טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentYunaev Anna
SubjectLysosomotropic Drugs Activate TFEB Via Lysosomal Membrane
Fluidization and Consequent Inhibition of mTORC1
Activity
DepartmentDepartment of Biology
Supervisor Professor Yehuda Assaraf
Full Thesis textFull thesis text - English Version


Abstract

The aim of the current research was to decipher the molecular mechanisms underlying the activation of TFEB via lysosomal sequestration of lysosomotropic anticancer drugs. TFEB is a member of the MiTF/TFE family of transcription factors and the master transcriptional regulator of lysosomal biogenesis by activating the CLEAR gene network (Coordinated Lysosomal Expression and Regulation). TFEB is phosphorylated by mTORC1 in the cytoplasm, which results in its sequestration in the cytoplasm via its binding by 14-3-3 proteins. Under stress conditions including amino acid starvation, Ca2 is released from lysosomes through Mucolipin-1, leading to calcineurin activation. It was previously suggested that the calcium-dependent phosphatase calcineurin mediates the dephosphorylation of pTFEB, facilitating its nuclear translocation and activation of the CLEAR gene network under conditions of lysosomal stress. Herein we demonstrate that lysosomal accumulation of lysosomotropic drugs induces fluidization of the lysosomal membrane, inhibiting the kinase activity of mTORC1, resulting in a rapid translocation of unphosphorylated TFEB to the nucleus. We further show that although lysosomotropic drug sequestration induces Ca2 release into the cytoplasm, and thus calcineurin activation, chelation of cytosolic Ca2 or direct inhibition of calcineurin activity, does not interfere with nuclear translocation of TFEB and CLEAR gene network activation. Our findings suggest that a Ca2+independent dephosphorylation of pTFEB occurs which leads to nuclear translocation of TFEB. Like TFEB, TFE3 is also a member of the MiTF/TFE family of transcription factors that binds to CLEAR promoter elements and induces lysosomal biogenesis. Like TFEB, TFE3 is also phosphorylated by the mTORC1 kinase and calcineurin is the phosphatase which was suggested to dephosphorylate pTFE3.  Our results demonstrate that these transcription factors activate the CLEAR pathway by forming homodimers and heterodimers in the nucleus. Moreover, TFEB and TFE3 also form heterodimers with TFE3 Isoform 2, which was suggested to act in a dominant negative manner. Surprisingly, dimerization with TFE3-isoform 2 did not inhibit TFEB or TFE3 activity, suggesting that TFE3 isoform 2 does not act in a dominant negative manner.
Thus, deciphering the mechanisms underlying TFEB activation and dimerization will facilitate the development of novel molecular interventions that abolish TFEB-dependent lysosomal biogenesis and exocytosis, hence resulting in the overcoming of cancer multidrug resistance.