Understanding the interaction of 14-3-3 proteins with hDMX and hDM2: a structural and biophysical study

p53 plays a critical role in regulating diverse biological processes: DNA repair, cell cycle arrest, apoptosis, and senescence. The p53 pathway has therefore served as the focus for drug-discovery efforts. p53 is negatively regulated by hDMX and hDM2; prior studies have identified 14-3-3 proteins as hDMX and hDM2 client proteins. 14-3-3 proteins are adaptor proteins that modulate localisation, degradation and interactions of their targets in response to phosphorylation. Thus 14-3-3 proteins may indirectly modulate the interaction between hDMX or hDM2 and p53 and represent potential targets for modulation of the p53 pathway. In this manuscript we report on the biophysical and structural characterization of peptide/protein interactions that are representative of the interaction between 14-3-3 and hDMX or hDM2. The data establish that proximal phosphosites spaced ~20-25 residues apart in both hDMX and hDM2 co-operate to facilitate high-affinity 14-3-3 binding and provide structural insight that can be utilized in future stabilizer/inhibitor discovery efforts.

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Exploring Proteomic Drug Targets, Therapeutic Strategies and Protein - Protein Interactions in Cancer: Mechanistic View

Current Cancer Drug Targets ◽

10.2174/1568009618666180803104631 ◽

2019 ◽

Vol 19 (6) ◽

pp. 430-448 ◽

Cited By ~ 1

Author(s):

Khalid Bashir Dar ◽

Aashiq Hussain Bhat ◽

Shajrul Amin ◽

Syed Anjum ◽

Bilal Ahmad Reshi ◽

...

Keyword(s):

Protein Interactions ◽

High Throughput Screening ◽

Critical Role ◽

Cancer Therapeutics ◽

Adaptor Proteins ◽

Therapeutic Strategies ◽

Small Gtpases ◽

Beta Catenin ◽

Protein Protein Interactions ◽

Apoptosis Protein

Protein-Protein Interactions (PPIs) drive major signalling cascades and play critical role in cell proliferation, apoptosis, angiogenesis and trafficking. Deregulated PPIs are implicated in multiple malignancies and represent the critical targets for treating cancer. Herein, we discuss the key protein-protein interacting domains implicated in cancer notably PDZ, SH2, SH3, LIM, PTB, SAM and PH. These domains are present in numerous enzymes/kinases, growth factors, transcription factors, adaptor proteins, receptors and scaffolding proteins and thus represent essential sites for targeting cancer. This review explores the candidature of various proteins involved in cellular trafficking (small GTPases, molecular motors, matrix-degrading enzymes, integrin), transcription (p53, cMyc), signalling (membrane receptor proteins), angiogenesis (VEGFs) and apoptosis (BCL-2family), which could possibly serve as targets for developing effective anti-cancer regimen. Interactions between Ras/Raf; X-linked inhibitor of apoptosis protein (XIAP)/second mitochondria-derived activator of caspases (Smac/DIABLO); Frizzled (FRZ)/Dishevelled (DVL) protein; beta-catenin/T Cell Factor (TCF) have also been studied as prospective anticancer targets. Efficacy of diverse molecules/ drugs targeting such PPIs although evaluated in various animal models/cell lines, there is an essential need for human-based clinical trials. Therapeutic strategies like the use of biologicals, high throughput screening (HTS) and fragment-based technology could play an imperative role in designing cancer therapeutics. Moreover, bioinformatic/computational strategies based on genome sequence, protein sequence/structure and domain data could serve as competent tools for predicting PPIs. Exploring hot spots in proteomic networks represents another approach for developing targetspecific therapeutics. Overall, this review lays emphasis on a productive amalgamation of proteomics, genomics, biochemistry, and molecular dynamics for successful treatment of cancer.

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Characterization of SMG7 14-3-3-like domain reveals phosphoserine binding-independent regulation of p53 and UPF1

Scientific Reports ◽

10.1038/s41598-019-49229-3 ◽

2019 ◽

Vol 9 (1) ◽

Author(s):

Lauren E. Cowen ◽

Hongwei Luo ◽

Yi Tang

Keyword(s):

Dna Damage ◽

Protein Interactions ◽

Cell Growth Arrest ◽

Damage Response ◽

Nonsense Mediated Mrna Decay ◽

Dependent Cell ◽

Dependent Protein ◽

Client Proteins

Abstract The 14-3-3-related protein SMG7 plays critical roles in regulation of DNA damage response and nonsense-mediated mRNA decay (NMD). Like 14-3-3, SMG7 engages phosphoserine-dependent protein interactions; however, the precise role of phosphorylation-mediated SMG7 binding remains unknown. Here, we show that DNA damage-induced SMG7-p53 binding requires phosphorylated Ser15 on p53, and that substitution of the conserved lysine residue K66 in the SMG7 14-3-3-like domain with the glutamic acid (E) abolishes interactions with its client proteins p53 and UPF1. Unexpectedly, loss of phosphoserine-dependent SMG7 binding does not significantly affect p53 stabilization/activation, and p53-dependent cell growth arrest or apoptosis upon DNA damage. Also surprisingly, cells expressing the SMG7 K66E-knockin mutant retain fully functional UPF1-mediated NMD. These findings are highly unusual, given that phosphorylation-mediated 14-3-3 binding has essential roles in numerous cellular signaling pathways. Thus, our studies suggest that 14-3-3-like proteins such as SMG7 likely function using additional distinct regulatory mechanisms besides phosphoserine-mediated protein interactions.

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Unbiased dynamic characterization of RNA-protein interactions by OOPS

10.1101/333336 ◽

2018 ◽

Cited By ~ 3

Author(s):

Rayner M. L. Queiroz ◽

Tom Smith ◽

Eneko Villanueva ◽

Mie Monti ◽

Mariavittoria Pizzinga ◽

...

Keyword(s):

Protein Interactions ◽

Mammalian Cells ◽

Rna Binding ◽

Rna Binding Proteins ◽

High Yield ◽

Cycle Arrest ◽

Bacterial Rna ◽

Unbiased Manner ◽

Cellular Compartments

AbstractCurrent methods for the identification of RNA–protein interactions require a quantity and quality of sample that hinders their application, especially for dynamic biological systems or when sample material is limiting. Here, we present a new approach to enrich RNA-Binding Proteins (RBPs): Orthogonal Organic Phase Separation (OOPS), which is compatible with downstream proteomics and RNA sequencing. OOPS enables recovery of RBPs and free protein, or protein-bound RNA and free RNA, from a single sample in an unbiased manner. By applying OOPS to human cell lines, we extract the majority of known RBPs, and importantly identify additional novel RBPs, including those from previously under-represented cellular compartments. The high yield and unbiased nature of OOPS facilitates its application in both dynamic and inaccessible systems. Thus, we have identified changes in RNA-protein interactions in mammalian cells following nocodazole cell-cycle arrest, and defined the first bacterial RNA-interactome. Overall, OOPS provides an easy-to-use and flexible technique that opens new opportunities to characterize RNA-protein interactions and explore their dynamic behaviour.

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A Novel Combined Scientific and Artistic Approach for the Advanced Characterization of Interactomes: The Akirin/Subolesin Model

Vaccines ◽

10.3390/vaccines8010077 ◽

2020 ◽

Vol 8 (1) ◽

pp. 77 ◽

Cited By ~ 6

Author(s):

Sara Artigas-Jerónimo ◽

Juan Pastor Comín ◽

Margarita Villar ◽

Marinela Contreras ◽

Pilar Alberdi ◽

...

Keyword(s):

Protein Interactions ◽

Functional Characterization ◽

Advanced Characterization ◽

Research Area ◽

Dependent Manner ◽

Biological Processes ◽

Visual Artists ◽

Protective Antigens ◽

Specific Regulation

The main objective of this study was to propose a novel methodology to approach challenges in molecular biology. Akirin/Subolesin (AKR/SUB) are vaccine protective antigens and are a model for the study of the interactome due to its conserved function in the regulation of different biological processes such as immunity and development throughout the metazoan. Herein, three visual artists and a music professor collaborated with scientists for the functional characterization of the AKR2 interactome in the regulation of the NF-κB pathway in human placenta cells. The results served as a methodological proof-of-concept to advance this research area. The results showed new perspectives on unexplored characteristics of AKR2 with functional implications. These results included protein dimerization, the physical interactions with different proteins simultaneously to regulate various biological processes defined by cell type-specific AKR–protein interactions, and how these interactions positively or negatively regulate the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway in a biological context-dependent manner. These results suggested that AKR2-interacting proteins might constitute suitable secondary transcription factors for cell- and stimulus-specific regulation of NF-κB. Musical perspective supported AKR/SUB evolutionary conservation in different species and provided new mechanistic insights into the AKR2 interactome. The combined scientific and artistic perspectives resulted in a multidisciplinary approach, advancing our knowledge on AKR/SUB interactome, and provided new insights into the function of AKR2–protein interactions in the regulation of the NF-κB pathway. Additionally, herein we proposed an algorithm for quantum vaccinomics by focusing on the model proteins AKR/SUB.

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Structural Insights into Ankyrin Repeat-Containing Proteins and Their Influence in Ubiquitylation

International Journal of Molecular Sciences ◽

10.3390/ijms22020609 ◽

2021 ◽

Vol 22 (2) ◽

pp. 609

Author(s):

Emma I. Kane ◽

Donald E. Spratt

Keyword(s):

Protein Interactions ◽

Neurological Diseases ◽

Critical Role ◽

Specific Protein ◽

Ankyrin Repeat ◽

Biological Processes ◽

Protein Protein Interactions ◽

Substrate Modification ◽

Eukaryotic Proteins ◽

Structural Insights

Ankyrin repeat (AR) domains are considered the most abundant repeat motif found in eukaryotic proteins. AR domains are predominantly known to mediate specific protein–protein interactions (PPIs) without necessarily recognizing specific primary sequences, nor requiring strict conformity within its own primary sequence. This promiscuity allows for one AR domain to recognize and bind to a variety of intracellular substrates, suggesting that AR-containing proteins may be involved in a wide array of functions. Many AR-containing proteins serve a critical role in biological processes including the ubiquitylation signaling pathway (USP). There is also strong evidence that AR-containing protein malfunction are associated with several neurological diseases and disorders. In this review, the structure and mechanism of key AR-containing proteins are discussed to suggest and/or identify how each protein utilizes their AR domains to support ubiquitylation and the cascading pathways that follow upon substrate modification.

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The p53 Family: A Role in Lipid and Iron Metabolism

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.715974 ◽

2021 ◽

Vol 9 ◽

Author(s):

Kyra Laubach ◽

Jin Zhang ◽

Xinbin Chen

Keyword(s):

Cell Cycle ◽

Cell Cycle Arrest ◽

Iron Metabolism ◽

Tumor Suppressors ◽

Tumor Suppression ◽

Critical Role ◽

Biological Processes ◽

P53 Family ◽

Cycle Arrest

The p53 family of tumor suppressors, which includes p53, p63, and p73, has a critical role in many biological processes, such as cell cycle arrest, apoptosis, and differentiation. In addition to tumor suppression, the p53 family proteins also participate in development, multiciliogenesis, and fertility, indicating these proteins have diverse roles. In this review, we strive to cover the relevant studies that demonstrate the roles of p53, p63, and p73 in lipid and iron metabolism.

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Molecular characterization of pear 14-3-3b gene regulated during fruit development

Canadian Journal of Plant Science ◽

10.1139/cjps-2015-0319 ◽

2016 ◽

Vol 96 (3) ◽

pp. 433-438

Author(s):

Haiyan Shi ◽

Yujing Zhao ◽

Xuemin An ◽

Yuxing Zhang

Keyword(s):

Protein Interactions ◽

Pcr Amplification ◽

Pcr Analysis ◽

Pyrus Pyrifolia ◽

Biological Processes ◽

Protein Protein Interactions ◽

Rt Pcr ◽

Developmentally Regulated ◽

Amplification Technique

Plant 14-3-3 proteins (14-3-3s) are known to function in protein–protein interactions that mediate signal transduction pathways regulating many biological processes. The cDNA encoding putative 14-3-3 protein was isolated from pear (Pyrus pyrifolia) and designated Pp14-3-3b. Using the PCR amplification technique, the genomic clone corresponding to Pp14-3-3b was isolated and shown to contain six introns. Phylogenetic analysis clearly demonstrated that Pp14-3-3b was classified into the non-ɛ class of 14-3-3 superfamilies. Quantitative RT-PCR analysis indicated that the expression of the Pp14-3-3b gene was developmentally regulated in fruit. This study suggested that Pp14-3-3b might be involved in fruit ripening and the senescence of pear.

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Characterization of Protein-Protein Interactions Critical for Poliovirus Replication: Analysis of 3AB and VPg Binding to the RNA-Dependent RNA Polymerase

Journal of Virology ◽

10.1128/jvi.02252-06 ◽

2007 ◽

Vol 81 (12) ◽

pp. 6369-6378 ◽

Cited By ~ 13

Author(s):

Daniel M. Strauss ◽

Deborah S. Wuttke

Keyword(s):

Rna Polymerase ◽

Protein Interactions ◽

Hot Spot ◽

Critical Role ◽

Rna Replication ◽

Titration Calorimetry ◽

Binding Interaction ◽

Rna Dependent Rna Polymerase ◽

Replication Complex

ABSTRACT Two critical interactions within the poliovirus RNA replication complex are those of the RNA-dependent RNA polymerase 3D with the viral proteins 3AB and VPg. 3AB is a membrane-binding protein responsible for the localization of the polymerase to the membranous vesicles at which replication occurs. VPg (a peptide comprising the 3B region of 3AB) is the 22-residue soluble product of 3AB cleavage and serves as the protein primer for RNA replication. The detailed interactions of these proteins with the RNA-dependent RNA polymerase 3D were analyzed to elucidate the precise roles of 3AB and VPg in the viral RNA replication complex. Using a membrane-based pull-down assay, we have identified a binding “hot-spot” spanning residues 100 to 104 in the 3B (VPg) region of 3AB which plays a critical role in mediating the interaction of 3AB with the polymerase. Isothermal titration calorimetry shows that the interaction of VPg with 3D is enthalpically driven, with a dissociation constant of 11 μM. Mutational analyses of VPg indicate that a subset of the residues important for 3AB-3D binding are also important for VPg-3D binding. Two residues in particular, P14 and R17, were shown to be absolutely critical for the binding interaction. This work provides the direct characterization of two binding interactions critical for the replication of this important class of viruses and identifies a conserved polymerase binding sequence responsible for targeting the polymerase.

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