scholarly journals Conserved residues in Ycf54 are required for protochlorophyllide formation in Synechocystis sp. PCC 6803

2017 ◽  
Vol 474 (5) ◽  
pp. 667-681 ◽  
Author(s):  
Sarah Hollingshead ◽  
Sophie Bliss ◽  
Patrick J. Baker ◽  
C. Neil Hunter
Keyword(s):  
Hydrogen Bonding ◽  
Protoporphyrin Ix ◽  
Wild Type ◽  
Structural Effects ◽  
Conserved Residues ◽  
Functional Differences ◽  
In Vivo Effects ◽  
Mgpme Cyclase ◽  
Synechocystis Sp

Chlorophylls (Chls) are modified tetrapyrrole molecules, essential for photosynthesis. These pigments possess an isocyclic E ring formed by the Mg-protoporphyrin IX monomethylester cyclase (MgPME–cyclase). We assessed the in vivo effects of altering seven highly conserved residues within Ycf54, which is required for MgPME–cyclase activity in the cyanobacterium Synechocystis. Synechocystis strains harbouring the Ycf54 alterations D39A, F40A and R82A were blocked to varying degrees at the MgPME–cyclase step, whereas the A9G mutation reduced Ycf54 levels by ∼75%. Wild-type (WT) levels of the cyclase subunit CycI are present in strains with D39A and F40A, but these strains have lowered cellular Chl and photosystem accumulation. CycI is reduced by ∼50% in A9G and R82A, but A9G has no perturbations in Chl or photosystem accumulation, whilst R82A contains very little Chl and few photosystems. When FLAG tagged and used as bait in pulldown experiments, the three mutants D39A, F40A and R82A were unable to interact with the MgPME–cyclase component CycI, whereas A9G pulled down a similar level of CycI as WT Ycf54. These observations suggest that a stable interaction between CycI and Ycf54 is required for unimpeded Pchlide biosynthesis. Crystal structures of the WT, A9G and R82A Ycf54 proteins were solved and analysed to investigate the structural effects of these mutations. A loss of the local hydrogen bonding network and a reversal in the surface charge surrounding residue R82 are probably responsible for the functional differences observed in the R82A mutation. We conclude that the Ycf54 protein must form a stable interaction with CycI to promote optimal Pchlide biosynthesis.

Gli1can rescue the in vivo function ofGli2

Development ◽  
2001 ◽  
Vol 128 (24) ◽  
pp. 5161-5172 ◽  
Author(s):  
Chunyang Brian Bai ◽  
Alexandra L. Joyner
Keyword(s):  
Spinal Cord ◽  
Sonic Hedgehog ◽  
Target Genes ◽  
Wild Type ◽  
Shh Signaling ◽  
Gli Proteins ◽  
Functional Differences ◽  
Expression Studies ◽  
Null Mutants

In mice, three Gli genes are thought to mediate sonic hedgehog (Shh) signaling collectively. Mis-expression studies and analysis of null mutants for each gene have indicated that the Gli proteins have different functions. In particular, Gli1 appears to be a constitutive activator, and Gli2 and Gli3 have repressor functions. To determine the precise functional differences between Gli1 and Gli2, we have expressed Gli1 in place of Gli2 from the endogenous Gli2 locus in mice. Strikingly, a low level of Gli1 can rescue all the Shh signaling defects in Gli2 mutants; however, only in the presence of a wild-type Shh gene. These studies demonstrate that only the activator function of Gli2 is actually required, and indicates that in specific situations, Shh can modulate the ability of Gli1 to activate target genes. Furthermore, expression of both copies of Gli1 in place of Gli2 does not disrupt spinal cord patterning, but does result in new gain-of-function defects that lead to lethality. We show that the defects are enhanced when Gli3 function is reduced, demonstrating that an important difference between Gli1 and Gli2 is the ability of Gli1 to antagonize Gli3 function.

scholarly journals Mutations in Saccharomyces cerevisiae GeneSIR2 Can Have Differential Effects on In Vivo Silencing Phenotypes and In Vitro Histone Deacetylation Activity

2002 ◽  
Vol 13 (4) ◽  
pp. 1427-1438 ◽  
Author(s):  
Christopher M. Armstrong ◽  
Matt Kaeberlein ◽  
Shin Ichiro Imai ◽  
Leonard Guarente
Keyword(s):  
Histone Deacetylase ◽  
Histone Deacetylases ◽  
Histone Deacetylation ◽  
Conserved Residues ◽  
Core Domain ◽  
Sir Complex ◽  
In Vivo Effects ◽  
The Relationship

The yeast SIR2 gene and many of its homologs have been identified as NAD+-dependent histone deacetylases. To get a broader view of the relationship between the histone deacetylase activity of Sir2p and its in vivo functions we have mutated eight highly conserved residues in the core domain ofSIR2. These mutations have a range of effects on the ability of Sir2p to deacetylate histones in vitro and to silence genes at the telomeres and HM loci. Interestingly, there is not a direct correlation between the in vitro and in vivo effects in some of these mutations. We also show that the histone deacetylase activity of Sir2p is necessary for the proper localiztion of the SIR complex to the telomeres.

scholarly journals Engineering Candida tenuis Xylose Reductase for Improved Utilization of NADH: Antagonistic Effects of Multiple Side Chain Replacements and Performance of Site-Directed Mutants under Simulated In Vivo Conditions

2005 ◽  
Vol 71 (10) ◽  
pp. 6390-6393 ◽  
Author(s):  
Barbara Petschacher ◽  
Bernd Nidetzky
Keyword(s):  
Xylose Reductase ◽  
Binding Pocket ◽  
Kinetic Mechanism ◽  
Side Chain ◽  
Multiple Site ◽  
Wild Type ◽  
Structural Effects ◽  
Phosphate Binding ◽  
And Performance

ABSTRACT Six single- and multiple-site variants of Candida tenuis xylose reductase that were engineered to have side chain replacements in the coenzyme 2′-phosphate binding pocket were tested for NADPH versus NADH selectivity (R sel) in the presence of physiological reactant concentrations. The experimental R sel values agreed well with predictions from a kinetic mechanism describing mixed alternative coenzyme utilization. The Lys-274→Arg and Arg-280→His substitutions, which individually improved wild-type R sel 50- and 20-fold, respectively, had opposing structural effects when they were combined in a double mutant.

scholarly journals Transglutaminase 2 Has Metabolic and Vascular Regulatory Functions Revealed by In Vivo Activation of Alpha1-Adrenergic Receptor

2020 ◽  
Vol 21 (11) ◽  
pp. 3865
Author(s):  
Kinga Lénárt ◽  
Attila Pap ◽  
Róbert Pórszász ◽  
Anna V. Oláh ◽  
László Fésüs ◽  
...  
Keyword(s):  
G Protein ◽  
Adrenergic Receptor ◽  
Tissue Transglutaminase ◽  
Lactate Concentration ◽  
Transglutaminase 2 ◽  
Cell Types ◽  
Wild Type ◽  
Regulatory Functions ◽  
In Vivo Effects

The multifunctional tissue transglutaminase has been demonstrated to act as α1-adrenergic receptor-coupled G protein with GTPase activity in several cell types. To explore further the pathophysiological significance of this function we investigated the in vivo effects of the α1-adrenergic receptor agonist phenylephrine comparing responses in wild type and TG2-/- mice. Injection of phenylephrine, but not a beta3-adrenergic agonist (CL-316,243), resulted in the long-term decline of the respiratory exchange ratio and lower lactate concentration in TG2-/- mice indicating they preferred to utilize fatty acids instead of glucose as fuels. Measurement of tail blood pressure revealed that the vasoconstrictive effect of phenylephrine was milder in TG2-/- mice leading to lower levels of lactate dehydrogenase (LDH) isoenzymes in blood. LDH isoenzyme patterns indicated more damage in lung, liver, kidney, skeletal, and cardiac muscle of wild type mice; the latter was confirmed by a higher level of heart-specific CK-MB. Our data suggest that TG2 as an α1-adrenergic receptor-coupled G protein has important regulatory functions in alpha1-adrenergic receptor-mediated metabolic processes and vascular functions.

Activity of the Multi-Targeted Kinase Inhibitor, AT9283 on Imatinib-Resistant CML Models.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1104-1104 ◽  
Author(s):  
Ruriko Tanaka ◽  
Matthew S Squires ◽  
Shinya Kimura ◽  
Asumi Yokota ◽  
Kirsty Mallett ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitors ◽  
Cell Lines ◽  
Kinase Inhibitors ◽  
Wild Type ◽  
Once Daily ◽  
Imatinib Resistant ◽  
Mutant Forms ◽  
In Vivo Effects

Abstract CML is caused by a consistent genetic abnormality, termed the Philadelphia chromosome, that results from a reciprocal (9;22) translocation leading to the expression of the BCR-ABL fusion protein. Although treatment has been revolutionized by the introduction of tyrosine kinase inhibitors which target Abl activity, reactivation of Abl signaling via several different point mutations remains problematic. In particular the mutation of Threonine 315 to Isoleucine (T315I) confers resistance to all existing therapies with tyrosine kinase inhibitors in the clinical settings. We describe the in vitro and in vivo effects of AT9283, a potent inhibitor of several protein kinases, including Abl kinase (wild type BCR-ABL and several of the drug resistant mutant variants that have arisen in clinical practice e.g. T315I), JAK2, JAK3 and Aurora kinases A and B, on imatinib-resistant CML cells including those harboring BCR-ABL (T315I). AT9283 has potent anti-proliferative activity in a panel of BaF3 and human cell lines expressing the BCR-ABL or its mutant forms. In BaF3 BCR-ABL wild-type and T315I mutant cells and K562 CML cells we observed inhibition of substrates of both BCR-ABL (STAT5) and Aurora B (Histone H3) at concentrations >300nM and <100nM, respectively, suggesting that AT9283 is capable of inhibiting Aurora and BCR-ABL simultaneously in these cell lines. The in vivo effects of AT9283 were examined in several mouse models engrafted either subcutaneously or intravenously with BaF3, human CML cell lines or primary CML patient samples expressing the BCR-ABL or its mutant forms. Specifically AT9283 prolonged the survival of mice engrafted intravenously with either BaF3 BCR-ABL T315I, or E255K cells when administered intraperitoneally twice daily at doses of either 6.25 or 10mg/kg or once daily at 15mg/kg when administered 5 days in every week repeated twice. Maximal survival advantage was conferred at either 10mg/kg twice daily or 15mg/kg once a day. Similar data were obtained in an intravenous model using primary CML cells taken from a patient harbouring the BCR-ABL E255K mutation. We also present data from ongoing studies showing increased survival rates in these in vivo model systems following multiple cycles of AT9283 administered on the 15mg/kg once daily schedule. These data together support further clinical investigation of AT9283 in patients with treatment resistant CML.

scholarly journals Granule-bound starch synthase I in isolated starch granules elongates malto-oligosaccharides processively

1999 ◽  
Vol 340 (1) ◽  
pp. 183-191 ◽  
Author(s):  
Kay DENYER ◽  
Darren WAITE ◽  
Saddik MOTAWIA ◽  
Birger Lindberg MØLLER ◽  
Alison M. SMITH
Keyword(s):  
Starch Synthase ◽  
Kinetic Properties ◽  
Starch Granules ◽  
Wild Type ◽  
Lower Affinity ◽  
Functional Differences ◽  
Glucose Molecule ◽  
Granule Bound Starch Synthase

Isoforms of starch synthase belonging to the granule-bound starch synthase I (GBSSI) class synthesize the amylose component of starch in plants. Other granule-bound isoforms of starch synthase, such as starch synthase II (SSII), are unable to synthesize amylose. The kinetic properties of GBSSI and SSII that are responsible for these functional differences have been investigated using starch granules from embryos of wild-type peas and rug5 and lam mutant peas, which contain, respectively, both GBSSI and SSII, GBSSI but not SSII and SSII but not GBSSI. We show that GBSSI in isolated granules elongates malto-oligosaccharides processively, adding more than one glucose molecule for each enzyme-glucan encounter. Granule-bound SSII can elongate malto-oligosaccharides, but has a lower affinity for these than GBSSI and does not elongate processively. As a result of these properties GBSSI synthesizes longer malto-oligosaccharides than SSII. The significance of these results with respect to the roles of GBSSI and SSII in vivo is discussed.

scholarly journals Functional Differences between Keratins of Stratified and Simple Epithelia

1998 ◽  
Vol 143 (2) ◽  
pp. 487-499 ◽  
Author(s):  
Elizabeth Hutton ◽  
Rudolph D. Paladini ◽  
Qian-Chun Yu ◽  
Mei Yen ◽  
Pierre A. Coulombe ◽  
...  
Keyword(s):  
Type I ◽  
Fundamental Issue ◽  
Transgenic Technology ◽  
Wild Type ◽  
Functional Differences ◽  
Wild Type Phenotype ◽  
Cellular Integrity ◽  
10 Nm Filaments

Dividing populations of stratified and simple epithelial tissues express keratins 5 and 14, and keratins 8 and 18, respectively. It has been suggested that these keratins form a mechanical framework important to cellular integrity, since their absence gives rise to a blistering skin disorder in neonatal epidermis, and hemorrhaging within the embryonic liver. An unresolved fundamental issue is whether different keratins perform unique functions in epithelia. We now address this question using transgenic technology to express a K16-14 hybrid epidermal keratin transgene and a K18 simple epithelial keratin transgene in the epidermis of mice null for K14. Under conditions where the hybrid epidermal keratin restored a wild-type phenotype to newborn epidermis, K18 partially but not fully rescued. The explanation does not appear to reside in an inability of K18 to form 10-nm filaments with K5, which it does in vitro and in vivo. Rather, it appears that the keratin network formed between K5 and K18 is deficient in withstanding mechanical stress, leading to perturbations in the keratin network in regions of the skin that are subjected either to natural or to mechanically induced trauma. Taken together, these findings suggest that the loss of a type I epidermal keratin cannot be fully compensated by its counterpart of simple epithelial cells, and that in vivo, all keratins are not equivalent.

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