Common regulatory control of CTP synthase enzyme activity and filament formation

The ability of enzymes to assemble into visible supramolecular complexes is a widespread phenomenon. Such complexes have been hypothesized to play a number of roles; however, little is known about how the regulation of enzyme activity is coupled to the assembly/disassembly of these cellular structures. CTP synthase is an ideal model system for addressing this question because its activity is regulated via multiple mechanisms and its filament-forming ability is evolutionarily conserved. Our structure–function studies of CTP synthase in Saccharomyces cerevisiae reveal that destabilization of the active tetrameric form of the enzyme increases filament formation, suggesting that the filaments comprise inactive CTP synthase dimers. Furthermore, the sites responsible for feedback inhibition and allosteric activation control filament length, implying that multiple regions of the enzyme can influence filament structure. In contrast, blocking catalysis without disrupting the regulatory sites of the enzyme does not affect filament formation or length. Together our results argue that the regulatory sites that control CTP synthase function, but not enzymatic activity per se, are critical for controlling filament assembly. We predict that the ability of enzymes to form supramolecular structures in general is closely coupled to the mechanisms that regulate their activity.

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Coupled structural transitions enable highly cooperative regulation of human CTPS2 filaments

10.1101/770594 ◽

2019 ◽

Author(s):

Eric M. Lynch ◽

Justin M. Kollman

Keyword(s):

Enzyme Activity ◽

Active Sites ◽

Large Scale ◽

Conformational State ◽

Ctp Synthase ◽

Widespread Phenomenon ◽

And Control ◽

Allosteric Regulators ◽

Low Activity

Many enzymes assemble into defined oligomers, providing a mechanism for cooperatively regulating enzyme activity. Recent studies in tissues, cells, and in vitro have described a mode of regulation in which enzyme activity is modulated by polymerization into large-scale filaments1–5. Enzyme polymerization is often driven by binding to substrates, products, or allosteric regulators, and tunes enzyme activity by locking the enzyme in high or low activity states1–5. Here, we describe a unique, ultrasensitive form of polymerization-based regulation employed by human CTP synthase 2 (CTPS2). High-resolution cryoEM structures of active and inhibited CTPS2 filaments reveal the molecular basis of this regulation. Rather than selectively stabilizing a single conformational state, CTPS2 filaments dynamically switch between active and inactive filament forms in response to changes in substrate and product levels. Linking the conformational state of many CTPS2 subunits in a filament results in highly cooperative regulation, greatly exceeding the limits of cooperativity for the CTPS2 tetramer alone. The structures also reveal a link between conformational state and control of ammonia channeling between the enzyme’s two active sites. This filament-based mechanism of enhanced cooperativity demonstrates how the widespread phenomenon of enzyme polymerization can be adapted to achieve different regulatory outcomes.

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Filament Formation by Slime Mould Myosin Isolated at Low Ionic Strength

Journal of Cell Science ◽

10.1242/jcs.15.1.113 ◽

1974 ◽

Vol 15 (1) ◽

pp. 113-129

Author(s):

H. HINSSEN ◽

J. D'HAESE

Keyword(s):

Ionic Strength ◽

Striated Muscle ◽

Divalent Cations ◽

Filament Formation ◽

Selective Precipitation ◽

Slime Mould ◽

Preparation Conditions ◽

Filament Structure ◽

Low Ionic Strength

Myosin was isolated and purified from plasmodia of the slime mould Physarum polycephalum by a new method. This method is based on actomyosin extraction at low ionic strength after extensive washing, followed by the selective precipitation of myosin at pH 6.1 under relaxing conditions. The yield of myosin was 3-5 times higher than reported for other methods. In contrast to earlier studies a remarkably strong tendency to filament formation was found for slime mould myosin, probably due to a better preservation of some structural properties during preparation. Conditions were worked out under which numerous filaments up to 4 µm in length can be produced. It was established that not only a gradual decrease of ionic strength may influence filament formation, but also pH, ATP concentration and the presence of divalent cations. Compared to the current filament models a difference exists in the structure of the filaments. No central bare zone can be found, and thus, they lack an apparent bipolarity. Along the entire filament there are lateral projections representing the head portion of myosin molecules. A clear periodicity with an axial repeat of about 14 nm was observed, indicating a highly ordered arrangement of these projections. In this paper it is shown for the first time that myosin from one of the primitive motile systems is able to form aggregates of high structural order, indicating that the contraction of non-muscular actomyosin systems is not necessarily effected with oligomeric or randomly aggregated myosin. The possible role of myosin aggregation in vivo and the similarity of filament structure to that recently reported for myosin from vertebrate smooth muscle and striated muscle are discussed.

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The embryo makes red blood cell progenitors in every tissue simultaneously with blood vessel morphogenesis

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00543.2002 ◽

2003 ◽

Vol 284 (4) ◽

pp. R1126-R1137 ◽

Cited By ~ 35

Author(s):

Maria Luisa S. Sequeira Lopez ◽

Daniel R. Cherñavvsky ◽

Takayo Nomasa ◽

Lee Wall ◽

Masashi Yanagisawa ◽

...

Keyword(s):

Blood Cells ◽

Fetal Liver ◽

Extramedullary Hematopoiesis ◽

Simultaneous Generation ◽

Long Time ◽

Embryonic Life ◽

Multiple Regions ◽

Widespread Phenomenon ◽

Laser Capture ◽

Transplant Experiments

During embryonic life, hematopoiesis occurs first in the yolk sac, followed by the aorto-gonado-mesonephric region, the fetal liver, and the bone marrow. The possibility of hematopoiesis in other embryonic sites has been suspected for a long time. With the use of different methodologies (transgenic mice, electron microscopy, laser capture microdissection, organ culture, and cross-transplant experiments), we show that multiple regions within the embryo are capable of forming blood before and during organogenesis. This widespread phenomenon occurs by hemo-vasculogenesis, the formation of blood vessels accompanied by the simultaneous generation of red blood cells. Erythroblasts develop within aggregates of endothelial cell precursors. When the lumen forms, the erythroblasts “bud” from endothelial cells into the forming vessel. The extensive hematopoietic capacity found in the embryo helps explain why, under pathological circumstances such as severe anemia, extramedullary hematopoiesis can occur in any adult tissue. Understanding the intrinsic ability of tissues to manufacture their own blood cells and vessels has the potential to advance the fields of organogenesis, regeneration, and tissue engineering.

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Regulation of enzyme activities in Drosophila: I. The detection of regulatory loci by gene dosage responses

Genetics Research ◽

10.1017/s001667230001507x ◽

1974 ◽

Vol 24 (1) ◽

pp. 59-72 ◽

Cited By ~ 33

Author(s):

John M. Rawls ◽

John C. Lucchesi

Keyword(s):

Enzyme Activity ◽

Drosophila Melanogaster ◽

Alcohol Dehydrogenase ◽

Gene Dosage ◽

Regulatory Elements ◽

Gpdh Activity ◽

Regulatory Loci ◽

Chromosome Ii ◽

Chromosome Segments ◽

Regulatory Sites

SUMMARYIn order to detect regulatory genetic sites in the autosomes of Drosophila melanogaster, the levels of X-linked glucose-6-phosphate dehydro-genase and autosomally linked α-glycerophosphate and isocitrate dehydrogenases have been monitored in extracts of flies aneuploid for regions of chromosomes II and III. In addition to expected structural gene dosage responses of α-GPDH and IDH, flies hyperploid for several autosome regions were found to display altered levels of one or more of the enzymes studied. While IDH activity was increased in flies hyperploid for segments of both chromosomes II and III, α-GPDH activity was decreased in specific hyperploids for chromosome II regions only. The latter group of segmental aneuploids were normal with respect to levels of chromosome II-linked alcohol dehydrogenase. To test if the observed responses were due to dosage changes of discrete genes lying within the larger effective segments, flies aneuploid for subdivisions of the chromosome segments 21A-25CD, 35A–40, and 70CD–71B were assayed. For two of these large segments so analysed, the apparent effects were attributable to specific small subdivisions, suggesting the presence of discrete regulatory sites within the latter. For the 35A–40 region the α-GPDH effect observed for subdivisions was not sufficient to account for the large α-GPDH decrease seen in flies hyperploid for the large, inclusive region. These observations are discussed with respect to the possible bases of effect of regulatory elements on enzyme activity.

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The Ability of CBFβ-SMMHC to Increase RUNX1 Binding Affinity to Target DNA Correlates with Its Leukemogenic Potential

Blood ◽

10.1182/blood-2021-146193 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1157-1157

Author(s):

Tao Zhen ◽

Tongyi Dou ◽

Yun Chen ◽

Wei Yang ◽

Jiansen Jiang ◽

...

Keyword(s):

Binding Affinity ◽

Conflicts Of Interest ◽

Fusion Gene ◽

Negative Staining ◽

Filament Formation ◽

Consistent Finding ◽

E Coli ◽

Filament Structure ◽

Target Dna ◽

Complex Forms

Abstract Inversion of chromosome 16 is a consistent finding in patients with acute myeloid leukemia subtype M4 with eosinophilia (AML M4Eo), which generates a CBFB-MYH11 fusion gene. We recently showed that RUNX1 is indispensable for Cbfb-MYH11-induced leukemogenesis in a mouse model. We found that RUNX1 interacted with CBFβ-SMMHC, the fusion protein encoded by CBFB-MYH11, to directly regulate critical genes for leukemogenesis (Zhen et al., Blood, 2020). However, our current understanding of the interaction between CBFβ-SMMHC and RUNX1 does not provide adequate explanation on how the RUNX1-CBFβ-SMMHC complex forms and how the complex interacts with DNA for leukemogenesis as CBFβ-SMMHC without the RUNX1 high-affinity-binding-domain (CBFβ-SMMHC-ΔHABD) is also able to induce leukemia while CBFβ-SMMHC with mutations in the C-terminal multimerization domain (CBFβ-SMMHC-mDE) is not able to induce leukemia in mice. To address this question, we used RHD domain of RUNX1, CBFβ, CBFβ-SMMHC, CBFβ-SMMHC-ΔHABD and CBFβ-SMMHC-mDE proteins, which were purified from E. coli overexpressing these proteins, to explore how the HABD and DE domains affect the interactions between CBFβ-SMMHC, RHD and RUNX1-target DNA Bio-Layer Interferometry (BLI) and negative staining. As expected, deletion of the HABD domain significantly reduced CBFβ-SMMHC's binding affinity to RHD by BLI assay. Interestingly, differences in binding affinity between RHD and different versions of CBFβ-SMMHC did not correlate with their leukemogenic capability. On the other hand, the binding affinity between RHD and its target oligo was more significantly enhanced by CBFβ-SMMHC and CBFβ-SMMHC-ΔHABD that can induce leukemia than CBFβ-SMMHC-DE, which cannot. We also found that both CBFβ-SMMHC and CBFβ-SMMHC-ΔHABD, but not CBFβ-SMMHC-mDE, could form a filament structure by negative staining, suggesting the filament formation ability is important for leukemogenesis by CBFβ-SMMHC. In addition, RHD reduces filament formation by CBFβ-SMMHC, which was overcome when target oligo was added. In contrast, RHD could not inhibit filament formation by CBFβ-SMMHC-ΔHABD, suggesting that HABD interaction is required for RHD to disrupt filament formation by CBFβ-SMMHC. Overall, we found that leukemogenic capability of CBFβ-SMMHC correlates with its ability to enhance binding between RHD and its target DNA and to form multimerized filaments. The results also suggest that HABD and DE domains of CBFβ-SMMHC are required for the formation of the RUNX1-CBFβ-SMMHC complex with higher binding affinity to target DNA. Disclosures No relevant conflicts of interest to declare.

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

10.1101/798322 ◽

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.

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Studies on mutants affecting amidophosphoribosyltransferase activity in Saccharomyces cerevisiae

Canadian Journal of Microbiology ◽

10.1139/m83-111 ◽

1983 ◽

Vol 29 (6) ◽

pp. 681-688 ◽

Cited By ~ 1

Author(s):

Daniel J. Nieto ◽

Robin A. Woods

Keyword(s):

Saccharomyces Cerevisiae ◽

Enzyme Activity ◽

Ultraviolet Light ◽

Temperature Sensitivity ◽

De Novo ◽

Feedback Inhibition ◽

Temperature Sensitive ◽

Ethylmethane Sulphonate ◽

Interallelic Complementation ◽

Different Levels

Mutants at the ade4 locus of yeast were isolated following mutagenesis of ade+ and ade2 with ultraviolet light (UV), ethylmethane sulphonate, and the acridine half mustard ICR-170. Tests for interallelic complementation, osmotic remediality, temperature sensitivity, and mutagen-specific reversion were carried out on 19 mutants. Six mutants showed interallelic complementation and fell into four groups, defining three complons. Three mutants were osmotic remedial and the same three were temperature sensitive. Three mutants induced by ICR-170 gave purine-excreting revertants, designated Pur6 or ade4.RCF, after exposure to UV. Activity of amidophosphoribosyltransferase (PRPPAT) was assayed in the ade4 mutants and other alleles at this locus. The ade4 mutants lacked activity of the enzyme; the alleles su-pur+, su-pur, PUR6, and Pur6, showed different levels of activity. The enzyme was subject to feedback inhibition by AMP and IMP in su-pur+ and PUR6; su-pur was hypersensitive to inhibition by AMP, whereas Pur6 was slightly resistant. Purine synthesis de novo was shown to be repressible in su-pur+ and constitutive in PUR6 and Pur6 by following the accumulation of aminoimidazole ribotide in the presence and absence of cycloheximide. These observations were confirmed by direct assay of enzyme activity.

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Human CTP synthase filament structure reveals the active enzyme conformation

Nature Structural & Molecular Biology ◽

10.1038/nsmb.3407 ◽

2017 ◽

Vol 24 (6) ◽

pp. 507-514 ◽

Cited By ~ 65

Author(s):

Eric M Lynch ◽

Derrick R Hicks ◽

Matthew Shepherd ◽

James A Endrizzi ◽

Allison Maker ◽

...

Keyword(s):

Active Enzyme ◽

Filament Structure ◽

Ctp Synthase

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Quantitative control analysis of branched-chain 2-oxo acid dehydrogenase complex activity by feedback inhibition

Biochemical Journal ◽

10.1042/bj2710523 ◽

1990 ◽

Vol 271 (2) ◽

pp. 523-528 ◽

Cited By ~ 10

Author(s):

B Boyer ◽

R Odessey

Keyword(s):

Feedback Inhibition ◽

Regulatory Control ◽

Control Factor ◽

Control Analysis ◽

Complex Activity ◽

Acid Dehydrogenase ◽

Branched Chain ◽

Acid Dehydrogenase Complex ◽

Dehydrogenase Complex

The potential for branched-chain 2-oxo acid dehydrogenase complex (BCOADC) activity to be controlled by feedback inhibition was investigated by calculating the Elasticity Coefficients for several feedback inhibitors. We suggest that feedback inhibition is a quantitatively important regulatory mechanism by which branched-chain 2-oxo acid dehydrogenase activity is regulated. The potential for control of enzyme activity is greater for NADH than for the acyl-CoA products, and suggests that factors that alter the redox potential may physiologically regulate BCOADC activity through a feedback inhibitory mechanism in vivo. Local pH may also be an important regulatory control factor.

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