Identification of LAMTOR1 as A Novel Regulator of NAADP-TPC2-mediated Calcium Release and Lysosomal Trafficking

鑑定LAMTOR1作為NAADP-TPC2介導的鈣釋放和溶酶體運輸的新型調節劑

Student thesis: Doctoral Thesis

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Award date12 Dec 2023

Abstract

Calcium ions (Ca2+) lie at the center of multiple fundamental biological processes. Ca2+ is one of the most important second messengers as it plays a crucial role in almost every aspect of cell metabolism, including the cell cycle, lipid synthesis, transcription, translation, and cell proliferation. Another second messenger, nicotinic acid adenine dinucleotide phosphate (NAADP), was found to induce lysosomal Ca2+ release and regulate subsequent protein turnover and other lysosomal degradation events. Two pore channel type 2 (TPC2) was first identified as a binding target of NAADP to release Ca2+ from the lysosomal lumen. TPC2 is predominantly located on the membrane of late endosomes and lysosomes, and reports indicate that NAADP-mediated TPC2 Ca2+ events are essential for maintaining functional endocytosis. Indeed, a loss of TPC2 or inhibition of NAADP-mediated Ca2+ release disrupts the fusion of late endosomes and lysosomes, as well as endolysosomal degradation. However, the mechanism underlying such regulation remains elusive. It has been reported that the small GTPase, RAB7, interacts with and regulates TPC2 activity as it participates in the fusion of late endosomes and lysosomes. It has been suggested that TPC2 might regulate endolysosomal fusion by contributing to RAB7-mediated membrane fusion. Nevertheless, NAADP-mediated TPC2 Ca2+ release is crucial for maintaining functional lysosome-mediated events.

However, the detailed mechanism of NAADP-induced TPC2 Ca2+ release is still largely unknown. There has been some debate about the ion permeability of TPC2. This is because although it was primarily identified as an NAADP-targeted Ca2+ channel, subsequent publications claimed that TPC2 was only stimulated by PI(3,5)P2 and only released Na+ from the lysosomes. More recently, reports suggested that additional adaptor protein(s) might help TPC2 associate with NAADP to mediate the Ca2+ flux. The identification of such TPC2-NAADP adaptor protein(s) caught a lot of attention. Recently, two NAADP-binding proteins, JPT2 and Lsm12 were identified. JPT2 was shown to interact with TPC1, whereas Lsm12 interacts with both TPC1 and TPC2. However, the specific mechanism of NAADP and TPC2 association has still not been explained. To fill this gap in our knowledge, members of our research team have been looking for NAADP and TPC2 binding candidates to reveal the detailed mechanism of NAADP-mediated TPC2 Ca2+ release.

Using mass spectrometry analysis, members of our team previously identified LAMTOR1 (a scaffold protein of ragulator), as a TPC2 binding protein. Therefore, in Chapter 3, I confirmed the binding affinity of LAMTOR1 and TPC2 and validated the regulatory role of LAMTOR1 on TPC2 Ca2+ release. Then, I generated LAMTOR1 deletions to test the TPC2 binding site and found that LAMTOR1 interacted with TPC2 and regulated TPC2 Ca2+ release via the N-terminus. Moreover, via the use of immobilized NAADP-conjunct agarose beads, I confirmed that LAMTOR1 can directly interact with NAADP. Then, I found that the association of TPC2 with NAADP requires the presence of LAMTOR1, which further confirmed the regulatory role of LAMTOR1 in NAADP-mediated TPC2 Ca2+ release. To fully understand the mechanism of LAMTOR1 regulation on TPC2, I showed that LAMTOR1 can interact with another TPC2 and NAADP binding protein, Lsm12, and that TPC2, LAMTOR1, and Lsm12 seem to be in the same complex. Thus, together my results suggest that LAMTOR1 regulates NAADP-induced TPC2 Ca2+ currents by directly binding to NAADP and that LAMTOR1 and Lsm12 cooperate to mediate the association between NAADP and TPC2.

In Chapter 4, I describe the data I obtained to better understand the regulatory role of LAMTOR1 in TPC2 activity by examining the role of LAMTOR1 in TPC2-related lysosomal events. In LAMTOR1 knockout cells (like following TPC2 knockdown or inhibition), I found that the fusion between late endosomes and lysosomes was interrupted. Then, to investigate the mechanism of LAMTOR1 regulation on lysosomal events in more detail, I examined whether LAMTOR1 regulated RAB7 activity. My results showed that LAMTOR1 can interact with RAB7 and regulate its activity. Thus, my data suggest that LAMTOR1 plays a role in lysosome-regulated endocytosis and the degradation of proteins and lipids via RAB7.

In Chapter 5, I investigated the involvement of TPC2 in LAMTOR1-regulated mTORC1 activity. I showed that a loss of TPC2 or inhibition of TPC2 Ca2+ release inhibited mTORC1 translocation to the lysosomes by cholesterol induction. I also showed that stimulation of TPC2 Ca2+ activity overcame the inhibitory effects on mTORC1 translocation and activation by cholesterol depletion. This suggests that TPC2 Ca2+ activity might be downstream of cholesterol-induced mTORC1 activation.

To sum up, I first described the mechanism of LAMTOR1 regulation on NAADP-mediated TPC2 Ca2+ release. I found that LAMTOR1 can directly interact with NAADP and cooperate with Lsm12 to regulate the association between TPC2 and NAADP. I also described the regulatory role of LAMTOR1 in lysosome-mediated protein and lipid trafficking and degradation, to further investigate the regulatory role of LAMTOR1 on TPC2-related activity. Similar to TPC2, LAMTOR1 was found to be crucial for endolysosomal fusion and lysosomal degradation. Finally, I found that TPC2 is required for cholesterol-induced mTORC1 translocation and activation, which makes the relationship between TPC2 and LAMTOR1 much clearer.

In general, with the identification of a new NAADP-binding protein, LAMTOR1, my data provide new insights into the mechanism of NAADP-mediated TPC2 Ca2+ release. I also discovered new details about the lysosome-mediated protein and lipid trafficking mechanism and the (often overlooked) role of LAMTOR1 in lysosomal Ca2+ flux and its related events.

    Research areas

  • LAMTOR1, TPC2, Ca2+, Ca2+channels, endocytosis, lipid degradation, mTORC1