BackgroundThe mechanistic (or mammalian) target of rapamycin (mTOR), a Ser/Thr kinase, associates with different subunits forming two functionally distinct complexes, mTORC1 and mTORC2, regulating a diverse set of cellular functions in response to growth factors, cellular energy levels, and nutrients. The mechanisms regulating mTORC1 activity are well characterized; regulation of mTORC2 activity, however, remains obscure. While studies conducted in Dictyostelium suggest a possible role of Ras protein as a potential upstream regulator of mTORC2, definitive studies delineating the underlying molecular mechanisms, particularly in mammalian cells, are still lacking.MethodsProtein levels were measured by Western blotting and kinase activity of mTORC2 was analyzed by in vitro kinase assay. In situ Proximity ligation assay (PLA) and co-immunoprecipitation assay was performed to detect protein-protein interaction. Protein localization was investigated by immunofluorescence and subcellular fractionation while cellular function of mTORC2 was assessed by assaying extent of cell migration and invasion.ResultsHere, we present experimental evidence in support of the role of Ras activation as an upstream regulatory switch governing mTORC2 signaling in mammalian cancer cells. We report that active Ras through its interaction with mSIN1 accounts for mTORC2 activation, while disruption of this interaction by genetic means or via peptide-based competitive hindrance, impedes mTORC2 signaling.ConclusionsOur study defines the regulatory role played by Ras during mTORC2 signaling in mammalian cells and highlights the importance of Ras-mSIN1 interaction in the assembly of functionally intact mTORC2.
Diabetes is a group of metabolic disorders characterized
by elevated
blood sugar levels, leading to many undesirable health consequences.
There are many herbal formulations, traditionally used by the Northeast
Indian population for disease management. These formulations require
scientific validations to optimize their efficacy and increase their
popularity. In this study, we attempt to scientifically validate a
polyherbal formulation traditionally used for the management of diabetes
through preliminary phytochemicals investigation, characterization
of potential phytochemicals using Fourier transform infrared (FT-IR)
spectroscopy, high-resolution liquid chromatography mass spectrometry
(HR-LC/MS) analysis, and
in silico
characterization
of physiochemical, drug-likeness, and pharmacokinetic properties of
identified phytochemical compounds. Qualitative phytochemical screening
of various extracts of the formulation confirmed the presence of alkaloids,
phenols and tannins, flavonoids, fats, and oils. Phytochemical quantification
of the various extracts showed that the highest total phenolic content
is present in the ethanolic extract (35.61 ± 0.15 mg GAE/g),
while the highest total flavonoid content is present in the chloroform
extract (76.33 ± 2.96 mg QE/g) of the formulation. FT-IR spectroscopic
analysis revealed various characteristic band values with various
functional groups in the formulation extract such as amines, alcohol,
fluoro compounds, phenol, alkane, alkene, and conjugated acid groups.
HR-LC/MS analyses identified nearly 51 compounds including 9 small
peptides and 42 potential phytochemical compounds.
In silico
SwissADME analysis of identified compounds revealed 25 potential
compounds following Lipinski’s rule and showing drug-like characteristics,
and out of them, 16 compounds exhibited good oral bioavailability,
as revealed in the bioavailability radar. The overall study showed
that the presented polyherbal formulation is enriched with bio-active
phytochemical compounds with good pharmaceutical values.
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