scholarly journals Disease Mutation Study Identifies Critical Residues for Phosphatidylserine Flippase ATP11A

2020 ◽  
Vol 2020 ◽  
pp. 1-9
Author(s):  
Kuanxiang Sun ◽  
Wanli Tian ◽  
Xiao Li ◽  
Wenjing Liu ◽  
Yeming Yang ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Amino Acid ◽  
Lipid Bilayer ◽  
Cellular Localization ◽  
Biological Activities ◽  
Amino Acid Residues ◽  
Mutant Proteins ◽  
Myotube Formation ◽  
Critical Residues ◽  
Insight Into

Phosphatidylserine flippase (P4-ATPase) transports PS from the outer to the inner leaflet of the lipid bilayer in the membrane to maintain PS asymmetry, which is important for biological activities of the cell. ATP11A is expressed in multiple tissues and plays a role in myotube formation. However, the detailed cellular function of ATP11A remains elusive. Mutation analysis revealed that I91, L308, and E897 residues in ATP8A2 are important for flippase activity. In order to investigate the roles of these corresponding amino acid residues in ATP11A protein, we assessed the expression and cellular localization of the respective ATP11A mutant proteins. ATP11A mainly localizes to the Golgi and plasma membrane when coexpressed with the β-subunit of the complex TMEM30A. Y300F mutation causes reduced ATP11A expression, and Y300F and D913K mutations affect correct localization of the Golgi and plasma membrane. In addition, Y300F and D913K mutations also affect PS flippase activity. Our data provides insight into important residues of ATP11A.

scholarly journals Disease mutation study identifies essential residues for phosphatidylserine flippase ATP11A

2020 ◽  
Author(s):  
Kuanxiang Sun ◽  
Wanli Tian ◽  
Wenjing Liu ◽  
Yeming Yang ◽  
Xianjun Zhu
Keyword(s):  
Biological Activity ◽  
Amino Acid ◽  
Lipid Bilayer ◽  
Mutation Analysis ◽  
Cellular Function ◽  
Amino Acid Residues ◽  
Β Subunit ◽  
Mutant Proteins ◽  
Myotube Formation ◽  
Insight Into

AbstractPS flippase (P4-ATPase) transports PS from the outer to the inner leaflet of the lipid bilayer in the membrane to maintain PS asymmetry, which is important for biological activity of the cell. ATP11A is expressed in multiple tissues and plays a role in myotube formation. However, detailed cellular function of ATP11A remains elusive. Mutation analysis revealed that I91, L308 and E897 residues in ATP8A2 are important for flippase activity. In order to investigate the roles of these corresponding amino acid residues in ATP11A, we assessed the expression and flippase activity of the respective ATP11A mutant proteins. ATP11A mainly localizes to the Golgi when co-expressed with TMEM30A, the β-subunit of the complex. Y300F and D913K mutations affect correct Golgi localization and PS stimulated flippase activity. In addition, Y300F mutation causes reduced ATP11A expression. Our data provides insight into essential residues for expression and flippase activity of ATP11A.

scholarly journals Enzyme characterization and biological activities of a resuscitation promoting factor from an oil degrading bacterium Rhodococcus erythropolis KB1

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e6951 ◽  
Author(s):  
Dan Luo ◽  
Jixiang Chen ◽  
Gang Xie ◽  
Liang Yue ◽  
Yonggang Wang
Keyword(s):  
Amino Acid ◽  
Affinity Chromatography ◽  
Growth Promotion ◽  
Biological Activities ◽  
Rhodococcus Erythropolis ◽  
Enzyme Characterization ◽  
Amino Acid Residues ◽  
Mutant Proteins ◽  
Bacteria Growth ◽  
Degrading Bacterium

Resuscitation-promoting factors (Rpf) are a class of muralytic enzymes, which participate in recovery of dormant cells and promoting bacteria growth in poor media. In the present study the expression vector of the rpf-1 gene from an oil-degrading bacterium Rhodococcus erythropolis KB1 was constructed and expressed in Escherichia coli. The expressed protein was purified by Ni2+-affinity chromatography, and showed muralytic activity when measured with 4-methylumbelliferyl-β-D-N,N′,N″-triacetyl chitotrioside as substrate. Addition of purified Rpf-1 to R. erythropolis culture efficiently improved bacterial cell growth. The purified protein also increased resuscitation of viable but nonculturable cells of R. erythropolis to culturable state. The conserved amino acid residues including Asp45, Glu51, Cys50, Thr60, Gln69, Thr74, Trp75 and Cys114 of the Rpf-1 were replaced with different amino acids. The mutant proteins were also expressed and purified with Ni2+-affinity chromatography. The muralytic activities of the mutant proteins decreased to different extents when compared with that of the wild type Rpf-1. Gln69 was found to play the most important role in the enzyme activity, substitution of Gln69 with lysine (Q69K) resulted in the greatest decrease of muralytic activity. The other amino acid residues such as Asp45, Glu51, Cys50 and Cys114 were also found to be very important in maintaining muralytic activity and biological function of the Rpf-1. Our results indicated that Rpf-1 from R. erythropolis showed muralytic activities and weak protease activity, but the muralytic activity was responsible for its growth promotion and resuscitation activity.

scholarly journals Amino Acid Residues in the ω-Minus Region Participate in Cellular Localization of Yeast Glycosylphosphatidylinositol-Attached Proteins

1999 ◽  
Vol 181 (13) ◽  
pp. 3886-3889 ◽  
Author(s):  
Kenji Hamada ◽  
Hiromichi Terashima ◽  
Mikio Arisawa ◽  
Nami Yabuki ◽  
Kunio Kitada
Keyword(s):  
Amino Acids ◽  
Cell Wall ◽  
Plasma Membrane ◽  
Amino Acid ◽  
Cellular Localization ◽  
Attachment Site ◽  
Basic Amino Acid ◽  
Amino Acid Residues ◽  
Specific Amino Acid ◽  
Final Destination

ABSTRACT The final destination of glycosylphosphatidylinositol (GPI)-attached proteins in Saccharomyces cerevisiae is the plasma membrane or the cell wall. Two kinds of signals have been proposed for their cellular localization: (i) the specific amino acid residues V, I, or L at the site 4 or 5 amino acids upstream of the GPI attachment site (the ω site) and Y or N at the site 2 amino acids upstream of the ω site for cell wall localization and (ii) dibasic residues in the region upstream of the ω site (the ω-minus region) for plasma membrane localization. The relationships between these amino acid residues and efficiencies of cell wall incorporation were examined by constructing fusion reporter proteins from open reading frames encoding putative GPI-attached proteins. The levels of incorporation were high in the constructs containing the specific amino acid residues and quite low in those containing two basic amino acid residues in the ω-minus region. With constructs that contained neither specific residues nor two basic residues, levels of incorporation were moderate. These correlations clearly suggest that GPI-attached proteins have two different signals which act positively or negatively in cell wall incorporation for their cellular localization.

Analogues of the cholecystokinin octapeptide modified in the central part of the molecule

1991 ◽  
Vol 56 (9) ◽  
pp. 1963-1970 ◽  
Author(s):  
Jan Hlaváček ◽  
Václav Čeřovský ◽  
Jana Pírková ◽  
Pavel Majer ◽  
Lenka Maletínská ◽  
...  
Keyword(s):  
Amino Acid ◽  
Biological Activities ◽  
Point Of View ◽  
Biologically Active ◽  
Side Chain ◽  
Amino Acid Residues ◽  
Cholecystokinin Octapeptide ◽  
Biological Tests ◽  
Biological Potency ◽  
Biologically Active Conformation

In a series of analogues of the cholecystokinin octapeptide (CCK-8) the amino acid residues were gradually modified by substituting Gly by Pro in position 4, Trp by His in position 5, Met by Cle in position 6, or the Gly residue was inserted between Tyr and Met in positions 2 and 3 of the peptide chain, and in the case of the cholecystokinin heptapeptide (CCK-7) the Met residues were substituted by Nle or Aib. These peptides were investigated from the point of view of their biological potency in the peripheral and central region. From the results of the biological tests it follows that the modifications carried out in these analogues and in their Nα-Boc derivatives mean a suppression of the investigated biological activities by 2-3 orders of magnitude (at a maximum dose of the tested substance of 2 . 10-2 mg per animal).This means that a disturbance of the assumed biologically active conformation of CCK-8, connected with a considerable decrease of the biological potency of the molecule, takes place not only after introduction of the side chain into its centre (substitution of Gly4), but also after the modification of the side chains of the amino acids or by extension of the backbone in further positions around this central amino acid.

Synthesis of Fragments of the Vasoactive Intestinal Peptide (VIP) and Analysis of Their Residual Biological Activities

1995 ◽  
Vol 60 (7) ◽  
pp. 1229-1235 ◽  
Author(s):  
Ivana Zoulíková ◽  
Ivan Svoboda ◽  
Jiří Velek ◽  
Václav Kašička ◽  
Jiřina Slaninová ◽  
...  
Keyword(s):  
Amino Acid ◽  
Biological Activities ◽  
Residual Activity ◽  
Detailed Examination ◽  
Amino Acid Residues ◽  
Linear Peptide ◽  
Zone Electrophoresis ◽  
Capillary Zone

The vasoactive intestinal (poly)peptide (VIP) is a linear peptide containing 28 amino acid residues, whose primary structure indicates a low metabolic stability. The following VIP fragments, as potential metabolites, and their analogues were prepared by synthesis on a solid: [His(Dnp)1]VIP(1-10), VIP(11-14), [D-Arg12]VIP(11-14), [Lys(Pac)15,21,Arg20]VIP(15-22), and VIP(23-28). After purification, the peptides were characterized by amino acid analysis, mass spectrometry, RP HPLC, and capillary zone electrophoresis. In some tests, detailed examination of the biological activity of the substances in vivo and in vitro gave evidence of a low, residual activity of some fragments, viz. a depressoric activity in vivo for [His(Dnp)1]VIP(1-10) and a stimulating activity for the release of α-amylase in vitro and in vivo for [Lys(Pac)15,21,Arg20]VIP(15-22) and VIP(23-28).

scholarly journals Insight into the mechanism of thermostabilization of GH10 xylanase from Bacillus sp. strain TAR-1 by the mutation of S92 to E

2021 ◽  
Vol 85 (2) ◽  
pp. 386-390
Author(s):  
Manami Suzuki ◽  
Teisuke Takita ◽  
Kohei Kuwata ◽  
Kota Nakatani ◽  
Tongyang Li ◽  
...  
Keyword(s):  
Amino Acid ◽  
Thermodynamic Analysis ◽  
Thermal Inactivation ◽  
Entropy Change ◽  
Crystallographic Analysis ◽  
Bacillus Sp ◽  
Amino Acid Residues ◽  
Gh10 Xylanase ◽  
Insight Into

ABSTRACT The mechanism of thermostabilization of GH10 xylanase, XynR, from Bacillus sp. strain TAR-1 by the mutation of S92 to E was investigated. Thermodynamic analysis revealed that thermostabilization was driven by the decrease in entropy change of activation for thermal inactivation. Crystallographic analysis suggested that this mutation suppressed the fluctuation of the amino acid residues at position 92-95.

scholarly journals Sea urchin (Anthocidaris crassispina) egg zinc-binding protein. Cellular localization, purification and characterization

1983 ◽  
Vol 211 (1) ◽  
pp. 109-118 ◽  
Author(s):  
H Ohtake ◽  
T Suyemitsu ◽  
M Koga
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Sea Urchin ◽  
Cellular Localization ◽  
Binding Protein ◽  
Gel Permeation Chromatography ◽  
Total Amino Acid ◽  
Amino Acid Residues ◽  
Gel Permeation ◽  
Anthocidaris Crassispina

Gel-filtration analysis of cytosol fraction obtained from unfertilized sea-urchin (Anthocidaris crassispina) eggs on Sephadex G-75 revealed the presence of two Zn-binding-protein fractions. The major Zn-binding protein fraction had a low molecular weight and a low absorbance at 280 nm, properties similar to those of the metallothionein found in the regenerating rat liver. These fractions were further purified by DEAE-cellulose and Sephadex G-50 chromatography. Homogeneity of the Zn-binding protein was judged by polyacrylamide-disc-gel electrophoresis and gel-permeation chromatography in the presence of 6 M-guanidinium chloride. The molecular weight determined by gel-permeation chromatography was 3900. This value is in good agreement with the minimum molecular weight calculated from the amino acid composition, which was 3655. Zn-binding protein is composed of 36 amino acid residues and the distinctive features include an extremely high content of cysteine, which accounted for one-third of the total amino acid residues, and a complete absence of aromatic amino acids, as well as of methionine, histidine and arginine. Zn-binding protein contained 4.1 g-atoms of zinc per mol and a trace of cadmium, but no copper, iron or calcium. The molar ratio of reactive thiol groups to metal ion was calculated to be 2.73:1. Possible roles of this Zn-binding protein in the homoeostasis of zinc in unfertilized sea-urchin eggs are discussed.

scholarly journals Location of antigenic sites recognized by monoclonal antibodies in the influenza A virus nucleoprotein molecule

2009 ◽  
Vol 90 (7) ◽  
pp. 1730-1733 ◽  
Author(s):  
Natalia L. Varich ◽  
Konstantin S. Kochergin-Nikitsky ◽  
Evgeny V. Usachev ◽  
Olga V. Usacheva ◽  
Alexei G. Prilipov ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Amino Acid ◽  
Influenza A Virus ◽  
Influenza A ◽  
Antigenic Variation ◽  
Strain Differences ◽  
Antigenic Structure ◽  
Amino Acid Residues ◽  
Mutant Proteins ◽  
Antigenic Sites

The locations of amino acid positions relevant to antigenic variation in the nucleoprotein (NP) of influenza virus are not conclusively known. We analysed the antigenic structure of influenza A virus NP by introducing site-specific mutations at amino acid positions presumed to be relevant for the differentiation of strain differences by anti-NP monoclonal antibodies. Mutant proteins were expressed in a prokaryotic system and analysed by performing ELISA with monoclonal antibodies. Four amino acid residues were found to determine four different antibody-binding sites. When mapped in a 3D X-ray model of NP, the four antigenically relevant amino acid positions were found to be located in separate physical sites of the NP molecule.

scholarly journals Structure–function studies of human deoxyhypusine synthase: identification of amino acid residues critical for the binding of spermidine and NAD

2001 ◽  
Vol 355 (3) ◽  
pp. 841-849 ◽  
Author(s):  
Chang Hoon LEE ◽  
Patrick Y. UM ◽  
Myung Hee PARK
Keyword(s):  
Crystal Structure ◽  
Enzyme Activity ◽  
Amino Acid ◽  
Binding Sites ◽  
Binding Pocket ◽  
Complete Loss ◽  
Amino Acid Residues ◽  
Deoxyhypusine Synthase ◽  
Positive Identification ◽  
Insight Into

Deoxyhypusine synthase catalyses the first step in the biosynthesis of hypusine [Nε-(4-amino-2-hydroxybutyl)lysine]. The crystal structure of human deoxyhypusine synthase in complex with NAD revealed four NAD-binding sites per enzyme tetramer, and led to a prediction of the spermidine-binding pocket. We have replaced each of the seven amino acid residues at the predicted spermidine-binding site, and eleven residues that contact NAD, on an individual basis with alanine. Of the amino acid residues at the spermidine site, substitution of Asp-243, Trp-327, His-288, Asp-316 or Glu-323 with alanine caused an almost complete loss of spermidine binding and enzyme activity; only the mutation Tyr-305 → Ala showed partial binding and activity. His-288 → Ala was also deficient in terms of binding NAD. NAD binding was significantly reduced in all of the NAD-site mutant enzymes, except for Glu-137 → Ala, which showed a normal binding of NAD, but was totally lacking in spermidine binding. Of the NAD-site mutant enzymes, Asp-342 → Ala, Asp-313 → Ala and Asp-238 → Ala displayed the lowest binding of NAD. These enzymes and His-288Ala also showed a reduced binding of spermidine, presumably because spermidine binding is dependent on NAD. These findings permit the positive identification of amino acid residues critical for binding of spermidine and NAD, and provide a new insight into the complex molecular interactions involved in the deoxyhypusine synthase reaction.

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