Improving fold activation of small transcription activating RNAs (STARs) with rational RNA engineering strategies

Hybrid hexamers were made by refolding mixtures of two mutant forms of clostridial glutamate dehydrogenase. Mutant Cys320Ser (C320S) has a similar activity to the wild-type enzyme, but is unreactive with Ellman's reagent, 5,5′-dithiobis(2-nitrobenzoate) (DTNB). The triple mutant Lys89Leu/Ala163Gly/Ser380Ala (K89L/A163G/S380A), active with norleucine but not glutamate, is inactivated by DTNB, since the amino acid residue at position 320 is a cysteine residue. The chosen ratio favoured 1:5 hybrids of the triple mutant and C320S. The renatured mixture was treated with DTNB and separated on an NAD+–agarose column to which only C320S subunits bind tightly. Fractions were monitored for glutamate and norleucine activity and for releasable thionitrobenzoate to establish subunit stoichiometry. A fraction highly enriched in the 1:5 hybrid was identified. Homohexamers (C320S with 40mM glutamate and 1mM NAD+ at pH8.8, or K89L/A163G/S380A with 70mM norleucine and 1mM NAD+ at pH8.5) showed allosteric activation; succinate activated C320S approx. 50-fold (EC50 = 70mM, h = 2.4), and glutarate gave approx. 30-fold activation (EC50 = 35mM, h = 2.3). For the triple mutant, corresponding values were 80mM and 2.2 for succinate, and 75mM and 1.7 for glutarate, but maximal activation was only about 2-fold. In the 1:5 hybrid, with only one norleucine-active subunit per hexamer, responses to glutarate and succinate were still co-operative, and activation was more extensive than in the triple mutant homohexamer. A single norleucine-active subunit can thus respond co-operatively to a substrate analogue at the other five inactive sites. On the other hand, similar hyperbolic dependence on the norleucine concentration for the hybrid and the triple mutant homohexamer reflected the inability of C320S subunits to bind norleucine. With glutamate at pH8.8, an h value of 3.6 was obtained for the 1:5 hybrid, in contrast with an h value of 5.2 for the C320S homohexamer. The ‘foreign’ subunit evidently impedes inter-subunit communication to some extent.

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Abstract MP13: TRPM7 Downregulation Contributes To Cardiovascular Injury And Hypertension Induced By Aldosterone And Salt

Hypertension ◽

10.1161/hyp.76.suppl_1.mp13 ◽

2020 ◽

Vol 76 (Suppl_1) ◽

Author(s):

Francisco J Rios ◽

ZhiGuo Zou ◽

Karla B Neves ◽

Sarah S Nichol ◽

Livia L Camargo ◽

...

Keyword(s):

Molecular Mechanisms ◽

Cardiac Fibroblasts ◽

Protective Role ◽

Internal Diameter ◽

Resistance Arteries ◽

Cross Sectional ◽

Vessel Structure ◽

Cardiovascular Fibrosis ◽

Fold Activation

TRPM7 has cation channel and kinase properties, is permeable to Mg 2+ , Ca 2+ , and Zn 2+ and is protective in the cardiovascular system. Hyperaldosteronism, which induces hypertension and cardiovascular fibrosis, is associated with Mg 2+ wasting. Here we questioned whether TRPM7 plays a role in aldosterone- induced hypertension and fibrosis and whether it influences cation regulation. Wild-type (WT) and TRPM7-deficient (M7+/Δ) mice were treated with aldosterone (600μg/Kg/day) and/or 1% NaCl (drinking water) (aldo, salt or aldo-salt) for 4 weeks. Blood pressure (BP) was evaluated by tail-cuff. Vessel structure was assessed by pressure myography. Molecular mechanisms were investigated in cardiac fibroblasts (CF) from WT and M7+/Δ mice. Protein expression was assessed by western-blot and histology. M7+/Δ mice exhibited reduced TRPM7 expression (30%) and phosphorylation (62%), levels that were recapitulated in WT aldo-salt mice. M7+/Δ exhibited increased BP by aldo, salt and aldo-salt (135-140mmHg) vs M7+/Δ-veh (117mmHg) (p<0.05), whereas in WT, BP was increased only by aldo-salt (134mmHg). Mesenteric resistance arteries from WT aldo-salt exhibited increased wall/lumen ratio (80%) and reduced internal diameter (15%) whereas vessels from M7+/Δ exhibited thinner walls by reducing cross-sectional area (35%) and increased internal diameter (23%) after aldo-salt. Aldo-salt induced greater collagen deposition in hearts (68%), kidneys (126%) and aortas (45%) from M7+/Δ vs WT. Hearts from M7+/Δ veh exhibited increased TGFβ, IL-11 and IL-6 (1.9-fold), p-Smad3 and p-Stat1 (1.5-fold) whereas in WT these effects were only found after aldo-salt. Cardiac expression of protein phosphatase magnesium-dependent 1A (PPM1A), a Mg 2+ -dependent phosphatase, was reduced (3-fold) only in M7+/Δ mice. M7+/Δ CF showed reduced proliferation (30%) and PPM1A (4-fold) and increased expression of TGFβ, IL-11 and IL-6 (2-3-fold), activation of Stat1 (2-fold), Smad3 (9-fold) and ERK1/2 (8-fold) compared with WT. Mg 2+ supplementation normalized cell proliferation and reduced protein phosphorylation in M7+/Δ CF (p<0.05). Our findings indicate a protective role of TRPM7 in aldosterone-salt induced cardiovascular injury through Mg 2+ -dependent mechanisms.

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Mechanisms of Human Blood VII Activation in Plasma During Contact Activation, Clotting and Exposure to Cold

10.1055/s-0039-1684637 ◽

1979 ◽

Author(s):

U. Seligsohn ◽

B. østerud ◽

S.F. Brown ◽

J.H. Griffin ◽

S.I. Rapaport

Keyword(s):

Molecular Weight ◽

Tissue Factor ◽

High Molecular Weight ◽

Human Blood ◽

Factor Vii ◽

High Molecular Weight Kininogen ◽

Contact Activation ◽

Partial Activation ◽

No Activation ◽

Fold Activation

Factor VII(VII) is activated, giving shorter clotting times with tissue factor, when plasma is exposed to kaolin, is clotted or exposed to cold. The mechanisms involved were studied. Incubation of plasma with kaolin resulted in: No activation in XII deficiency plasma (dp), partial activation (2.5 fold) in Prekallikrein (PK) dp and High Molecular Weight Kininogen (HMWK) dp, and 4.5-9 fold activation in normal or other dp. Clotting plasma by recalcification resulted in: No activation with XII dp, HMWK dp, XI dp and IX dp, and 4-5 fold activation with VIII dp, X dp and V dp. The mechanism of cold promoted activation of VII in plasma was studied by adding purified 125-XII or 125I-IX to plasma before storage at 4° and observing the extent of their proteolysis (a measure of activation) from their radioactivity profiles on reduced Polyacrylamide gels following electrophoresis in the presence of SDS. Significantly greater 125I-IX and 125I-XII proteolysis was observed in plasma from 4 subjects whose VII activated in the cold, than in plasma from 5 subjects whose VII was not activated in the cold. Addition of anti-IX antiserum inhibited 50% of the observed cold activation of VII. Thus, with kaolin XIIa was the principal activator of VII; after clotting IXa was the principal activator and in cold activation both XIIa and IXa played roles.

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Functional interaction of the K-Cl cotransporter (KCC1) with the Na-K-Cl cotransporter in HEK-293 cells

AJP Cell Physiology ◽

10.1152/ajpcell.1999.276.2.c328 ◽

1999 ◽

Vol 276 (2) ◽

pp. C328-C336 ◽

Cited By ~ 89

Author(s):

Christopher M. Gillen ◽

Bliss Forbush

Keyword(s):

Cell Swelling ◽

Primary Role ◽

Functional Interaction ◽

Hek 293 ◽

Hek 293 Cells ◽

Hek Cells ◽

293 Cells ◽

Hypotonic Cell Swelling ◽

The Relationship ◽

Fold Activation

We have studied the regulation of the K-Cl cotransporter KCC1 and its functional interaction with the Na-K-Cl cotransporter. K-Cl cotransporter activity was substantially activated in HEK-293 cells overexpressing KCC1 (KCC1-HEK) by hypotonic cell swelling, 50 mM external K, and pretreatment with N-ethylmaleimide (NEM). Bumetanide inhibited 86Rb efflux in KCC1-HEK cells after cell swelling [inhibition constant ( K i) ∼190 μM] and pretreatment with NEM ( K i ∼60 μM). Thus regulation of KCC1 is consistent with properties of the red cell K-Cl cotransporter. To investigate functional interactions between K-Cl and Na-K-Cl cotransporters, we studied the relationship between Na-K-Cl cotransporter activation and intracellular Cl concentration ([Cl]i). Without stimulation, KCC1-HEK cells had greater Na-K-Cl cotransporter activity than controls. Endogenous Na-K-Cl cotransporter of KCC1-HEK cells was activated <2-fold by low-Cl hypotonic prestimulation, compared with 10-fold activation in HEK-293 cells and >20-fold activation in cells overexpressing the Na-K-Cl cotransporter (NKCC1-HEK). KCC1-HEK cells had lower resting [Cl]i than HEK-293 cells; cell volume was not different among cell lines. We found a steep relationship between [Cl]i and Na-K-Cl cotransport activity within the physiological range, supporting a primary role for [Cl]iin activation of Na-K-Cl cotransport and in apical-basolateral cross talk in ion-transporting epithelia.

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Mechanistic Deficits Of a Leukemogenic GATA-2 Mutant

Blood ◽

10.1182/blood.v122.21.3668.3668 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3668-3668

Author(s):

Koichi Ricardo Katsumura ◽

Chenxi Yang ◽

Jing Zhang ◽

Lingjun Li ◽

Kirby D Johnson ◽

...

Keyword(s):

Target Genes ◽

Conflicts Of Interest ◽

Phosphorylation Site ◽

Spectrometric Analysis ◽

Ras Signaling ◽

Dependent Manner ◽

Subnuclear Localization ◽

Transactivation Activity ◽

N Terminus ◽

Fold Activation

Abstract Recent studies have demonstrated a role for the master regulator of hematopoiesis GATA-2 in MonoMAC Syndrome, a human immunodeficiency disorder associated with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Though GATA2 coding region and cis-regulatory element mutations underlie MonoMAC syndrome, many questions remain unanswered regarding how GATA-2 is controlled physiologically and how it is dysregulated in pathological contexts. We dissected how a T354M mutation in the GATA-2 DNA binding zinc finger, which is frequently detected in MonoMAC syndrome and familial MDS/AML, alters GATA-2 activity. The T354M mutation reduced GATA-2 chromatin occupancy, induced GATA-2 hyperphosphorylation, and disrupted GATA-2 subnuclear localization. These molecular phenotypes also characterized an additional familial MDS/AML-associated GATA-2 mutant (Δ355T). T354M hyperphosphorylation and ectopic subnuclear localization were detected in hematopoietic and non-hematopoietic cell lines. We developed a new model system in mouse aortic endothelial (MAE) cells to quantitate GATA-2 activity to regulate endogenous target genes. T354M exhibited significantly reduced activity in this assay (GATA-2: 200-fold activation; T354M: 7.7-fold activation). Mass spectrometric analysis of the phosphorylation states of GATA-2 and T354M revealed that the T354M mutation enhanced phosphorylation at several GATA-2 residues. Analysis of single phosphorylation site mutants indicated that only mutation of S192 (S192A) abolished T354M-induced hyperphosphorylation. The S192A mutation attenuated phosphorylation of sites within wild-type GATA-2 and reduced transactivation activity (50% decrease, p < 0.01). A distinct 60 amino acid (aa) region within the GATA-2 N-terminus was required for T354M hyperphosphorylation and ectopic subnuclear localization. Deletion of this sequence decreased GATA-2 transactivation activity (60 aa deletion: 85% decrease, p < 0.01; 10 aa deletion: 45% decrease, p < 0.05). GATA-1 lacks an analogous subnuclear targeting sequence, and accordingly, a GATA-1(T263M) mutant, which corresponds to the GATA-2(T354M) mutant, localized normally and was not hyperphosphorylated. However, a GATA-1 chimera containing the GATA-2 subnuclear targeting sequence localized to ectopic subnuclear foci in a T263M-dependent manner. The GATA-2 N-terminus endowed GATA-1 with the capacity to induce GATA-2 target genes. By contrast, a GATA-2 chimera containing the GATA-1 N-terminus exhibited normal subnuclear localization. Thus, the leukemogenic T354M mutation utilizes the GATA-2-specific subnuclear targeting sequence to disrupt the normal subnuclear localization pattern, and this disruption is associated with S192-dependent hyperphosphorylation. In addition to its involvement in AML, GATA-2 interfaces with RAS signaling to promote the development of non-small cell lung cancer. We discovered that RAS signaling promotes S192-dependent GATA-2 hyperphosphorylation and ectopic subnuclear localization and propose that GATA-2 is an important component in oncogenic RAS-dependent leukemogenesis, which is being formally tested using innovative mouse models. In summary, dissecting the mechanistic deficits of a leukemogenic GATA-2 mutant revealed unexpected insights into mechanisms underlying physiological GATA-2 function and GATA-2-dependent hematologic pathologies. Disclosures: No relevant conflicts of interest to declare.

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The effects of calcium ions and adenine nucleotides on the activity of pig heart 2-oxoglutarate dehydrogenase complex

Biochemical Journal ◽

10.1042/bj1800533 ◽

1979 ◽

Vol 180 (3) ◽

pp. 533-544 ◽

Cited By ~ 294

Author(s):

J G McCormack ◽

R M Denton

Keyword(s):

Adenine Nucleotide ◽

Adenine Nucleotides ◽

Product Inhibition ◽

Maximal Velocity ◽

Brown Adipose ◽

Dehydrogenase System ◽

Oxoglutarate Dehydrogenase ◽

Ph Range ◽

Fold Activation ◽

Dehydrogenase Complex

1. The effects of Ca2+ (mainly by using EGTA buffers), pH, ATP and ADP on the activity of the 2-oxoglutarate dehydrogenase complex from pig heart were explored. 2. Ca2+ (about 30 micrometer) resulted in a decrease in the apparent Km for 2-oxoglutarate from 2.1 to 0.16 mM (at pH 7) without altering the maximal velocity. At 0.1 mM-oxoglutarate there was a 4–5-fold activation by Ca2+, with an apparent Km for Ca2+ of 1.2 micrometer. A similar activation was also observed with Sr2+ (Km 15.1 micrometer), but not wised markedly from pH 7.4 TO 6.6. The effects of Ca2+ remained evident over this pH range. 4. In the presence of Mg2+, ATP resulted in a marked increase in the apparent Km for oxoglutarate, whereas ADP greatly decreased thisp arameter. The concentrations of adenine nucleotide required for half-maximal effects were about 10 micrometer in each case. 5. The effects of the adenine nucleotides and Ca2+ on the apparent Km for oxoglutarate appeared to be essentially independent of each other, reversible, and demonstrable in the presence of end product inhibition by NADH and obtained. 6. Effects similar to those described above were also observed on the activity of 2-oxoglutarate dehydrogenase from rat heart and brown adipose tissue. 7. We discuss the mechanisms controlling this enzyme's activity and compare these regulatory features with those of NAD-isocitrate dehydrogenase and the pyruvate dehydrogenase system, which are also sensitive to Ca2+ and adenine nucleotides.

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Analysis of mitogen-activated protein kinase pathways used by interleukin 1 in tissues in vivo: activation of hepatic c-Jun N-terminal kinases 1 and 2, and mitogen-activated protein kinase kinases 4 and 7

Biochemical Journal ◽

10.1042/bj3530275 ◽

2001 ◽

Vol 353 (2) ◽

pp. 275-281 ◽

Cited By ~ 8

Author(s):

Andrew FINCH ◽

W. DAVIS ◽

Wayne G. CARTER ◽

Jeremy SAKLATVALA

Keyword(s):

Protein Kinase ◽

Cultured Cells ◽

Interleukin 1 ◽

Mitogen Activated Protein Kinase ◽

Signalling Pathways ◽

Jnk Pathway ◽

Mitogen Activated Protein ◽

Jnk Isoforms ◽

Fold Activation

The effects of interleukin 1 (IL-1) are mediated by the activation of protein kinase signalling pathways, which have been well characterized in cultured cells. We have investigated the activation of these pathways in rabbit liver and other tissues after the systemic administration of IL-1α. In liver there was 30Ő40-fold activation of c-Jun N-terminal kinase (JNK) and 5-fold activation of both JNK kinases, mitogen-activated protein kinase (MAPK) kinase (MKK)4 and MKK7. IL-1α also caused 2Ő3-fold activation of p38 MAPK and degradation of the inhibitor of nuclear factor κB (‘IκB’), although no activation of extracellular signal-regulated protein kinase (ERK) (p42/44 MAPK) was observed. The use of antibodies against specific JNK isoforms showed that, in liver, short (p46) JNK1 and long (p54) JNK2 are the predominant forms activated, with smaller amounts of long JNK1 and short JNK2. No active JNK3 was detected. A similar pattern of JNK activation was seen in lung, spleen, skeletal muscle and kidney. Significant JNK3 activity was detectable only in the brain, although little activation of the JNK pathway in response to IL-1α was observed in this tissue. This distribution of active JNK isoforms probably results from a different expression of JNKs within the tissues, rather than from a selective activation of isoforms. We conclude that IL-1α might activate a more restricted set of signalling pathways in tissues in vivo than it does in cultured cells, where ERK and JNK3 activation are often observed. Cultured cells might represent a ‘repair’ phenotype that undergoes a broader set of responses to the cytokine.

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Osmotic stress activates Rac and Cdc42 in neutrophils: role in hypertonicity-induced actin polymerization

AJP Cell Physiology ◽

10.1152/ajpcell.00427.2001 ◽

2002 ◽

Vol 282 (2) ◽

pp. C271-C279 ◽

Cited By ~ 53

Author(s):

Alison Lewis ◽

Caterina Di Ciano ◽

Ori D. Rotstein ◽

András Kapus

Keyword(s):

Actin Polymerization ◽

Cell Movement ◽

Small Gtpases ◽

Small Gtpase ◽

Phosphatidylinositol 3 Kinase ◽

Cytoskeleton Remodeling ◽

Clostridium Difficile Toxin ◽

Clostridium Difficile Toxin B ◽

Gtpase Activation ◽

Fold Activation

Hypertonicity inhibits a variety of neutrophil functions through poorly defined mechanisms. Our earlier studies suggest that osmotically induced actin polymerization and cytoskeleton remodeling is a key component in the hypertonic block of exocytosis and cell movement. To gain insight into the signaling mechanisms underlying the hyperosmotic F-actin response, we investigated whether hypertonicity stimulates Rac and Cdc42 and, if so, whether their activation contributes to the hypertonic rise in F-actin. Using a recently developed pull-down assay that specifically captures the active forms of these small GTPases, we found that hypertonicity caused an ∼2.5- and ∼7.2-fold activation of Rac and Cdc42, respectively. This response was rapid and sustained. Small GTPase activation was not mediated by the osmotic stimulation of Src kinases, heterotrimeric G proteins, or phosphatidylinositol 3-kinase. Interestingly, an increase in intracellular ionic strength was sufficient to activate Rac even in the absence of cell shrinkage. Inhibition of Rac and Cdc42 by Clostridium difficile toxin B substantially reduced but did not abolish the hypertonicity-induced F-actin response. Thus hypertonicity is a potent activator of Rac and Cdc42, and this effect seems to play an important but not exclusive role in the hyperosmolarity-triggered cytoskeleton remodeling.

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A Novel Class of Activating Mutations in NOTCH1 in T-ALL.

Blood ◽

10.1182/blood.v110.11.694.694 ◽

2007 ◽

Vol 110 (11) ◽

pp. 694-694 ◽

Cited By ~ 3

Author(s):

Maria Luisa Sulis ◽

Odette Williams ◽

Valeria Tosello ◽

Sasikala Pallippukam ◽

Teresa Palomero ◽

...

Keyword(s):

Amino Acids ◽

T Cell ◽

Mutant Allele ◽

Gamma Secretase ◽

Activating Mutations ◽

Notch1 Signaling ◽

Juxtamembrane Region ◽

Human T Cell ◽

Fold Activation ◽

Notch1 Receptor

Abstract Aberrant activation of NOTCH1 signaling induces transformation of T-cell progenitors and plays a prominent role in the pathogenesis of over 50% of human T-cell acute lymphoblastic leukemias (T-ALL), which harbor activating mutations in the heterodimerization (HD) and PEST domains of NOTCH1. Here we report a new class of activating mutations in NOTCH1 in human T-ALL. These so called JuxtaMembrane Expansion (JME) mutants consist of internal tandem duplications of exon 28 and adjacent intronic sequences in the NOTCH1 gene, which result in expansions of the extracellular juxtamembrane region of the NOTCH1 receptor. Western blot analysis of T-ALL cell lines lacking known NOTCH1 mutations demonstrated high levels of activated NOTCH1 protein in Jurkat T-ALL cells, suggesting the presence of an as yet unidentified activating NOTCH1 mutation in this cell line. Sequence analysis of Jurkat NOTCH1 transcripts revealed an internal tandem duplication in exon 28 of NOTCH1, resulting in the insertion of 17 amino acids at position 1740 in the extracelullar juxtamembrane region of the receptor. Subsequent PCR amplification of NOTCH1 exon 28 sequences from 194 primary T-ALL lymphoblast samples identified seven additional in frame insertion mutations ranging from 11 to 36 amino acids in length, all of which were located in the vicinity of codon 1740 in the extracelullar juxtamembrane region of the NOTCH1 receptor. Luciferase assays showed that expression of the NOTCH1 Jurkat JME17 mutant allele induced over 200 fold activation of a NOTCH1 reporter construct compared to controls. Activation of NOTCH1 signaling requires proteolytic cleavage of the receptor, first by an ADAM metalloprotease (S2 clevage) and subsequently by the gamma-secretase complex. NOTCH1 signaling induced by the Jurkat JME17 mutant was completely abrogated by incubation with CompE, a highly active gamma-secretase inhibitor. Consistently, treatment of Jurkat T-ALL cells with CompE resulted in rapid clearance of activated NOTCH1 protein and in marked downregulation of NOTCH1 target genes such as HES1 and DELTEX1. Interestingly, and in contrast with previously described HD mutations, JME NOTCH1 alleles retain an intact HD domain and a protected canonical S2 metalloprotease cleavage site. Thus, we hypothesized that activation of NOTCH1 by JME mutations could be mediated by aberrant metalloprotease cleavage at ectopic S2 sites within the JME insertion sequence. However, mutation of the canonical S2 cleavage abrogated the function of the NOTCH1 Jurkat JME17 mutant allele. Furthermore, analysis of artificially generated JME insertions containing sequences unrelated to the leukemia-derived JME alleles, showed that activation of NOTCH1 by JME mutations depends primarily on the length of the inserted peptides and not on their specific amino acid sequences. Thus, shorter insertions of 5 to 13 amino acids in length induced moderate (5–10 fold) activation of the NOTCH1 receptor, while insertions of 14 amino acids or longer induced marked (>70 fold) increases in NOTCH1 signaling. Overall, these results provide further insight in the mechanisms that control the activation of the NOTCH1 receptor in T-ALL.

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