Multi-Tox: Application of the ToxR-transcriptional reporter assay to the study of multi-pass protein transmembrane domain oligomerization

Joce, Catherine; Wiener, A.; Yin, Hang

doi:10.1016/j.bbamem.2011.07.008

Cited by 11 publications

(12 citation statements)

References 27 publications

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“…We utilized a previously established ToxR assay as the platform for screening TMD disruptors in cellular membranes (Figure 3a) [11,12,13]. This E. coli -based assay uses a chimeric construct made of the N-terminal DNA binding domain of ToxR and the monomeric anchor maltose binding protein (MBP) fused to the LMP-1 TMD-5 cytoplasmic and periplasmic termini, respectively.…”

Section: Resultsmentioning

confidence: 99%

“…ToxR7-TMD-5, ToxR7–TMD of diacylglycerol kinase (DAGK) and ToxR7-TMD of integrin αIIb plasmids were constructed as described previously [11,12,13]. Diversity Set II library (1364 compounds) was provided by the Developmental Therapeutic Program, NCI/NIH.…”

Section: Methodsmentioning

confidence: 99%

“…ToxR7-TMD-5 plasmid (200 ng) was transformed into 200 μL of FHK12 competent cells (kindly provided by D. Langosch, Technische Universität München, Germany) with heat shock at 42 °C for 90 s and incubation on ice for 2 minutes, followed by addition of 800 μL SOC media and incubation with shaking at 37 °C for 1 h. 5 μL of the transformation mixture was used to inoculate 100 μL LB + arabinose (0.0025%) and chloramphenicol (30 μg/mL) and 100 μM compound. Cultures were incubated with shaking at 37 °C for 20 h and β-galactosidase activity was measured using a Beckman Coulter DTX 880 plate reader (Beckman, Coulter, Fullerton, CA, USA) as described previously [11,12]. Briefly, 5 μL of culture was transferred to the wells of a Costar 3596 polystyrene 96-well plate (Corning, NY, USA) containing 100 μL Z buffer/chloroform (1% β-mercaptoethanol, 10% chloroform, 89% A buffer: 1 M sodium phosphate, 10 mM KCl, 1 mM MgSO4 and pH 7.0).…”

Section: Methodsmentioning

confidence: 99%

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Targeting the lateral interactions of transmembrane domain 5 of Epstein–Barr virus latent membrane protein 1

Wang

Saludes

Zhao

et al. 2012

Biochimica et Biophysica Acta (BBA) - Biomembranes

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The lateral transmembrane protein-protein interaction has been regarded as “undruggable” despite its importance in many biological processes. The homo-trimerization of transmembrane domain 5 (TMD-5) of latent membrane protein 1 (LMP-1) is critical for the constitutive oncogenic activation of the Epstein-Barr virus (EBV). Herein, we report a small molecule agent, NSC 259242 (compound 1), to be a TMD-5 self-association disruptor. Both the positively charged acetimidamide functional groups and the stilbene backbone of compound 1 are essential for its inhibitory activity. Furthermore, cell-based assays revealed that compound 1 inhibits full-length LMP-1 signaling in EBV infected B cells. These studies demonstrated a new strategy for identifying small molecule disruptors for investigating transmembrane protein-protein interactions.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Targeting the lateral interactions of transmembrane domain 5 of Epstein–Barr virus latent membrane protein 1

Wang

Saludes

Zhao

et al. 2012

Biochimica et Biophysica Acta (BBA) - Biomembranes

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“…Dimerization of the heterologous TM domain results in a LexA fusion capable of repressing ␤-galactosidase expression in Escherichia coli (41,42). Similar bacterial two-hybrid systems have also been developed to examine homo-and heterotypic interactions between TM domains (43)(44)(45)(46)(47)(48).…”

mentioning

confidence: 99%

Hepatitis C Virus RNA Replication and Virus Particle Assembly Require Specific Dimerization of the NS4A Protein Transmembrane Domain

Kohlway

Pirakitikulr

Barrera

et al. 2014

J Virol

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e Hepatitis C virus (HCV) NS4A is a single-pass transmembrane (TM) protein essential for viral replication and particle assembly. The sequence of the NS4A TM domain is highly conserved, suggesting that it may be important for protein-protein interactions. To test this hypothesis, we measured the potential dimerization of the NS4A TM domain in a well-characterized two-hybrid TM protein interaction system. The NS4A TM domain exhibited a strong homotypic interaction that was comparable in affinity to glycophorin A, a well-studied human blood group antigen that forms TM homodimers. Several mutations predicted to cluster on a common surface of the NS4A TM helix caused significant reductions in dimerization, suggesting that these residues form an interface for NS4A dimerization. Mutations in the NS4A TM domain were further examined in the JFH-1 genotype 2a replicon system; importantly, all mutations that destabilized NS4A dimers also caused defects in RNA replication and/or virus assembly. Computational modeling of NS4A TM interactions suggests a right-handed dimeric interaction of helices with an interface that is consistent with the mutational effects. Furthermore, defects in NS4A oligomerization and virus particle assembly of two mutants were rescued by NS4A A15S, a TM mutation recently identified through forward genetics as a cell culture-adaptive mutation. Together, these data provide the first example of a functionally important TM dimer interface within an HCV nonstructural protein and reveal a fundamental role of the NS4A TM domain in coordinating HCV RNA replication and virus particle assembly. Hepatitis C virus (HCV) is an enveloped, positive-sense RNA virus in the Flaviviridae family (1). HCV has been grouped into seven major genotypes and several subtypes (2, 3). The 9.6-kb genome is translated into a single ϳ3,000-amino-acid polypeptide by cap-independent translation through an internal ribosome entry site (1). The polypeptide is cleaved into three structural proteins-core, E1, and E2-as well as seven nonstructural (NS) proteins-p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B (4). The structural proteins and p7 are cleaved from the polypeptide by cellular signal peptidases, whereas cleavage of the NS proteins is accomplished by the NS2-NS3 (NS2-3) cysteine autoprotease and the NS3-NS4A (NS3-4A) serine protease (4).HCV NS4A is a 54-amino-acid polypeptide that anchors NS3 to the endoplasmic reticulum (ER) (1, 5, 6). NS4A encompasses an N-terminal, alpha-helical, single-pass transmembrane (TM) region required for association with the ER membrane (6-8), a central region that mediates interaction with the NS3 protease domain (5, 6, 9-11), and a C-terminal region implicated in both RNA replication and virus particle assembly (12-15). NS4A functions as a cofactor for both serine protease and RNA helicase activities of NS3 and exhibits genetic and physical interactions with several other NS proteins, including NS2, NS4B, NS5A, and NS5B (15-19). Additional roles for NS4A include translation inhibition (20, 21) through in...

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“…Upon TMD-driven heterodimerization, CAT expression decreases, leading to a corresponding decrease in bacterial growth. Although the ToxR system was initially developed for studies of single-pass MPs, Yin and colleagues (93) have also extended it to studies of MPs with multiple TMDs.…”

Section: Membrane Proteins Emerging From “Undruggable” Targetsmentioning

confidence: 99%

Drugging Membrane Protein Interactions

Yin¹,

Flynn

2016

Annu. Rev. Biomed. Eng.

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285

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The majority of therapeutics target membrane proteins, accessible on the surface of cells, to alter cellular signaling. Cells use membrane proteins to transduce signals into cells, transport ions and molecules, bind the cell to a surface or substrate, and catalyze reactions. Newly devised technologies allow us to drug conventionally “undruggable” regions of membrane proteins, enabling modulation of protein–protein, protein–lipid, and protein–nucleic acid interactions. In this review, we survey the state of the art in high-throughput screening and rational design in drug discovery, and we evaluate the advances in biological understanding and technological capacity that will drive pharmacotherapy forward against unorthodox membrane protein targets.

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Multi-Tox: Application of the ToxR-transcriptional reporter assay to the study of multi-pass protein transmembrane domain oligomerization

Cited by 11 publications

References 27 publications

Targeting the lateral interactions of transmembrane domain 5 of Epstein–Barr virus latent membrane protein 1

Targeting the lateral interactions of transmembrane domain 5 of Epstein–Barr virus latent membrane protein 1

Hepatitis C Virus RNA Replication and Virus Particle Assembly Require Specific Dimerization of the NS4A Protein Transmembrane Domain

Drugging Membrane Protein Interactions

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