Selective agonist of TRPML2 reveals direct role in chemokine release from innate immune cells
- Ludwig Maximilian University of Munich, Germany
- Telethon Institute of Genetics and Medicine, Italy
- Medical Faculty, Ludwig Maximilian University of Munich, Germany
- National Heart, Lung, and Blood Institute, National Institutes of Health, United States
- Martin Luther University of Halle-Wittenberg, Germany
- University Hospital Munich, Germany
- Hannover Medical School, Germany
Figures
Figure 1 with 3 supplements
Chemical synthesis strategies and functional evaluation of SN-2 analogous compounds using calcium imaging.
(A) Shown are synthesis strategies a and b used to generate most of the SN-2 analogous compounds shown in Suppl. Figure 1. R1 = alkyl/ halogen/nitro/ methoxy; R2 / R3 = (cyclo)alkyl/phenyl/ …
Figure 1—figure supplement 1
Structures of SN-2 analogues.
Shown are the chemical structures of all generated SN-2 analogous compounds. Changes in comparison to SN-2 are highlighted in red. (A) Structure of the lead compound SN-2. (B) Variations in the aryl …
Figure 1—figure supplement 2
Structures of SF-51/ML-SA1 analogues.
Shown are the chemical structures of all generated SF-51/ML-SA1 analogous compounds. Changes in comparison to SF-51/ML-SA1 are highlighted in red. (A) Structure of the lead compounds ML-SA1 and …
Figure 1—figure supplement 3
Chemical synthesis strategies and functional evaluation of SF-51/ML-SA1-analogous compounds using calcium imaging.
(A) Shown are synthesis strategies a and b used to generate the 35 SF-51/ML-SA1-analogous compounds shown in Figure 1—figure supplement 2. X = Cl/ Br; Y = CO/SO2 / CH2; R1 / R2 / R3 / R4=H/(cyclo)alk…
Figure 2 with 3 supplements
Effect of ML2-SA1 on TRPML channels.
(A) Cartoon depicting chemical structures of SN-2 and ML2-SA1. (B) Representative ML2-SA1 or ML-SA1 (10 μM) elicited currents from YM201636-enlarged LE/LY isolated from hTRPML2 expressing HEK293 …
Figure 2—figure supplement 1
Effect of ML2-SA1 on TRPML2 under different pH conditions.
(A) Representative basal, PI(3,5)P2 (1 μM) or ML2-SA1 (10 μM) elicited currents from YM201636-enlarged LE/LY isolated from hTRPML2 expressing HEK293 cells (neutral luminal pH). (B) Representative …
Figure 2—figure supplement 2
Co-transfection of HEK293 cells with fluorescently labelled TRPML2 and vesicle-specific markers of the endolysosomal system.
(A) Shown are representative images of TRPML2 (green) transiently cotransfected with either TRPML2 (red) or markers of the endolysosomal system (red): Rab5 (early endosomes), TfR (early/recycling …
Figure 2—figure supplement 3
DRC of ML2-SA1 effect on mTRPML2, effects of ML2-SA1, SN2, and ML-SA1 on mTRPML channel isoforms, and cytotoxicity of ML2-SA1.
(A) Dose-response curve (DRC) for ML2-SA1 effect on mouse TRPML2 in HEK293 cells, obtained from fura-2 calcium imaging experiments. The calculated EC50 value for mTRPML2 is: 2.38 ± 0.001 μM (mean ± S…
Figure 3 with 1 supplement
Molecular modeling of ML2-SA1 and ML-SA1 binding.
(A) Binding mode of ML-SA1 (green colored carbon atoms) at hTRPML1, showing residues within 5 Å of ML-SA1, as observed in one of the four identical binding pockets of the cryo-EM structure (PDB ID: …
Figure 3—figure supplement 1
Additional molecular modeling of ML2-SA1 and ML-SA1 binding.
(A) Comparison of the experimentally derived binding mode of ML-SA1 (green colored carbon atoms) and the docking pose (obtained with program GLIDE) at the hTRPML1 binding pocket (orange colored …
Figure 4
Effect of ML2-SA1 on channel currents in endolysosomal organelles isolated from different primary mouse macrophages.
(A) Representative currents from vacuolin-enlarged/Tf+ vesicles isolated from murine (LPS 6 hr or LPS 0 hr) primary WT BMDMΦ, basal or elicited by an application of 10 μM ML2-SA1. All currents are …
Figure 5
Effect of ML2-SA1 on chemokine release from primary mouse macrophages.
(A) Shown are data obtained from primary WT and TRPML2-/- mouse bone marrow macrophages (BMDMΦ) with and without LPS treatment for 4 hr and 8 hr, respectively. The fraction treated with LPS and 10 …
Figure 6 with 2 supplements
Effect of ML2-SA1 on macrophage migration.
(A) Shown are representative images obtained from a modified Boyden chamber experiment. Images show fixed and crystal violet stained BMDMΦ after 3 hr migration through a transwell chamber along a …
Figure 6—figure supplement 1
Modified and classical Boyden chamber setup.
In the modified Boyden chamber setup (A–C) BMDMΦ plated on poly-L-lysine coated cover slips in a twenty-four well plate are treated with 1 µg/mL LPS or equal volume of DMSO for 6 hr (A). After 6 hr …
Figure 6—figure supplement 2
Migration assay without cells in the lower compartment of the classical Boyden chamber.
(A) Representative images obtained from a classical Boyden chamber setup showing fixed and crystal violet stained BMDMΦ after 3 hr migration through a transwell chamber. The respective conditions in …
Author response image 1
Tables
Key resources table
| Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|
| HEK293 | DSMZ | ACC 305 | |
| HEK 293 stable stably expressing TRPML3-YFP | Grimm et al. (2010); PMID: 20189104 | ||
| HEK 293 stable stably expressing TRPML1-YFP | Chen et al. (2014), PMID: 25119295 | ||
| TRPML1 (encoded by the Mcoln1 gene) KO mouse; Mcoln1tm1Sasl, C57BL/6 | Venugopal et al. (2007); PMID: 17924347 | MGI ID: 3794204 | |
| TRPML2 (encoded by the Mcoln2 gene) KO mouse; C57BL/6 | Sun et al. (2015); PMID: 26432893 | MGI: 1915529 | |
| TRPML3 (encoded by the Mcoln3 gene) KO mouse; Mcoln3tm1. 1Hels, FVB/NJ | Jörs et al. (2010); PMID: 21179200 | MGI ID: 5319089 | |
| anti-LAMP-1 (1D4B) (rat monoclonal) | Santa Cruz | Cat#A-11006; RRID: AB_2134495 | (1:100) |
| Goat anti-Rat IgG (H + L) Secondary Antibody, Alexa Fluor 488 | ThermoFisher | Cat#sc-19992; RRID: AB_2534074 | (1:1000) |
| mcherry-Transferrin Receptor 20 (plasmid) | N/A | Addgene Plasmid #55144 | |
| DsRed-Rab11 (plasmid) | Choudhury et al. (2002); PMID: 12070301 | Addgene Plasmid #12679 | |
| TRPML1-YFP (plasmid) | Grimm et al. (2010), PMID: 20189104 | ||
| TRPML2-YFP (plasmid) | Grimm et al. (2010), PMID: 20189104 | ||
| TRPML3-YFP (plasmid) | Grimm et al. (2010), PMID: 20189104 | ||
| Quikchange primers for TRPML2:YFP A422C | this paper | forward: CTTCGGTTTTGTTGTTGTG CTGGTATGATTTATCTGGG reverse: CCCAGATAAATCATACCAGC ACAACAACAAAACCGAAG | |
| Quikchange primers for TRPML2:YFP A424V | this paper | forward: CGGTTTTGTGCTTG TGTTGGTATGATTTATCTGGGTTACAC reverse: GTGTAACCCAGATAAATCAT ACCAACACAAGCACAAAACCG | |
| Quikchange primers for TRPML2:YFP G425A | this paper | forward: CGGTTTTGTGCTTGT GCTGCTATGATTTATCTGGGTTACAC reverse: GTGTAACCCAGATAAATCA TAGCAGCACAAGCACAAAACCG | |
| Quikchange primers for TRPML2:YFP A453S | this paper | forward: CTGAACACAGTTTCTG AGTGTCTGTTTTCTCTGG reverse: CCAGAGAAAACAGACA CTCAGAAACTGTGTTCAG | |
| Quikchange primers for TRPML2:YFP V460I | this paper | forward: TGTCTGTTTTCTCTGATCA ACGGTGATGACATG reverse: CATGTCATCACCGTTGATC AGAGAAAACAGACA | |
| Quikchange primers for TRPML2:YFP I498V | this paper | forward: CCTTCATCAGCCTTTTTATATATA TGGTTCTCAGTCTTTTTATTGC reverse: GCAATAAAAAGACTGAGAACCA TATATATAAAAAGGCTGATGAAGG | |
| qPCR Primer for TRPML1 (NM_053177) | www.pga.mgh.harvard.edu/primerbank | PrimerBankID: 16716462 c2 | forward: GCCTTGGGCCAATGGATCA reverse: CCCTTGGATCAATGTCAAAGGTA |
| qPCR Primer for TRPML2 (NM_026656) | this paper | forward: AATTTGGGGTCACGTCATGC reverse: AGAATCGAGAGACGCCATCG | |
| qPCR Primer for TRPML3 (NM_134160) | this paper | forward: GAGTTACCTGGTGTGGCTGT reverse: TGCTGGTAGTGCTTAATTGTTTCG | |
| qPCR Primer for HPRT (NM_013556) | Hruz et al. (2011); PMID: 21418615 | N/A | forward: GCTCGAGATGTCATGAAGGAGAT reverse: AAAGAACTTATAGCCCCCCTTGA |
| Lipopolysaccharides (LPS) from Escherichia coli O26:B6 | Sigma-Aldrich | Cat#L2762 | |
| Lipopolysaccharides (LPS) from Escherichia coli O111:B4 | Sigma-Aldrich | Cat#L4391 | |
| Fura-2, AM, cell permeant | ThermoFisher | Cat#F1201 | |
| Mouse M-CSF, premium grade | Miltenyi Biotech | Cat#130-101-703 | |
| Transferrin from human serum, Alexa FluorTM 546-conjugated | TermoFisher | Cat# T23364 | |
| Transferrin from human serum, Alexa FluorTM 555-conjugated | Thermo Fisher | Cat#T35352 | |
| JE/MCP-1/CCL2 from mouse, recombinant | Sigma-Aldrich | Cat# SRP4207 | |
| YM201636 | Chemdea | Cat#CD0181 | |
| MLSA-1 | Sigma-Aldrich | Cat#SML0627 | |
| PI(3,5)P2 | AG Scientific | Cat#P-1123 | |
| Wortmannin | Sigma-Aldrich | Cat#W1628 | |
| LatrunculinB | Sigma-Aldrich | Cat#L5288 | |
| Vacuolin | Santa Cruz | Cat# sc-216045 | |
| Calcium ionophore A23187 | Sigma-Aldrich | Cat#C7522 | |
| 4-Methylumbelliferyl N-acetyl-b-D-glucosaminide | Sigma-Aldrich | Cat#M2133 | |
| RNeasy Plus Mini Kit | Qiagen | Cat# 74134 | |
| RevertAid first strand cDNA synthesis Kit | ThermoScientific | Cat# K1621 | |
| CD11b MicroBeads, human and mouse | Miltenyi Biotech | Cat#130-049-601 | |
| QuikChange II Site-Directed Mutagenesis Kit | Agilent | Cat#200523 | |
| Mouse/rat CCL2/ JE/MCP-1 Quantikine ELISA Kit | BioLegend | Cat#432707 | |
| Origin8 | OriginLab | ||
| GraphPad Prism | GraphPad Software Inc. |
Additional files
-
Supplementary file 1
Synthesis details and analytical data
- https://doi.org/10.7554/eLife.39720.017
-
Supplementary file 2
Summary of characteristics of TRPML channels
- https://doi.org/10.7554/eLife.39720.018
-
Transparent reporting form
- https://doi.org/10.7554/eLife.39720.019
