TMBIM6 (transmembrane BAX inhibitor motif containing 6) enhances autophagy through regulation of lysosomal calcium 3 1 Hyun-Kyoung Kim , Geum-Hwa Lee , Kashi Raj Bhattarai , Myung-Shik Lee , Sung Hoon Back , Hyung-Ryong Kim , Han-Jung Chae 1 1 1 2 4 1 Department of Pharmacology and New Drug Development Research Institute, Chonbuk National University Medical School, Jeonju, 2 Severance Biomedical Science Institute and Department of Internal Medicine, Yonsei University College of Medicine, Seoul, School of 3 4 Biological Sciences, University of Ulsan, Ulsan, College of Dentistry, Dankook
University, Cheonan, Republic of Korea BACKGROUND AIM TMBIM6 (transmembrane BAX inhibitor motif containing 6), a highly Recent findings indicated that TMBIM6 interacts with ITPR, which conserved multi-transmembrane protein, has been identified as a may regulate steady-state [Ca ]ER, leading to the relatively low 2+ 2+ suppressor of BAX-mediated cell death. TMBIM6 has been suggested mitochondrial calcium ([Ca ]mito) levels and reduced mitochondrial to be a Ca 2+ channel-like protein that is integral to the intracellular bioenergetics, and ultimately autophagy. Independently, TMBIM6- membranes
of ER. The calcium-binding activity has been found to be specific regulation of a specific arm of ER stress involving responsive to protons and other cations. The conserved aspartyl dyad ERN1/IRE-1α has also been reported in the context of secretory (Asp171-Asp195) in an uncharacterized protein YetJ from Bacillus protein IgG and autophagy studies. Although there have been a subtilis (BsYetJ) among TMBIM members regulates pH-dependent few studies on TMBIM6-associated autophagy regulation, the effect calcium-binding and manages the channel pore opening and closing, of TMBIM6 on ER and lysosomal
Ca 2+ signaling-associated and Ca2+ translocation. The Ca permeating role of TMBIM6 lowers autophagy has not been studied yet. In the present study, we have 2+- 2+ the steady-state [Ca ]ER. investigated the role of TMBIM6 in lysosomal Ca 2+ signaling and related autophagy. METHODS GCaMP3-ML-1 Ca 2+ imaging, Fura-2 Ca 2+ imaging, Oregon green 488 BAPTA-1 dextran (OG-BAPTA-dextran) or Rhod-dextran imaging, Immunofluorescence assays, Immunohistochemistry, Proximity ligation assay (PLA), Real-time PCR analysis, and Autophagy flux detection were performed. RESULTS Figure 1. TMBIM6 enhances
lysosomal calcium levels. (A-B) Vector/HT1080 and TMBIM6/HT1080 (A) or Tmbim6+/+ and tmbim6-/- MEF Figure 2. TMBIM6 enhances TFEB nuclear localization independent of Figure 3. TMBIM6 enhances autophagy flux. (A) Lysosomal staining was cells (B) were treated with the indicated agent. (C-D) ER Ca 2+ stores were MTORC1 activity. (A) Proximity ligation assay (PLA) between TFEB and performed with 100 nM LysoTracker for 30 min. (B) Autophagic flux was emptied with 1-5 μM thapsigargin or and 1 μM ionomycin before inducing PPP3CA (red dots) in TMBIM6- or TMBIM6D213A expressing HT1080 determined using
cyto-ID under microplate reader. (C) Immunoblotting of Ca 2+ release from acidic stores by GPN in vector/HT1080 and cells and vector cells under starvation or torin or PP242-treatment. (B) cell lysates against LC3B and SQSTM1 was performed and quantified TMBIM6/HT1080 cells (C) or Tmbim6+/+ and tmbim6-/- MEF cells (D). (E- Fluorescence images of endogenous TFEB after 3 h of starvation or torin or (bottom). N, vector; B, TMBIM6; M, TMBIM6D213A. (D) RFP-GFP-LC3 F) Fluorescence images of intraluminal Ca 2+ in vector/HT1080 and PP242-treatment TFEB nuclear translocation. (C) Fluorescence images of
puncta formation was analyzed in vector, TMBIM6, and TMBIM6/HT1080 cells. Representative images were showing the cells endogenous TFEB in siRNA of TMBIM6 and MCOLN1-pretreated cells TMBIM6D213A/HT1080 cells under starvation or torin or PP242-treatment loaded with low-affinity Rhod-dextran (LA-RhodDx, E) and OG-BAPTA- under starvation or torin or PP242-treatment after 3 h. (D) qPCR analysis of for 3 h. The yellow puncta (autophagosome) and red puncta dextran (F). (G) Time-lapse images of GPN-treated GCaMP3-ML1- TMBIM6 and MCOLN1 was performed to confirm the efficacy of siRNA- (autolysosome)
formation were quantified (bottom). Asterisks indicate expressing vector/HT1080 and TMBIM6/HT1080 cells during the indicated mediated silencing. Asterisks indicate significant differences from vector or significant differences from the vector treatment. Hash indicates significant time periods. The GPN responses were quantified (bottom). Representative scramble siRNA treatments. The hash indicates significant differences differences between TMBIM6 and TMBIM6D213A. fluorescence image of GCaMP3-ML1-expressing (green) and LysoTracker- between TMBIM6 and TMBIM6D213A. loaded (red) vector/HT1080 and
TMBIM6/HT1080 cells (up, right). CONCLUSION REFERENCES TMBIM6 induces autophagy through a Xu Q, Reed JC. Bax inhibitor-1, a mammalian apoptosis suppressor identified by functional screening in process that involves lysosomal Ca 2+ and yeast. Molecular cell 1998; 1:337-46. TFEB activation. These autophagy- Chae HJ, Kim HR, Xu C, Bailly-Maitre B, Krajewska M, Krajewski S, Banares S, Cui J, Digicaylioglu M, Ke N, enhancing characteristics might explain et al. BI-1 regulates an apoptosis pathway linked to endoplasmic reticulum stress. Molecular cell 2004; 15:355-66. TMBIM6-associated
anti-apoptosis, ER Castillo K, Rojas-Rivera D, Lisbona F, Caballero B, Nassif M, Court FA, Schuck S, Ibar C, Walter P, Sierralta stress regulation, and enhanced lysosomal J, et al. BAX inhibitor-1 regulates autophagy by controlling the IRE1alpha branch of the unfolded protein activity. This study contributes to the response. The EMBO journal 2011; 30:4465-78. understanding of the cellular response Sano R, Hou YC, Hedvat M, Correa RG, Shu CW, Krajewska M, Diaz PW, Tamble CM, Quarato G, Gottlieb associated with ER Ca leak and its clinical RA, et al. Endoplasmic reticulum protein BI-1 regulates
Ca(2)(+)-mediated bioenergetics to promote 2+ relevance in diseases such as autophagy. Genes & development 2012; 26:1041-54. neurodegenerative diseases. Correspondence to Han-Jung Chae, PhD; Email: Contact information hjchae@jbnu.ac.kr [F. Cell biology-1] TMBIM6 (transmembrane BAX inhibitor motif containing 6) enhances autophagy through regulation of lysosomal calcium Hyun-Kyoung Kim¹, Hyung-Ryong Kim⁴, Geum-Hwa Lee¹, Kashi Raj Bhattarai¹, Myung-Shik Lee², Sung Hoon Back³, Han-Jung Chae¹ ¹Department of Pharmacology and New Drug Development Research Institute, Chonbuk National University
Medical School, Jeonju 54896, Republic of Korea, ²Severance Biomedical Science Institute and Department of Internal Medicine, Yonsei University College of Medicine, Seoul 03722, Republic of Korea, ³School of Biological Sciences, University of Ulsan, Ulsan 44610, Republic of Korea, ⁴College of Dentistry, Dankook University, Cheonan 31116, Republic of Korea Lysosomal Ca2+ contributes to macroautophagy/autophagy, an intracellular process for the degradation of cytoplasmic material and organelles in the lysosomes to protect cells against stress responses. TMBIM6 (transmembrane BAX inhibitor motif
containing 6) is a Ca2+ channel-like protein known to regulate ER stress response and apoptosis. In this study, we examined the as yet unknown role of TMBIM6 in regulating lysosomal Ca2+ levels. The Ca2+ efflux from the ER through TMBIM6 was found to increase the resting lysosomal Ca2+ level, in which ITPR-independent regulation of Ca2+ status was observed. Further, TMBIM6 regulated the local release of Ca2+ through lysosomal MCOLN1/TRPML1 channels under nutrient starvation or MTOR inhibition. The local Ca2+ efflux through MCOLN1 channels was found to activate PPP3/calcineurin, triggering TFEB
(transcription factor EB) nuclear translocation, autophagy induction, and lysosome biogenesis. Upon genetic inactivation of TMBIM6, lysosomal Ca2+ and the associated TFEB nuclear translocation were decreased. Together, our observations indicated that under stress conditions, TMBIM6 increases lysosomal Ca2+ release, leading to PPP3/calcineurinmediated TFEB activation and subsequently enhanced autophagy. Tubby like protein family is an adaptor for ciliary G protein-coupled receptor trafficking Kyoungeun Kim¹ ¹, Seok Jun Moon¹ ¹ , , Oral Biology, Yonsei University College of Dentistry, Seoul
03722, Republic of Korea Introduction Cilia are highly specialized antennae-like cellular organelles serves as mediator for cell signaling cascade. Dysfunction of cilia due to diverse factor leads to clinical phenotype such as retinal degeneration, polycystic kidney disease and obesity. An elaborate mechanism for establishing and maintaining the compartmentalization of cilia is necessary for proper ciliary function, which includes the entry, localization, and exit of specific signaling molecules to and from the ciliary compartment. In the previous study, TULP3 is known as adapters for the
ciliary trafficking of G protein-coupled receptors (GPCRs). Since Tubby like protein family (TULP) shares N-terminal IFT binding domain and C-terminal Tubby domain, it is intriguing to speculate that all TULPs function as adaptors for ciliary membrane cargo trafficking. This study investigate whether TULPs regulate ciliary protein trafficking. hTERT RPE-1(RPE1) cell is generally known for cell line that doesn’t express TUB, we additionally identified TULP1 and TULP2 are not expressed in RPE1cell and used CRISPR/Cas9 system to generate Tulp3 knockout cell, namely TULPs abolished cell.
Subsequently, the primary Fig. 2. Ciliary formation of TULP3 knockout cell cilia of wild type cell, Tulp3 knockout cells and each TULP rescued Tulp3 knockout cells were visualized by immunocytochemistry test. Ciliary formation Tulp3 knockout cells were downregulated comparing to wild type cells and length of cilia was also unambiguously shorter than wild type cells. Interestingly, only TULP3 was able to fully rescue ciliary length and frequency in Tulp3 knockout cells. TULP1, TULP2 and TULP3 were not able to rescue ciliary length or formation. Next, candidate ciliary GPCRs selected based on
previous data were transfected to Tulp3 knockout cells. Ciliary GPCRs which failed to localize cilia in Tulp3 knockout cells tend to recover ciliary trafficking depending on TULP1, TULP2, TULP3 and TUB. This result indicates TULPs are functionally redundant in ciliary GPCR trafficking but selectively involved in maintenance of primary cilium. Further, regarding the distinct tissue distribution between TULPs, TULPs might have tissue-specific functions. This result indicates that TULPs share similar functions but also might have functional diversity through its differential tissue distribution.
Materials and Methods Fig. 3. Effect of TULPs in ciliary GPCRs trafficking. Conclusions • TULP3 serve as core factor that regulate primary cilia development among TULPs. • TULP1, TULP2, TULP3 and TUB function as adaptor for ciliary molecule trafficking. References • Badgandi, H. B., Hwang, S. H., Shimada, I. S., Loriot, E., & Mukhopadhyay, S. (2017). Tubby family proteins are adapters for ciliary trafficking of integral membrane proteins. Journal of Cell Biology, 216(3), 743-760. Results • Han, S., Miyoshi, K., Shikada, S., Amano, G., Wang, Y., Yoshimura, T., & Katayama, T. (2019). TULP3 is
required for localization of membrane- associated proteins ARL13B and INPP5E to primary cilia. Biochemical and biophysical research communications, 509(1), 227-234. • Loktev, A. V., & Jackson, P. K. (2013). Neuropeptide Y family receptors traffic via the Bardet-Biedl syndrome pathway to signal in neuronal primary cilia. Cell reports, 5(5), 1316-1329. • Omori, Y., Chaya, T., Yoshida, S., Irie, S., Tsujii, T., & Furukawa, T. (2015). Identification of G protein-coupled receptors (GPCRs) in primary cilia and their possible involvement in body weight control. PLoS One, 10(6). Acknowledgments This
work was supported by the BK21 plus program through the Natio
