significant decrease in GTP and the total MNL guanine nucleotide pool in the TN-MMF group (P<0.05) compared with control, CRF-HD and TN-AZA groups, although no significant differences were found between any of the other groups. Adenine nucleotide concentrations in MNLs were decreased in the TN-AZA group, but not in the TN-MMF group compared with the CRF-HD group and controls. There were no differences in CTP concentrations, but UTP concentrations were decreased in the CRF-HD, TN-AZA and TN-MMF groups compared with controls. MMF caused a significant and sustained decrease in the guanine nucleotide pool in MNLs from renal transplant recipients. This decrease contrasts with the elevation in GTP reported in erythrocytes of MMF-treated patients.

scholarly journals Tight links between adenine and guanine nucleotide pools in mouse pancreatic islets: a study with mycophenolic acid

1997 ◽  
Vol 324 (2) ◽  
pp. 467-471 ◽  
Author(s):  
Philippe DETIMARY ◽  
Changqing XIAO ◽  
Jean-Claude HENQUIN
Keyword(s):  
Insulin Secretion ◽  
Pancreatic Islets ◽  
Mycophenolic Acid ◽  
Adenine Nucleotide ◽  
Guanine Nucleotides ◽  
Specific Role ◽  
Guanine Nucleotide ◽  
Isolated Pancreatic Islets ◽  
Nucleotide Pools

Glucose metabolism in pancreatic B-cells leads to an increase in the ATP/ADP ratio that might participate in the regulation of insulin secretion. Good correlations have also been observed between guanine nucleotide levels in isolated pancreatic islets and insulin secretion. To assess whether guanine nucleotides have a specific role in stimulus–secretion coupling, their concentration should be modified selectively. This was attempted by culturing mouse islets overnight in the presence of mycophenolic acid (MPA), an inhibitor of GMP synthesis at the level of IMP dehydrogenase. The drug (25–50 μg/ml) did not affect the insulin content but decreased the GTP content of the islets and inhibited insulin secretion during subsequent incubation in the presence of 15 mM glucose. However, MPA also decreased the ATP/ADP ratio in the islets. The addition of guanine to the culture medium (to stimulate the salvage pathway of GTP synthesis) restored normal GTP levels, corrected the ATP/ADP ratio and partly prevented the inhibition of insulin release. In contrast, attempts to stimulate ATP synthesis specifically (by provision of adenine or adenosine) failed to reverse any of the effects of MPA. It is concluded that guanine and adenine nucleotide pools are tightly linked and cannot be specifically affected by MPA in pancreatic islet cells, probably because of the activity of nucleoside diphosphate kinase and because of the role of GTP in several reactions leading to adenine nucleotide generation. Contrary to previous claims, MPA is not an adequate tool for evaluating a specific role of guanine nucleotides in the control of insulin secretion.

scholarly journals Signaling through polymerization and degradation: Analysis and simulations of T cell activation mediated by Bcl10

2021 ◽  
Vol 17 (5) ◽  
pp. e1007986
Author(s):  
Leonard Campanello ◽  
Maria K. Traver ◽  
Hari Shroff ◽  
Brian C. Schaefer ◽  
Wolfgang Losert
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Defense Mechanism ◽  
Three Dimensional ◽  
Adaptive Immune System ◽  
Cell Receptor ◽  
Regulatory Control ◽  
Effector T Cell ◽  
Filament Assembly

The adaptive immune system serves as a potent and highly specific defense mechanism against pathogen infection. One component of this system, the effector T cell, facilitates pathogen clearance upon detection of specific antigens by the T cell receptor (TCR). A critical process in effector T cell activation is transmission of signals from the TCR to a key transcriptional regulator, NF-κB. The transmission of this signal involves a highly dynamic process in which helical filaments of Bcl10, a key protein constituent of the TCR signaling cascade, undergo competing processes of polymeric assembly and macroautophagy-dependent degradation. Through computational analysis of three-dimensional, super-resolution optical micrographs, we quantitatively characterize TCR-stimulated Bcl10 filament assembly and length dynamics, and demonstrate that filaments become shorter over time. Additionally, we develop an image-based, bootstrap-like resampling method that demonstrates the preferred association between autophagosomes and both Bcl10-filament ends and punctate-Bcl10 structures, implying that autophagosome-driven macroautophagy is directly responsible for Bcl10 filament shortening. We probe Bcl10 polymerization-depolymerization dynamics with a stochastic Monte-Carlo simulation of nucleation-limited filament assembly and degradation, and we show that high probabilities of filament nucleation in response to TCR engagement could provide the observed robust, homogeneous, and tunable response dynamic. Furthermore, we demonstrate that the speed of filament disassembly preferentially at filament ends provides effective regulatory control. Taken together, these data suggest that Bcl10 filament growth and degradation act as an excitable system that provides a digital response mechanism and the reliable timing critical for T cell activation and regulatory processes.

Allosteric theory

Hemoglobin ◽  
2018 ◽  
pp. 42-57
Author(s):  
Jay F. Storz
Keyword(s):  
Active Site ◽  
Binding Sites ◽  
Allosteric Regulation ◽  
Theoretical Models ◽  
State Model ◽  
Regulatory Control ◽  
Indirect Interaction ◽  
Protein Activity ◽  
Conformational States ◽  
Two State Model

Chapter 3 provides a brief overview of allostery, the modulation of protein activity that is caused by an indirect interaction between structurally remote binding sites. In this mode of intramolecular regulatory control, the binding of ligand at a protein’s active site is influenced by the binding of another ligand at a different site in the same protein. This interaction at a distance is mediated by a ligation-induced transition between alternative conformational states. Hemoglobin is regarded as the “allosteric paradigm,” and the oxygenation-linked transition between alternative quaternary conformations provides a textbook example of how allostery works. This chapter reviews different theoretical models, such as the Monod-Wyman-Changeux “two-state” model, to explain the allosteric regulation of hemoglobin function.

scholarly journals The Gfi-1 proto-oncoprotein contains a novel transcriptional repressor domain, SNAG, and inhibits G1 arrest induced by interleukin-2 withdrawal.

1996 ◽  
Vol 16 (11) ◽  
pp. 6263-6272 ◽  
Author(s):  
H L Grimes ◽  
T O Chan ◽  
P A Zweidler-McKay ◽  
B Tong ◽  
P N Tsichlis
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Transcriptional Repression ◽  
Cell Activation ◽  
Cellular Proliferation ◽  
Single Point ◽  
Interleukin 2 ◽  
Transcriptional Repressor ◽  
Single Point Mutation ◽  
G1 Arrest

The Gfi-1 proto-oncogene is activated by provirus insertion in T-cell lymphoma lines selected for interleukin-2 (IL-2) independence in culture and in primary retrovirus-induced thymomas and encodes a nuclear, sequence-specific DNA-binding protein. Here we show that Gfi-1 is a position- and orientation-independent active transcriptional repressor, whose activity depends on a 20-amino-acid N-terminal repressor domain, coincident with a nuclear localization motif. The sequence of the Gfi-1 repressor domain is related to the sequence of the repressor domain of Gfi-1B, a Gfi-1-related protein, and to sequences at the N termini of the insulinoma-associated protein, IA-1, the homeobox protein Gsh-1, and the vertebrate but not the Drosophila members of the Snail-Slug protein family (Snail/Gfi-1, SNAG domain). Although not functionally characterized, these SNAG-related sequences are also likely to mediate transcriptional repression. Therefore, the Gfi-1 SNAG domain may be the prototype of a novel family of evolutionarily conserved repressor domains that operate in multiple cell lineages. Gfi-1 overexpression in IL-2-dependent T-cell lines allows the cells to escape from the G1 arrest induced by IL-2 withdrawal. Since a single point mutation in the SNAG domain (P2A) inhibits both the Gfi-1-mediated transcriptional repression and the G1 arrest induced by IL-2 starvation, we conclude that the latter depends on the repressor activity of the SNAG domain. Induction of Gfi-1 may therefore contribute to T-cell activation and tumor progression by repressing the expression of genes that inhibit cellular proliferation.

scholarly journals Nucleotide Profiles of the Formed Elements of Human Blood Determined by High-Pressure Liquid Chromatography

Blood ◽  
1973 ◽  
Vol 41 (6) ◽  
pp. 927-936 ◽  
Author(s):  
E. M. Scholar ◽  
Phyllis R. Brown ◽  
R. E. Parks ◽  
P. Calabresi
Keyword(s):  
High Pressure ◽  
Liquid Chromatography ◽  
High Pressure Liquid Chromatography ◽  
Human Blood ◽  
Peripheral Blood ◽  
Human Peripheral Blood ◽  
Guanine Nucleotide ◽  
Disease States ◽  
Nucleotide Pools ◽  
Unique Potential

Abstract The nucleotide patterns of the acid soluble extracts of the formed elements of human peripheral blood were determined by high-pressure liquid chromatography. The patterns are remarkably reproducible for each of the respective elements, and the concentration of each major nucleotide present was determined. Erythrocytes are characterized by relatively simple nucleotide patterns with high adenine and very low guanine concentrations. The chromatograms of granulocytes and lymphocytes are difficult to distinguish from one another and demonstrate substantially greater UTP and guanine nucleotide levels than are observed in erythrocytes. The nucleotide patterns of platelets revealed a significantly lower ATP/ADP ratio than did either leukocytes or erythrocytes. The unique potential of this new technique for monitoring changes in nucleotide pools in normal and disease states is discussed.

scholarly journals Adenine nucleotide levels in Rhodospirillum rubrum during switch-off of whole-cell nitrogenase activity

1984 ◽  
Vol 224 (3) ◽  
pp. 961-969 ◽  
Author(s):  
T D Paul ◽  
P W Ludden
Keyword(s):  
Protein Modification ◽  
Rhodospirillum Rubrum ◽  
Adenine Nucleotide ◽  
Nitrogenase Activity ◽  
Energy Charge ◽  
Whole Cell ◽  
Fe Protein ◽  
Nucleotide Pools ◽  
Phenazine Methosulphate

Adenine nucleotide pools were measured in Rhodospirillum rubrum cultures that contained nitrogenase. The average energy charge [([ATP] + 1/2[ADP])/([ATP] + [ADP] + [AMP])] was found to be 0.66 and 0.62 in glutamate-grown and N-limited cultures respectively. Treatment of glutamate-grown cells with darkness, ammonia, glutamine, carbonyl cyanide m-chlorophenylhydrazone, or phenazine methosulphate resulted in perturbations in the adenine nucleotide pools, and led to loss of whole-cell nitrogenase activity and modification in vivo of the Fe protein. Treatment of N-limited cells resulted in similar changes in adenine nucleotide pools but not enzyme modification. No correlations were found between changes in adenine nucleotide pools or ratios of these pools and switch-off of nitrogenase activity by Fe protein modification in vivo. Phenazine methosulphate inhibited whole-cell activity at low concentrations. The effect on nitrogenase activity was apparently independent of Fe protein modification.

Single Stranded Polydeoxyribonucleotides Compete with the Incorporation of [3H] Thymidine or [3H]dAMP into DNA.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4444-4444
Author(s):  
Ting Yang ◽  
Raymund Buhmann ◽  
Michaela Svihla ◽  
Hans-Jochem Kolb
Keyword(s):  
T Cells ◽  
Fluorescence Microscopy ◽  
Cellular Uptake ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cellular Proliferation ◽  
Trypan Blue Exclusion ◽  
Activation Markers ◽  
Cytotoxic Effects ◽  
Current Survey

Abstract Defibrotide, the sodium salt of a single-stranded polydeoxyribonucleotide is prepared by a controlled depolymerisation of deoxyribonucleic acid (DNA) which is obtained from mammalian organs. In previous studies it was shown to mediate immunosuppressive effects (Ferraresso et al. 1993)1. In the current survey we investigated whether randomly chemically synthesized single-stranded polydeoxyribonucleotides of different length and composition could provide similar effects. For this purpose, purified T-cells were stimulated in the presence of dNTP’s or single-stranded polydeoxyribonucleotides with irradiated, allogeneic PBMC’s, PHA or anti-CD3/CD28 Dynabeads. Cellular proliferation was assessed by incorporation of tritium-labelled thymidine ([3H]thymidine), respectively [3H]dAMP or by staining with CFSE (carboxyfluorescein succinimidyl ester). After 72h or 120h of incubation, the incorporation of [3H]thymidine, or [3H]dAMP as well as the CFSE distribution was assessed. Cell viability was measured by trypan blue exclusion. T-cell activation was measured after 72h by quantifying the number of CD3+ T-cells expressing the activation markers CD25 and CD69. Cellular uptake of fluorochrome-labelled oligos was detected by fluorescence microscopy using an AxioCam HR and visualized by AxioVison. Each experiment was performed at least three times. Polydeoxyribonucleotides of different length, composition or concentrations (up to 5mM) did not cause cytotoxic effects to lymphocytes. But the incorporation of [3H]thymidine or [3H]dAMP was competed by polydeoxyribonucleotides. These effects were found to be dependent on length, concentration and base-composition of the nucleic acids. The proliferative capacity of the T-cells, as assessed by CFSE-staining, seemed to be unaffected. In context with data obtained by fluorescence microscopy, we hypothesise that there might be a cellular uptake of the polydeoxyribonucleotides followed by a subsequent cytoplasmatic degradation and a intracellular reutilisation via the salvage pathway for synthesis of nucleotides. The standard approach to detect cellular proliferation by incorporation of tritium-labelled nucleotides or derivatives is not useful to assess changes in cellular metabolism or proliferation in context with polydeoxyribonucleotides. Moreover, and that might be even more important, treatment approaches using nucleoside analogues like fludarabine, cytarabine e.g. in context with polydeoxyribonucleotides might be critical and diminish the efficacy of these drugs. Further experiments are necessary to elucidate the precise mechanism of these effects.

A phase I trial of AVN944 in patients with advanced hematologic malignancies

2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 14026-14026 ◽  
Author(s):  
R. B. Klisovic ◽  
G. Tricot ◽  
S. Coutre ◽  
T. Kovacsovics ◽  
F. Giles ◽  
...  
Keyword(s):  
Gene Expression ◽  
Phase I ◽  
Hematologic Malignancies ◽  
Guanine Nucleotide ◽  
Inosine Monophosphate Dehydrogenase ◽  
Open Label ◽  
Serious Adverse Events ◽  
Peripheral Blood Mononuclear ◽  
Nucleotide Synthesis

14026 Background: AVN-944 is an inhibitor of inosine monophosphate dehydrogenase (IMPDH), an enzyme that catalyzes the rate- limiting step in guanine nucleotide synthesis, and induces apoptosis in malignant hematopoietic cell lines in vitro. Methods: This phase I study employed open-label dose escalations in patients (pts) with relapsed, refractory hematologic cancers with safety, pharmacokinetic (PK), pharmacodynamic, & efficacy endpoints. Between 12/05 and 1/07 a total of 70 cycles of AVN944 at 25 (7pts), 50 (6pts), 75 (7pts), 100 (7pts) or 125 mg (3pts) b.i.d. orally X 21d every 28d were administered to 30 pts with AML (12), ALL (2), CLL (3), and multiple myeloma (13). Peripheral blood mononuclear cell (PBMC) or leukemic blast samples were obtained from all pts pre and post-receiving AVN944 to determine effects on GTP pools, IMPDH activity, and to correlate these changes to response in a 32-gene set that relates directly to cellular pathways dependent upon guanine nucleotide biosynthesis. Results: Pharmacokinetics were dose proportional with mean Tmax=1 hour, T1/2=1.5 hours, Cmax=2800 ng/ml and AUC=7228 hr.ng/ml at the 100 mg b.i.d. dose. Toxicities were generally mild-moderate and/or not attributed to AVN944. Twelve serious adverse events (SAEs) occurred in 8 pts; 7 of 8 had AML. No SAEs was attributed to AVN944. DLT was not seen. No protocol defined responses were seen. Twelve of 24 assessable pts had stable disease of 2 to 10 months duration. Changes in the gene expression set correlated with disease stability. Conclusions: AVN944 is well tolerated with oral b.i.d. dosing. Stabilization of disease was observed in half of the pts. Gene expression correlated with stable versus progressive disease. [Table: see text]

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