Sushil Kumar

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a Ca ²⁺ -releasing second messenger that activates two-pore channels (TPCs) on endosomes and lysosomes. Rather than binding TPCs directly, NAADP acts through cytoplasmic NAADP-binding proteins (NAADP-BPs) which are essential for endolysosomal Ca ²⁺ release. Here we characterized the properties of two recombinant, purified NAADP-BPs: Jupiter Microtubule Associated Homolog 2 (JPT2) and like-Sm protein 12 (LSM12). In contrast to LSM12, JPT2 is predicted to be an intrinsically disordered protein, a feature confirmed by circular dichroism and NMR spectroscopy. Under conditions of low Na ⁺ concentration or molecular crowding, JPT2 underwent phase separation, as demonstrated by multiple orthogonal approaches. JPT2 condensates displayed liquid-like behavior and efficiently recruited LSM12, a novel fluorescent NAADP analog, as well as tubulin. JPT2 condensates also interacted with polymerized microtubules and lysosomes isolated from human cell lines. These findings reveal an unexpected capability of NAADP-BPs to undergo phase separation, and segregate with components needed for NAADP-dependent Ca ²⁺ release. We speculate that these signaling condensates dictate cellular NAADP sensitivity, desensitization of NAADP responses, as well as NAADP targeting to TPCs at membrane contact sites between acidic organelles and the endoplasmic reticulum. Graphical

During prolonged starvation and exhaustive exercise, when there is low availability of carbohydrates, the liver breaks down fatty acids to generate ketone bodies, which are utilized by peripheral tissues as an alternative fuel source. The transcription factor MEF2D undergoes regulated alternative splicing in the postnatal period to produce a highly conserved, muscle specific MEF2Dα2 protein isoform. Here, we discover that compared to WT mice, MEF2Dα2 exon knockout (Eko) mice display reduced running capacity and muscle expression of all three ketolytic enzymes: BDH1, OXCT1, and ACAT1. MEF2Dα2 Eko mice consistently show increased blood ketone body levels in a tolerance test, after exercise, and when fed a ketogenic diet. Lastly, using mitochondria isolated from skeletal muscle, Eko mice show reduced ketone body utilization compared to WT mice. Collectively, our findings identify a new role for the MEF2Dα2 protein isoform in regulating skeletal muscle ketone body oxidation, exercise capacity, and systemic ketone body levels.

The second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) evokes calcium ion (Ca ²⁺ ) release from endosomes and lysosomes by activating two-pore channels (TPCs) on these organelles. Rather than directly binding to TPCs, NAADP associates with proteins that indirectly confer NAADP sensitivity to the TPC complex. We investigated whether and how the NAADP-binding proteins Jupiter microtubule–associated homolog 2 (JPT2) and like-Sm protein 12 (LSM12) contributed to NAADP-TPC-Ca ²⁺ signaling in human cells. Biochemical and functional analyses revealed that recombinant JPT2 and LSM12 both bound to NAADP with high affinity and that endogenous JPT2 and LSM12 independently associated with TPC1 and TPC2. On the basis of knockout and rescue analyses, both NAADP-binding proteins were required to support NAADP-evoked Ca ²⁺ signaling and contributed to endolysosomal trafficking of pseudotyped coronavirus particles. These data reveal that the NAADP-binding proteins JPT2 and LSM12 convergently regulate NAADP-evoked Ca ²⁺ release and function through TPCs.

TPC2 is a pathophysiologically relevant lysosomal ion channel that is activated directly by the phosphoinositide PI(3,5)P ₂ and indirectly by the calcium ion (Ca ²⁺ )–mobilizing molecule NAADP through accessory proteins that associate with the channel. TPC2 toggles between PI(3,5)P ₂ -induced, sodium ion (Na ⁺ )–selective and NAADP-induced, Ca ²⁺ –permeable states in response to these cues. To address the molecular basis of polymodal gating and ion-selectivity switching, we investigated the mechanism by which NAADP and its synthetic functional agonist, TPC2-A1-N, induced Ca ²⁺ release through TPC2 in human cells. Whereas NAADP required the NAADP-binding proteins JPT2 and LSM12 to evoke endogenous calcium ion signals, TPC2-A1-N did not. Residues in TPC2 that bind to PI(3,5)P ₂ were required for channel activation by NAADP but not for activation by TPC2-A1-N. The cryptic voltage-sensing region of TPC2 was required for the actions of TPC2-A1-N and PI(3,5)P ₂ but not for those of NAADP. These data mechanistically distinguish natural and synthetic agonist action at TPC2 despite convergent effects on Ca ²⁺ permeability and delineate a route for pharmacologically correcting impaired NAADP-evoked Ca ²⁺ signals.