Diazirine-AIOC-NAADP, a Clickable-Photoactive NAADP Analog for Sea Urchin NAADP Binding Proteins.

Calcium ions (Ca) play a vital role as intracellular messengers, regulating essential cellular processes. Nicotinic acid adenine dinucleotide phosphate (NAADP) serves as a potent second messenger, responsible for releasing Ca in both mammals and echinoderms. Despite identification of two human NAADP receptor proteins, their counterparts in sea urchins remain elusive.

Mechanistic Characterization of Cancer-associated Fibroblast Depletion via an Antibody-Drug Conjugate Targeting Fibroblast Activation Protein.

Unlabelled: Cancer-associated fibroblasts (CAF) are a prominent cell type within the tumor microenvironment (TME) where they are known to promote cancer cell growth and survival, angiogenesis, drug resistance, and immunosuppression. The transmembrane prolyl protease fibroblast activation protein (FAP) is expressed on the surface of highly protumorigenic CAFs found in the stroma of nearly every cancer of epithelial origin. The widespread expression of FAP has made it an attractive therapeutic target based on the underlying hypothesis that eliminating protumorigenic CAFs will disrupt the cross-talk between components of TME resulting in cancer cell death and immune infiltration.

Development of an Engineered Single-Domain Antibody for Targeting MET in Non-Small Cell Lung Cancer.

The Mesenchymal Epithelial Transition (MET) receptor tyrosine kinase is upregulated or mutated in 5% of non-small-cell lung cancer (NSCLC) patients and overexpressed in multiple other cancers. We sought to develop a novel single-domain camelid antibody with high affinity for MET that could be used to deliver conjugated payloads to MET expressing cancers. From a naïve camelid variable-heavy-heavy (VHH) domain phage display library, we identified a VHH clone termed 1E7 that displayed high affinity for human MET and was cross-reactive with MET across multiple species.

Progesterone receptor membrane component 1 facilitates Ca signal amplification between endosomes and the endoplasmic reticulum.

Membrane contact sites (MCSs) between endosomes and the endoplasmic reticulum (ER) are thought to act as specialized trigger zones for Ca signaling, where local Ca released via endolysosomal ion channels is amplified by ER Ca-sensitive Ca channels into global Ca signals. Such amplification is integral to the action of the second messenger, nicotinic acid adenine dinucleotide phosphate (NAADP). However, functional regulators of inter-organellar Ca crosstalk between endosomes and the ER remain poorly defined.

Convergent activation of Ca permeability in two-pore channel 2 through distinct molecular routes.

TPC2 is a pathophysiologically relevant lysosomal ion channel that is activated directly by the phosphoinositide PI(3,5)P and indirectly by the calcium ion (Ca)-mobilizing molecule NAADP through accessory proteins that associate with the channel. TPC2 toggles between PI(3,5)P-induced, sodium ion (Na)-selective and NAADP-induced, Ca-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 release through TPC2 in human cells.

Convergent activation of two-pore channels mediated by the NAADP-binding proteins JPT2 and LSM12.

The second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) evokes calcium ion (Ca) 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 signaling in human cells.

Vitamin C transport in neurons and epithelia is regulated by secretory carrier-associated membrane protein-2 (SCAMP2).

The human sodium-dependent vitamin C transporter-1 (hSVCT1) is localized at the apical membrane domain of polarized intestinal and renal epithelial cells to mediate ascorbic acid (AA) uptake. Currently, little is known about the array of interacting proteins that aid hSVCT1 trafficking and functional expression at the cell surface. Here we used an affinity tagging ('One-STrEP') and proteomic approach to identify hSVCT1 interacting proteins, which resolved secretory carrier-associated membrane protein-2 (SCAMP2) as a novel accessary protein partner.

Synthesis and biological evaluation of novel photo-clickable adenosine and cyclic ADP-ribose analogs: 8-N-2'-O-propargyladenosine and 8-N-2'-O-propargyl-cADPR.

A photo-clickable analog of adenosine was devised and synthesized in which the photoactive functional group (8-azidoadenosine) and the click moiety (2'-O-propargyl-ether) were compactly combined within the structure of the adenosine nucleoside itself. We synthesized 8-N-2'-O-propargyl adenosine in four steps starting from adenosine. This photo-clickable adenosine was 5'-phosphorylated and coupled to nicotinamide mononucleotide to form the NAD analog 8-N-2'-O-propargyl-NAD.

Electrophysiology of Endolysosomal Two-Pore Channels: A Current Account.

Two-pore channels TPC1 and TPC2 are ubiquitously expressed pathophysiologically relevant proteins that reside on endolysosomal vesicles. Here, we review the electrophysiology of these channels. Direct macroscopic recordings of recombinant TPCs expressed in enlarged lysosomes in mammalian cells or vacuoles in plants and yeast demonstrate gating by the Ca-mobilizing messenger NAADP and/or the lipid PI(3,5)P.

Activation of endo-lysosomal two-pore channels by NAADP and PI(3,5)P. Five things to know.

Two-pore channels are ancient members of the voltage-gated ion channel superfamily that are expressed predominantly on acidic organelles such as endosomes and lysosomes. Here we review recent advances in understanding how TPCs are activated by their ligands and identify five salient features: (1) TPCs are Ca-permeable non-selective cation channels gated by NAADP. (2) NAADP activation is indirect through associated NAADP receptors.

Identification of a dihydropyridine scaffold that blocks ryanodine receptors.

Ryanodine receptors (RyRs) are large, intracellular ion channels that control Ca release from the sarco/endoplasmic reticulum. Dysregulation of RyRs in skeletal muscle, heart, and brain has been implicated in various muscle pathologies, arrhythmia, heart failure, and Alzheimer's disease. Therefore, there is considerable interest in therapeutically targeting RyRs to normalize Ca homeostasis in scenarios involving RyR dysfunction.

The ins and outs of virus trafficking through acidic Ca stores.

Many viruses exploit host-cell Ca signaling processes throughout their life cycle. This is especially relevant for viruses that translocate through the endolysosomal system, where cellular infection is keyed to the microenvironment of these acidic Ca stores and Ca-dependent trafficking pathways. As regulators of the endolysosomal ionic milieu and trafficking dynamics, two families of endolysosomal Ca-permeable cation channels - two pore channels (TPCs) and transient receptor potential mucolipins (TRPMLs) - have emerged as important host-cell factors in viral entry.

Mechanisms of SARS-CoV-2 neutralization by shark variable new antigen receptors elucidated through X-ray crystallography.

Single-domain Variable New Antigen Receptors (VNARs) from the immune system of sharks are the smallest naturally occurring binding domains found in nature. Possessing flexible paratopes that can recognize protein motifs inaccessible to classical antibodies, VNARs have yet to be exploited for the development of SARS-CoV-2 therapeutics. Here, we detail the identification of a series of VNARs from a VNAR phage display library screened against the SARS-CoV-2 receptor binding domain (RBD).

NAADP-binding proteins find their identity.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a second messenger that releases Ca 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.

Calsyntenin-3 interacts with the sodium-dependent vitamin C transporter-2 to regulate vitamin C uptake.

Ascorbic acid (AA) uptake in neurons occurs via a Na-dependent carrier-mediated process mediated by the sodium-dependent vitamin C transporter-2 (SVCT2). Relatively little information is available concerning the network of interacting proteins that support human (h)SVCT2 trafficking and cell surface expression in neuronal cells. Here we identified the synaptogenic adhesion protein, calsyntenin-3 (CLSTN3) as an hSVCT2 interacting protein from yeast two-hybrid (Y2H) screening of a human adult brain cDNA library.

Essential requirement for JPT2 in NAADP-evoked Ca signaling.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a second messenger that releases Ca from acidic organelles through the activation of two-pore channels (TPCs) to regulate endolysosomal trafficking events. NAADP action is mediated by NAADP-binding protein(s) of unknown identity that confer NAADP sensitivity to TPCs. Here, we used a "clickable" NAADP-based photoprobe to isolate human NAADP-binding proteins and identified Jupiter microtubule-associated homolog 2 (JPT2) as a TPC accessory protein required for endogenous NAADP-evoked Ca signaling.

Chemo-enzymatic synthesis of adenine substituted nicotinic acid adenine dinucleotide phosphate (NAADP) analogs.

Nicotinamide adenine dinucleotide phosphate (NADP) is an indispensable metabolic co-substrate and nicotinic acid adenine dinucleotide phosphate (NAADP) is an important Ca releasing intracellular second messenger. Exploration of the NADP and NAADP interactome often requires the synthesis of NADP derivatives substituted on the adenosine nucleoside. The introduction of the 2'-phosphate of NADP makes the synthesis of substituted NADP derivatives difficult.

The anthelmintic drug praziquantel activates a schistosome transient receptor potential channel.

The anthelmintic drug praziquantel (PZQ) is used to treat schistosomiasis, a neglected tropical disease that affects over 200 million people worldwide. PZQ causes Ca influx and spastic paralysis of adult worms and rapid vacuolization of the worm surface. However, the mechanism of action of PZQ remains unknown even after 40 years of clinical use.

The synthesis and characterization of a clickable-photoactive NAADP analog active in human cells.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca mobilizing second messenger which triggers Ca release in both sea urchin egg homogenates and in mammalian cells. The NAADP binding protein has not been identified and the regulation of NAADP mediated Ca release remains controversial. To address this issue, we have synthesized an NAADP analog in which 3-azido-5-azidomethylbenzoic acid is attached to the amino group of 5-(3-aminopropyl)-NAADP to produce an NAADP analog which is both a photoaffinity label and clickable.

TMEM33 regulates intracellular calcium homeostasis in renal tubular epithelial cells.

Mutations in the polycystins cause autosomal dominant polycystic kidney disease (ADPKD). Here we show that transmembrane protein 33 (TMEM33) interacts with the ion channel polycystin-2 (PC2) at the endoplasmic reticulum (ER) membrane, enhancing its opening over the whole physiological calcium range in ER liposomes fused to planar bilayers. Consequently, TMEM33 reduces intracellular calcium content in a PC2-dependent manner, impairs lysosomal calcium refilling, causes cathepsins translocation, inhibition of autophagic flux upon ER stress, as well as sensitization to apoptosis.

Probing Ca release mechanisms using sea urchin egg homogenates.

Sea urchin eggs have been extensively used to study Ca release through intracellular Ca-permeable channels. Their amenability to homogenization yields a robust, cell-free preparation that was central to establishing the Ca mobilizing actions of cyclic ADP-ribose and NAADP. Egg homogenates have continued to provide insight into the basic properties and pharmacology of intracellular Ca release channels.

5-Azido-8-ethynyl-NAADP: A bifunctional, clickable photoaffinity probe for the identification of NAADP receptors.

Nicotinic acid adenine dinucleotide phosphate is an evolutionarily conserved second messenger, which mobilizes Ca from acidic stores. The molecular identity of the NAADP receptor has yet to be defined. In pursuit of isolating and identifying NAADP-binding proteins, we synthesized and characterized a bifunctional probe that incorporates both a photoactivatable crosslinking azido moiety at the 5-position of the nicotinic ring and a 'clickable' ethynyl moiety to the 8-adenosyl position in NAADP.

A screening campaign in sea urchin egg homogenate as a platform for discovering modulators of NAADP-dependent Ca signaling in human cells.

The Ca mobilizing second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) regulates intracellular trafficking events, including translocation of certain enveloped viruses through the endolysosomal system. Targeting NAADP-evoked Ca signaling may therefore be an effective strategy for discovering novel antivirals as well as therapeutics for other disorders. To aid discovery of novel scaffolds that modulate NAADP-evoked Ca signaling in human cells, we have investigated the potential of using the sea urchin egg homogenate system for a screening campaign.

NAADP-dependent Ca signaling regulates Middle East respiratory syndrome-coronavirus pseudovirus translocation through the endolysosomal system.

Middle East Respiratory Syndrome coronavirus (MERS-CoV) infections are associated with a significant mortality rate, and existing drugs show poor efficacy. Identifying novel targets/pathways required for MERS infectivity is therefore important for developing novel therapeutics. As an enveloped virus, translocation through the endolysosomal system provides one pathway for cellular entry of MERS-CoV.