scholarly journals Positive and Negative Regulation of Poly(A) Nuclease

2004 ◽  
Vol 24 (12) ◽  
pp. 5521-5533 ◽  
Author(s):  
David A. Mangus ◽  
Matthew C. Evans ◽  
Nathan S. Agrin ◽  
Mandy Smith ◽  
Preetam Gongidi ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Protein Interactions ◽  
Binding Pocket ◽  
Negative Regulation ◽  
Single Amino Acid ◽  
Carboxy Terminus ◽  
Protein Protein Interactions ◽  
C Terminus

ABSTRACT PAN, a yeast poly(A) nuclease, plays an important nuclear role in the posttranscriptional maturation of mRNA poly(A) tails. The activity of this enzyme is dependent on its Pan2p and Pan3p subunits, as well as the presence of poly(A)-binding protein (Pab1p). We have identified and characterized the associated network of factors controlling the maturation of mRNA poly(A) tails in yeast and defined its relevant protein-protein interactions. Pan3p, a positive regulator of PAN activity, interacts with Pab1p, thus providing substrate specificity for this nuclease. Pab1p also regulates poly(A) tail trimming by interacting with Pbp1p, a factor that appears to negatively regulate PAN. Pan3p and Pbp1p both interact with themselves and with the C terminus of Pab1p. However, the domains required for Pan3p and Pbp1p binding on Pab1p are distinct. Single amino acid changes that disrupt Pan3p interaction with Pab1p have been identified and define a binding pocket in helices 2 and 3 of Pab1p's carboxy terminus. The importance of these amino acids for Pab1p-Pan3p interaction, and poly(A) tail regulation, is underscored by experiments demonstrating that strains harboring substitutions in these residues accumulate mRNAs with long poly(A) tails in vivo.

Engineering an acetyllysine reader with photocrosslinking amino acid for interactome profiling

2021 ◽  
Author(s):  
Babu Sudhamalla ◽  
Anirban Roy ◽  
Soumen Barman ◽  
Jyotirmayee Padhan
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Protein Interactions ◽  
Unnatural Amino Acids ◽  
Protein Protein Interactions ◽  
Site Specific ◽  
Powerful Approach

The site-specific installation of light-activable crosslinker unnatural amino acids offers a powerful approach to trap transient protein-protein interactions both in vitro and in vivo. Herein, we engineer a bromodomain to...

scholarly journals ArabidopsisImmunophilin-like TWD1 Functionally Interacts with Vacuolar ABC Transporters

2004 ◽  
Vol 15 (7) ◽  
pp. 3393-3405 ◽  
Author(s):  
Markus Geisler ◽  
Marjolaine Girin ◽  
Sabine Brandt ◽  
Vincent Vincenzetti ◽  
Sonia Plaza ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Abc Transporters ◽  
Loss Of Function ◽  
Protein Protein Interactions ◽  
Binding Motifs ◽  
Calmodulin Binding ◽  
C Terminus ◽  
Domain Mapping ◽  
Tetratricopeptide Repeat Domain

Previously, the immunophilin-like protein TWD1 from Arabidopsis has been demonstrated to interact with the ABC transporters AtPGP1 and its closest homologue, AtPGP19. Physiological and biochemical investigation of pgp1/pgp19 and of twd1 plants suggested a regulatory role of TWD1 on AtPGP1/AtPGP19 transport activities. To further understand the dramatic pleiotropic phenotype that is caused by loss-of-function mutation of the TWD1 gene, we were interested in other TWD1 interacting proteins. AtMRP1, a multidrug resistance-associated (MRP/ABCC)-like ABC transporter, has been isolated in a yeast two-hybrid screen. We demonstrate molecular interaction between TWD1 and ABC transporters AtMRP1 and its closest homologue, AtMRP2. Unlike AtPGP1, AtMRP1 binds to the C-terminal tetratricopeptide repeat domain of TWD1, which is well known to mediate protein-protein interactions. Domain mapping proved that TWD1 binds to a motif of AtMRP1 that resembles calmodulin-binding motifs; and calmodulin binding to the C-terminus of MRP1 was verified. By membrane fractionation and GFP-tagging, we localized AtMRP1 to the central vacuolar membrane and the TWD1-AtMRP1 complex was verified in vivo by coimmunoprecipitation. We were able to demonstrate that TWD1 binds to isolated vacuoles and has a significant impact on the uptake of metolachlor-GS and estradiol-β-glucuronide, well-known substrates of vacuolar transporters AtMRP1 and AtMRP2.

scholarly journals Single Amino Acid Replacements in RocA Disrupt Protein-Protein Interactions To Alter the Molecular Pathogenesis of Group A Streptococcus

2020 ◽  
Vol 88 (11) ◽  
Author(s):  
Paul E. Bernard ◽  
Amey Duarte ◽  
Mikhail Bogdanov ◽  
James M. Musser ◽  
Randall J. Olsen
Keyword(s):  
Amino Acid ◽  
Protein Interactions ◽  
Molecular Pathogenesis ◽  
Single Amino Acid ◽  
Accessory Protein ◽  
Protein Protein Interactions ◽  
Tissue Destruction ◽  
Intact Cells ◽  
Group A

ABSTRACT Group A Streptococcus (GAS) is a human-specific pathogen and major cause of disease worldwide. The molecular pathogenesis of GAS, like many pathogens, is dependent on the coordinated expression of genes encoding different virulence factors. The control of virulence regulator/sensor (CovRS) two-component system is a major virulence regulator of GAS that has been extensively studied. More recent investigations have also involved regulator of Cov (RocA), a regulatory accessory protein to CovRS. RocA interacts, in some manner, with CovRS; however, the precise molecular mechanism is unknown. Here, we demonstrate that RocA is a membrane protein containing seven transmembrane helices with an extracytoplasmically located N terminus and cytoplasmically located C terminus. For the first time, we demonstrate that RocA directly interacts with itself (RocA) and CovS, but not CovR, in intact cells. Single amino acid replacements along the entire length of RocA disrupt RocA-RocA and RocA-CovS interactions to significantly alter the GAS virulence phenotype as defined by secreted virulence factor activity in vitro and tissue destruction and mortality in vivo. In summary, we show that single amino acid replacements in a regulatory accessory protein can affect protein-protein interactions to significantly alter the virulence of a major human pathogen.

scholarly journals Monoclonal Antibodies Detect a Single Amino Acid Difference Between the Coat Proteins of Soilborne Wheat Mosaic Virus Isolates: Implications for Virus Structure

1997 ◽  
Vol 87 (3) ◽  
pp. 295-301 ◽  
Author(s):  
Jianping Chen ◽  
Lesley Torrance ◽  
Graham H. Cowan ◽  
Stuart A. MacFarlane ◽  
Gerald Stubbs ◽  
...  
Keyword(s):  
Amino Acids ◽  
Monoclonal Antibodies ◽  
Amino Acid ◽  
Mosaic Virus ◽  
Single Amino Acid ◽  
Computer Assisted ◽  
Amino Acid Difference ◽  
Coat Proteins ◽  
C Terminus ◽  
Readthrough Protein

Four monoclonal antibodies (MAbs) were prepared against an isolate of soilborne wheat mosaic furovirus from Oklahoma (SBWMV Okl-7). Three MAbs had different reactivities in tests on SBWMV isolates from Nebraska (Lab1), France, and Japan. One MAb (SCR 133) also reacted with oat golden stripe furovirus. None of the MAbs cross-reacted with other rod-shaped viruses including beet necrotic yellow vein furovirus, potato mop-top furovirus, and tobacco rattle tobravirus. Sequence analysis of nucleotides between 334 and 1,000 of RNA 2, the region that encodes the coat protein (CP) and the first 44 amino acids of a readthrough protein, of the four SBWMV isolates revealed up to 27 base changes from the published sequence of a Nebraska field isolate of SBWMV. Most changes were translationally silent, but some caused differences of one to three amino acids in residues located near either the N- or C-terminus of the CPs of the different isolates. Two further single amino acid changes were found at the beginning of the readthrough domain of the CP-readthrough protein. Some of these amino acid changes could be discriminated by MAbs SCR 132, SCR 133, and SCR 134. Peptide scanning (Pepscan) analysis indicated that the epitope recognized by SCR 134 is located near the N-terminus of the CP. SCR 132 was deduced to react with a discontinuous CP epitope near the C-terminus, and SCR 133 reacted with a surface-located continuous epitope also near the C-terminus. Predictions of CP structure from computer-assisted three-dimensional model building, by comparison with the X-ray fiber diffraction structure of tobacco mosaic virus, suggested that the three CP amino acids found to differ between isolates of SBWMV were located near the viral surface and were in regions predicted to be antigenic.

scholarly journals Structure/Function Analysis of the Vaccinia Virus F18 Phosphoprotein, an Abundant Core Component Required for Virion Maturation and Infectivity

2010 ◽  
Vol 84 (13) ◽  
pp. 6846-6860 ◽  
Author(s):  
Nadi T. Wickramasekera ◽  
Paula Traktman
Keyword(s):  
Amino Acids ◽  
Structure Function ◽  
Protein Interactions ◽  
Function Analysis ◽  
Aromatic Amino Acids ◽  
Protein Protein Interactions ◽  
Core Component ◽  
Virion Morphogenesis

ABSTRACT Poxvirus virions, whose outer membrane surrounds two lateral bodies and a core, contain at least 70 different proteins. The F18 phosphoprotein is one of the most abundant core components and is essential for the assembly of mature virions. We report here the results of a structure/function analysis in which the role of conserved cysteine residues, clusters of charged amino acids and clusters of hydrophobic/aromatic amino acids have been assessed. Taking advantage of a recombinant virus in which F18 expression is IPTG (isopropyl-β-d-thiogalactopyranoside) dependent, we developed a transient complementation assay to evaluate the ability of mutant alleles of F18 to support virion morphogenesis and/or to restore the production of infectious virus. We have also examined protein-protein interactions, comparing the ability of mutant and WT F18 proteins to interact with WT F18 and to interact with the viral A30 protein, another essential core component. We show that F18 associates with an A30-containing multiprotein complex in vivo in a manner that depends upon clusters of hydrophobic/aromatic residues in the N′ terminus of the F18 protein but that it is not required for the assembly of this complex. Finally, we confirmed that two PSSP motifs within F18 are the sites of phosphorylation by cellular proline-directed kinases in vitro and in vivo. Mutation of both of these phosphorylation sites has no apparent impact on virion morphogenesis but leads to the assembly of virions with significantly reduced infectivity.

scholarly journals A Bifunctional Amino Acid Enables Both Covalent Chemical Capture and Isolation of in Vivo Protein-Protein Interactions

ChemBioChem ◽  
2016 ◽  
Vol 18 (2) ◽  
pp. 181-184 ◽  
Author(s):  
Cassandra M. Joiner ◽  
Meghan E. Breen ◽  
James Clayton ◽  
Anna K. Mapp
Keyword(s):  
Amino Acid ◽  
Protein Interactions ◽  
Protein Protein Interactions

scholarly journals The C-terminal Domain Is the Primary Determinant of Histone H1 Binding to Chromatinin Vivo

2004 ◽  
Vol 279 (19) ◽  
pp. 20028-20034 ◽  
Author(s):  
Michael J. Hendzel ◽  
Melody A. Lever ◽  
Ellen Crawford ◽  
John P. H. Th'ng
Keyword(s):  
Amino Acid ◽  
Fluorescent Protein ◽  
Point Mutations ◽  
Histone H1 ◽  
Single Amino Acid ◽  
Kinetic Measurements ◽  
Terminal Domain ◽  
C Terminus ◽  
Green Fluorescent

We have used a combination of kinetic measurements and targeted mutations to show that the C-terminal domain is required for high-affinity binding of histone H1 to chromatin, and phosphorylations can disrupt binding by affecting the secondary structure of the C terminus. By measuring the fluorescence recovery after photo-bleaching profiles of green fluorescent protein-histone H1 proteins in living cells, we find that the deletion of the N terminus only modestly reduces binding affinity. Deletion of the C terminus, however, almost completely eliminates histone H1.1 binding. Specific mutations of the C-terminal domain identified Thr-152 and Ser-183 as novel regulatory switches that control the binding of histone H1.1in vivo. It is remarkable that the single amino acid substitution of Thr-152 with glutamic acid was almost as effective as the truncation of the C terminus to amino acid 151 in destabilizing histone H1.1 bindingin vivo. We found that modifications to the C terminus can affect histone H1 binding dramatically but have little or no influence on the charge distribution or the overall net charge of this domain. A comparison of individual point mutations and deletion mutants, when reviewed collectively, cannot be reconciled with simple charge-dependent mechanisms of C-terminal domain function of linker histones.

scholarly journals Mutational Asymmetries in the SARS-CoV-2 Genome May Lead to Increased Hydrophobicity of Virus Proteins

Genes ◽  
2021 ◽  
Vol 12 (6) ◽  
pp. 826
Author(s):  
Roman Matyášek ◽  
Kateřina Řehůřková ◽  
Kristýna Berta Marošiová ◽  
Aleš Kovařík
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Protein Interactions ◽  
Amino Acid Level ◽  
Biochemical Properties ◽  
Genomic Diversity ◽  
Protein Protein Interactions ◽  
Data Set ◽  
Virion Assembly ◽  
Codon Composition

The genomic diversity of SARS-CoV-2 has been a focus during the ongoing COVID-19 pandemic. Here, we analyzed the distribution and character of emerging mutations in a data set comprising more than 95,000 virus genomes covering eight major SARS-CoV-2 lineages in the GISAID database, including genotypes arising during COVID-19 therapy. Globally, the C>U transitions and G>U transversions were the most represented mutations, accounting for the majority of single-nucleotide variations. Mutational spectra were not influenced by the time the virus had been circulating in its host or medical treatment. At the amino acid level, we observed about a 2-fold excess of substitutions in favor of hydrophobic amino acids over the reverse. However, most mutations constituting variants of interests of the S-protein (spike) lead to hydrophilic amino acids, counteracting the global trend. The C>U and G>U substitutions altered codons towards increased amino acid hydrophobicity values in more than 80% of cases. The bias is explained by the existing differences in the codon composition for amino acids bearing contrasting biochemical properties. Mutation asymmetries apparently influence the biochemical features of SARS CoV-2 proteins, which may impact protein–protein interactions, fusion of viral and cellular membranes, and virion assembly.

scholarly journals Coordination of RNA and protein condensation by the P granule protein MEG-3

2020 ◽  
Author(s):  
Helen Schmidt ◽  
Andrea Putnam ◽  
Dominique Rasoloson ◽  
Geraldine Seydoux
Keyword(s):  
Protein Interactions ◽  
Intrinsically Disordered Protein ◽  
Protein Protein Interactions ◽  
C Elegans ◽  
Intrinsically Disordered ◽  
Disordered Protein ◽  
C Terminus ◽  
Necessary And Sufficient

ABSTRACTGerm granules are RNA-protein condensates in germ cells. The mechanisms that drive germ granule assembly are not fully understood. MEG-3 is an intrinsically-disordered protein required for germ (P) granule assembly in C. elegans. MEG-3 forms gel-like condensates on liquid condensates assembled by PGL proteins. MEG-3 is related to the GCNA family and contains an N-terminal disordered region (IDR) and a predicted ordered C-terminus featuring an HMG-like motif (HMGL). Using in vitro and in vivo experiments, we find the MEG-3 C-terminus is necessary and sufficient to build MEG-3/PGL co-condensates independent of RNA. The HMGL domain is required for high affinity MEG-3/PGL binding in vitro and for assembly of MEG-3/PGL co-condensates in vivo. The MEG-3 IDR binds RNA in vitro and is required but not sufficient to recruit RNA to P granules. Our findings suggest that P granule assembly depends in part on protein-protein interactions that drive condensation independent of RNA.

Sign in / Sign up

Export Citation Format

Share Document