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

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.

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

Open Biology ◽

10.1098/rsob.120173 ◽

2013 ◽

Vol 3 (10) ◽

pp. 120173 ◽

Cited By ~ 23

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.

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Regulatory Role of PopN and Its Interacting Partners in Type III Secretion of Pseudomonas aeruginosa

Journal of Bacteriology ◽

10.1128/jb.01680-06 ◽

2007 ◽

Vol 189 (7) ◽

pp. 2599-2609 ◽

Cited By ~ 29

Author(s):

Hongjing Yang ◽

Zhiying Shan ◽

Jaewha Kim ◽

Weihui Wu ◽

Wei Lian ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Protein Interactions ◽

Type Iii Secretion ◽

Calcium Binding ◽

High Capacity ◽

Calcium Signal ◽

Dependent Manner ◽

Protein Protein Interactions ◽

Type Iii

ABSTRACT The type III secretion system (T3SS) of Pseudomonas aeruginosa plays a significant role in pathogenesis. We have previously identified type III secretion factor (TSF), which is required for effective secretion of the type III effector molecules, in addition to the low calcium signal. TSF includes many low-affinity high-capacity calcium binding proteins, such as serum albumin and casein. A search for the TSF binding targets on the bacterial outer membrane resulted in identification of PopN, a component of the T3SS that is readily detectable on the bacterial cell surface. PopN specifically interacts with Pcr1, and both popN and pcr1 mutants have a constitutive type III secretion phenotype, suggesting that the two proteins form a complex that functions as a T3SS repressor. Further analysis of the popN operon genes resulted in identification of protein-protein interactions between Pcr1 and Pcr4 and between Pcr4 and Pcr3, as well as between PopN and Pcr2 in the presence of PscB. Unlike popN and pcr1 mutants, pcr3 and pcr4 mutants are totally defective in type III secretion, while a pcr2 mutant exhibits reduced type III secretion. Interestingly, PopN, Pcr1, Pcr2, and Pcr4 are all secreted in a type III secretion machinery-dependent manner, while Pcr3 is not. These findings imply that these components have important regulatory roles in controlling type III secretion.

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A novel pathway for human endothelial cell activation by antiphospholipid/anti-β2 glycoprotein I antibodies

Blood ◽

10.1182/blood-2011-03-344671 ◽

2012 ◽

Vol 119 (3) ◽

pp. 884-893 ◽

Cited By ~ 89

Author(s):

Kristi L. Allen ◽

Fabio V. Fonseca ◽

Venkaiah Betapudi ◽

Belinda Willard ◽

Jainwei Zhang ◽

...

Keyword(s):

Protein Interactions ◽

Cell Activation ◽

Transmembrane Protein ◽

Critical Role ◽

Fetal Loss ◽

Annexin A2 ◽

Dependent Manner ◽

Protein Protein Interactions ◽

Endothelial Cell Activation ◽

Signaling Complex

Abstract Antiphospholipid Abs (APLAs) are associated with thrombosis and recurrent fetal loss. These Abs are primarily directed against phospholipid-binding proteins, particularly β2GPI, and activate endothelial cells (ECs) in a β2GPI-dependent manner after binding of β2GPI to EC annexin A2. Because annexin A2 is not a transmembrane protein, the mechanisms of APLA/anti-β2GPI Ab–mediated EC activation are uncertain, although a role for a TLR4/myeloid differentiation factor 88–dependent pathway leading to activation of NF-κB has been proposed. In the present study, we confirm a critical role for TLR4 in anti-β2GPI Ab–mediated EC activation and demonstrate that signaling through TLR4 is mediated through the assembly of a multiprotein signaling complex on the EC surface that includes annexin A2, TLR4, calreticulin, and nucleolin. An essential role for each of these proteins in cell activation is suggested by the fact that inhibiting the expression of each using specific siRNAs blocked EC activation mediated by APLAs/anti-β2GPI Abs. These results provide new evidence for novel protein-protein interactions on ECs that may contribute to EC activation and the pathogenesis of APLA/anti-β2GPI–associated thrombosis and suggest potential new targets for therapeutic intervention in antiphospholipid syndrome.

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Identification of Ca2+-dependent binding partners for the neuronal calcium sensor protein neurocalcin δ: interaction with actin, clathrin and tubulin

Biochemical Journal ◽

10.1042/bj3630599 ◽

2002 ◽

Vol 363 (3) ◽

pp. 599-608 ◽

Cited By ~ 25

Author(s):

Lenka IVINGS ◽

Stephen R. PENNINGTON ◽

Roz JENKINS ◽

Jamie L. WEISS ◽

Robert D. BURGOYNE

Keyword(s):

Protein Interactions ◽

Protein Identification ◽

Specific Interaction ◽

Coated Vesicle ◽

Dependent Manner ◽

Protein Protein Interactions ◽

Vesicle Traffic ◽

Calcium Sensors ◽

Neuronal Calcium Sensor Protein ◽

Neurocalcin Δ

The neuronal calcium sensors are a family of EF-hand-containing Ca2+-binding proteins expressed predominantly in retinal photoreceptors and neurons. One of the family members is neurocalcin δ, the function of which is unknown. As an approach to elucidating the protein interactions made by neurocalcin δ, we have identified brain cytosolic proteins that bind to neurocalcin δ in a Ca2+-dependent manner. We used immobilized recombinant myristoylated neurocalcin δ combined with protein identification using MS. We demonstrate a specific interaction with clathrin heavy chain, α- and β-tubulin, and actin. These interactions were dependent upon myristoylation of neurocalcin δ indicating that the N-terminal myristoyl group may be important for protein—protein interactions in addition to membrane association. Direct binding of neurocalcin δ to clathrin, tubulin and actin was confirmed using an overlay assay. These interactions were also demonstrated for endogenous neurocalcin δ by co-immunoprecipitation from rat brain cytosol. When expressed in HeLa cells, neurocalcin δ was cytosolic at resting Ca2+ levels but translocated to membranes, including a perinuclear compartment (trans-Golgi network) where it co-localized with clathrin, following Ca2+ elevation. These data suggest the possibility that neurocalcin δ functions in the control of clathrin-coated vesicle traffic.

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GAC63, a GRIP1-Dependent Nuclear Receptor Coactivator

Molecular and Cellular Biology ◽

10.1128/mcb.25.14.5965-5972.2005 ◽

2005 ◽

Vol 25 (14) ◽

pp. 5965-5972 ◽

Cited By ~ 50

Author(s):

Yong-Heng Chen ◽

Jeong Hoon Kim ◽

Michael R. Stallcup

Keyword(s):

Estrogen Receptor ◽

Protein Interactions ◽

Transcriptional Activation ◽

Target Genes ◽

Gene Promoter ◽

Dependent Manner ◽

Protein Protein Interactions ◽

Promoter Regions ◽

Nuclear Receptor Coactivator ◽

Binding Domains

ABSTRACT Nuclear receptors (NRs) regulate target gene transcription through the recruitment of multiple coactivator complexes to the promoter regions of target genes. One important coactivator complex includes a p160 coactivator (GRIP1, SRC-1, or ACTR) and its downstream coactivators (e.g., p300, CARM1, CoCoA, and Fli-I), which contribute to transcriptional activation by protein acetylation, protein methylation, and protein-protein interactions. In this study, we identified a novel NR coactivator, GAC63, which binds to the N-terminal region of p160 coactivators as well as the ligand binding domains of some NRs. GAC63 enhanced transcriptional activation by NRs in a hormone-dependent and GRIP1-dependent manner in transient transfection assays and cooperated synergistically and selectively with other NR coactivators, including GRIP1 and CARM1, to enhance estrogen receptor function. Endogenous GAC63 was recruited to the estrogen-responsive pS2 gene promoter of MCF-7 cells in response to the hormone. Reduction of the endogenous GAC63 level by small interfering RNA inhibited transcriptional activation by the hormone-activated estrogen receptor. Thus, GAC63 is a physiologically relevant part of the p160 coactivator signaling pathway that mediates transcriptional activation by NRs.

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Tribbles Pseudokinase 3 Regulation and Contribution to Cancer

Cancers ◽

10.3390/cancers13081822 ◽

2021 ◽

Vol 13 (8) ◽

pp. 1822

Author(s):

Bojana Stefanovska ◽

Fabrice André ◽

Olivia Fromigué

Keyword(s):

Gene Expression ◽

Amino Acids ◽

Catalytic Activity ◽

Protein Interactions ◽

Kinase Activity ◽

Cancer Development ◽

Dependent Manner ◽

Protein Protein Interactions ◽

Cellular Processes ◽

Signaling Components

The first Tribbles protein was identified as critical for the coordination of morphogenesis in Drosophila melanogaster. Three mammalian homologs were subsequently identified, with a structure similar to classic serine/threonine kinases, but lacking crucial amino acids for the catalytic activity. Thereby, the very weak ATP affinity classifies TRIB proteins as pseudokinases. In this review, we provide an overview of the regulation of TRIB3 gene expression at both transcriptional and post-translational levels. Despite the absence of kinase activity, TRIB3 interferes with a broad range of cellular processes through protein–protein interactions. In fact, TRIB3 acts as an adaptor/scaffold protein for many other proteins such as kinase-dependent proteins, transcription factors, ubiquitin ligases, or even components of the spliceosome machinery. We then state the contribution of TRIB3 to cancer development, progression, and metastasis. TRIB3 dysregulation can be associated with good or bad prognosis. Indeed, as TRIB3 interacts with and regulates the activity of many key signaling components, it can act as a tumor-suppressor or oncogene in a context-dependent manner.

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Development of a Split Esterase 1 for Protein-Protein 2 Interaction Dependent Small Molecule Activation

10.26434/chemrxiv.8258633.v1 ◽

2019 ◽

Author(s):

Krysten A. Jones ◽

Kaitlin Kentala ◽

Michael Beck ◽

Weiwei An ◽

Alexander Lippert ◽

...

Keyword(s):

Synthetic Biology ◽

Small Molecule ◽

Protein Interactions ◽

Simultaneous Detection ◽

Cell Killing ◽

Dependent Manner ◽

Protein Protein Interactions ◽

Biological Studies ◽

Cytotoxic Molecule ◽

Measured Input

Split reporters based on fluorescent proteins and luciferases have emerged as valuable tools for measuring interactions in biological systems. Relatedly, biosensors that transduce measured input signals into outputs that influence the host system are key components of engineered gene circuits for synthetic biology applications. While small molecule-based imaging agents are widely used in biological studies, and small molecule-based drugs and chemical probes can target a range of biological processes, a general method for generating a target small molecule in a biological system based on a measured input signal is lacking. Here, we develop a proximity-dependent split esterase that selectively unmasks ester-protected small molecules in an interaction-dependent manner. Exploiting the versatility of an ester-protected small molecule output, we demonstrate fluorescent, chemiluminescent, and pharmacological probe generation, each created by masking key alcohol functional groups on a target small molecule. We show the split esterase system can be used in combination with ester-masked fluorescent or luminescent probes to measure a protein-protein interactions and protein-protein interaction inhibitor engagement. We demonstrate the esterase-based reporter system is compatible with other commonly-used split reporter imaging systems for the simultaneous detection of multiple protein-protein interactions. Finally, we develop a system for selective small molecule-dependent cell killing by unmasking a cytotoxic molecule using an inducible split esterase. Presaging utility in future synthetic biology-based therapeutic applications, we also show the system can be used for intercellular cell killing via a bystander effect, where one activated cell unmasks a cytotoxic molecule and kills cells physically adjacent to the activated cells. Collectively, this work illustrates that the split esterase system is a valuable new addition to the split protein toolbox, with particularly exciting potential in synthetic biology applications.

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Development of a Split Esterase for Protein-Protein Interaction Dependent Small Molecule Activation

10.26434/chemrxiv.8258633 ◽

2019 ◽

Cited By ~ 1

Author(s):

Krysten A. Jones ◽

Kaitlin Kentala ◽

Michael Beck ◽

Weiwei An ◽

Alexander Lippert ◽

...

Keyword(s):

Synthetic Biology ◽

Small Molecule ◽

Protein Interaction ◽

Protein Interactions ◽

Cell Killing ◽

Dependent Manner ◽

Protein Protein Interactions ◽

Protein Protein Interaction ◽

Cytotoxic Molecule ◽

Measured Input

Split reporters based on fluorescent proteins and luciferases have emerged as valuable tools for measuring interactions in biological systems. Relatedly, biosensors that transduce measured input signals into outputs that influence the host system are key components of engineered gene circuits for synthetic biology applications. While small molecule-based imaging agents are widely used in biological studies, and small molecule-based drugs and chemical probes can target a range of biological processes, a general method for generating a target small molecule in a biological system based on a measured input signal is lacking. Here, we develop a proximity-dependent split esterase that selectively unmasks ester-protected small molecules in an interaction-dependent manner. Exploiting the versatility of an ester-protected small molecule output, we demonstrate fluorescent, chemiluminescent, and pharmacological probe generation, each created by masking key alcohol functional groups on a target small molecule. We show the split esterase system can be used in combination with ester-masked fluorescent or luminescent probes to measure a protein-protein interactions and protein-protein interaction inhibitor engagement. We demonstrate the esterase-based reporter system is compatible with other commonly-used split reporter imaging systems for the simultaneous detection of multiple protein-protein interactions. Finally, we develop a system for selective small molecule-dependent cell killing by unmasking a cytotoxic molecule using an inducible split esterase. Presaging utility in future synthetic biology-based therapeutic applications, we also show the system can be used for intercellular cell killing via a bystander effect, where one activated cell unmasks a cytotoxic molecule and kills cells physically adjacent to the activated cells. Collectively, this work illustrates that the split esterase system is a valuable new addition to the split protein toolbox, with particularly exciting potential in synthetic biology applications.

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

Nucleic Acids Research ◽

10.1093/nar/gkz293 ◽

2019 ◽

Vol 47 (12) ◽

pp. 6269-6286 ◽

Cited By ~ 1

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.

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