LuMPIS – A modified luminescence‐based mammalian interactome mapping pull‐down assay for the investigation of protein–protein interactions encoded by GC‐low ORFs

Pinto, María Guadalupe Vizoso; Villegas, Josefina M.; Peter, J Clyne; Haase, Rudolf; Haas, Jürgen; Lotz, Amelie S.; Muntau, Ania C.; Baiker, Armin

doi:10.1002/pmic.200900298

Cited by 16 publications

(33 citation statements)

References 21 publications

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“…FATP4, a member of the family of the fatty acid transporters (39), transports LCFAs and VLCFAs. In addition, FATP4 is capable of activating both LCFAs and VLCFAs to their CoA derivatives (26,40,41). However, elongation of LCFAs is catalyzed by the fatty-acid elongase machinery, an endoplasmic reticulumbound enzyme complex (42).…”

Section: Discussionmentioning

confidence: 99%

Identification of a New Fatty Acid Synthesis-Transport Machinery at the Peroxisomal Membrane

Hillebrand

Gersting

Lotz-Havla

et al. 2012

Journal of Biological Chemistry

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Background: Dysfunction of the peroxisomal ABC transporter ALDP and elevated very long chain fatty acids are the hallmark of X-ALD. Results: Peroxisomal ABC transporters interact with proteins functioning in fatty acid synthesis and activation. Conclusion: We identified a new fatty acid synthesis-transport machinery adapted to different chain lengths and metabolic conditions. Significance: This machinery extends the knowledge on peroxisomal ␤-oxidation and X-ALD pathogenesis.

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Section: Discussionmentioning

confidence: 99%

Identification of a New Fatty Acid Synthesis-Transport Machinery at the Peroxisomal Membrane

Hillebrand

Gersting

Lotz-Havla

et al. 2012

Journal of Biological Chemistry

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“…Both LUMIER and LuMPIS assays also use Rluc-tagged proteins to determine PPIs [6], [7]. However, these assays only provide quantitative information about the Rluc-tagged protein among interacting protein pairs, similar to the results shown in the left of Figs.…”

Section: Discussionmentioning

confidence: 70%

“…Notably, these proteins strongly interact in the cellular nucleus via their basic leucine zipper regions, making human Jun and Fos classic model protein pairs for the development of novel PPI assays [7], [11]. To establish DLR-PD assays for the analysis of PPIs, biotinylated Fluc tag and Rluc tag were fused to the basic leucine zipper regions of Jun and Fos, respectively, through a flexible linker 4x(G 4 S).…”

Section: Resultsmentioning

confidence: 99%

“…This molar ratio is different from the native Fos-Jun interaction, which presumably is 1∶1. Furthermore, the DLR-PD assay may not be suited to measure the affinity between interacting proteins, as is the case with both the LUMIER and LuMPIS methods [6], [7].…”

Section: Discussionmentioning

confidence: 99%

“…Luminescence-based PPIs assays, such as luminescence-based mammalian interactome mapping (LUMIER) and the luminescence-based MBP pull-down interaction screening system (LuMPIS), only provide quantitative information about a Rluc-tagged protein among interacting protein pairs [6], [7]. Fluc and Rluc are regarded as dual luciferase reporters (DLRs), which are commonly combined to analyze relative protein expression levels.…”

Section: Introductionmentioning

confidence: 99%

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Relative Quantification of Protein-Protein Interactions Using a Dual Luciferase Reporter Pull-Down Assay System

Jia¹,

Peng²,

Gao³

et al. 2011

PLoS ONE

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The identification and quantitative analysis of protein-protein interactions are essential to the functional characterization of proteins in the post-proteomics era. The methods currently available are generally time-consuming, technically complicated, insensitive and/or semi-quantitative. The lack of simple, sensitive approaches to precisely quantify protein-protein interactions still prevents our understanding of the functions of many proteins. Here, we develop a novel dual luciferase reporter pull-down assay by combining a biotinylated Firefly luciferase pull-down assay with a dual luciferase reporter assay. The biotinylated Firefly luciferase-tagged protein enables rapid and efficient isolation of a putative Renilla luciferase-tagged binding protein from a relatively small amount of sample. Both of these proteins can be quantitatively detected using the dual luciferase reporter assay system. Protein-protein interactions, including Fos-Jun located in the nucleus; MAVS-TRAF3 in cytoplasm; inducible IRF3 dimerization; viral protein-regulated interactions, such as MAVS-MAVS and MAVS-TRAF3; IRF3 dimerization; and protein interaction domain mapping, are studied using this novel assay system. Herein, we demonstrate that this dual luciferase reporter pull-down assay enables the quantification of the relative amounts of interacting proteins that bind to streptavidin-coupled beads for protein purification. This study provides a simple, rapid, sensitive, and efficient approach to identify and quantify relative protein-protein interactions. Importantly, the dual luciferase reporter pull-down method will facilitate the functional determination of proteins.

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Investigating the biology of alpha herpesviruses with MS‐based proteomics

et al. 2015

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Viruses are intracellular parasites that can only replicate and spread in cells of susceptible hosts. Alpha herpesviruses (α-HVs) contain double stranded DNA genomes of at least 120 kb, encoding for 70 or more genes. The viral genome is contained in an icosahedral capsid that is surrounded by a proteinaceous tegument layer and a lipid envelope. Infection starts in epithelial cells and spreads to the peripheral nervous system (PNS). In the natural host, α-HVs establish a chronic latent infection that can be reactivated, rarely spread to the central nervous system (CNS). In the non-natural host, viral infection will in most cases spread to the CNS with often fatal outcome. The host response plays a crucial role in the outcome of viral infection. α-HVs do not encode all the genes required for viral replication and spread. They need a variety of host gene products including RNA polymerase, ribosomes, dynein, and kinesin. As a result, the infected cell is dramatically different from the uninfected cell revealing a complex and dynamic interplay of viral and host components required to complete the virus life cycle. In this review, we describe the pivotal contribution of mass spectrometry-based proteomics (MSBP) studies over the past 15 years to understanding the complicated life cycle and pathogenesis of four α-HV species from the alphaherpesvirinae subfamily: Herpes simplex virus-1 (HSV-1), varicella zoster virus (VZV), pseudorabies virus (PRV) and bovine herpes virus (BHV-1). We describe the viral proteome dynamics during host infection and the host proteomic response to counteract such pathogens.

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LuMPIS – A modified luminescence‐based mammalian interactome mapping pull‐down assay for the investigation of protein–protein interactions encoded by GC‐low ORFs

Cited by 16 publications

References 21 publications

Identification of a New Fatty Acid Synthesis-Transport Machinery at the Peroxisomal Membrane

Identification of a New Fatty Acid Synthesis-Transport Machinery at the Peroxisomal Membrane

Relative Quantification of Protein-Protein Interactions Using a Dual Luciferase Reporter Pull-Down Assay System

Investigating the biology of alpha herpesviruses with MS‐based proteomics

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