The Ras Recruitment System (RRS) for the Identification and Characterization of Protein–Protein Interactions

2018 ◽  
pp. 61-73 ◽  
Author(s):  
Ami Aronheim
Keyword(s):  
Protein Interactions ◽  
Protein Protein Interactions ◽  
Recruitment System ◽  
Identification And Characterization

scholarly journals Early Intermediates of mariner Transposition: Catalysis without Synapsis of the Transposon Ends Suggests a Novel Architecture of the Synaptic Complex

2004 ◽  
Vol 24 (18) ◽  
pp. 8301-8311 ◽  
Author(s):  
Karen Lipkow ◽  
Nicolas Buisine ◽  
David J. Lampe ◽  
Ronald Chalmers
Keyword(s):  
Protein Interactions ◽  
Reaction Mechanisms ◽  
Protein Protein Interactions ◽  
Dna Protection ◽  
Synaptic Complex ◽  
Insertion Elements ◽  
Identification And Characterization

ABSTRACT The mariner family is probably the most widely distributed family of transposons in nature. Although these transposons are related to the well-studied bacterial insertion elements, there is evidence for major differences in their reaction mechanisms. We report the identification and characterization of complexes that contain the Himar1 transposase bound to a single transposon end. Titrations and mixing experiments with the native transposase and transposase fusions suggested that they contain different numbers of transposase monomers. However, the DNA protection footprints of the two most abundant single-end complexes are identical. This indicates that some transposase monomers may be bound to the transposon end solely by protein-protein interactions. This would mean that the Himar1 transposase can dimerize independently of the second transposon end and that the architecture of the synaptic complex has more in common with V(D)J recombination than with bacterial insertion elements. Like V(D)J recombination and in contrast to the case for bacterial elements, Himar1 catalysis does not appear to depend on synapsis of the transposon ends, and the single-end complexes are active for nicking and probably for cleavage. We discuss the role of this single-end activity in generating the mutations that inactivate the vast majority of mariner elements in eukaryotes.

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 TULP1 and TUB Are Required for Specific Localization of PRCD to Photoreceptor Outer Segments

2020 ◽  
Vol 21 (22) ◽  
pp. 8677
Author(s):  
Lital Remez ◽  
Ben Cohen ◽  
Mariela J. Nevet ◽  
Leah Rizel ◽  
Tamar Ben-Yosef
Keyword(s):  
Protein Interactions ◽  
Mammalian Cells ◽  
Outer Segment ◽  
Protein Protein Interactions ◽  
Photoreceptor Outer Segment ◽  
Photoreceptor Outer Segments ◽  
Outer Segments ◽  
Yeast Two Hybrid System ◽  
Specific Localization ◽  
Recruitment System

Photoreceptor disc component (PRCD) is a small protein which is exclusively localized to photoreceptor outer segments, and is involved in the formation of photoreceptor outer segment discs. Mutations in PRCD are associated with retinal degeneration in humans, mice, and dogs. The purpose of this work was to identify PRCD-binding proteins in the retina. PRCD protein-protein interactions were identified when implementing the Ras recruitment system (RRS), a cytoplasmic-based yeast two-hybrid system, on a bovine retina cDNA library. An interaction between PRCD and tubby-like protein 1 (TULP1) was identified. Co-immunoprecipitation in transfected mammalian cells confirmed that PRCD interacts with TULP1, as well as with its homolog, TUB. These interactions were mediated by TULP1 and TUB highly conserved C-terminal tubby domain. PRCD localization was altered in the retinas of TULP1- and TUB-deficient mice. These results show that TULP1 and TUB, which are involved in the vesicular trafficking of several photoreceptor proteins from the inner segment to the outer segment, are also required for PRCD exclusive localization to photoreceptor outer segment discs.

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 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.

The Discovery and Characterization of Protein-Protein Interactions

2010 ◽  
pp. 1-21
Author(s):  
C. W. Bertoncini ◽  
A. Higueruelo ◽  
X. Salvatella
Keyword(s):  
Protein Interactions ◽  
Protein Protein Interactions
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