An affinity tool for the isolation of endogenous active mTORC1 from various cellular sources.

The mechanistic target of rapamycin complex 1 (mTORC1) is a central regulator of mammalian cell growth that is dysregulated in a number of human diseases, including metabolic syndromes, aging, and cancer. Structural, biochemical, and pharmacological studies that have increased our understanding of how mTORC1 executes growth control often relied upon purified mTORC1 protein. However, current immunoaffinity-based purification methods are expensive, inefficient, and do not necessarily isolate endogenous mTORC1, hampering their overall utility in research. Here we present a simple tool to isolate endogenous mTORC1 from various cellular sources. By recombinantly expressing and isolating mTORC1-binding Rag GTPases from Escherichia coli and using them as affinity probes, we demonstrate that mTORC1 can be isolated from mouse, bovine, and human sources. Our results indicate that mTORC1 isolated by this relatively inexpensive method is catalytically active and amenable to scaling. Collectively, this tool may be utilized to isolate mTORC1 from various cellular sources, organs, and disease contexts, aiding mTORC1-related research.

CRISPR/Cas9-mediated generic protein tagging in mammalian cells.

Systematic protein localization and protein-protein interaction studies to characterize specific protein functions are most effectively performed using tag-based assays. Ideally, protein tags are introduced into a gene of interest by homologous recombination to ensure expression from endogenous control elements. However, inefficient homologous recombination makes this approach difficult in mammalian cells. Although gene targeting efficiency by homologous recombination increased dramatically with the development of designer endonuclease systems such as CRISPR/Cas9 capable of inducing DNA double-strand breaks with unprecedented accuracy, the strategies still require synthesis or cloning of homology templates for every single gene. Recent developments have shown that endogenous protein tagging can be achieved efficiently in a homology independent manner. Hence, combinations between CRISPR/Cas9 and generic tag-donor plasmids have been used successfully for targeted gene modifications in mammalian cells. Here, we developed a tool kit comprising a CRISPR/Cas9 expression vector with several EGFP encoding plasmids that should enable tagging of almost every protein expressed in mammalian cells. By performing protein-protein interaction and subcellular localization studies of mTORC1 signal transduction pathway-related proteins expressed in HEK293T cells, we show that tagged proteins faithfully reflect the behavior of their native counterparts under physiological conditions.

Noninvasive Measurement of mTORC1 Signaling with 89Zr-Transferrin.

Purpose: mTOR regulates many normal physiological processes and when hyperactive can drive numerous cancers and human diseases. However, it is very challenging to detect and quantify mTOR signaling noninvasively in clinically relevant animal models of disease or man. We hypothesized that a nuclear imaging tool measuring intracellular mTOR activity could address this unmet need.Experimental Design: Although the biochemical activity of mTOR is not directly amenable to nuclear imaging probe development, we show that the transferrin receptor can be used to indirectly measure intracellular changes in mTOR activity.Results: After verifying that the uptake of radiolabeled transferrin (the soluble ligand of the transferrin receptor) is stimulated by active mTORC1 in vitro, we showed that 89Zr-labeled transferrin (Tf) can measure mTORC1 signaling dynamics in normal and cancerous mouse tissues with PET. Finally, we show that 89Zr-Tf can detect the upregulation of mTORC1 by tumor cells to escape the antitumor effects of a standard-of-care antiandrogen, which is to our knowledge the first example of applying PET to interrogate the biology of treatment resistant cancer.Conclusions: In summary, we have developed the first quantitative assay to provide a comprehensive measurement of mTOR signaling dynamics in vivo, in specific normal tissues, and during tumor development in genetically engineered animal models using a nuclear imaging tool that is readily translatable to man. Clin Cancer Res; 23(12); 3045-52. ©2016 AACR.