“HACSαβγδφε"
|导读：
Overexpression or Downregulation of mTOR in Mammalian Cells
This chapter presents an overview of the methods that have been used to overexpress or downregulate the level of mTOR isoforms in mammalian cells.
The techniques of transient overexpression, generation of stable cell lines, retroviral- and lentiviral-mediated overexpression or downregulation are discussed.
|背景介绍：
The mammalian target of rapamycin (mTOR) was identified at the same time by several independent groups as the cellular target for the immunosuppressant drug rapamycin, a naturally occurring antifungal macrolide.
These studies were preceded by the pioneering discovery of two TOR genes in yeast, TOR1 and TOR2, by M. Hall and colleagues .
In mammals, there is one mTOR gene which encodes two splicing isoforms, mTOR α and mTOR β.
The domain organization and regulatory sites of phosphorylation of mTOR isoforms are shown in Fig. 1a.
Fig. 1. Domain organization, signaling complexes, and expression pattern of mTOR splicing isoforms.
( a ) Structural features of mTOR α and mTOR β splicing isoforms. HEAT ( H untingtin, E F3, PP2 A , and T OR1); FAT ( F RAP/TOR- A TM- T RRAP); FRB ( F KBP12- R apamycin- B inding); KD ( K inase D omain).
( b ) Schematic presentation of TORC1 and TORC2, and key downstream signaling effectors.
( c ) Western blot analysis of the mTOR expression in rat tissues. 30 μ g of total protein extracts were immunoblotted with the C-terminal mTOR polyclonal antibodies from Cell Signaling (mTOR-CS) ( top panel ). The membrane was reprobed with antiactin antibodies ( lower panel ).
( d ) Northern blot analysis of mTOR transcripts in human tissues. The 3 ¢ mTOR probe was used to probe the blot ( top panel ) and then the expression β -actin analyzed by reprobing the membrane ( lower panel ) . Expression analysis of both mTOR isoforms at mRNA and protein levels was published by Panasyuk et al.
Since mTOR β was identified only a year ago, most studies on defining the role of mTOR in signal transduction and the regulation of cellular functions have solely involved originally cloned mTOR α isoform.
Northern and Western blot analysis indicate that both isoforms are ubiquitously expressed in mammalian tissues, while mTOR α is the predominantly expressed isoform at both RNA and protein levels (Fig. 1c).
Since Northern blot analysis has revealed at least four mRNA transcripts with the mTOR specific probe, the existence of yet undiscovered mTOR splicing isoforms is feasible.
Interestingly, the overexpression of mTOR α in mammalian cells does not result in the upregulation of TOR signaling, nor the induction of cell growth, proliferation, or survival.
By contrast, the overexpression of mTOR β isoform significantly shortens the G1/S transition of the cell cycle, induces cell proliferation and survival.
As a consequence, mTOR β, but not mTOR α, has the potential to induce oncogenic transformation when stably overexpressed in immortalized cell lines and to promote the growth of tumors in a xenograft model.
The inability of overexpressed mTOR α to induce mTOR-mediated signaling and cellular functions could be explained by the complexity of its activation and substrate presentation in two mutually exclusive complexes, mTOR complex 1 (mTORC1) and mTORC2, which coordinate cellular functions in response to mitogenic stimulation, nutrient and energy sufficiency.
The main components of TORC1 and TORC2 signaling complexes and their key downstream signaling effectors are shown in Fig. 1b.
As mTOR β has the ability to signal via both TORC1 and TORC2, and is also sensitive to rapamycin, the existence of yet unidentified regulators/downstream effectors of mTOR β signaling might explain the observed differences in overexpression studies.
Interaction between rapamycin and the immunophilin FKBP12 generates a highly potent and specific inhibitor of mTORC1.
The mTORC1 complex signals to eIF4E-binding protein 1 (4E-BP) and S6 kinases (S6Ks), which are key regulatory components of protein synthesis .
It has been demonstrated that the activity of mTOR in the mTORC2 complex can also be inhibited by prolong (24h) treatment of cells with rapamycin.
It appears that the rapamycin–FKBP12 complex does not bind directly to mTORC2, but long-term treatment of cells with rapamycin disrupts the assembly of functional mTORC2 through an unknown mechanism.
It has been proposed that the FKBP12–rapamycin interaction with mTOR might block subsequent binding of other components of TORC2, such as Rictor and SIN1.
However, it is not clear why the rapamycin-mediated inhibition of mTORC2 assembly only occurs in certain cell types.
Genetic studies in model organisms targeting different components of TOR regulatory complexes and the use of rapamycin/ rapalogs uncovered a critical role of mTOR in integrating signaling information from growth factors, nutrient and energy sufficiency to coordinate cell growth, proliferation and survival via the regulation of cellular biosynthetic processes and autophagy.
Deregulation of the mTOR signaling pathway has been implicated in a diverse range of human pathologies, including cancer, autoimmunity, cardiovascular, and neurodegenerative diseases and metabolic disorders such as diabetes.
This has prompted researchers from academia and pharmaceutical companies to develop novel mTOR inhibitors, ranging from derivatives of rapamycin to ATPcompetitive compounds .
At present, sirolimus (a rapamycin homolog) is used in the clinic for treating patients with coronary stenosis (sirolimus-eluting stents), while temsirolimus is indicated as the first line therapy for patients with renal cell carcinoma.
