Light-Controlled Affinity Purification of Protein Complexes Exemplified by the Resting ZAP70 Interactome

Hörner, Maximilian; Eble, Julian; Yousefi, O. Sascha; Schwarz, Jennifer; Warscheid, Bettina; Weber, Wilfried; Schamel, Wolfgang W. A.

doi:10.3389/fimmu.2019.00226

Cited by 15 publications

(13 citation statements)

References 44 publications

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“…Importantly, we did not observe binding of Syk to ribosomal proteins, suggesting that this property of ZAP-70 is not shared with Syk (Supplement Figure 3B). Interestingly, a recent study using light-affinity purification of ZAP-70 complexes from Jurkat cells also incidentally reported binding to ribosomes in T cells (Supplement Figure 4A) 21 .…”

Section: Zap-70 Binding To Ribosomal Proteins Is Associated With Incr...mentioning

confidence: 95%

ZAP-70 constitutively regulates gene expression and protein synthesis in chronic lymphocytic leukemia

Chen

Sathiaseelan

Moore

et al. 2021

Blood

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The expression of ZAP-70 in a subset of CLL patients strongly correlates with a more aggressive clinical course, though the exact underlying mechanisms remain elusive. The ability of ZAP-70 to enhance B cell receptor (BCR) signaling, independently of its kinase function, is considered to contribute. Here we employed RNA-sequencing and proteomic analyses of primary cells differing only in their expression of ZAP-70 to further define how ZAP-70 increases aggressiveness of CLL. We identified that ZAP-70 is directly required for cell survival in the absence of an overt BCR signal, which can compensate for ZAP-70 deficiency as an anti-apoptotic signal. In addition, the expression of ZAP-70 regulates the transcription of factors regulating recruitment and activation of T cells, such as CCL3, CCL4 and IL4I1. Quantitative mass spectrometry of double-cross linked ZAP-70 complexes further demonstrated constitutive and direct protein-protein interactions between ZAP-70 and BCR-signaling components. Unexpectedly, ZAP-70 also binds to ribosomal proteins, which is not dependent on, but further increased by BCR-stimulation. Importantly, decreased expression of ZAP-70 significantly reduced MYC-expression and global protein synthesis, providing evidence that ZAP-70 contributes to translational dysregulation in CLL. In conclusion, ZAP-70 constitutively promotes cell survival, microenvironment-interactions and protein synthesis in CLL cells, likely to improve cellular fitness and to further drive disease progression.

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Section: Zap-70 Binding To Ribosomal Proteins Is Associated With Incr...mentioning

confidence: 95%

ZAP-70 constitutively regulates gene expression and protein synthesis in chronic lymphocytic leukemia

Chen

Sathiaseelan

Moore

et al. 2021

Blood

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“…We show that one of these chimeras preferentially binds to the SH2 domain in the dark, allowing us to reversibly control its binding both in vitro and in mammalian cells. We then harness the ~330-fold change in binding affinity between lit and dark states to implement light-based protein purification 26 using an OptoMB resin in what we call “light-controlled affinity chromatography” (LCAC). This work represents the first demonstration of light control over the binding activity of a monobody and the first example of this capability in a synthetic non-immunoglobulin protein binder.…”

Section: Introductionmentioning

confidence: 99%

Development of light-responsive protein binding in the monobody non-immunoglobulin scaffold

et al. 2020

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Monobodies are synthetic non-immunoglobulin customizable protein binders invaluable to basic and applied research, and of considerable potential as future therapeutics and diagnostic tools. The ability to reversibly control their binding activity to their targets on demand would significantly expand their applications in biotechnology, medicine, and research. Here we present, as proof-of-principle, the development of a light-controlled monobody (OptoMB) that works in vitro and in cells and whose affinity for its SH2-domain target exhibits a 330-fold shift in binding affinity upon illumination. We demonstrate that our αSH2-OptoMB can be used to purify SH2-tagged proteins directly from crude E. coli extract, achieving 99.8% purity and over 40% yield in a single purification step. By virtue of their ability to be designed to bind any protein of interest, OptoMBs have the potential to find new powerful applications as light-switchable binders of untagged proteins with the temporal and spatial precision afforded by light.

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“…Recently, Horner et al have used the PhyB/PIF system to isolate the tyrosine kinase ZAP70 [33]. Biotinylated PhyB is immobilized on NeutrAvidin (N)functionalized agarose beads to pull down the ZAP70-PIF fusion protein when illuminated with 660 nm red light and released when exposed to 740 nm farred light.…”

Section: Discussionmentioning

confidence: 99%

mem-iLID, a fast and economic protein purification method

et al. 2021

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Protein purification is the vital basis to study the function, structure and interaction of proteins. Widely used methods are affinity chromatography-based purifications, which require different chromatography columns and harsh conditions, such as acidic pH and/or adding imidazole or high salt concentration, to elute and collect the purified proteins. Here we established an easy and fast purification method for soluble proteins under mild conditions, based on the light-induced protein dimerization system iLID, which regulates protein binding and release with light. We utilize the biological membrane, which can be easily separated by centrifugation, as the port to anchor the target proteins. In Xenopus laevis oocyte and Escherichia coli, the blue light-sensitive part of iLID, AsLOV2-SsrA, was targeted to the plasma membrane by different membrane anchors. The other part of iLID, SspB, was fused with the protein of interest (POI) and expressed in the cytosol. The SspB-POI can be captured to the membrane fraction through light-induced binding to AsLOV2-SsrA and then released purely to fresh buffer in the dark after simple centrifugation and washing. This method, named mem-iLID, is very flexible in scale and economic. We demonstrate the quickly obtained yield of two pure and fully functional enzymes: a DNA polymerase and a light-activated adenylyl cyclase. Furthermore, we also designed a new SspB mutant for better dissociation and less interference with the protein of interest, which could potentially facilitate other optogenetic manipulations of protein-protein interaction.

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Light-Controlled Affinity Purification of Protein Complexes Exemplified by the Resting ZAP70 Interactome

Cited by 15 publications

References 44 publications

ZAP-70 constitutively regulates gene expression and protein synthesis in chronic lymphocytic leukemia

ZAP-70 constitutively regulates gene expression and protein synthesis in chronic lymphocytic leukemia

Development of light-responsive protein binding in the monobody non-immunoglobulin scaffold

mem-iLID, a fast and economic protein purification method

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