scholarly journals Structural characterization of human aryl sulphotransferases

1999 ◽  
Vol 337 (2) ◽  
pp. 337-343 ◽  
Author(s):  
Lulu A. BRIX ◽  
Ronald G. DUGGLEBY ◽  
Andrea GAEDIGK ◽  
Michael E. McMANUS
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Change ◽  
Substrate Binding ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid ◽  
Wild Type ◽  
Substrate Specificities ◽  
Loop Region ◽  
Single Amino Acid Change

Human aryl sulphotransferase (HAST) 1, HAST3, HAST4 and HAST4v share greater than 90% sequence identity, but vary markedly in their ability to catalyse the sulphonation of dopamine and p-nitrophenol. In order to investigate the amino acid(s) involved in determining differing substrate specificities of HASTs, a range of chimaeric HAST proteins were constructed. Analysis of chimaeric substrate specificities showed that enzyme affinities are mainly determined within the N-terminal end of each HAST protein, which includes two regions of high sequence divergence, termed Regions A (amino acids 44–107) and B (amino acids 132–164). To investigate the substrate-binding sites of HASTs further, site-directed mutagenesis was performed on HAST1 to change 13 individual residues within these two regions to the HAST3 equivalent. A single amino acid change in HAST1 (A146E) was able to change the specificity for p-nitrophenol to that of HAST3. The substrate specificity of HAST1 towards dopamine could not be converted into that of HAST3 with a single amino acid change. However, compared with wild-type HAST1, a number of the mutations resulted in interference with substrate binding, as shown by elevated Ki values towards the co-substrate 3´-phosphoadenosine 5´-phosphosulphate, and in some cases loss of activity towards dopamine. These findings suggest that a co-ordinated change of multiple amino acids in HAST proteins is needed to alter the substrate specificities of these enzymes towards dopamine, whereas a single amino acid at position 146 determines p-nitrophenol affinity. A HAST1 mutant was constructed to express a protein with four amino acids deleted (P87–P90). These amino acids were hypothesized to correspond to a loop region in close proximity to the substrate-binding pocket. Interestingly, the protein showed substrate specificities more similar to wild-type HAST3 than HAST1 and indicates an important role of these amino acids in substrate binding.

scholarly journals Single amino acid changes in the mumps virus haemagglutinin–neuraminidase and polymerase proteins are associated with neuroattenuation

2009 ◽  
Vol 90 (7) ◽  
pp. 1741-1747 ◽  
Author(s):  
Tahir H. Malik ◽  
Candie Wolbert ◽  
Laura Nerret ◽  
Christian Sauder ◽  
Steven Rubin
Keyword(s):  
Amino Acid ◽  
Clinical Isolate ◽  
Amino Acid Change ◽  
Mumps Virus ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid ◽  
Supporting Evidence ◽  
L Protein ◽  
Single Amino Acid Change

It has previously been shown that three amino acid changes, one each in the fusion (F; Ala/Thr-91→Thr), haemagglutinin–neuraminidase (HN; Ser-466→Asn) and polymerase (L; Ile-736→Val) proteins, are associated with attenuation of a neurovirulent clinical isolate of mumps virus (88-1961) following serial passage in vitro. Here, using full-length cDNA plasmid clones and site-directed mutagenesis, it was shown that the single amino acid change in the HN protein and to a lesser extent, the change in the L protein, resulted in neuroattenuation, as assessed in rats. The combination of both amino acid changes caused neuroattenuation of the virus to levels previously reported for the clinical isolate following attenuation in vitro. The amino acid change in the F protein, despite having a dramatic effect on protein function in vitro, was previously shown to not be involved in the observed neuroattenuation, highlighting the importance of conducting confirmatory in vivo studies. This report provides additional supporting evidence for the role of the HN protein as a virulence factor and, as far as is known, is the first report to associate an amino acid change in the L protein with mumps virus neuroattenuation.

scholarly journals A Single Amino Acid Change in the Newcastle Disease Virus Fusion Protein Alters the Requirement for HN Protein in Fusion

2000 ◽  
Vol 74 (11) ◽  
pp. 5101-5107 ◽  
Author(s):  
Theresa A. Sergel ◽  
Lori W. McGinnes ◽  
Trudy G. Morrison
Keyword(s):  
Amino Acid ◽  
Cell Surface ◽  
Protein Expression ◽  
Newcastle Disease ◽  
Amino Acid Change ◽  
Syncytium Formation ◽  
Single Amino Acid ◽  
Fusion Activity ◽  
Wild Type ◽  
Single Amino Acid Change

ABSTRACT The role of a leucine heptad repeat motif between amino acids 268 and 289 in the structure and function of the Newcastle disease virus (NDV) F protein was explored by introducing single point mutations into the F gene cDNA. The mutations affected either folding of the protein or the fusion activity of the protein. Two mutations, L275A and L282A, likely interfered with folding of the molecule since these proteins were not proteolytically cleaved, were minimally expressed at the cell surface, and formed aggregates. L268A mutant protein was cleaved and expressed at the cell surface although the protein migrated slightly slower than wild type on polyacrylamide gels, suggesting an alteration in conformation or processing. L268A protein was fusion inactive in the presence or absence of HN protein expression. Mutant L289A protein was expressed at the cell surface and proteolytically cleaved at better than wild-type levels. Most importantly, this protein mediated syncytium formation in the absence of HN protein expression although HN protein enhanced fusion activity. These results show that a single amino acid change in the F1 portion of the NDV F protein can alter the stringent requirement for HN protein expression in syncytium formation.

Pre-Donor Evaluation of an HLA Matched Sibling Identifies a Novel Inherited RUNX1 Mutation Encoding a Missense Mutation Found Outside of the RUNT Domain in Familial Platelet Disorder

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2709-2709 ◽  
Author(s):  
Jacqueline S Garcia ◽  
Jozef Madzo ◽  
Devin Cooper ◽  
Sarah A Jackson ◽  
Kenan Onel ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Amino Acid ◽  
Dna Binding ◽  
Amino Acid Change ◽  
Single Amino Acid ◽  
Wild Type ◽  
Single Amino Acid Change ◽  
Platelet Disorder ◽  
Familial Platelet Disorder ◽  
Runx1 Mutation

Abstract Abstract 2709 Introduction: RUNX1 is a critical transcription factor in the regulation of normal hematopoiesis. Inherited RUNX1 mutations have been identified as the culprit genetic lesion in Familial Platelet Disorder (FPD; OMIM 601399), a rare autosomal dominant condition with a propensity to myeloid malignancy. The spectrum of RUNX1 mutations causing the FPD/acute myeloid leukemia (AML) syndrome includes frameshift and termination mutations detected throughout the gene, and missense mutations clustered within the highly conserved RUNT homology domain (RHD), which is responsible for both DNA binding and heterodimerization with CBFβ/PEBP2β, the non-DNA binding regulatory subunit. We present a new FPD/AML pedigree with a novel missense mutation leading to a single amino acid change, L56S. This L56S mutation is the first reported point mutation in this syndrome to be found outside of the RHD. Patients and Methods: Our new pedigree involves a 41-year-old man (proband) diagnosed with myelodysplastic syndrome (MDS, specifically refractory anemia with excess blasts type-2) with a normal karyotype. He was initiated on azacitidine, which was administered on a seven-day treatment schedule every four weeks. Bone marrow biopsy analysis after six monthly cycles of azacitidine showed persistent MDS, with similar findings after a total of ten monthly cycles. Given his lack of a clinical response, his young age and good performance status, he was referred to The University of Chicago for allogeneic hematopoietic stem cell transplantation (HCT). Routine pre-transplant evaluation revealed mild thrombocytopenia (platelets = 123,000 K/μl) in his HLA-matched brother. In addition, his father was reported to have thrombocytopenia. Clinical concern for an inherited condition initiated the investigation for a RUNX1 mutation in the family. Results: We sequenced full-length cDNA synthesized from leukocyte-derived RNA collected from the proband's sibling with thrombocytopenia, and detected a novel missense germline mutation in exon 4 at nucleotide position 371, causing a T to C mutation leading to a single amino acid change in the RUNX1 protein, L56S. This amino acid substitution is located N-terminal to the RHD (aa 76–209). RUNX1 sequencing of the proband with MDS demonstrated the same mutation. The RUNX1 RHD and the transactivation domain remain intact in this mutant. Initial transactivation assays using a luciferase reporter assay performed in triplicate demonstrated similar levels of activation as wild-type RUNX1. Corresponding Western blot analysis showed similar levels of protein expression of both wild-type RUNX1 and mutant RUNX1 transfected cell lines using an anti-RUNX1-antibody. Current studies include determination of the transactivation ability of mutant RUNX1 with its heterodimerization partner, CBFβ/PEBP2β, testing the DNA binding ability of this RUNX1 mutant by electrophoretic mobility shift assay, and analysis of the RUNX1 cDNA for an acquired biallelic mutation in leukocytes collected from the proband's bone marrow aspirate at the time of diagnosis of bone marrow malignancy. Conclusions: FPD/AML is likely an underreported condition. Clinical suspicion for this inherited syndrome may be raised by the presence of mild to moderate thrombocytopenia in healthy siblings, and should lead to prompt screening for germline RUNX1 mutations to confirm an inherited predisposition and to prevent siblings carrying RUNX1 mutations from being selected as HCT donors. In vitro studies of identified RUNX1 mutations may elucidate potential mechanisms involved in the pathogenesis of the FPD/AML syndrome. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Structural and functional analyses of Saccharomyces cerevisiae wild-type and mutant RNA1 genes.

1989 ◽  
Vol 9 (7) ◽  
pp. 2989-2999 ◽  
Author(s):  
H M Traglia ◽  
N S Atkinson ◽  
A K Hopper
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Open Reading Frame ◽  
Single Amino Acid ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Reading Frame ◽  
Genomic Deletions ◽  
Single Amino Acid Change ◽  
Carboxy Terminal

The yeast gene RNA1 has been defined by the thermosensitive rna1-1 lesion. This lesion interferes with the processing and production of all major classes of RNA. Each class of RNA is affected at a distinct and presumably unrelated step. Furthermore, RNA does not appear to exit the nucleus. To investigate how the RNA1 gene product can pleiotropically affect disparate processes, we undertook a structural analysis of wild-type and mutant RNA1 genes. The wild-type gene was found to contain a 407-amino-acid open reading frame that encodes a hydrophilic protein. No clue regarding the function of the RNA1 protein was obtained by searching banks for similarity to other known gene products. Surprisingly, the rna1-1 lesion was found to code for two amino acid differences from wild type. We found that neither single-amino-acid change alone resulted in temperature sensitivity. The carboxy-terminal region of the RNA1 open reading frame contains a highly acidic domain extending from amino acids 334 to 400. We generated genomic deletions that removed C-terminal regions of this protein. Deletion of amino acids 397 to 407 did not appear to affect cell growth. Removal of amino acids 359 to 397, a region containing 24 acidic residues, caused temperature-sensitive growth. This allele, rna1-delta 359-397, defines a second conditional lesion of the RNA1 locus. We found that strains possessing the rna1-delta 359-397 allele did not show thermosensitive defects in pre-rRNA or pre-tRNA processing. Removal of amino acids 330 to 407 resulted in loss of viability.

scholarly journals A Point Mutation in the Rhesus Rotavirus VP4 Protein Generated through a Rotavirus Reverse Genetics System Attenuates Biliary Atresia in the Murine Model

2017 ◽  
Vol 91 (15) ◽  
Author(s):  
Sujit K. Mohanty ◽  
Bryan Donnelly ◽  
Phylicia Dupree ◽  
Inna Lobeck ◽  
Sarah Mowery ◽  
...  
Keyword(s):  
Amino Acid ◽  
Biliary Atresia ◽  
Amino Acid Change ◽  
Reverse Genetics ◽  
Single Amino Acid ◽  
Wild Type ◽  
Neonatal Mice ◽  
Single Amino Acid Change

ABSTRACT Rotavirus infection is one of the most common causes of diarrheal illness in humans. In neonatal mice, rhesus rotavirus (RRV) can induce biliary atresia (BA), a disease resulting in inflammatory obstruction of the extrahepatic biliary tract and intrahepatic bile ducts. We previously showed that the amino acid arginine (R) within the sequence SRL (amino acids 445 to 447) in the RRV VP4 protein is required for viral binding and entry into biliary epithelial cells. To determine if this single amino acid (R) influences the pathogenicity of the virus, we generated a recombinant virus with a single amino acid mutation at this site through a reverse genetics system. We demonstrated that the RRV mutant (RRVVP4-R446G) produced less symptomatology and replicated to lower titers both in vivo and in vitro than those seen with wild-type RRV, with reduced binding in cholangiocytes. Our results demonstrate that a single amino acid change in the RRV VP4 gene influences cholangiocyte tropism and reduces pathogenicity in mice. IMPORTANCE Rotavirus is the leading cause of diarrhea in humans. Rhesus rotavirus (RRV) can also lead to biliary atresia (a neonatal human disease) in mice. We developed a reverse genetics system to create a mutant of RRV (RRVVP4-R446G) with a single amino acid change in the VP4 protein compared to that of wild-type RRV. In vitro, the mutant virus had reduced binding and infectivity in cholangiocytes. In vivo, it produced fewer symptoms and lower mortality in neonatal mice, resulting in an attenuated form of biliary atresia.

scholarly journals Evolution of New Function through a Single Amino Acid Change in the Yeast Repressor Sum1p

2008 ◽  
Vol 28 (8) ◽  
pp. 2567-2578 ◽  
Author(s):  
Alexias Safi ◽  
Kelley A. Wallace ◽  
Laura N. Rusche
Keyword(s):  
Amino Acid ◽  
Origin Recognition Complex ◽  
Amino Acid Change ◽  
Single Amino Acid ◽  
Wild Type ◽  
Gain Of Function ◽  
Critical Residue ◽  
Single Amino Acid Change ◽  
Interaction Domains ◽  
Self Association

ABSTRACT The SUM1-1 mutation is an example of a single amino acid change that results in new function. Wild-type Sum1p in Saccharomyces cerevisiae is a DNA-binding repressor that acts locally, whereas mutant Sum1-1p forms an extended repressive chromatin structure. By characterizing a panel of mutations in which various amino acids replaced the critical residue, threonine 988, we found that threonine was required for wild-type function and that in the absence of threonine the association of Sum1p with DNA was reduced. Isoleucine, the amino acid in mutant Sum1-1p, was required for the novel spreading property. Thus, the SUM1-1 mutation results in both a loss and a gain of function. The presence of isoleucine caused Sum1-1p to self-associate, a property that may promote spreading. In addition, isoleucine enabled Sum1-1p to associate with the origin recognition complex (ORC) and accumulate near ORC binding sites. Thus, both threonine and isoleucine at position 988 enable Sum1p to form intermolecular interactions. We propose that interaction domains may be hotspots for gain-of-function mutations because alterations in such domains have the potential to redirect a protein to new sets of binding partners. In addition, self-association of chromatin proteins may promote the formation of extended chromatin structures.

scholarly journals FlexibilityIn Vitroof Amino Acid 226 in the Receptor-Binding Site of an H9 Subtype Influenza A Virus and Its EffectIn Vivoon Virus Replication, Tropism, and Transmission

2018 ◽  
Vol 93 (6) ◽  
Author(s):  
Adebimpe O. Obadan ◽  
Jefferson Santos ◽  
Lucas Ferreri ◽  
Andrew J. Thompson ◽  
Silvia Carnaccini ◽  
...  
Keyword(s):  
Public Health ◽  
Amino Acids ◽  
Influenza Virus ◽  
Amino Acid ◽  
Influenza A ◽  
Amino Acid Change ◽  
Single Amino Acid ◽  
Receptor Binding Site ◽  
Single Amino Acid Change

ABSTRACTInfluenza A viruses (IAVs) remain a significant public health threat, causing more than 300,000 hospitalizations in the United States during the 2015–2016 season alone. While only a few IAVs of avian origin have been associated with human infections, the ability of these viruses to cause zoonotic infections further increases the public health risk of influenza. Of these, H9N2 viruses in Asia are of particular importance as they have contributed internal gene segments to other emerging zoonotic IAVs. Notably, recent H9N2 viruses have acquired molecular markers that allow for a transition from avian-like to human-like terminal sialic acid (SA) receptor recognition via a single amino acid change at position 226 (H3 numbering), from glutamine (Q226) to leucine (L226), within the hemagglutinin (HA) receptor-binding site (RBS). We sought to determine the plasticity of amino acid 226 and the biological effects of alternative amino acids on variant viruses. We created a library of viruses with the potential of having any of the 20 amino acids at position 226 on a prototypic H9 HA subtype IAV. We isolated H9 viruses that carried naturally occurring amino acids, variants found in other subtypes, and variants not found in any subtype at position 226. Fitness studies in quails revealed that some natural amino acids conferred anin vivoreplication advantage. This study shows the flexibility of position 226 of the HA of H9 influenza viruses and the resulting effect of single amino acid changes on the phenotype of variantsin vivoandin vitro.IMPORTANCEA single amino acid change at position 226 in the hemagglutinin (HA) from glutamine (Q) to leucine (L) has been shown to play a key role in receptor specificity switching in various influenza virus HA subtypes, including H9. We tested the flexibility of amino acid usage and determined the effects of such changes. The results reveal that amino acids other than L226 and Q226 are well tolerated and that some amino acids allow for the recognition of both avian and human influenza virus receptors in the absence of other changes. Our results can inform better avian influenza virus surveillance efforts as well as contribute to rational vaccine design and improve structural molecular dynamics algorithms.

Structural and functional analyses of Saccharomyces cerevisiae wild-type and mutant RNA1 genes

1989 ◽  
Vol 9 (7) ◽  
pp. 2989-2999
Author(s):  
H M Traglia ◽  
N S Atkinson ◽  
A K Hopper
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Open Reading Frame ◽  
Single Amino Acid ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Reading Frame ◽  
Genomic Deletions ◽  
Single Amino Acid Change ◽  
Carboxy Terminal

The yeast gene RNA1 has been defined by the thermosensitive rna1-1 lesion. This lesion interferes with the processing and production of all major classes of RNA. Each class of RNA is affected at a distinct and presumably unrelated step. Furthermore, RNA does not appear to exit the nucleus. To investigate how the RNA1 gene product can pleiotropically affect disparate processes, we undertook a structural analysis of wild-type and mutant RNA1 genes. The wild-type gene was found to contain a 407-amino-acid open reading frame that encodes a hydrophilic protein. No clue regarding the function of the RNA1 protein was obtained by searching banks for similarity to other known gene products. Surprisingly, the rna1-1 lesion was found to code for two amino acid differences from wild type. We found that neither single-amino-acid change alone resulted in temperature sensitivity. The carboxy-terminal region of the RNA1 open reading frame contains a highly acidic domain extending from amino acids 334 to 400. We generated genomic deletions that removed C-terminal regions of this protein. Deletion of amino acids 397 to 407 did not appear to affect cell growth. Removal of amino acids 359 to 397, a region containing 24 acidic residues, caused temperature-sensitive growth. This allele, rna1-delta 359-397, defines a second conditional lesion of the RNA1 locus. We found that strains possessing the rna1-delta 359-397 allele did not show thermosensitive defects in pre-rRNA or pre-tRNA processing. Removal of amino acids 330 to 407 resulted in loss of viability.

scholarly journals Resistance to Colistin Associated with a Single Amino Acid Change in Protein PmrB among Klebsiella pneumoniae Isolates of Worldwide Origin

2014 ◽  
Vol 58 (8) ◽  
pp. 4762-4766 ◽  
Author(s):  
Aurélie Jayol ◽  
Laurent Poirel ◽  
Adrian Brink ◽  
Maria-Virginia Villegas ◽  
Mesut Yilmaz ◽  
...  
Keyword(s):  
Amino Acid ◽  
Klebsiella Pneumoniae ◽  
Amino Acid Change ◽  
Sequence Type ◽  
Single Amino Acid ◽  
Wild Type ◽  
Two Component System ◽  
Content Type ◽  
Single Amino Acid Change ◽  
Two Component

ABSTRACTA series of colistin-resistantKlebsiella pneumoniaeisolates recovered from different countries was investigated in order to evaluate the involvement of the PmrA/PmrB two-component system in this resistance. Six isolates possessed a mutated PmrB protein, which is encoded by thepmrBgene, part of thepmrCABoperon involved in lipopolysaccharide modification. The same amino acid substitution (Thr157Pro) in PmrB was identified in the six isolates. The six isolates belonged to four distinct clonal groups, recovered in South Africa (sequence type 14 [ST14]), Turkey (ST101), and Colombia (ST258 and ST15). Three out of the four clones produced a carbapenemase, OXA-181, OXA-48, or KPC-3, while a single isolate did not produce any carbapenemase. Expression assays revealed an overexpression of thepmrA(70-fold),pmrB(70-fold),pmrC(170-fold), andpmrK(40-fold) genes in thepmrB-mutated isolate compared to expression of thepmrBwild-type isogenicK. pneumoniaeisolate, confirming that the PmrB substitution was responsible for increased expression levels of those genes. Complementation assays leading to the expression of a wild-type PmrB protein restored the susceptibility to colistin in all isolates, confirming that the substitution in PmrB was responsible for the resistance phenotype. This study identified a key amino acid located in the PmrB protein as being responsible for the overexpression ofpmrCABandpmrHFIJKLMoperons, leading to resistance to colistin.

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