Characterization of Conformational Changes and Protein-Protein Interactions of Rod Photoreceptor Phosphodiesterase (PDE6)

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.

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Proteomic Characterization of Protein-Protein Interactions

Austin Biology ◽

10.26420/austinbiol.2021.1027 ◽

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.

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POT1-TPP1 differentially regulates telomerase via POT1 His266 and as a function of single-stranded telomere DNA length

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1905381116 ◽

2019 ◽

Vol 116 (47) ◽

pp. 23527-23533 ◽

Cited By ~ 9

Author(s):

Mengyuan Xu ◽

Janna Kiselar ◽

Tawna L. Whited ◽

Wilnelly Hernandez-Sanchez ◽

Derek J. Taylor

Keyword(s):

Conformational Changes ◽

Protein Interactions ◽

Environmental Changes ◽

Lymphocytic Leukemia ◽

Protein Protein Interactions ◽

Cellular Environment ◽

Multiple Protein ◽

Linear Chromosomes ◽

Hydroxyl Radical Footprinting ◽

Regulate Telomere Length

Telomeres cap the ends of linear chromosomes and terminate in a single-stranded DNA (ssDNA) overhang recognized by POT1-TPP1 heterodimers to help regulate telomere length homeostasis. Here hydroxyl radical footprinting coupled with mass spectrometry was employed to probe protein–protein interactions and conformational changes involved in the assembly of telomere ssDNA substrates of differing lengths bound by POT1-TPP1 heterodimers. Our data identified environmental changes surrounding residue histidine 266 of POT1 that were dependent on telomere ssDNA substrate length. We further determined that the chronic lymphocytic leukemia-associated H266L substitution significantly reduced POT1-TPP1 binding to short ssDNA substrates; however, it only moderately impaired the heterodimer binding to long ssDNA substrates containing multiple protein binding sites. Additionally, we identified a telomerase inhibitory role when several native POT1-TPP1 proteins coat physiologically relevant lengths of telomere ssDNA. This POT1-TPP1 complex-mediated inhibition of telomerase is abrogated in the context of the POT1 H266L mutation, which leads to telomere overextension in a malignant cellular environment.

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Quantitative analysis of the interactions between HIV-1 integrase and retroviral reverse transcriptases

Biochemical Journal ◽

10.1042/bj20071279 ◽

2008 ◽

Vol 412 (1) ◽

pp. 163-170 ◽

Cited By ~ 21

Author(s):

Alon Herschhorn ◽

Iris Oz-Gleenberg ◽

Amnon Hizi

Keyword(s):

Conformational Changes ◽

Protein Interactions ◽

Molecular Mechanisms ◽

Reaction Model ◽

Protein Protein Interactions ◽

Common Site ◽

Leukaemia Virus ◽

Reverse Transcriptases ◽

Interaction Kinetics ◽

Hiv 1

The RT (reverse transcriptase) of HIV-1 interacts with HIV-1 IN (integrase) and inhibits its enzymatic activities. However, the molecular mechanisms underling these interactions are not well understood. In order to study these mechanisms, we have analysed the interactions of HIV-1 IN with HIV-1 RT and with two other related RTs: those of HIV-2 and MLV (murine-leukaemia virus). All three RTs inhibited HIV-1 IN, albeit to a different extent, suggesting a common site of binding that could be slightly modified for each one of the studied RTs. Using surface plasmon resonance technology, which monitors direct protein–protein interactions, we performed kinetic analyses of the binding of HIV-1 IN to these three RTs and observed interesting binding patterns. The interaction of HIV-1 RT with HIV-1 IN was unique and followed a two-state reaction model. According to this model, the initial IN–RT complex formation was followed by a conformational change in the complex that led to an elevation of the total affinity between these two proteins. In contrast, HIV-2 and MLV RTs interacted with IN in a simple bi-molecular manner, without any apparent secondary conformational changes. Interestingly, HIV-1 and HIV-2 RTs were the most efficient inhibitors of HIV-1 IN activity, whereas HIV-1 and MLV RTs showed the highest affinity towards HIV-1 IN. These modes of direct protein interactions, along with the apparent rate constants calculated and the correlations of the interaction kinetics with the capacity of the RTs to inhibit IN activities, are all discussed.

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Molecular dynamics shows complex interplay and long-range effects of post-translational modifications in yeast protein interactions

PLoS Computational Biology ◽

10.1371/journal.pcbi.1008988 ◽

2021 ◽

Vol 17 (5) ◽

pp. e1008988

Author(s):

Nikolina ŠoŠtarić ◽

Vera van Noort

Keyword(s):

Molecular Dynamics ◽

Conformational Changes ◽

Protein Interactions ◽

Protein Structures ◽

Cellular Protein ◽

Free Energy Calculations ◽

Protein Protein Interactions ◽

Post Translational Modifications ◽

Protein Properties ◽

Dynamics Simulations

Post-translational modifications (PTMs) play a vital, yet often overlooked role in the living cells through modulation of protein properties, such as localization and affinity towards their interactors, thereby enabling quick adaptation to changing environmental conditions. We have previously benchmarked a computational framework for the prediction of PTMs’ effects on the stability of protein-protein interactions, which has molecular dynamics simulations followed by free energy calculations at its core. In the present work, we apply this framework to publicly available data on Saccharomyces cerevisiae protein structures and PTM sites, identified in both normal and stress conditions. We predict proteome-wide effects of acetylations and phosphorylations on protein-protein interactions and find that acetylations more frequently have locally stabilizing roles in protein interactions, while the opposite is true for phosphorylations. However, the overall impact of PTMs on protein-protein interactions is more complex than a simple sum of local changes caused by the introduction of PTMs and adds to our understanding of PTM cross-talk. We further use the obtained data to calculate the conformational changes brought about by PTMs. Finally, conservation of the analyzed PTM residues in orthologues shows that some predictions for yeast proteins will be mirrored to other organisms, including human. This work, therefore, contributes to our overall understanding of the modulation of the cellular protein interaction networks in yeast and beyond.

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Identification and Characterization of Protein‐Protein Interactions between Yeast Rap1p and the TFIID Complex

The FASEB Journal ◽

10.1096/fasebj.22.1_supplement.782.20 ◽

2008 ◽

Vol 22 (S1) ◽

Author(s):

Justin Harrison Layer ◽

Peter Anthony Weil

Keyword(s):

Protein Interactions ◽

Protein Protein Interactions ◽

Tfiid Complex ◽

Identification And Characterization

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

10.32920/ryerson.14668284.v1 ◽

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.

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

Biochemical Journal ◽

10.1042/bj20051664 ◽

2006 ◽

Vol 398 (1) ◽

pp. 63-71 ◽

Cited By ~ 61

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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