Single Amino Acid Substitutions at the N-Terminus of a Recombinant Cytotoxic Ribonuclease Markedly Influence Biochemical and Biological Properties†

Studies of conditionally lethal mutants can help delineate the structure-function relationships of biomolecules. Temperature-sensitive (ts) mammalian reovirus (MRV) mutants were isolated and characterized many years ago. Two of the most well-defined MRV ts mutants are tsC447, which contains mutations in the S2 gene encoding viral core protein σ2, and tsG453, which contains mutations in the S4 gene encoding major outer-capsid protein σ3. Because many MRV ts mutants, including both tsC447 and tsG453, encode multiple amino acid substitutions, the specific amino acid substitutions responsible for the ts phenotype are unknown. We used reverse genetics to recover recombinant reoviruses containing the single amino acid polymorphisms present in ts mutants tsC447 and tsG453 and assessed the recombinant viruses for temperature-sensitivity by efficiency-of-plating assays. Of the three amino acid substitutions in the tsG453 S4 gene, Asn16-Lys was solely responsible for the tsG453ts phenotype. Additionally, the mutant tsC447 Ala188-Val mutation did not induce a temperature-sensitive phenotype. This study is the first to employ reverse genetics to identify the dominant amino acid substitutions responsible for the tsC447 and tsG453 mutations and relate these substitutions to respective phenotypes. Further studies of other MRV ts mutants are warranted to define the sequence polymorphisms responsible for temperature sensitivity.

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Natural variants in SARS-CoV-2 Spike protein pinpoint structural and functional hotspots with implications for prophylaxis and therapeutic strategies

Scientific Reports ◽

10.1038/s41598-021-92641-x ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Suman Pokhrel ◽

Benjamin R. Kraemer ◽

Scott Burkholz ◽

Daria Mochly-Rosen

Keyword(s):

Amino Acid ◽

Severe Disease ◽

Single Amino Acid ◽

Amino Acid Substitutions ◽

Severe Respiratory Distress ◽

S Protein ◽

Individual Sequences ◽

Natural Variants ◽

Novel Coronavirus ◽

The Individual

AbstractIn December 2019, a novel coronavirus, termed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was identified as the cause of pneumonia with severe respiratory distress and outbreaks in Wuhan, China. The rapid and global spread of SARS-CoV-2 resulted in the coronavirus 2019 (COVID-19) pandemic. Earlier during the pandemic, there were limited genetic viral variations. As millions of people became infected, multiple single amino acid substitutions emerged. Many of these substitutions have no consequences. However, some of the new variants show a greater infection rate, more severe disease, and reduced sensitivity to current prophylaxes and treatments. Of particular importance in SARS-CoV-2 transmission are mutations that occur in the Spike (S) protein, the protein on the viral outer envelope that binds to the human angiotensin-converting enzyme receptor (hACE2). Here, we conducted a comprehensive analysis of 441,168 individual virus sequences isolated from humans throughout the world. From the individual sequences, we identified 3540 unique amino acid substitutions in the S protein. Analysis of these different variants in the S protein pinpointed important functional and structural sites in the protein. This information may guide the development of effective vaccines and therapeutics to help arrest the spread of the COVID-19 pandemic.

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Influence of single amino acid substitutions on electrophoretic mobility of sodium dodecyl sulfate-protein complexes

Biochemical and Biophysical Research Communications ◽

10.1016/0006-291x(78)90907-5 ◽

1978 ◽

Vol 82 (2) ◽

pp. 532-539 ◽

Cited By ~ 155

Author(s):

Wilfried W. de Jong ◽

Anneke Zweers ◽

Louis H. Cohen

Keyword(s):

Amino Acid ◽

Sodium Dodecyl Sulfate ◽

Electrophoretic Mobility ◽

Protein Complexes ◽

Sodium Dodecyl ◽

Single Amino Acid ◽

Amino Acid Substitutions ◽

Dodecyl Sulfate

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Further characterization and determination of the single amino acid change in thetsI138reovirus thermosensitive mutant

Canadian Journal of Microbiology ◽

10.1139/w2012-033 ◽

2012 ◽

Vol 58 (5) ◽

pp. 589-595

Author(s):

Guy Lemay ◽

Martin Bisaillon

Keyword(s):

Amino Acid ◽

Amino Acid Change ◽

Genetic Alterations ◽

Biological Properties ◽

Single Amino Acid ◽

Temperature Sensitive ◽

Nonpermissive Temperature ◽

Gene Encoding ◽

Viral Particles ◽

Single Amino Acid Change

Many temperature-sensitive mutants have been isolated in early studies of mammalian reovirus. However, the biological properties and nature of the genetic alterations remain incompletely explored for most of these mutants. The mutation harbored by the tsI138 mutant was already assigned to the L3 gene encoding the λ1 protein. In the present study, this mutant was further studied as a possible tool to establish the role of the putative λ1 enzymatic activities in viral multiplication. It was observed that synthesis of viral proteins is only marginally reduced, while it was difficult to recover viral particles at the nonpermissive temperature. A single nucleotide substitution resulting in an amino acid change was found; the position of this amino acid is consistent with a probable defect in assembly of the inner capsid at the nonpermissive temperature.

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Single Amino Acid Substitutions Disrupt Tetramer Formation in the Dihydroneopterin Aldolase Enzyme ofPneumocystis carinii†

Biochemistry ◽

10.1021/bi980540x ◽

1998 ◽

Vol 37 (33) ◽

pp. 11629-11636 ◽

Cited By ~ 8

Author(s):

M. Carmen Thomas ◽

Stuart P. Ballantine ◽

Susanne S. Bethell ◽

Satty Bains ◽

Paul Kellam ◽

...

Keyword(s):

Amino Acid ◽

Single Amino Acid ◽

Amino Acid Substitutions ◽

Tetramer Formation ◽

Dihydroneopterin Aldolase

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Identification of critical amino acid residues in the regulatory N-terminal domain of PMEL

Scientific Reports ◽

10.1038/s41598-021-87259-y ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Susan M. Mitchell ◽

Morven Graham ◽

Xinran Liu ◽

Ralf M. Leonhardt

Keyword(s):

Amino Acid ◽

Pigment Cell ◽

Specific Protein ◽

Single Amino Acid ◽

Amyloid Formation ◽

Amino Acid Residues ◽

Proteolytic Fragment ◽

The Core ◽

N Terminus ◽

In Cis

AbstractThe pigment cell-specific protein PMEL forms a functional amyloid matrix in melanosomes onto which the pigment melanin is deposited. The amyloid core consists of a short proteolytic fragment, which we have termed the core-amyloid fragment (CAF) and perhaps additional parts of the protein, such as the PKD domain. A highly O-glycosylated repeat (RPT) domain also derived from PMEL proteolysis associates with the amyloid and is necessary to establish the sheet-like morphology of the assemblies. Excluded from the aggregate is the regulatory N-terminus, which nevertheless must be linked in cis to the CAF in order to drive amyloid formation. The domain is then likely cleaved away immediately before, during, or immediately after the incorporation of a new CAF subunit into the nascent amyloid. We had previously identified a 21 amino acid long region, which mediates the regulatory activity of the N-terminus towards the CAF. However, many mutations in the respective segment caused misfolding and/or blocked PMEL export from the endoplasmic reticulum, leaving their phenotype hard to interpret. Here, we employ a saturating mutagenesis approach targeting the motif at single amino acid resolution. Our results confirm the critical nature of the PMEL N-terminal region and identify several residues essential for PMEL amyloidogenesis.

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Pathogenic alleles in microtubule, secretory granule and extracellular matrix-related genes in familial keratoconus

Human Molecular Genetics ◽

10.1093/hmg/ddab075 ◽

2021 ◽

Author(s):

Vishal Shinde ◽

Nara Sobreira ◽

Elizabeth S Wohler ◽

George Maiti ◽

Nan Hu ◽

...

Keyword(s):

Amino Acid ◽

Cell Cultures ◽

Cell Biology ◽

Stromal Cell ◽

Primary Cilia ◽

Single Amino Acid ◽

European Ancestry ◽

Base Substitution ◽

Amino Acid Substitutions ◽

Functional Link

Abstract Keratoconus is a common corneal defect with a complex genetic basis. By whole exome sequencing of affected members from 11 multiplex families of European ancestry, we identified 23 rare, heterozygous, potentially pathogenic variants in 8 genes. These include nonsynonymous single amino acid substitutions in HSPG2, EML6 and CENPF in two families each, and in NBEAL2, LRP1B, PIK3CG and MRGPRD in three families each; ITGAX had nonsynonymous single amino acid substitutions in two families and an indel with a base substitution producing a nonsense allele in the third family. Only HSPG2, EML6 and CENPF have been associated with ocular phenotypes previously. With the exception of MRGPRD and ITGAX, we detected the transcript and encoded protein of the remaining genes in the cornea and corneal cell cultures. Cultured stromal cells showed cytoplasmic punctate staining of NBEAL2, staining of the fibrillar cytoskeletal network by EML6, while CENPF localized to the basal body of primary cilia. We inhibited the expression of HSPG2, EML6, NBEAL2 and CENPF in stromal cell cultures and assayed for the expression of COL1A1 as a readout of corneal matrix production. An upregulation in COL1A1 after siRNA inhibition indicated their functional link to stromal cell biology. For ITGAX, encoding a leukocyte integrin, we assayed its level in the sera of 3 affected families compared with 10 unrelated controls to detect an increase in all affecteds. Our study identified genes that regulate the cytoskeleton, protein trafficking and secretion, barrier tissue function and response to injury and inflammation, as being relevant to keratoconus.

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Identification of separate domains in the adenovirus E1A gene for immortalization activity and the activation of virus early genes.

Molecular and Cellular Biology ◽

10.1128/mcb.6.10.3470 ◽

1986 ◽

Vol 6 (10) ◽

pp. 3470-3480 ◽

Cited By ~ 98

Author(s):

E Moran ◽

B Zerler ◽

T M Harrison ◽

M B Mathews

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Point Mutations ◽

Apparent Molecular Weight ◽

Amino Acid Position ◽

Cysteine Residue ◽

Transformation Function ◽

Single Amino Acid ◽

Amino Acid Substitutions ◽

E1a Gene

The transformation and early adenovirus gene transactivation functions of the E1A region were analyzed with deletion and point mutations. Deletion of amino acids from position 86 through 120 had little effect on the lytic or transforming functions of the E1A products, while deletion of amino acids from position 121 through 150 significantly impaired both functions. The sensitivity of the transformation function to alterations in the region from amino acid position 121 to 150 was further indicated by the impairment of transforming activity resulting from single amino acid substitutions at positions 124 and 135. Interestingly, conversion of a cysteine residue at position 124 to glycine severely impaired the transformation function without affecting the early adenovirus gene activating functions. Single amino acid substitutions in a different region of the E1A gene had the converse effect. All the mutants produced polypeptides of sufficient stability to be detected by Western immunoblot analysis. The single amino acid substitutions at positions 124 and 135, although impairing the transformation functions, did not detectably alter the formation of the higher-apparent-molecular-weight forms of the E1A products.

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