|M.Sc Student||Yunaev Anna|
|Subject||Lysosomotropic Drugs Activate TFEB Via Lysosomal Membrane|
Fluidization and Consequent Inhibition of mTORC1
|Department||Department of Biology||Supervisor||Professor Yehuda Assaraf|
|Full Thesis text|
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.