scholarly journals The Glycoprotein Cytoplasmic Tail of Uukuniemi Virus (Bunyaviridae) Interacts with Ribonucleoproteins and Is Critical for Genome Packaging

2007 ◽  
Vol 81 (7) ◽  
pp. 3198-3205 ◽  
Author(s):  
Anna K. Överby ◽  
Ralf F. Pettersson ◽  
Etienne P. A. Neve
Keyword(s):  
Amino Acids ◽  
Protein Interactions ◽  
Mutational Analysis ◽  
Cytoplasmic Tail ◽  
Protein Protein Interactions ◽  
Genome Packaging ◽  
Glycoprotein Precursor ◽  
Glycoprotein G ◽  
Rescue System ◽  
Polymerase I

ABSTRACT We have analyzed the importance of specific amino acids in the cytoplasmic tail of the glycoprotein GN for packaging of ribonucleoproteins (RNPs) into virus-like particles (VLPs) of Uukuniemi virus (UUK virus), a member of the Bunyaviridae family. In order to study packaging, we added the GN/GC glycoprotein precursor (p110) to a polymerase I-driven minigenome rescue system to generate VLPs that are released into the supernatant. These particles can infect new cells, and reporter gene expression can be detected. To determine the role of UUK virus glycoproteins in RNP packaging, we performed an alanine scan of the glycoprotein GN cytoplasmic tail (amino acids 1 to 81). First, we discovered three regions in the tail (amino acids 21 to 25, 46 to 50, and 71 to 81) which are important for minigenome transfer by VLPs. Further mutational analysis identified four amino acids that were important for RNP packaging. These amino acids are essential for the binding of nucleoproteins and RNPs to the glycoprotein without affecting the morphology of the particles. No segment-specific interactions between the RNA and the cytoplasmic tail could be observed. We propose that VLP systems are useful tools for analyzing protein-protein interactions important for packaging of viral genome segments, assembly, and budding of other members of the Bunyaviridae family.

NPXY motifs control the recruitment of the alpha5beta1 integrin in focal adhesions independently of the association of talin with the beta1 chain

1997 ◽  
Vol 110 (12) ◽  
pp. 1421-1430 ◽  
Author(s):  
L. Vignoud ◽  
C. Albiges-Rizo ◽  
P. Frachet ◽  
M.R. Block
Keyword(s):  
Amino Acids ◽  
Protein Interactions ◽  
Focal Adhesions ◽  
Cytoplasmic Tail ◽  
Cytoplasmic Domain ◽  
Membrane Receptors ◽  
Sufficient Information ◽  
Protein Protein Interactions ◽  
Exact Function ◽  
Beta1 Subunit

With the exception of the divergent beta4 and beta8 chains, the integrin beta subunit cytoplasmic domains are short and highly conserved sequences. Consensus motifs are found among the different cytoplasmic beta chains. Experiments using chimeric receptors demonstrated that the 47 amino acids of the beta1 subunit cytoplasmic domain contain sufficient information to target integrins to adhesion plaques. Three clusters of amino acids, named cyto-1, cyto-2 and cyto-3, seem to contribute to this localization. Cyto-2 and cyto-3 exhibit NPXY motifs. At present, the exact function of these motifs remains unknown but it is likely that these sequences are involved in protein-protein interactions. Although NPXY motifs often act as internalization signals at the cytoplasmic tail of membrane receptors, our previous results showed that the two NPXY motifs are not responsible for the alpha5beta1 integrin endocytosis. Herein, we address the question of the role of the two highly conserved NPXY motifs found in the beta1 cytoplasmic domain, and which correspond to the conserved domains cyto-2 and cyto-3. We demonstrate that, within the integrin beta1 cytoplasmic tail, the two NPXY motifs are required for the recruitment of the integrin in focal adhesions. In addition, our results indicate that these two motifs control but do not belong to the talin-binding sites. Finally, the analysis of the phenotypes of NPXY mutants reveals that the interaction of talin with the beta1 cytosolic domain is not sufficient to target the integrins to focal adhesions.

scholarly journals Mapping of a Domain Required for Protein-Protein Interactions and Inhibitory Activity of a Helicobacter pylori Dominant-Negative VacA Mutant Protein

2006 ◽  
Vol 74 (4) ◽  
pp. 2093-2101 ◽  
Author(s):  
Victor J. Torres ◽  
Mark S. McClain ◽  
Timothy L. Cover
Keyword(s):  
Amino Acids ◽  
Helicobacter Pylori ◽  
Protein Interactions ◽  
Inhibitory Activity ◽  
Mammalian Cells ◽  
Mutational Analysis ◽  
Dominant Negative ◽  
Mutant Protein ◽  
Wild Type ◽  
Protein Protein Interactions

ABSTRACT The Helicobacter pylori VacA toxin is an 88-kDa secreted protein that causes multiple alterations in mammalian cells and is considered an important virulence factor in the pathogenesis of peptic ulcer disease and gastric cancer. We have shown previously that a VacA mutant protein lacking amino acids 6 to 27 (Δ6-27p88 VacA) is able to inhibit many activities of wild-type VacA in a dominant-negative manner. Analysis of a panel of C-terminally truncated Δ6-27p88 VacA proteins indicated that a fragment containing amino acids 1 to 478 (Δ6-27p48) exhibited a dominant-negative phenotype similar to that of the full-length Δ6-27p88 VacA protein. In contrast, a shorter VacA fragment lacking amino acids 6 to 27 (Δ6-27p33) did not exhibit detectable inhibitory activity. The Δ6-27p48 protein physically interacted with wild-type p88 VacA, whereas the Δ6-27p33 protein did not. Mutational analysis indicated that amino acids 351 to 360 are required for VacA protein-protein interactions and for dominant-negative inhibitory activity. The C-terminal portion (p55 domain) of wild-type p88 VacA could complement either Δ6-27p33 or Δ(6-27/351-360)p48, reconstituting dominant-negative inhibitory activity. Collectively, our data provide strong evidence that the inhibitory properties of dominant-negative VacA mutant proteins are dependent on interactions between the mutant VacA proteins and wild-type VacA, and they allow mapping of a domain involved in the formation of oligomeric VacA complexes.

Leucine periodicity of U2 small nuclear ribonucleoprotein particle (snRNP) A' protein is implicated in snRNP assembly via protein-protein interactions

1991 ◽  
Vol 11 (3) ◽  
pp. 1578-1589
Author(s):  
L D Fresco ◽  
D S Harper ◽  
J D Keene
Keyword(s):  
Protein Interactions ◽  
Leucine Zipper ◽  
Tandem Repeats ◽  
Mutational Analysis ◽  
Particle Density ◽  
Protein Protein Interactions ◽  
Small Nuclear Ribonucleoprotein ◽  
Cell Extracts ◽  
Associated Proteins ◽  
Nuclear Ribonucleoprotein

Recombinant A' protein could be reconstituted into U2 small nuclear ribonucleoprotein particles (snRNPs) upon addition to HeLa cell extracts as determined by coimmunoprecipitation and particle density; however, direct binding to U2 RNA could not be demonstrated except in the presence of the U2 snRNP B" protein. Mutational analysis indicated that a central core region of A' was required for particle reconstitution. This region consists of five tandem repeats of approximately 24 amino acids each that exhibit a periodicity of leucine and asparagine residues that is distinct from the leucine zipper. Similar leucine-rich (Leu-Leu motif) repeats are characteristic of a diverse array of soluble and membrane-associated proteins from yeasts to humans but have not been reported previously to reside in nuclear proteins. Several of these proteins, including Toll, chaoptin, RNase/angiogenin inhibitors, lutropin-choriogonadotropin receptor, carboxypeptidase N, adenylyl cyclase, CD14, and human immunodeficiency virus type 1 Rev, may be involved in protein-protein interactions. Our findings suggest that in cell extracts the Leu-Leu motif of A' is required for reconstitution with U2 snRNPs and perhaps with other components involved in splicing through protein-protein interactions.

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 Human U4/U6 snRNP Recycling Factor p110: Mutational Analysis Reveals the Function of the Tetratricopeptide Repeat Domain in Recycling

2004 ◽  
Vol 24 (17) ◽  
pp. 7392-7401 ◽  
Author(s):  
Jan Medenbach ◽  
Silke Schreiner ◽  
Sunbin Liu ◽  
Reinhard Lührmann ◽  
Albrecht Bindereif
Keyword(s):  
Amino Acids ◽  
Protein Interactions ◽  
Rna Binding ◽  
Mutational Analysis ◽  
Protein A ◽  
Tetratricopeptide Repeat ◽  
Terminal Sequence ◽  
Recognition Motifs ◽  
Tpr Domain

ABSTRACT After each spliceosome cycle, the U4 and U6 snRNAs are released separately and are recycled to the functional U4/U6 snRNP, requiring in the mammalian system the U6-specific RNA binding protein p110 (SART3). Its domain structure is made up of an extensive N-terminal domain with at least seven tetratricopeptide repeat (TPR) motifs, followed by two RNA recognition motifs (RRMs) and a highly conserved C-terminal sequence of 10 amino acids. Here we demonstrate under in vitro recycling conditions that U6-p110 is an essential splicing factor. Recycling activity requires both the RRMs and the TPR domain but not the highly conserved C-terminal sequence. For U6-specific RNA binding, the two RRMs with some flanking regions are sufficient. Yeast two-hybrid assays reveal that p110 interacts through its TPR domain with the U4/U6-specific 90K protein, indicating a specific role of the TPR domain in spliceosome recycling. On the 90K protein, a short internal region (amino acids 416 to 550) suffices for the interaction with p110. Together, these data suggest a model whereby p110 brings together U4 and U6 snRNAs through both RNA-protein and protein-protein interactions.

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.

scholarly journals Leucine periodicity of U2 small nuclear ribonucleoprotein particle (snRNP) A' protein is implicated in snRNP assembly via protein-protein interactions.

1991 ◽  
Vol 11 (3) ◽  
pp. 1578-1589 ◽  
Author(s):  
L D Fresco ◽  
D S Harper ◽  
J D Keene
Keyword(s):  
Protein Interactions ◽  
Leucine Zipper ◽  
Tandem Repeats ◽  
Mutational Analysis ◽  
Particle Density ◽  
Protein Protein Interactions ◽  
Small Nuclear Ribonucleoprotein ◽  
Cell Extracts ◽  
Associated Proteins ◽  
Nuclear Ribonucleoprotein

Recombinant A' protein could be reconstituted into U2 small nuclear ribonucleoprotein particles (snRNPs) upon addition to HeLa cell extracts as determined by coimmunoprecipitation and particle density; however, direct binding to U2 RNA could not be demonstrated except in the presence of the U2 snRNP B" protein. Mutational analysis indicated that a central core region of A' was required for particle reconstitution. This region consists of five tandem repeats of approximately 24 amino acids each that exhibit a periodicity of leucine and asparagine residues that is distinct from the leucine zipper. Similar leucine-rich (Leu-Leu motif) repeats are characteristic of a diverse array of soluble and membrane-associated proteins from yeasts to humans but have not been reported previously to reside in nuclear proteins. Several of these proteins, including Toll, chaoptin, RNase/angiogenin inhibitors, lutropin-choriogonadotropin receptor, carboxypeptidase N, adenylyl cyclase, CD14, and human immunodeficiency virus type 1 Rev, may be involved in protein-protein interactions. Our findings suggest that in cell extracts the Leu-Leu motif of A' is required for reconstitution with U2 snRNPs and perhaps with other components involved in splicing through protein-protein interactions.

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.

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