scholarly journals Stability and sub-cellular localization of DNA polymerase β is regulated by interactions with NQO1 and XRCC1 in response to oxidative stress

2019 ◽  
Vol 47 (12) ◽  
pp. 6269-6286 ◽  
Author(s):  
Qingming Fang ◽  
Joel Andrews ◽  
Nidhi Sharma ◽  
Anna Wilk ◽  
Jennifer Clark ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Dna Polymerase ◽  
Protein Interactions ◽  
Cellular Localization ◽  
Somatic Mutations ◽  
Dependent Manner ◽  
Protein Protein Interactions ◽  
Dna Polymerase Β ◽  
Enzyme Active Site ◽  
The Stability

Abstract Protein–protein interactions regulate many essential enzymatic processes in the cell. Somatic mutations outside of an enzyme active site can therefore impact cellular function by disruption of critical protein–protein interactions. In our investigation of the cellular impact of the T304I cancer mutation of DNA Polymerase β (Polβ), we find that mutation of this surface threonine residue impacts critical Polβ protein–protein interactions. We show that proteasome-mediated degradation of Polβ is regulated by both ubiquitin-dependent and ubiquitin-independent processes via unique protein–protein interactions. The ubiquitin-independent proteasome pathway regulates the stability of Polβ in the cytosol via interaction between Polβ and NAD(P)H quinone dehydrogenase 1 (NQO1) in an NADH-dependent manner. Conversely, the interaction of Polβ with the scaffold protein X-ray repair cross complementing 1 (XRCC1) plays a role in the localization of Polβ to the nuclear compartment and regulates the stability of Polβ via a ubiquitin-dependent pathway. Further, we find that oxidative stress promotes the dissociation of the Polβ/NQO1 complex, enhancing the interaction of Polβ with XRCC1. Our results reveal that somatic mutations such as T304I in Polβ impact critical protein–protein interactions, altering the stability and sub-cellular localization of Polβ and providing mechanistic insight into how key protein–protein interactions regulate cellular responses to stress.

scholarly journals SET7/9-dependent methylation of ARTD1 at K508 stimulates poly-ADP-ribose formation after oxidative stress

Open Biology ◽  
2013 ◽  
Vol 3 (10) ◽  
pp. 120173 ◽  
Author(s):  
Ingrid Kassner ◽  
Anneli Andersson ◽  
Monika Fey ◽  
Martin Tomas ◽  
Elisa Ferrando-May ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Protein Interactions ◽  
Protein Function ◽  
Dependent Manner ◽  
Protein Protein Interactions ◽  
Post Translational Modification ◽  
Target Proteins ◽  
Protein Methyltransferase

ADP-ribosyltransferase diphtheria toxin-like 1 (ARTD1, formerly PARP1) is localized in the nucleus, where it ADP-ribosylates specific target proteins. The post-translational modification (PTM) with a single ADP-ribose unit or with polymeric ADP-ribose (PAR) chains regulates protein function as well as protein–protein interactions and is implicated in many biological processes and diseases. SET7/9 (Setd7, KMT7) is a protein methyltransferase that catalyses lysine monomethylation of histones, but also methylates many non-histone target proteins such as p53 or DNMT1. Here, we identify ARTD1 as a new SET7/9 target protein that is methylated at K508 in vitro and in vivo . ARTD1 auto-modification inhibits its methylation by SET7/9, while auto-poly-ADP-ribosylation is not impaired by prior methylation of ARTD1. Moreover, ARTD1 methylation by SET7/9 enhances the synthesis of PAR upon oxidative stress in vivo . Furthermore, laser irradiation-induced PAR formation and ARTD1 recruitment to sites of DNA damage in a SET7/9-dependent manner. Together, these results reveal a novel mechanism for the regulation of cellular ARTD1 activity by SET7/9 to assure efficient PAR formation upon cellular stress.

scholarly journals E. coli primase and DNA polymerase III holoenzyme are able to bind concurrently to a primed template during DNA replication

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Andrea Bogutzki ◽  
Natalie Naue ◽  
Lidia Litz ◽  
Andreas Pich ◽  
Ute Curth
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Protein Interactions ◽  
Dna Polymerase Iii ◽  
Protein Protein Interactions ◽  
Single Stranded Dna ◽  
E Coli ◽  
Polymerase Iii ◽  
C Terminus ◽  
Pol Iii

Abstract During DNA replication in E. coli, a switch between DnaG primase and DNA polymerase III holoenzyme (pol III) activities has to occur every time when the synthesis of a new Okazaki fragment starts. As both primase and the χ subunit of pol III interact with the highly conserved C-terminus of single-stranded DNA-binding protein (SSB), it had been proposed that the binding of both proteins to SSB is mutually exclusive. Using a replication system containing the origin of replication of the single-stranded DNA phage G4 (G4ori) saturated with SSB, we tested whether DnaG and pol III can bind concurrently to the primed template. We found that the addition of pol III does not lead to a displacement of primase, but to the formation of higher complexes. Even pol III-mediated primer elongation by one or several DNA nucleotides does not result in the dissociation of DnaG. About 10 nucleotides have to be added in order to displace one of the two primase molecules bound to SSB-saturated G4ori. The concurrent binding of primase and pol III is highly plausible, since even the SSB tetramer situated directly next to the 3′-terminus of the primer provides four C-termini for protein-protein interactions.

scholarly journals Identification and Biochemical Characterization of High Mobility Group Protein 20A as a Novel Ca2+/S100A6 Target

Biomolecules ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 510
Author(s):  
Maho Yamamoto ◽  
Rina Kondo ◽  
Haruka Hozumi ◽  
Seita Doi ◽  
Miwako Denda ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Biochemical Characterization ◽  
S100 Protein ◽  
High Mobility ◽  
Dependent Manner ◽  
Protein Signaling ◽  
Protein Protein Interactions ◽  
High Mobility Group Protein ◽  
Pulldown Assay

During screening of protein-protein interactions, using human protein arrays carrying 19,676 recombinant glutathione s-transferase (GST)-fused human proteins, we identified the high-mobility protein group 20A (HMG20A) as a novel S100A6 binding partner. We confirmed the Ca2+-dependent interaction of HMG20A with S100A6 by the protein array method, biotinylated S100A6 overlay, and GST-pulldown assay in vitro and in transfected COS-7 cells. Co-immunoprecipitation of S100A6 with HMG20A from HeLa cells in a Ca2+-dependent manner revealed the physiological relevance of the S100A6/HMG20A interaction. In addition, HMG20A has the ability to interact with S100A1, S100A2, and S100B in a Ca2+-dependent manner, but not with S100A4, A11, A12, and calmodulin. S100A6 binding experiments using various HMG20A mutants revealed that Ca2+/S100A6 interacts with the C-terminal region (residues 311–342) of HMG20A with stoichiometric binding (HMG20A:S100A6 dimer = 1:1). This was confirmed by the fact that a GST-HMG20A mutant lacking the S100A6 binding region (residues 311–347, HMG20A-ΔC) failed to interact with endogenous S100A6 in transfected COS-7 cells, unlike wild-type HMG20A. Taken together, these results identify, for the first time, HMG20A as a target of Ca2+/S100 proteins, and may suggest a novel linkage between Ca2+/S100 protein signaling and HMG20A function, including in the regulation of neural differentiation.

scholarly journals Crosstalk between β-catenin and WT1 signalling activity in acute myeloid leukemia

2021 ◽  
Author(s):  
Megan Payne ◽  
Olga Tsaponina ◽  
Gillian Caalim ◽  
Hayley Greenfield ◽  
Leanne Milton-Harris ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Myeloid Leukemia ◽  
Normal Development ◽  
Signal Transduction Pathway ◽  
Myeloid Cell ◽  
Wnt Signalling ◽  
Protein Protein Interactions ◽  
The Stability ◽  
First Time ◽  
Acute Myeloid

Wnt signalling is an evolutionary conserved signal transduction pathway heavily implicated in normal development and disease. The central mediator of this pathway, β-catenin, is frequently overexpressed, mislocalised and overactive in acute myeloid leukaemia (AML) where it mediates the establishment, maintenance and drug resistance of leukaemia stem cells. Critical to the stability, localisation and activity of β-catenin are the protein-protein interactions it forms, yet these are poorly defined in AML. We recently performed the first β-catenin interactome study in blood cells of any kind and identified a plethora of novel interacting partners. This study shows for the first time that β-catenin interacts with Wilms tumour protein (WT1), a protein frequently overexpressed and mutated in AML, in both myeloid cell lines and also primary AML samples. We demonstrate crosstalk between the signalling activity of these two proteins in myeloid cells, and show that modulation of either protein can affect expression of the other. Finally, we demonstrate that WT1 mutations frequently observed in AML can increase stabilise β-catenin and augment Wnt signalling output. This study has uncovered new context-dependent molecular interactions for β-catenin which could inform future therapeutic strategies to target this dysregulated molecule in AML.

scholarly journals The functional impact of alternative splicing in cancer

2016 ◽  
Author(s):  
Héctor Climente-González ◽  
Eduard Porta-Pardo ◽  
Adam Godzik ◽  
Eduardo Eyras
Keyword(s):  
Alternative Splicing ◽  
Negative Correlation ◽  
Protein Interactions ◽  
Somatic Mutations ◽  
Protein Domain ◽  
Protein Protein Interactions ◽  
Systematic Analysis ◽  
Functional Impact ◽  
Functional Consequences ◽  
Functional Transformations

SummaryAlternative splicing changes are frequently observed in cancer and are starting to be recognized as important signatures for tumor progression and therapy. However, their functional impact and relevance to tumorigenesis remains mostly unknown. We carried out a systematic analysis to characterize the potential functional consequences of alternative splicing changes in thousands of tumor samples. This analysis revealed that a subset of alternative splicing changes affect protein domain families that are frequently mutated in tumors and potentially disrupt protein protein interactions in cancer-related pathways. Moreover, there was a negative correlation between the number of these alternative splicing changes in a sample and the number of somatic mutations in drivers. We propose that a subset of the alternative splicing changes observed in tumors may represent independent oncogenic processes that could be relevant to explain the functional transformations in cancer and some of them could potentially be considered alternative splicing drivers (AS-drivers).

scholarly journals Analysis of IFITM-IFITM Interactions by a Flow Cytometry-Based FRET Assay

2019 ◽  
Vol 20 (16) ◽  
pp. 3859 ◽  
Author(s):  
Michael Winkler ◽  
Florian Wrensch ◽  
Pascale Bosch ◽  
Maike Knoth ◽  
Michael Schindler ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Protein Interactions ◽  
Viral Entry ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Protein Protein Interactions

The interferon-induced transmembrane proteins 1–3 (IFITM1–3) inhibit host cell entry of several viruses. However, it is incompletely understood how IFITM1–3 exert antiviral activity. Two phenylalanine residues, F75 and F78, within the intramembrane domain 1 (IM1) were previously shown to be required for IFITM3/IFITM3 interactions and for inhibition of viral entry, suggesting that IFITM/IFITM interactions might be pivotal to antiviral activity. Here, we employed a fluorescence resonance energy transfer (FRET) assay to analyze IFITM/IFITM interactions. For assay calibration, we equipped two cytosolic, non-interacting proteins, super yellow fluorescent protein (SYFP) and super cyan fluorescent protein (SCFP), with signals that target proteins to membrane rafts and also analyzed a SCFP-SYFP fusion protein. This strategy allowed us to discriminate background signals resulting from colocalization of proteins at membrane subdomains from signals elicited by protein–protein interactions. Coexpression of IFITM1–3 and IFITM5 fused to fluorescent proteins elicited strong FRET signals, and mutation of F75 and F78 in IFITM3 (mutant IFITM3-FF) abrogated antiviral activity, as expected, but did not alter cellular localization and FRET signals. Moreover, IFITM3-FF co-immunoprecipitated efficiently with wild type (wt) IFITM3, lending further support to the finding that lack of antiviral activity of IFITM3-FF was not due to altered membrane targeting or abrogated IFITM3-IFITM3 interactions. Collectively, we report an assay that allows quantifying IFITM/IFITM interactions. Moreover, we confirm residues F75 and F78 as critical for antiviral activity but also show that these residues are dispensable for IFITM3 membrane localization and IFITM3/IFITM3 interactions.

Carnoquinolines Target Copper Dyshomeostasis, Aberrant Protein–Protein Interactions, and Oxidative Stress

2020 ◽  
Vol 26 (70) ◽  
pp. 16690-16705
Author(s):  
Francesco Bellia ◽  
Giuseppa Ida Grasso ◽  
Ikhlas Mohamed Mohamud Ahmed ◽  
Valentina Oliveri ◽  
Graziella Vecchio
Keyword(s):  
Oxidative Stress ◽  
Protein Interactions ◽  
Protein Protein Interactions ◽  
Aberrant Protein ◽  
Copper Dyshomeostasis ◽  
And Oxidative Stress

scholarly journals The BH3 α-Helical Mimic BH3-M6 Disrupts Bcl-XL, Bcl-2, and MCL-1 Protein-Protein Interactions with Bax, Bak, Bad, or Bim and Induces Apoptosis in a Bax- and Bim-dependent Manner

2010 ◽  
Vol 286 (11) ◽  
pp. 9382-9392 ◽  
Author(s):  
Aslamuzzaman Kazi ◽  
Jiazhi Sun ◽  
Kenichiro Doi ◽  
Shen-Shu Sung ◽  
Yoshinori Takahashi ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Dependent Manner ◽  
Protein Protein Interactions

scholarly journals Targeting protein–protein interactions by rational design: mimicry of protein surfaces

2006 ◽  
Vol 3 (7) ◽  
pp. 215-233 ◽  
Author(s):  
Steven Fletcher ◽  
Andrew D Hamilton
Keyword(s):  
Protein Interactions ◽  
Active Site ◽  
Rational Design ◽  
Considerable Progress ◽  
Biological Processes ◽  
Protein Protein Interactions ◽  
Protein Surfaces ◽  
Enzyme Active Site ◽  
Novel Therapeutics ◽  
Interfacial Areas

Protein–protein interactions play key roles in a range of biological processes, and are therefore important targets for the design of novel therapeutics. Unlike in the design of enzyme active site inhibitors, the disruption of protein–protein interactions is far more challenging, due to such factors as the large interfacial areas involved and the relatively flat and featureless topologies of these surfaces. Nevertheless, in spite of such challenges, there has been considerable progress in recent years. In this review, we discuss this progress in the context of mimicry of protein surfaces: targeting protein–protein interactions by rational design.

Sign in / Sign up

Export Citation Format

Share Document