Effects of 27 CYP3A4 Protein Variants on saxagliptin metabolism in vitro

2021 ◽  
Author(s):  
Qian liu ◽  
Qiu‐Geng Ou‐Yang ◽  
Qian‐Meng Lin ◽  
Xiang‐Ran Lu ◽  
Ya‐Qing Ma ◽  
...  
Keyword(s):  
Protein Variants

scholarly journals Weaver Syndrome‐Associated EZH2 Protein Variants Show Impaired Histone Methyltransferase Function In Vitro

2016 ◽  
Vol 37 (3) ◽  
pp. 301-307 ◽  
Author(s):  
Ana S.A. Cohen ◽  
Damian B. Yap ◽  
M.E. Suzanne Lewis ◽  
Chieko Chijiwa ◽  
Maria A. Ramos‐Arroyo ◽  
...  
Keyword(s):  
Histone Methyltransferase ◽  
Weaver Syndrome ◽  
Protein Variants

In-vitro Selection of Highly Stabilized Protein Variants with Optimized Surface

2001 ◽  
Vol 309 (3) ◽  
pp. 717-726 ◽  
Author(s):  
Andreas Martin ◽  
Volker Sieber ◽  
Franz X. Schmid
Keyword(s):  
In Vitro Selection ◽  
Protein Variants ◽  
Selection Of

Estimation of mutation rates based on the analysis of polypeptide constituents of cultured human lymphoblastoid cells.

Genetics ◽  
1988 ◽  
Vol 119 (3) ◽  
pp. 693-703
Author(s):  
E H Chu ◽  
M Boehnke ◽  
S M Hanash ◽  
R D Kuick ◽  
B J Lamb ◽  
...  
Keyword(s):  
Structural Gene ◽  
Somatic Mutations ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gene Mutations ◽  
Apparent Molecular Weight ◽  
Cell Generation ◽  
Mutation Rates ◽  
Cell Clones ◽  
Protein Variants

Abstract A subclone of a human diploid lymphoblastoid cell line, TK-6, with consistently high cloning efficiency has been used to estimate the rates of somatic mutations on the basis of protein variation detected by two-dimensional polyacrylamide gel electrophoresis. A panel of 267 polypeptide spots per gel was screened, representing the products of approximately 263 unselected loci. The rate of human somatic mutation in vitro was estimated by measuring the proportion of protein variants among cell clones isolated at various times during continuous exponential growth of a TK-6 cell population. Three mutants of spontaneous origin were observed, giving an estimated spontaneous rate of 6 x 10(-8) electrophoretic mutations per allele per cell generation (i.e., 1.2 x 10(-7) per locus per cell generation). Following treatment of cells with N-ethyl-N-nitrosourea, a total of 74 confirmed variants at 54 loci were identified among 1143 clones analyzed (approximately 601,000 allele tests). The induced variants include 65 electromorphs which exhibit altered isoelectric charge and/or apparent molecular weight and nine nullimorphs for each of which a gene product was not detected at its usual location on the gel. The induced frequency for these 65 structural gene mutants is 1.1 x 10(-4) per allele. An excess of structural gene mutations at ten known polymorphic loci and repeat mutations at these and other loci suggest nonrandomness of mutation in human somatic cells. Nullimorphs occurring at three heterozygous loci in TK-6 cells may be caused by genetic processes other than structural gene mutation.

scholarly journals Targeting Human Thrombus by Liposomes Modified with Anti-Fibrin Protein Binders

Pharmaceutics ◽  
2019 ◽  
Vol 11 (12) ◽  
pp. 642 ◽  
Author(s):  
Hana Petroková ◽  
Josef Mašek ◽  
Milan Kuchař ◽  
Andrea Vítečková Wünschová ◽  
Jana Štikarová ◽  
...  
Keyword(s):  
Amino Acid ◽  
Large Scale ◽  
Enzyme Linked Immunosorbent Assay ◽  
Ribosome Display ◽  
Amino Acid Peptide ◽  
His Tag ◽  
Insoluble Form ◽  
Protein Binders ◽  
Protein Variants

Development of tools for direct thrombus imaging represents a key step for diagnosis and treatment of stroke. Nanoliposomal carriers of contrast agents and thrombolytics can be functionalized to target blood thrombi by small protein binders with selectivity for fibrin domains uniquely formed on insoluble fibrin. We employed a highly complex combinatorial library derived from scaffold of 46 amino acid albumin-binding domain (ABD) of streptococcal protein G, and ribosome display, to identify variants recognizing fibrin cloth in human thrombus. We constructed a recombinant target as a stretch of three identical fibrin fragments of 16 amino acid peptide of the Bβ chain fused to TolA protein. Ribosome display selection followed by large-scale Enzyme-Linked ImmunoSorbent Assay (ELISA) screening provided four protein variants preferentially binding to insoluble form of human fibrin. The most specific binder variant D7 was further modified by C-terminal FLAG/His-Tag or double His-tag for the attachment onto the surface of nanoliposomes via metallochelating bond. D7-His-nanoliposomes were tested using in vitro flow model of coronary artery and their binding to fibrin fibers was demonstrated by confocal and electron microscopy. Thus, we present here the concept of fibrin-targeted binders as a platform for functionalization of nanoliposomes in the development of advanced imaging tools and future theranostics.

Human SP-A protein variants derived from one or both genes stimulate TNF-α production in the THP-1 cell line

2000 ◽  
Vol 278 (5) ◽  
pp. L946-L954 ◽  
Author(s):  
Guirong Wang ◽  
David S. Phelps ◽  
Todd M. Umstead ◽  
Joanna Floros
Keyword(s):  
Tumor Necrosis ◽  
Single Gene ◽  
Surfactant Protein ◽  
Functional Genes ◽  
Gene Products ◽  
Functional Differences ◽  
Tnf Α ◽  
Protein Variants ◽  
Necrosis Factor

In humans, two functional genes of surfactant protein (SP) A, SP-A1 and SP-A2, and several alleles of each functional gene have been characterized. SP-A is a multimeric molecule consisting of six trimers. Each trimer contains two SP-A1 molecules and one SP-A2 molecule. Until now, it has been unclear whether a single SP-Agene product is functional or whether there are functional differences either among alleles or between single-gene SP-A products and SP-A products derived from both genes. We tested the ability of in vitro expressed SP-A variants to stimulate tumor necrosis factor (TNF)-α production by THP-1 cells. We observed that 1) single-gene products and products derived from both genes stimulate TNF-α production, 2) there are differences among SP-A1 and SP-A2 alleles in their ability to stimulate TNF-α production, and 3) the increases in TNF-α production are lower after treatment with the SP-A1 alleles than after treatment with the SP-A2 alleles. Furthermore, coexpressed SP-As from SP-A1 and SP-A2 genes have a higher activity compared with SP-As from individual alleles or mixed SP-As from SP-A1and SP-A2 genes. These data suggest that the SP-A-induced increases in TNF-α levels differ among SP-A variants and appear to be affected by SP-A genotype and whether SP-A is derived from one or both genes.

scholarly journals Comparison of the ability of mammalian eEF1A1 and its oncogenic variant eEF1A2 to interact with actin and calmodulin

2017 ◽  
Vol 398 (1) ◽  
pp. 113-124 ◽  
Author(s):  
Oleksandra Novosylna ◽  
Annette Doyle ◽  
Dmytro Vlasenko ◽  
Mark Murphy ◽  
Boris Negrutskii ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Elongation Factor ◽  
Translation Elongation Factor ◽  
Translation Elongation ◽  
Oncogenic Potential ◽  
Regulatory Systems ◽  
Protein Variants ◽  
Homologous Tissue ◽  
Trna Binding

Abstract The question as to why a protein exerts oncogenic properties is answered mainly by well-established ideas that these proteins interfere with cellular signaling pathways. However, the knowledge about structural and functional peculiarities of the oncoproteins causing these effects is far from comprehensive. The 97.5% homologous tissue-specific A1 and A2 isoforms of mammalian translation elongation factor eEF1A represent an interesting model to study a difference between protein variants of a family that differ in oncogenic potential. We propose that the different oncogenic impact of A1 and A2 might be explained by differences in their ability to communicate with their respective cellular partners. Here we probed this hypothesis by studying the interaction of eEF1A with two known partners – calmodulin and actin. Indeed, an inability of the A2 isoform to interact with calmodulin is shown, while calmodulin is capable of binding A1 and interferes with its tRNA-binding and actin-bundling activities in vitro. Both A1 and A2 variants revealed actin-bundling activity; however, the form of bundles formed in the presence of A1 or A2 was distinctly different. Thus, a potential inability of A2 to be controlled by Ca2+-mediated regulatory systems is revealed.

scholarly journals Severe Biallelic Loss-of-function Mutations in Nicotinamide Mononucleotide Adenylyltransferase 2 (NMNAT2) in Two Fetuses with Fetal Akinesia Deformation Sequence

2019 ◽  
Author(s):  
Marshall Lukacs ◽  
Jonathan Gilley ◽  
Yi Zhu ◽  
Giuseppe Orsomando ◽  
Carlo Angeletti ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Wallerian Degeneration ◽  
Molecular Evidence ◽  
Compound Heterozygous ◽  
Loss Of Function ◽  
Nicotinamide Mononucleotide ◽  
Nicotinamide Mononucleotide Adenylyltransferase ◽  
Protein Variants ◽  
Axon Survival

AbstractThe three nicotinamide mononucleotide adenylyltransferase (NMNAT) family members synthesize the electron carrier nicotinamide adenine dinucleotide (NAD+) and are essential for cellular metabolism. In mammalian axons, NMNAT activity appears to be required for axon survival and is predominantly provided by NMNAT2. NMNAT2 has recently been shown to also function as a chaperone to aid in the refolding of misfolded proteins. Nmnat2 deficiency in mice, or in its ortholog dNmnat in Drosophila, results in axon outgrowth and survival defects. Peripheral nerve axons in NMNAT2-deficient mice fail to extend and innervate targets, and skeletal muscle is severely underdeveloped. In addition, removing NMNAT2 from established axons initiates axon death by Wallerian degeneration. We report here on two stillborn siblings with fetal akinesia deformation sequence (FADS), severely reduced skeletal muscle mass and hydrops fetalis. Clinical exome sequencing identified compound heterozygous NMNAT2 variant alleles in both cases. Both protein variants are incapable of supporting axon survival in mouse primary neuron cultures when overexpressed. In vitro assays demonstrate altered protein stability and/or defects in NAD+ synthesis and chaperone functions. Thus, both patient NMNAT2 alleles are null or severely hypo-morphic. These data indicate a previously unknown role for NMNAT2 in human neurological development and provide the first direct molecular evidence to support the involvement of Wallerian degeneration in a human axonal disorder.

scholarly journals Functional Characterization of 22 CYP3A4 Protein Variants to Metabolize Ibrutinib In Vitro

2017 ◽  
Vol 122 (4) ◽  
pp. 383-387 ◽  
Author(s):  
Ren-ai Xu ◽  
Jian Wen ◽  
Pengfei Tang ◽  
Chenchen Wang ◽  
Saili Xie ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Protein Variants

scholarly journals A combination of RBD and NTD neutralizing antibodies limits the generation of SARS-CoV-2 spike neutralization-escape mutants

2021 ◽  
Author(s):  
Denise Haslwanter ◽  
M. Eugenia Dieterle ◽  
Anna Z. Wec ◽  
Cecilia M. O'Brien ◽  
Mrunal Sakharkar ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Yeast Display ◽  
Cell Repertoire ◽  
Neutralization Potential ◽  
S Protein ◽  
B Cell Repertoire ◽  
Escape Mutants ◽  
Vesicular Stomatitis ◽  
Protein Variants

Most known SARS-CoV-2 neutralizing antibodies (nAbs), including those approved by the FDA for emergency use, inhibit viral infection by targeting the receptor-binding domain (RBD) of the spike (S) protein. Variants of concern (VOC) carrying mutations in the RBD or other regions of S reduce the effectiveness of many nAbs and vaccines by evading neutralization. Therefore, therapies that are less susceptible to resistance are urgently needed. Here, we characterized the memory B-cell repertoire of COVID-19 convalescent donors and analyzed their RBD and non-RBD nAbs. We found that many of the non-RBD-targeting nAbs were specific to the N-terminal domain (NTD). Using neutralization assays with authentic SARS-CoV-2 and a recombinant vesicular stomatitis virus carrying SARS-CoV-2 S protein (rVSV-SARS2), we defined a panel of potent RBD and NTD nAbs. Next, we used a combination of neutralization-escape rVSV-SARS2 mutants and a yeast display library of RBD mutants to map their epitopes. The most potent RBD nAb competed with hACE2 binding and targeted an epitope that includes residue F490. The most potent NTD nAb epitope included Y145, K150 and W152. As seen with some of the natural VOC, the neutralization potencies of COVID-19 convalescent sera were reduced by 4-16-fold against rVSV-SARS2 bearing Y145D, K150E or W152R spike mutations. Moreover, we found that combining RBD and NTD nAbs modestly enhanced their neutralization potential. Notably, the same combination of RBD and NTD nAbs limited the development of neutralization-escape mutants in vitro, suggesting such a strategy may have higher efficacy and utility for mitigating the emergence of VOC.

scholarly journals Functional Analysis of the Influenza Virus H5N1 Nucleoprotein Tail Loop Reveals Amino Acids That Are Crucial for Oligomerization and Ribonucleoprotein Activities

2010 ◽  
Vol 84 (14) ◽  
pp. 7337-7345 ◽  
Author(s):  
Wai-Hon Chan ◽  
Andy Ka-Leung Ng ◽  
Nicole C. Robb ◽  
Mandy Ka-Han Lam ◽  
Paul Kay-Sheung Chan ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Influenza A ◽  
Influenza Virus Infection ◽  
Structural Framework ◽  
Loop Region ◽  
Oligomeric Form ◽  
Protein Variants ◽  
Loop Conformation ◽  
Transcription And Replication

ABSTRACT Homo-oligomerization of the nucleoprotein (NP) of influenza A virus is crucial for providing a major structural framework for the assembly of viral ribonucleoprotein (RNP) particles. The nucleoprotein is also essential for transcription and replication during the virus life cycle. In the H5N1 NP structure, the tail loop region is important for NP to form oligomers. Here, by an RNP reconstitution assay, we identified eight NP mutants that had different degrees of defects in forming functional RNPs, with the RNP activities of four mutants being totally abolished (E339A, V408S P410S, R416A, and L418S P419S mutants) and the RNP activities of the other four mutants being more than 50% decreased (R267A, I406S, R422A, and E449A mutants). Further characterization by static light scattering showed that the totally defective protein variants existed as monomers in vitro, deviating from the trimeric/oligomeric form of wild-type NP. The I406S, R422A, and E449A variants existed as a mixture of unstable oligomers, thus resulting in a reduction of RNP activity. Although the R267A variant existed as a monomer in vitro, it resumed an oligomeric form upon the addition of RNA and retained a certain degree of RNP activity. Our data suggest that there are three factors that govern the NP oligomerization event: (i) interaction between the tail loop and the insertion groove, (ii) maintenance of the tail loop conformation, and (iii) stabilization of the NP homo-oligomer. The work presented here provides information for the design of NP inhibitors for combating influenza virus infection.

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