NAADP-binding proteins find their identity

Abstract

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a second messenger that releases Ca²⁺ from endosomes and lysosomes by activating ion channels called two-pore channels (TPCs). However, no NAADP-binding site has been identified on TPCs. Rather, NAADP activates TPCs indirectly by engaging NAADP-binding proteins (NAADP-BPs) that form part of the TPC complex. After a decade of searching, two different NAADP-BPs were recently identified: Jupiter microtubule associated homolog 2 (JPT2) and like-Sm protein 12 (LSM12). These discoveries bridge the gap between NAADP generation and NAADP activation of TPCs, providing new opportunity to understand and manipulate the NAADP-signaling pathway. The unmasking of these NAADP-BPs will catalyze future studies to define the molecular choreography of NAADP action.

Keywords: calcium signaling; cancer; endosomes; lysosomes; photoaffinity probes.

Figure 1.. The action of nicotinic acid adenine dinucleotide phosphate (NAADP)-binding proteins (BPs) and emerging questions enabled by their identification.

(A) Two-pore channels (TPCs) are ion channels expressed on endolysosomes. They are subject to polymodal activation by NAADP, PI(3,5)P₂, and/or voltage (V). (B) Models for TPC activation by NAADP. Top: the originally envisioned model that TPCs are directly activated by the binding of NAADP (white) has not received experimental support. Rather, NAADP has been proposed to activate TPCs indirectly by engaging NAADP-BPs (purple) that are essential components for NAADP-triggered Ca²⁺ release. Bottom, this may occur in several ways, for example, by (i) NAADP association with the NAADP-BP causing a translocation of NAADP-liganded NAADP-BPs to the TPC complex or (ii) by NAADP engaging NAADP-BPs already associated with the TPC complex. (iii) Dissociation of the NAADP-BP from the channel complex, or dissociation of NAADP from the NAADP-BP, could serve to terminate NAADP action. (C) Future areas for NAADP-BP research enabled by the recent identification of two NAADP-BPs, JPT2 and LSM12. These include (1) the mechanism of JPT2 interaction of NAADP and JPT2 association with different TPC isoforms, (2) regulation of JPT2 expression levels, for example, through degradation, to set cellular NAADP sensitivity, (3) characterization of other functions of JPT2 and the broader JPT2 interactome as a roadmap for understanding new facets of NAADP biology, (4) identification and characterization of additional vertebrate and invertebrate NAADP-BPs, and (5) development of novel tools to manipulate this signaling pathway. Each of these areas is discussed in the main text. This figure was created at BioRender.com. Abbreviations: JPT2, Jupiter microtubule associated homolog 2; LSM12, ‘like-Sm’ protein 12; PI(3,5)P_{2, phosphatidylinositol 3,5-bisphosphate; RyR, ryanodine receptor}.

Figure 2.. JPT2 structure and evolution.

(A) Schematic representation of human JPT2 splice isoforms. Length in amino acids for JPT2.3 is shown. (B) Cladogram of JPT sequences from animals with bootstrap values shown. Phlylogenetics was performed as described in [97] using PHYML (version 3.1) with the JTT amino acid substitution model, estimated proportion of invariable sites, and the four-category discrete gamma model (JTT + I + G) selected by ProtTest (version 3.4.2). Deuterostome branches are shaded in brown, and protostome branches are shaded in yellow. Abbreviations: CteJPT (ELU08900.1), Capitella teleta; HroJPT (XP_009029441.1), Helobdella robusta; AcaJPT (XP_005102887.1), Aplysia californica; PmaJPT (XP_033742783.1), Pecten maximus; OvuJPT (XP_029648722.1), Octopus vulgaris; TnaJPT (OUC50045.1), Trichinella nativa; DmeJPT, (Q9I7K0) Drosophila melanogaster; NpiJPT, (AII97726.1) Nephila pilipes; CscJPT (XP_023222178.1), Centruroides sculpturatus; DmaJPT (XP_032791280.1), Daphnia magna; AruJPT (XP_033631490.1), Asterias rubens; AcanJPT (XP_022098988.1), Acanthaster planci; SpuJPT (XP_011684190.1), Strongylocentrotus purpuratus; AjaJPT (PIK56568.1), Apostichopus japonicus; AjapJPT (XP_033109709.1), Anneissia japonica; BflJPT (XP_002611678.1), Branchiostoma floridae; CinJPT (XP_009861954.1), Ciona intestinalis; PmamJPT (CAB3257823.1), Phallusia mammillata; OdiJPT (CBY20678.1), Oikopleura dioica; PmarJPT (XP_032826119.1), Petromyzon marinus; AraJPT1 (XP_032900329.1), AraJPT2 (XP_032896233.1), Amblyraja radiata; DreJPT1a (NP_001082982.1), DreJPT1b (NP_991176.1), DreJPT2 (NP_955869.2), Danio rerio; LchJPT1 (XP_005998303.1), LchJPT2 (XP_005997850.1), Latimeria chalumnae; XtrJPT1 (NP_001139220.1), XtrJPT2 (XP_012825626.1), Xenopus tropicalis; AcarJPT1 (XP_003217263.1), AcarJPT2 (XP_008120328.1), Anolis carolinensis; GgaJPT1 (XP_015150886.1), GgaJPT2 (NP_001265082.1), Gallus gallus; CluJPT1 (NP_001093413.1), CluJPT2 (XP_022275981.1), Canis lupus familiaris; MmuJPT1 (NP_032284.1), MmuJPT2 (NP_945175.1), Mus musculus; HsaJPT1 (NP_001002032.1), HsaJPT2 (NP_653171.1), Homo sapiens; JPT2, Jupiter microtubule associated homolog 2.

Figure 3.. NAADP-BP expression.

(A) Violin plot showing expression values [transcripts per million (TPM)] of JPT2 (brown) and LSM12 (blue) across a variety of tissues, calculated from a gene model where all isoforms are collapsed to a single gene. Box plots show median as well as 25th and 75th percentiles. TPM values were produced with RNA-SeQC v1.1.9. Noticeably, expression of both NAADP-BPs shows a broad tissue distribution, with increased JPT2 expression in the spinal cord, as described in the main text. (B) Heatmap depicting median TPMs for NAADP-BPs (JPT2, LSM12) as well as two-pore channels (TPCs; TPC1 and TPC2). Tissues are arrayed alphabetically. These data show JPT2 expression is better correlated with TPC1 and LSM12 expression is better correlated with TPC2. Data for Figure 3 are reproduced from the Genotype-Tissue Expression project (GTEx, v8 [45]). Abbreviations: JPT2, Jupiter microtubule associated homolog 2; LSM12, ‘like-Sm’ protein 12; NAADP-BP, NAADP-binding protein.

Figure 4.. JPT2 expression in cancer.

(A) Expression plot detailing JPT2 transcript levels in 33 different tumors (red) compared with controls (green). Different cancers are color coded to indicate statistically higher JPT2 expression in cancerous (red) or normal tissue (green) or no statistical difference (black). Mean expression (bar). Cancer abbreviations: ACC, adrenocortical carcinoma; BLCA, bladder urothelial carcinoma; BRCA, breast invasive carcinoma; CESC, cervical squamous cell carcinoma, endocervical adenocarcinoma; CHOL, cholangiocarcinoma; COAD, colon adenocarcinoma; DLBC, lymphoid neoplasm diffuse large B cell lymphoma; ESCA, esophageal carcinoma; GBM, glioblastoma multiforme; HNSC, head and neck squamous cell carcinoma; KICH, kidney chromophobe; KIRC, kidney renal clear cell carcinoma; KIRP, kidney renal papillary cell carcinoma; LAML, acute myeloid leukemia; LGG, brain lower grade glioma; LIHC, liver hepatocellular carcinoma; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; MESO, mesothelioma; OV, ovarian serous cystadenocarcinoma; PAAD, pancreatic adenocarcinoma; PCPG, pheochromocytoma and paraganglioma; PRAD, prostate adenocarcinoma; READ, rectum adenocarcinoma; SARC, sarcoma; SKCM, skin cutaneous melanoma; STAD, stomach adenocarcinoma; TGCT, testicular germ cell tumors; THCA, thyroid carcinoma; THYM, thymoma; UCEC, uterine corpus endometrial carcinoma; UCS, uterine carcinosarcoma; UVM, uveal melanoma. (B) Disease-free survival plot for high versus low JPT2 expression levels (median ± 50% cutoffs) in LGG (n = 257). Dotted lines (95% confidence). (C and D) Similar analyses for (C) LSM12 expression and (D) outcomes in ACC (n = 38). Expression and survival data for Figure 4 are reproduced from GEPIA2 [69,70]. Abbreviations: JPT2, Jupiter microtubule associated homolog 2; LSM12, ‘like-Sm’ protein 12.

Figure I.

[³²P]-Nicotinic acid adenine dinucleotide phosphate (NAADP; top left) represents one version of a radioisotope (red) used to study NAADP binding in cells and tissues. [³²P]-5-N₃-NAADP was the original photoaffinity probe used to characterize NAADP targets [24,25]. The probe incorporates a photoreactive azide group (blue) to crosslink binding partners following ultraviolet irradiation. [³²P]-alkyne-’all-in-one-click’ (AIOC)-NAADP is a bifunctional probe that incorporates an additional clickable alkynyl moiety (magenta) to couple photolabeling to NAADP-BP enrichment. For example, to isolate JPT2 [14], photoprobe-bound NAADP-BPs were biotinylated via a copper-catalyzed alkyne–azide cycloaddition (CuAAC) using ‘click chemistry’ such that the biotinylated proteins could then be captured using neutravidin. Tetrandrine is a bisbenzylisoquinoline alkaloid originally shown to block two-pore channels (TPCs) [57], with other derivatives of this chemotype also blocking TPC function [58,87]. TPC2-A1-N is a novel chemotype recently identified in a drug screen profiling TPC2 that mimics NAADP-evoked Ca²⁺ signals and NAADP-evoked TPC currents [10].