For the inheritance of genetic information and organism maintenance, accurate and complete DNA replication and separation are required. Defects in DNA duplication is the major cause of genetic instability therefore understanding DNA duplication regulation is essential to elucidate the processes behind human genetic disorders, including cancer so that effective therapeutic regimens could be developed for them.
It is clear that the processes of cell mass and size growth through macromolecular biosynthesis, and cell division are all entirely distinct from one another and may be distinguished in certain circumstances. However, because of the close relationship between both processes, each cell division cycle results in a doubling of both cell mass and DNA content. A mechanism like this ensures that appropriately sized daughter cells are produced following mitosis. Although the mechanisms governing cell growth and cell cycle division are unknown, the signalling protein TOR regulates both cell growth and cell cycle progression in species ranging from yeast to flies to mammals, and is thus recognized as an evolutionarily conserved central coordinator of these fundamental biological processes.
Rapamycin is a macrolide that has strong anticancer and immunosuppressive activity. It strongly suppresses downstream signalling from TOR proteins. TOR protein kinase is an amino acid that seves as a nutrient sensor and when nutrient is abundant it blocks pathways of authophagy. From yeast to mice, the longevity of many species is increased by inhibiting the TOR system. Apart from regulating autophagy, TOR system governs many other functions including the translation, ribosome biosynthesis, amino acid import, and transcription of several enzymes engaged in multiple metabolic pathways. It is established that the mammalian target of rapamycin (mTOR) protein incorporates food and mitogen signals to control cell growth (increasing cell mass and size) and division. Although mTOR is inhibited by the immunosuppressive medication rapamycin, the signalling mechanisms by which mTOR controls cell cycle progression are still poorly understood.
While there has been a tremendous progress in understanding the makeup and structure of the TOR protein kinase complexes, major concerns remain about the relevance of particular sequences within mTOR that are crucial for complex formation and activity. To fill these gaps and support the ongoing structural studies, investigators Jennifer Tsverov, Kristina Yegorov, and led by Professor Ted Powers from the Department of Molecular and Cellular Biology at the College of Biological Sciences, University of California, utilized elegant molecular genetic experiments to investigate the significance of specific elements within TOR1 and TOR2 by taking advantage of their distinct behaviour in yeast (Saccharomyces cerevisiae). The findings of the study was published in the journal Molecular Biology of the Cell.
The research team developed a set of TOR1-TOR2 chimeras by previously identifying ∼500-amino-acid domain corresponding to the N-HEAT domain. The scientists used these chimaeras to evaluate the importance of expected quaternary interactions for TORC2 assembly and function using a synthetic sick/lethal (SSL) genetic interaction technique. The researchers wanted to test the function of TOR2 and, by extension, TORC2 by introducing chimeric TOR genes into certain heterozygous diploid strains, followed by sporulation, tetrad dissection, and phenotypic analysis.
The authors findings made it possible to understand that importance of the presence of TOR2-specific determinants over the whole length of the protein, including the FRB and kinase domains, which are necessary for optimum TORC2 function in cells with a shortened allele of AVO3. Moreover, the researchers for the first time successfully developed “Minimal” (pPL628) and “Sub-Minimal” (pPL629) TOR2 MAS domain chimeras. However, it was observed that Minimal TOR2 MAS domain (pPL628) only provided extremely limited TOR2 function, while the Sub-Minimal TOR2 MAS domain (pPL629) had no effect at all on TOR2. When rapamycin resistance given by both chimeras and the normal protein production they exhibited are consistent with a loss in TORC2 activity alone. This research lead the investigators to the conclusion that effective TORC2 function necessitates sequences outside of the TOR2 MAS domain’s central core. Surprisingly, authors found that the Minimal MAS+C (pPL636) chimaera also caused a substantial decrease in the steady state level of TOR protein and a decrease in rapamycin resistance.
In summary, The study by Professor Ted Powers lab emphasizes the cooperative aspect of TOR complex assembly. By using synthetic lethal interaction studies, several subdomains were revealed in this study that are critical for TORC2 function. Authors were able to pinpoint certain areas by making them necessary for TORC2 identification by using TOR1-TOR2 chimeras. These findings pave the door for further investigation into their role in TORC2 assembly and function. Developed technique demonstrates the value of examining paralogues in understanding the molecular basis of big protein complex construction and evolution.
Tsverov J, Yegorov K, Powers T. Identification of defined structural elements within TOR2 kinase required for TOR complex 2 assembly and function in Saccharomyces cerevisiae. Molecular Biology of the Cell. 2022;33(5):ar44.