scholarly journals Amino Acid Polymorphism and Rare Electrophoretic Variants of G6PD From Natural Populations of Drosophila melanogaster

Genetics ◽  
1996 ◽  
Vol 143 (1) ◽  
pp. 401-406
Author(s):  
Walter F Eanes ◽  
Michele Kirchner ◽  
Daniel R Taub ◽  
Jeanne Yoon ◽  
Jiang-tian Chen
Keyword(s):  
Drosophila Melanogaster ◽  
Amino Acid ◽  
Quaternary Structure ◽  
Rare Variants ◽  
Protein Domain ◽  
Allozyme Polymorphism ◽  
Electrophoretic Variants ◽  
Function Classes ◽  
Amino Acid Mutations

Abstract Identifing the amino acid changes responsible for electrophoretic variants is essential to understanding the significance of allozyme polymorphism in adaptation. The amino acid mutations responsible for the common G6PD allozyme polymorphisms in Drosophila melanogaster have been recently described. This study characterizes the amino acid changes associated with 11 rare electrophoretic G6PD variants. The 11 rare electrophoretic variants result from six independent amino acid mutations. The in vivo function of the rare variants was determined in an earlier study and most variants fell into one of two function classes. It is shown here that the function of the rare variants reflects the state of the Pro/Leu mutation responsible for the A / B allozyme polymorphism in each variant. Two mutations destabilize quaternary structure resulting in shifts from tetrameric to dimeric alleles, and one of these also results in a variant with in vivo function intermediate to A and B. That mutation is an aspartic-acid-to-asparagine change that is two residues away from the Pro/Leu polymorphism responsible for the A / B dimertetramer quaternary shift. Structure-function relationships based on studies of human G6PD deficency-associated mutations predict that these last two amino acid changes fall within the protein domain responsible for NADP-binding.

scholarly journals Casein Kinase I Regulates Membrane Binding by ARF GAP1

2002 ◽  
Vol 13 (8) ◽  
pp. 2559-2570 ◽  
Author(s):  
Sidney Yu ◽  
Michael G. Roth
Keyword(s):  
Amino Acid ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Membrane Binding ◽  
Casein Kinase ◽  
Protein Domain ◽  
Amino Terminal ◽  
Early Secretory Pathway

ARF GAP1, a 415-amino acid GTPase activating protein (GAP) for ADP-ribosylation factor (ARF) contains an amino-terminal 115-amino acid catalytic domain and no other recognizable features. Amino acids 203–334 of ARF GAP1 were sufficient to target a GFP-fusion protein to Golgi membranes in vivo. When overexpressed in COS-1 cells, this protein domain inhibited protein transport between the ER and Golgi and, in vitro, competed with the full-length ARF GAP1 for binding to membranes. Membrane binding by ARF GAP1 in vitro was increased by a factor in cytosol and this increase was inhibited by IC261, an inhibitor selective for casein kinase Iδ (CKIδ), or when cytosol was treated with antibody to CKIδ. The noncatalytic domain of ARF GAP1 was phosphorylated both in vivo and in vitro by CKI. IC261 blocked membrane binding by ARF GAP1 in vivo and inhibited protein transport in the early secretory pathway. Overexpression of a catalytically inactive CKIδ also inhibited the binding of ARF GAP1 to membranes and interfered with protein transport. Thus, a CKI isoform is required for protein traffic through the early secretory pathway and can modulate the amount of ARF GAP1 that can bind to membranes.

Alanine-170 and proline-172 are critical determinants for extracellular CD20 epitopes; heterogeneity in the fine specificity of CD20 monoclonal antibodies is defined by additional requirements imposed by both amino acid sequence and quaternary structure

Blood ◽  
2002 ◽  
Vol 99 (9) ◽  
pp. 3256-3262 ◽  
Author(s):  
Maria J. Polyak ◽  
Julie P. Deans
Keyword(s):  
Monoclonal Antibodies ◽  
Amino Acid ◽  
Quaternary Structure ◽  
Fine Specificity ◽  
Human Sequence ◽  
Cellular Aggregation ◽  
Membrane Compartment ◽  
Anti Cd20 ◽  
High Degree

Abstract In vivo ablation of malignant B cells can be achieved using antibodies directed against the CD20 antigen. Fine specificity differences among CD20 monoclonal antibodies (mAbs) are assumed not to be a factor in determining their efficacy because evidence from antibody-blocking studies indicates limited epitope diversity with only 2 overlapping extracellular CD20 epitopes. However, in this report a high degree of heterogeneity among antihuman CD20 mAbs is demonstrated. Mutation of alanine and proline at positions 170 and 172 (AxP) (single-letter amino acid codes; x indicates the identical amino acid at the same position in the murine and human CD20 sequences) in human CD20 abrogated the binding of all CD20 mAbs tested. Introduction of AxP into the equivalent positions in the murine sequence, which is not otherwise recognized by antihuman CD20 mAbs, fully reconstituted the epitope recognized by B1, the prototypic anti-CD20 mAb. 2H7, a mAb previously thought to recognize the same epitope as B1, did not recognize the murine AxP mutant. Reconstitution of the 2H7 epitope was achieved with additional mutations replacing VDxxD in the murine sequence for INxxN (positions 162-166 in the human sequence). The integrity of the 2H7 epitope, unlike that of B1, further depends on the maintenance of CD20 in an oligomeric complex. The majority of 16 antihuman CD20 mAbs tested, including rituximab, bound to murine CD20 containing the AxP mutations. Heterogeneity in the fine specificity of these antibodies was indicated by marked differences in their ability to induce homotypic cellular aggregation and translocation of CD20 to a detergent-insoluble membrane compartment previously identified as lipid rafts.

scholarly journals Direct measurement of in vivo flux differences between electrophoretic variants of G6PD from Drosophila melanogaster.

Genetics ◽  
1992 ◽  
Vol 132 (3) ◽  
pp. 783-787
Author(s):  
J Labate ◽  
W F Eanes
Keyword(s):  
Drosophila Melanogaster ◽  
Steady State ◽  
Adaptive Significance ◽  
Electrophoretic Variants ◽  
Naturally Occurring ◽  
Steady State Kinetics ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
In Vivo Flux ◽  
Pathway Flux

Abstract Demonstrating that naturally occurring enzyme polymorphisms significantly impact metabolic pathway flux is a fundamental step in examining the possible adaptive significance of such polymorphisms. In earlier studies of the glucose-6-phosphate dehydrogenase (G6PD) polymorphism in Drosophila melanogaster, we used two different methods, exploiting both genotype-dependent interactions with the 6Pgd locus, and conventional steady-state kinetics to examine activity differences between the two common allozymes. In this report we use 1-14C- and 6-14C-labeled glucose to estimate directly genotype-dependent flux differences through the pentose shunt. Our results show that G6pdA genotype possesses statistically lower pentose shunt flux than G6pdB at 25 degrees. We estimate this to be about a 32% reduction, which is consistent with the two former studies. These results reflect a significant responsiveness of pentose shunt flux to activity variation at the G6PD-catalyzed step, and predict that the G6PD allozymes generate a polymorphism for pentose shunt flux.

Identification of key residues in the formate channel FocA that control import and export of formate

2014 ◽  
Vol 395 (7-8) ◽  
pp. 813-825 ◽  
Author(s):  
Doreen Hunger ◽  
Claudia Doberenz ◽  
R. Gary Sawers
Keyword(s):  
Amino Acid ◽  
Quaternary Structure ◽  
Salt Bridge ◽  
Structural Features ◽  
Amino Acid Residues ◽  
Form Part ◽  
Import And Export ◽  
Key Residues ◽  
Acid Exchange

Abstract The formate-nitrite transporter (FNT) family comprises pentameric channels that transport monovalent anions. The prototype of this family is the formate channel (FocA), which was originally identified as a formate channel in Escherichia coli. Each protomer in the channel has a pore with structural features that include periplasmic and cytoplasmic constriction sites, which are likely important for bi-directional gating of substrate passage. Highly conserved amino acid residues within FocA previously identified in structural studies are predicted to be important in the control of formate translocation. Here we present a first detailed in vivo analysis of these residues using a combined targeted amino acid exchange and formate-responsive lacZ fusion-based reporter approach. Sixteen exchanges were made and each variant was shown to be largely unaffected in its secondary and quaternary structure. The invariant H209 and T91 residues, which form part of the lower constriction site linking the Ω-loop with the pore cavity, proved to be important in governing the directionality of formate passage through the pore. A predicted salt-bridge triad of E208-K156-N213 along with the cytoplasmically-oriented N-terminal helix are also involved in pH-dependent gating of the channel. Together, our data are consistent with passive export and import of formate or formic acid through the channel.

scholarly journals Rebuilding Ring-Type Assembly of Peroxiredoxin by Chemical Modification

2020 ◽  
Author(s):  
Tomoki Himiyama ◽  
Yuko Tsuchiya ◽  
Yasushige Yonezawa ◽  
Tsutomu Nakamura
Keyword(s):  
Amino Acid ◽  
Protein Interactions ◽  
Hot Spots ◽  
Quaternary Structure ◽  
Protein Assembly ◽  
Protein Protein Interactions ◽  
Aeropyrum Pernix ◽  
Amino Acid Mutations ◽  
Regulate Protein ◽  
Additional Amino Acid

Direct control of protein quaternary structure (QS) is challenging owing to the complexity of protein structure. As a protein with a characteristic QS, peroxiredoxin from <i>Aeropyrum pernix</i> K1 (ApPrx) forms a decamer, wherein five dimers associate to form a ring. Here, we disrupted and reconstituted ApPrx QS via amino acid mutations and chemical modifications targeting hot spots for protein assembly. The decameric QS of an ApPrx* mutant, wherein all cysteine residues in wild-type ApPrx were mutated to serine, was destructed to dimers via an F80C mutation. The dimeric ApPrx*F80C mutant was then modified with a small molecule and successfully assembled as a decamer. Structural analysis confirmed that an artificially installed chemical moiety potentially facilitates suitable protein-protein interactions to rebuild a native structure. Rebuilding of dodecamer was also achieved through an additional amino acid mutation. This study describes a facile method to regulate protein assembly state.

scholarly journals Attenuation and Immunogenicity of a Live High Pathogenic PRRSV Vaccine Candidate with a 32-Amino Acid Deletion in the nsp2 Protein

2014 ◽  
Vol 2014 ◽  
pp. 1-11 ◽  
Author(s):  
Wenhui Lu ◽  
Baoli Sun ◽  
Jianyue Mo ◽  
Xiduo Zeng ◽  
Guanqun Zhang ◽  
...  
Keyword(s):  
Amino Acid ◽  
Vaccine Candidate ◽  
Clinical Signs ◽  
Respiratory Syndrome Virus ◽  
Amino Acid Deletion ◽  
Lethal Challenge ◽  
Nsp2 Gene ◽  
Amino Acid Mutations

A porcine reproductive and respiratory syndrome virus (PRRSV) QY1 was serially passed on Marc-145 cells. Virulence of different intermediate derivatives of QY1 (P5, P60, P80, and P100) were determined. The study found that QY1 had been gradually attenuated during the in vitro process. Pathogenicity study showed that pigs inoculated with QY1 P100 and P80 did not develop any significant PRRS clinic symptoms. However, mild-to-moderate clinical signs and acute HP-PRRSV symptoms of infection were observed in pigs inoculated with QY1 P60 and P5, respectively. Furthermore, we determined the whole genome sequences of these four intermediate viruses. The results showed that after 100 passages, compared to QY1 P5, a total of 32 amino acid mutations were found. Moreover, there were one nucleotide deletion and a unique 34-amino acid deletion found at 5′UTR and in nsp2 gene during the attenuation process, respectively. Such deletions were genetically stable in vivo. Following PRRSV experimental challenge, pigs inoculated with a single dose of QY1 P100 developed no significant clinic symptoms and well tolerated lethal challenge, while QY1 P80 group still developed mild fever in the clinic trial after challenge. Thus, we concluded that QY1 P100 was a promising and highly attenuated PRRSV vaccine candidate.

IN VIVO FUNCTION OF RARE G6pd VARIANTS FROM NATURAL POPULATIONS OF DROSOPHILA MELANOGASTER

Genetics ◽  
1986 ◽  
Vol 113 (3) ◽  
pp. 679-693
Author(s):  
Walter F Eanes ◽  
Jody Hey
Keyword(s):  
Rare Variants ◽  
Specific Activity ◽  
Natural Populations ◽  
P Element ◽  
X Chromosomes ◽  
Electrophoretic Variants ◽  
The Common ◽  
Glucose 6 Phosphate Dehydrogenase

ABSTRACT From 1981 to 1983, 15,097 X-chromosomes were genetically extracted from a number of North American populations of D. melanogaster and were electrophoretically screened for rare mobility and activity variants of glucose-6-phosphate dehydrogenase (G6PD). Overall, 13 rare variants were recovered for a frequency of about 10-3. Eleven variants affect electrophoretic mobility and are apparently structural, and two variants exhibit low G6PD activity. One low activity variant is closely associated with a P-element insertion at 18D12-13—all of the variants were subjected to the previously described genetic scheme used to identify relative in vivo activity differences between the two common electrophoretic variants associated with the global polymorphism. Most of the rare variants exhibit apparent in vivo activities that are similar to one or the other of the common variants, and these specific rare variants appear to be geographically widespread. Several variants have significantly reduced function. All of the variants were measured for larval specific activity for G6PD as a first measure of in vitro activity. It appears that specific activity alone is not a sufficient predictor for G6PD in vivo function.

GENETIC LIMITS OF THE XANTHINE DEHYDROGENASE STRUCTURAL ELEMENT WITHIN THE ROSY LOCUS IN DROSOPHILA MELANOGASTER

Genetics ◽  
1974 ◽  
Vol 78 (3) ◽  
pp. 869-886
Author(s):  
William M Gelbart ◽  
Margaret McCarron ◽  
Janardan Pandey ◽  
Arthur Chovnick
Keyword(s):  
Drosophila Melanogaster ◽  
Amino Acid ◽  
Structural Information ◽  
Xanthine Dehydrogenase ◽  
Gene Organization ◽  
Null Mutant ◽  
Electrophoretic Variation ◽  
Structural Element ◽  
Electrophoretic Variants ◽  
Left And Right

Abstract Experiments are described that provide an opportunity to estimate the genetic limits of the structural (amino acid coding) portion of the rosy locus (3: 52.0) in Drosophila melanogaster, which controls the enzyme, xanthine dehydrogenase (XDH) . This is accomplished by mapping experiments which localize sites responsible for electrophoretic variation in the enzyme on the known genetic map of null-XDH rosy mutants. Electrophoretic sites are distributed along a large portion of the null mutant map. A cis-trans test involving electrophoretic variants in the left- and right-hand portions of the map leads to the conclusion that the entire region between these variants is also structural. Hence most, if not all, of the null mutant map of the rosy locus contains structural information for the amino acid sequence of the XDH polypeptide. Consideration is given to the significance of the present results for the general problem of gene organization in higher eukaryotes.

scholarly journals WldS requires Nmnat1 enzymatic activity and N16–VCP interactions to suppress Wallerian degeneration

2009 ◽  
Vol 184 (4) ◽  
pp. 501-513 ◽  
Author(s):  
Michelle A. Avery ◽  
Amy E. Sheehan ◽  
Kimberly S. Kerr ◽  
Jing Wang ◽  
Marc R. Freeman
Keyword(s):  
Drosophila Melanogaster ◽  
Amino Acid ◽  
Enzymatic Activity ◽  
Wallerian Degeneration ◽  
Protective Activity ◽  
Protective Function ◽  
Nicotinamide Mononucleotide ◽  
Severed Axons ◽  
Drosophila Cells

Slow Wallerian degeneration (WldS) encodes a chimeric Ube4b/nicotinamide mononucleotide adenylyl transferase 1 (Nmnat1) fusion protein that potently suppresses Wallerian degeneration, but the mechanistic action of WldS remains controversial. In this study, we characterize WldS-mediated axon protection in vivo using Drosophila melanogaster. We show that Nmnat1 can protect severed axons from autodestruction but at levels significantly lower than WldS, and enzyme-dead versions of Nmnat1 and WldS exhibit severely reduced axon-protective function. Interestingly, a 16–amino acid N-terminal domain of WldS (termed N16) accounts for the differences in axon-sparing activity between WldS and Nmnat1, and N16-dependent enhancement of Nmnat1-protective activity in WldS requires the N16-binding protein valosin-containing protein (VCP)/TER94. Thus, WldS-mediated suppression of Wallerian degeneration results from VCP–N16 interactions and Nmnat1 activity converging in vivo. Surprisingly, mouse Nmnat3, a mitochondrial Nmnat enzyme that localizes to the cytoplasm in Drosophila cells, protects severed axons at levels indistinguishable from WldS. Thus, nuclear Nmnat activity does not appear to be essential for WldS-like axon protection.

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