scholarly journals Analysis of protein–protein interactions in the feline calicivirus replication complex

2006 ◽  
Vol 87 (2) ◽  
pp. 363-368 ◽  
Author(s):  
William J. Kaiser ◽  
Yasmin Chaudhry ◽  
Stanislav V. Sosnovtsev ◽  
Ian G. Goodfellow
Keyword(s):  
Capsid Protein ◽  
Protein Interactions ◽  
Feline Calicivirus ◽  
Protein Protein Interactions ◽  
Replication Complex ◽  
Orf2 Protein ◽  
The Family ◽  
Capsid Protein Vp1 ◽  
The Individual

Caliciviruses are a major cause of gastroenteritis in humans and cause a wide variety of other diseases in animals. Here, the characterization of protein–protein interactions between the individual proteins of Feline calicivirus (FCV), a model system for other members of the family Caliciviridae, is reported. Using the yeast two-hybrid system combined with a number of other approaches, it is demonstrated that the p32 protein (the picornavirus 2B analogue) of FCV interacts with p39 (2C), p30 (3A) and p76 (3CD). The FCV protease/RNA polymerase (ProPol) p76 was found to form homo-oligomers, as well as to interact with VPg and ORF2, the region encoding the major capsid protein VP1. A weak interaction was also observed between p76 and the minor capsid protein encoded by ORF3 (VP2). ORF2 protein was found to interact with VPg, p76 and VP2. The potential roles of the interactions in calicivirus replication are discussed.

scholarly journals Characterization of Nonstructural Protein Membrane Anchor Deletion Mutants Expressed in the Context of the Hepatitis C Virus Polyprotein

2005 ◽  
Vol 79 (12) ◽  
pp. 7911-7917 ◽  
Author(s):  
Rainer Gosert ◽  
Wiebke Jendrsczok ◽  
Jan Martin Berke ◽  
Volker Brass ◽  
Hubert E. Blum ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Protein Interactions ◽  
Nonstructural Protein ◽  
Protein Protein Interactions ◽  
Nonstructural Proteins ◽  
Membrane Anchor ◽  
Replication Complex ◽  
Cytosolic Domains

ABSTRACT Protein-protein interactions involved in formation of the membrane-associated hepatitis C virus (HCV) replication complex are poorly understood. Here, we investigated nonstructural proteins with deletions in their membrane anchor domains when expressed in the context of the entire HCV polyprotein. Interactions among cytosolic domains of HCV nonstructural proteins were found not to be sufficiently strong to rescue such mutants to the membrane. Thus, the membrane anchor domains of nonstructural proteins are essential for incorporation of these proteins into the HCV replication complex while interactions among the cytosolic domains appear to be relatively weak. This feature may provide the nonstructural proteins with a certain flexibility to perform their multiple functions during HCV replication.

scholarly journals Genetic and Molecular Characterization of the Caenorhabditis elegans Gene, mel-26, a Postmeiotic Negative Regulator of MEI-1, a Meiotic-Specific Spindle Component

Genetics ◽  
1998 ◽  
Vol 150 (1) ◽  
pp. 119-128
Author(s):  
M Rhys Dow ◽  
Paul E Mains
Keyword(s):  
Caenorhabditis Elegans ◽  
Protein Interactions ◽  
Negative Regulator ◽  
Meiotic Spindle ◽  
Loss Of Function ◽  
Protein Protein Interactions ◽  
Gain Of Function ◽  
Recessive Mutations ◽  
Female Germline

Abstract We have previously described the gene mei-1, which encodes an essential component of the Caenorhabditis elegans meiotic spindle. When ectopically expressed after the completion of meiosis, mei-1 protein disrupts the function of the mitotic cleavage spindles. In this article, we describe the cloning and the further genetic characterization of mel-26, a postmeiotic negative regulator of mei-1. mel-26 was originally identified by a gain-of-function mutation. We have reverted this mutation to a loss-of-function allele, which has recessive phenotypes identical to the dominant defects of its gain-of-function parent. Both the dominant and recessive mutations of mel-26 result in mei-1 protein ectopically localized in mitotic spindles and centrosomes, leading to small and misoriented cleavage spindles. The loss-of-function mutation was used to clone mel-26 by transformation rescue. As suggested by genetic results indicating that mel-26 is required only maternally, mel-26 mRNA was expressed predominantly in the female germline. The gene encodes a protein that includes the BTB motif, which is thought to play a role in protein-protein interactions.

Proteomic Characterization of Protein-Protein Interactions

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Veenstra TD ◽  
Keyword(s):  
Protein Interactions ◽  
Disease Diagnosis ◽  
Cell System ◽  
Protein Protein Interactions ◽  
Novel Technologies ◽  
Cell Functions ◽  
Single Protein ◽  
A Cell ◽  
Molecular Components

Identifying all the molecular components within a living cell is the first step into understanding how it functions. To further understand how a cell functions requires identifying the interactions that occur between these components. This fact is especially relevant for proteins. No protein within a human cell functions on its own without interacting with another biomolecule - usually another protein. While Protein-Protein Interactions (PPI) have historically been determined by examining a single protein per study, novel technologies developed over the past couple of decades are enabling high-throughput methods that aim to describe entire protein networks within cells. In this review, some of the technologies that have led to these developments are described along with applications of these techniques. Ultimately the goal of these technologies is to map out the entire circuitry of PPI within human cells to be able to predict the global consequences of perturbations to the cell system. This predictive capability will have major impacts on the future of both disease diagnosis and treatment.

scholarly journals Cytosolic localization and in vitro assembly of human de novo thymidylate synthesis complex

2020 ◽  
Author(s):  
Sharon Spizzichino ◽  
Dalila Boi ◽  
Giovanna Boumis ◽  
Roberta Lucchi ◽  
Francesca R. Liberati ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Protein Interactions ◽  
De Novo ◽  
Proximity Ligation Assay ◽  
Protein Protein Interactions ◽  
Aggregation State ◽  
Entire Complex ◽  
The Individual ◽  
Thymidylate Synthesis

ABSTRACTDe novo thymidylate synthesis is a crucial pathway for normal and cancer cells. Deoxythymidine monophosphate (dTMP) is synthesized by the combined action of three enzymes: thymidylate synthase (TYMS), serine hydroxymethyltransferase (SHMT) and dihydrofolate reductase (DHFR), targets of widely used chemotherapeutics such as antifolates and 5-fluorouracil. These proteins translocate to the nucleus after SUMOylation and are suggested to assemble in this compartment into the thymidylate synthesis complex (dTMP-SC). We report the intracellular dynamics of the complex in lung cancer cells by in situ proximity ligation assay, showing that it is also detected in the cytoplasm. We have successfully assembled the dTMP synthesis complex in vitro, employing tetrameric SHMT1 and a bifunctional chimeric enzyme comprising human TYMS and DHFR. We show that the SHMT1 tetrameric state is required for efficient complex assembly, indicating that this aggregation state is evolutionary selected in eukaryotes to optimize protein-protein interactions. Lastly, our results on the activity of the complete thymidylate cycle in vitro, provide a useful tool to develop drugs targeting the entire complex instead of the individual components.

scholarly journals Characterization of major chromatin assembly proteins in tetrahymena thermophile using proeomic and molecular evolutionary analyses

2021 ◽  
Author(s):  
Syed N Shah
Keyword(s):  
Protein Interactions ◽  
Protein Complexes ◽  
Affinity Purification ◽  
Regulation Of Gene Expression ◽  
Chromatin Assembly ◽  
Histone Chaperones ◽  
Protein Protein Interactions ◽  
Selective Forces ◽  
Assembly Proteins

Histones H3/H4 are deposited onto DNA in a replication-dependent or independent fashion by the CAF1 and HIRA protein complexes. Despite the identification of these protein complexes, mechanistic details remain unclear. Recently, we showed that in T. thermophila histone chaperones Nrp1, Asf1 and the Impβ6 importin function together to transport newly synthesized H3/H4 from the cytoplasm to the nucleus. To characterize chromatin assembly proteins in T.thermophila, I used affinity purification combined with mass spectrometry to identify protein-protein interactions of Nrp1, Cac2 subunit of CAF1, HIRA and histone modifying Hat1-complex in T. thermophila. I found that the three-subunit T.thermophila CAF1 complex interacts with Casein Kinase 2 (CKII), possibly accounting for previously reported human CAF1phosphorylation. I also found that Hat2 subunit of HAT1 complex is also shared by CAF1 complex as its Cac3 subunit. This suggests that Hat2/Cac3 might exist in two separate pools of protein complexes. Remarkably, proteomic analysis of Hat2/Cac3 in turn revealed that it forms several complexes with other proteins including SIN3, RXT3, LIN9 and TESMIN, all of which have known roles in the regulation of gene expression. Finally, I asked how selective forces might have impacted on the function of proteins involved in H3/H4 transport. Focusing on NASP which possesses several TPR motifs, I showed that its protein-protein interactions are conserved in T. thermophila. Using molecular evolutionary methods I show that different TPRs in NASP evolve at different rates possibly accounting for the functional diversity observed among different family members.

scholarly journals β-Subunit overexpression alters the stoicheometry of assembled Na-K-ATPase subunits in MDCK cells

2008 ◽  
Vol 295 (5) ◽  
pp. F1314-F1323 ◽  
Author(s):  
Rebecca J. Clifford ◽  
Jack H. Kaplan
Keyword(s):  
Protein Interactions ◽  
Mdck Cells ◽  
Β Subunit ◽  
Protein Protein Interactions ◽  
Cellular Mechanisms ◽  
Atpase Subunit ◽  
Subunit Expression ◽  
Atpase Subunits ◽  
Confocal Images ◽  
The Individual

In eukaryotic cells, the apparent maintenance of 1:1 stoicheometry between the Na-K-ATPase α- and β-subunits led us to question whether this was alterable and thus if some form of regulation was involved. We have examined the consequences of overexpressing Na-K-ATPase β1-subunits using Madin-Darby canine kidney (MDCK) cells expressing flag-tagged β1-subunits (β1flag) or Myc-tagged β1-subunits (β1myc) under the control of a tetracycline-dependent promoter. The induction of β1flag subunit synthesis in MDCK cells, which increases β1-subunit expression at the plasma membrane by more than twofold, while maintaining stable α1 expression levels, revealed that all mature β1-subunits associate with α1-subunits, and no evidence of “free” β1-subunits was obtained. Consequently, the ratio of assembled β1- to α1-subunits is significantly increased when “extra” β-subunits are expressed. An increased β1/α1 stoicheometry is also observed in cells treated with tunicamycin, suggesting that the protein-protein interactions involved in these complexes are not dependent on glycosylation. Confocal images of cocultured β1myc-expressing and β1flag-expressing MDCK cells show colocalization of β1myc and β1flag subunits at the lateral membranes of neighboring cells, suggesting the occurrence of intercellular interactions between the β-subunits. Immunoprecipitation using MDCK cells constitutively expressing β1myc and tetracycline-regulated β1flag subunits confirmed β-β-subunit interactions. These results demonstrate that the equimolar ratio of assembled β1/α1-subunits of the Na-K-ATPase in kidney cells is not fixed by the inherent properties of the interacting subunits. It is likely that cellular mechanisms are present that regulate the individual Na-K-ATPase subunit abundance.

scholarly journals Characterization of COMMD protein–protein interactions in NF-κB signalling

2006 ◽  
Vol 398 (1) ◽  
pp. 63-71 ◽  
Author(s):  
Prim de Bie ◽  
Bart van de Sluis ◽  
Ezra Burstein ◽  
Karen J. Duran ◽  
Ruud Berger ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Copper Metabolism ◽  
Nuclear Factor Κb ◽  
Inhibitory Effect ◽  
Protein Family ◽  
Amino Acid Residues ◽  
Protein Protein Interactions ◽  
Necrosis Factor

COMMD [copper metabolism gene MURR1 (mouse U2af1-rs1 region 1) domain] proteins constitute a recently identified family of NF-κB (nuclear factor κB)-inhibiting proteins, characterized by the presence of the COMM domain. In the present paper, we report detailed investigation of the role of this protein family, and specifically the role of the COMM domain, in NF-κB signalling through characterization of protein–protein interactions involving COMMD proteins. The small ubiquitously expressed COMMD6 consists primarily of the COMM domain. Therefore COMMD1 and COMMD6 were analysed further as prototype members of the COMMD protein family. Using specific antisera, interaction between endogenous COMMD1 and COMMD6 is described. This interaction was verified by independent techniques, appeared to be direct and could be detected throughout the whole cell, including the nucleus. Both proteins inhibit TNF (tumour necrosis factor)-induced NF-κB activation in a non-synergistic manner. Mutation of the amino acid residues Trp24 and Pro41 in the COMM domain of COMMD6 completely abolished the inhibitory effect of COMMD6 on TNF-induced NF-κB activation, but this was not accompanied by loss of interaction with COMMD1, COMMD6 or the NF-κB subunit RelA. In contrast with COMMD1, COMMD6 does not bind to IκBα (inhibitory κBα), indicating that both proteins inhibit NF-κB in an overlapping, but not completely similar, manner. Taken together, these data support the significance of COMMD protein–protein interactions and provide new mechanistic insight into the function of this protein family in NF-κB signalling.

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