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Mitochondrial calcium transport in nutrient sensing: does NCLX regulate mTORC1 activity?

Grant number: 22/08581-1
Support Opportunities:Scholarships abroad - Research Internship - Doctorate
Effective date (Start): December 01, 2022
Effective date (End): November 30, 2023
Field of knowledge:Biological Sciences - Biochemistry - Metabolism and Bioenergetics
Principal Investigator:Alicia Juliana Kowaltowski
Grantee:Vitor de Miranda Ramos
Supervisor: Viktor Korolchuk
Host Institution: Instituto de Química (IQ). Universidade de São Paulo (USP). São Paulo , SP, Brazil
Research place: Newcastle University, England  
Associated to the scholarship:19/18402-4 - Effects of mitochondrial calcium transport regulation in autophagic process of hepatocytes, BP.DR


Mitochondrial metabolism and mTORC1 activity are central in nutrient sensing and coordinating an adaptive cellular response. In fact, both pathways are interconnected by different intermediates, providing reciprocal regulation. Given the well-established role of mitochondria in shaping intracellular calcium signals and the clear participation of calcium in mTORC1 regulation, it is rational to speculate that calcium ions may be one of the many players in the communication between mitochondria and mTORC1. Importantly, we have previously demonstrated that pharmacological inhibition of mitochondrial calcium efflux prevents mTORC1 inhibition by serum and amino acids starvation. Thus, the focus of this project is to evaluate the participation of mitochondrial calcium efflux through the mitochondrial Ca2+/Li+/Na+ exchanger (NCLX) in the regulation of mTORC1 activity. The working plan for this project is divided into three phases. First, we will test different protocols for modulation of mTORC1 activity in the presence or absence of NCLX pharmacological inhibition. We may elucidate which pathways of mTORC1 regulation are more sensitive to NCLX activity, narrowing the conditions for future experiments. As an initial readout for mTORC1 activity, we will measure the phosphorylation levels of its canonical targets, S6K (T389), S6 (S235/236) or 4E-BP1 (T37/46), by western blot (WB) analysis. Importantly, the results obtained will be validated by using genetic knockdown (KD) of NCLX with small-interference RNA (siRNA) transfection. In the second phase of this project, we will focus on investigating the mechanism by which NCLX can alter mTORC1 activity. We will focus on three main factors involved in mTORC1 regulation: (1) mTORC1 recruitment to lysosome surface, (2) TSC complex recruitment to lysosome surface and (3) lysosome positioning within the cell. All these mechanisms will be investigated by immunostaining of either mTOR-Lamp1, TSC2-Lamp1 or Lamp1 alone and evaluated by confocal microscopy and colocalization analysis. In the third phase of the project, we intend to evaluate the possible outcomes of the regulatory pathways uncovered. For example, the lack of sensitivity of mTORC1 to nutrient starvation is associated with cellular senescence and age-related diseases. Thus, we intend to investigate if NCLX activity is also altered in senescent cells and how this may be related to mTORC1 dysfunction. To do so, we will evaluate NCLX expression by RT-qPCR and activity by measuring mitochondrial calcium efflux in intact cells using a model of stress- induced cellular senescence. If we observe alterations in NCLX activity by senescence, we may use genetic tools to either KD or overexpress NCLX, hence investigating the relationship of this transporter with mTORC1 in this context. (AU)

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