Abstract
Rapamycin (Rap) and its derivatives, called rapalogs, are being explored in clinical trials targeting cancer and neurodegeneration. The underlying mechanisms of Rap actions, however, are not well understood. Mechanistic target of rapamycin (mTOR), a lysosome-localized protein kinase that acts as a critical regulator of cellular growth, is believed to mediate most Rap actions. Here, we identified mucolipin 1 (transient receptor potential channel mucolipin 1 [TRPML1], also known as MCOLN1), the principle Ca2+ release channel in the lysosome, as another direct target of Rap. Patch-clamping of isolated lysosomal membranes showed that micromolar concentrations of Rap and some rapalogs activated lysosomal TRPML1 directly and specifically. Pharmacological inhibition or genetic inactivation of mTOR failed to mimic the Rap effect. In vitro binding assays revealed that Rap bound directly to purified TRPML1 proteins with a micromolar affinity. In both healthy and disease human fibroblasts, Rap and rapalogs induced autophagic flux via nuclear translocation of transcription factor EB (TFEB). However, such effects were abolished in TRPML1-deficient cells or by TRPML1 inhibitors. Hence, Rap and rapalogs promote autophagy via a TRPML1-dependent mechanism. Given the demonstrated roles of TRPML1 and TFEB in cellular clearance, we propose that lysosomal TRPML1 may contribute a significant portion to the in vivo neuroprotective and anti-aging effects of Rap via an augmentation of autophagy and lysosomal biogenesis.
Highlights
Rapamycin (Rap) is a natural macrocyclic compound that was initially isolated from Streptomyces hygroscopicus as an antifungal agent [1]
Whole-endolysosome recordings were performed in vacuoles that had been enlarged with vacuolin-1 and isolated manually from enhanced green fluorescent protein (EGFP)-transient receptor potential channel mucolipin 1 (TRPML1)–transfected CV-1 in Origin Simian-1 (COS1) cells [26] (Fig 1A)
We found that Rap induced robust activation of whole-endolysosomal TRPML1 current (ITRPML1; Fig 1B and 1C)
Summary
Rapamycin (Rap) is a natural macrocyclic compound that was initially isolated from Streptomyces hygroscopicus as an antifungal agent [1]. The first identified target protein of Rap was discovered in yeast and named target of rapamycin (TOR) [6, 7]. TOR, renamed mechanistic target of rapamycin (mTOR), is a serine and/or threonine kinase that is highly conserved in eukaryotes [6, 7]. In response to environmental changes, such as nutrient availability, mTOR kinase activity is switched on and off through the formation of alternate protein complexes—mTOR complex 1 (mTORC1) and mTORC2— and through association with and dissociation from lysosomal membranes. Rap acts as a high-affinity (nM range) allosteric inhibitor of mTORC1 (hereafter referred to as mTOR) that blocks mTOR substrate recruitment by binding to the FK506 binding protein (FKBP) and the rapamycin binding (FRB) domain of mTOR, forming a ternary FKBP12-Rap-mTOR complex [3, 4]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
