TRPML3/BK complex promotes autophagy and bacterial clearance by providing a positive feedback regulation of mTOR via PI3P.

TRPML3 is a Ca2+/Na+ release channel residing in both phagophores and endolysosomal membranes. It is activated by PI3P and PI3,5P2. Its activity can be enhanced by high luminal pH and by replacing luminal Na+ with K+. Here, we report that big-conductance Ca2+-activated potassium (BK) channels form a positive feedback loop with TRPML3. Ca2+ release via TRPML3 activates BK, which in turn facilitates TRPML3-mediated Ca2+ release, potentially through removing luminal Na+ inhibition. We further show that TRPML3/BK and mammalian target of rapamycin (mTOR) form another positive feedback loop to facilitate autophagy induction in response to nutrient starvation, i.e., mTOR inhibition upon nutrient starvation activates TRPML3/BK, and this further reduces mTOR activity, thereby increasing autophagy induction. Mechanistically, the feedback regulation between TRPML3/BK and mTOR is mediated by PI3P, an endogenous TRPML3 activator that is enriched in phagophores and is up-regulated by mTOR reduction. Importantly, bacterial infection activates TRPML3 in a BK-dependent manner, and both TRPML3 and BK are required for mTOR suppression and autophagy induction responding to bacterial infection. Suppressing either TRPML3 or BK helps bacteria survival whereas increasing either TRPML3 or BK favors bacterial clearance. Considering that TRPML3/BK is inhibited by low luminal pH but activated by high luminal pH and PI3P in phagophores, we suggest that TRPML3/BK and mTOR form a positive feedback loop via PI3P to ensure efficient autophagy induction in response to nutrient deprivation and bacterial infection. Our study reveals a role of TRPML3-BK coupling in controlling cellular homeostasis and intracellular bacterial clearance via regulating mTOR signaling.

TRPM2: bridging calcium and ROS signaling pathways-implications for human diseases.

TRPM2 is a versatile and essential signaling molecule that plays diverse roles in Ca2+ homeostasis and oxidative stress signaling, with implications in various diseases. Research evidence has shown that TRPM2 is a promising therapeutic target. However, the decision of whether to activate or inhibit TRPM2 function depends on the context and specific disease. A deeper understanding of the molecular mechanisms governing TRPM2 activation and regulation could pave the way for the development of innovative therapeutics targeting TRPM2 to treat a broad range of diseases. In this review, we examine the structural and biophysical details of TRPM2, its involvement in neurological and cardiovascular diseases, and its role in inflammation and immune system function. In addition, we provide a comprehensive overview of the current knowledge of TRPM2 signaling pathways in cancer, including its functions in bioenergetics, oxidant defense, autophagy, and response to anticancer drugs.

Inhibiting BCKDK in triple negative breast cancer suppresses protein translation, impairs mitochondrial function, and potentiates doxorubicin cytotoxicity.

Triple-negative breast cancers (TNBCs) are characterized by poor survival, prognosis, and gradual resistance to cytotoxic chemotherapeutics, like doxorubicin (DOX). The clinical utility of DOX is limited by its cardiotoxic and chemoresistant effects that manifest over time. To induce chemoresistance, TNBC rewires oncogenic gene expression and cell signaling pathways. Recent studies have demonstrated that reprogramming of branched-chain amino acids (BCAAs) metabolism facilitates tumor growth and survival. Branched-chain ketoacid dehydrogenase kinase (BCKDK), a regulatory kinase of the rate-limiting enzyme of the BCAA catabolic pathway, is reported to activate RAS/RAF/MEK/ERK signaling to promote tumor cell proliferation. However, it remains unexplored if BCKDK action remodels TNBC proliferation and survival per se and influences susceptibility to DOX-induced genotoxic stress. TNBC cells treated with DOX exhibited reduced BCKDK expression and intracellular BCKAs. Genetic and pharmacological inhibition of BCKDK in TNBC cell lines also showed a similar reduction in intracellular and secreted BCKAs. BCKDK silencing in TNBC cells downregulated mitochondrial metabolism genes, reduced electron complex protein expression, oxygen consumption, and ATP production. Transcriptome analysis of BCKDK silenced cells confirmed dysregulation of mitochondrial metabolic networks and upregulation of the apoptotic signaling pathway. Furthermore, BCKDK inhibition with concurrent DOX treatment exacerbated apoptosis, caspase activity, and loss of TNBC proliferation. Inhibition of BCKDK in TNBC also upregulated sestrin 2 and concurrently decreased mTORC1 signaling and protein synthesis. Overall, loss of BCKDK action in TNBC remodels BCAA flux, reduces protein translation triggering cell death, ATP insufficiency, and susceptibility to genotoxic stress.

Two positively charged amino acid side-chains in the inner vestibule of the CFTR channel pore play analogous roles in controlling anion binding and anion conductance.

Positively charged amino acid side-chains play important roles in anion binding and permeation through the CFTR chloride channel. One pore-lining lysine residue in particular (K95) has been shown to be indispensable for anion binding, conductance, and selectivity. Here, we use functional investigation of CFTR to show that a nearby arginine (R134) plays a functionally analogous role. Removal of this positive charge (in the R134Q mutant) drastically reduces single-channel conductance, weakens binding of both permeant and blocking anions, and abolishes the normal anion conductance selectivity pattern. Each of these functional effects was reversed by a second-site mutation (S1141K) that introduces an ectopic positive charge to a nearby pore-lining residue. Substituted cysteine accessibility experiments confirm that R134-but not nearby residues in the same transmembrane helix-is accessible within the pore lumen. These results suggest that K95 and R134, which are very close together within the inner vestibule of the pore, play analogous, important roles, and that both are required for the normal anion binding and anion conductance properties of the pore. Nevertheless, that fact that both positive charges can be "transplanted" to other sites in the inner vestibule with little effect on channel permeation properties indicates that it is the overall number of charges-rather than their exact locations-that controls pore function.

TRPML1-Emerging Roles in Cancer.

The mucolipin-1 (TRPML1) channel maintains lysosomal ionic homeostasis and regulates autophagic flux. Defects of TRPML1 lead to lysosomal storage diseases and neurodegeneration. In this report, we discuss emerging evidence pertaining to differential regulation of TRPML1 signaling pathways in cancer progression with the goal of leveraging the oncogenic potential of TRPML1 to inspire therapeutic interventions.

Getting Lost in the Cell-Lysosomal Entrapment of Chemotherapeutics.

Despite extensive research, resistance to chemotherapy still poses a major obstacle in clinical oncology. An exciting strategy to circumvent chemoresistance involves the identification and subsequent disruption of cellular processes that are aberrantly altered in oncogenic states. Upon chemotherapeutic challenges, lysosomes are deemed to be essential mediators that enable cellular adaptation to stress conditions. Therefore, lysosomes potentially hold the key to disarming the fundamental mechanisms of chemoresistance. This review explores modes of action of classical chemotherapeutic agents, adaptive response of the lysosomes to cell stress, and presents physiological and pharmacological insights pertaining to drug compartmentalization, sequestration, and extracellular clearance through the lens of lysosomes.

Branched-chain ketoacid overload inhibits insulin action in the muscle.

Branched-chain α-keto acids (BCKAs) are catabolites of branched-chain amino acids (BCAAs). Intracellular BCKAs are cleared by branched-chain ketoacid dehydrogenase (BCKDH), which is sensitive to inhibitory phosphorylation by BCKD kinase (BCKDK). Accumulation of BCKAs is an indicator of defective BCAA catabolism and has been correlated with glucose intolerance and cardiac dysfunction. However, it is unclear whether BCKAs directly alter insulin signaling and function in the skeletal and cardiac muscle cell. Furthermore, the role of excess fatty acids (FAs) in perturbing BCAA catabolism and BCKA availability merits investigation. By using immunoblotting and ultra-performance liquid chromatography MS/MS to analyze the hearts of fasted mice, we observed decreased BCAA-catabolizing enzyme expression and increased circulating BCKAs, but not BCAAs. In mice subjected to diet-induced obesity (DIO), we observed similar increases in circulating BCKAs with concomitant changes in BCAA-catabolizing enzyme expression only in the skeletal muscle. Effects of DIO were recapitulated by simulating lipotoxicity in skeletal muscle cells treated with saturated FA, palmitate. Exposure of muscle cells to high concentrations of BCKAs resulted in inhibition of insulin-induced AKT phosphorylation, decreased glucose uptake, and mitochondrial oxygen consumption. Altering intracellular clearance of BCKAs by genetic modulation of BCKDK and BCKDHA expression showed similar effects on AKT phosphorylation. BCKAs increased protein translation and mTORC1 activation. Pretreating cells with mTORC1 inhibitor rapamycin restored BCKA's effect on insulin-induced AKT phosphorylation. This study provides evidence for FA-mediated regulation of BCAA-catabolizing enzymes and BCKA content and highlights the biological role of BCKAs in regulating muscle insulin signaling and function.

Exploring the Therapeutic Potential of Membrane Transport Proteins: Focus on Cancer and Chemoresistance.

Improving the therapeutic efficacy of conventional anticancer drugs represents the best hope for cancer treatment. However, the shortage of druggable targets and the increasing development of anticancer drug resistance remain significant problems. Recently, membrane transport proteins have emerged as novel therapeutic targets for cancer treatment. These proteins are essential for a plethora of cell functions ranging from cell homeostasis to clinical drug toxicity. Furthermore, their association with carcinogenesis and chemoresistance has opened new vistas for pharmacology-based cancer research. This review provides a comprehensive update of our current knowledge on the functional expression profile of membrane transport proteins in cancer and chemoresistant tumours that may form the basis for new cancer treatment strategies.

Electrostatic Tuning of Anion Attraction from the Cytoplasm to the Pore of the CFTR Chloride Channel.

Anions enter from the cytoplasm into the channel pore of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel not via a central pathway but via a single lateral portal or fenestration. High Cl- conductance is dependent on electrostatic attraction of cytoplasmic Cl- ions by four positively charged amino acid side-chains located within this portal. Here we use a mutagenic approach to investigate the functional effects of transplanting or supplementing these positive charges at nearby portal-lining sites. Using patch clamp recording, we find that the functionally important positive charges at K190 and R303 can be transplanted to four nearby sites (N186, L197, W356, and A367) with little loss of Cl- conductance. Introduction of additional positive charge at these sites had almost no effect on Cl- conductance, but did increase the sensitivity to channel block by intracellular suramin and Pt(NO2)42- anions. We suggest that it is the number of positive charges within the portal, rather than their exact location, that is the most important factor influencing Cl- conductance. The portal appears well optimized in terms of charge distribution to maximize Cl- conductance.

A lysosome independent role for TFEB in activating DNA repair and inhibiting apoptosis in breast cancer cells.

Transcription factor EB (TFEB) is a master regulator of lysosomal biogenesis and autophagy with critical roles in several cancers. Lysosomal autophagy promotes cancer survival through the degradation of toxic molecules and the maintenance of adequate nutrient supply. Doxorubicin (DOX) is the standard of care treatment for triple-negative breast cancer (TNBC); however, chemoresistance at lower doses and toxicity at higher doses limit its usefulness. By targeting pathways of survival, DOX can become an effective antitumor agent. In this study, we examined the role of TFEB in TNBC and its relationship with autophagy and DNA damage induced by DOX. In TNBC cells, TFEB was hypo-phosphorylated and localized to the nucleus upon DOX treatment. TFEB knockdown decreased the viability of TNBC cells while increasing caspase-3 dependent apoptosis. Additionally, inhibition of the TFEB-phosphatase calcineurin sensitized cells to DOX-induced apoptosis in a TFEB dependent fashion. Regulation of apoptosis by TFEB was not a consequence of altered lysosomal function, as TFEB continued to protect against apoptosis in the presence of lysosomal inhibitors. RNA-Seq analysis of MDA-MB-231 cells with TFEB silencing identified a down-regulation in cell cycle and homologous recombination genes while interferon-γ and death receptor signaling genes were up-regulated. In consequence, TFEB knockdown disrupted DNA repair following DOX, as evidenced by persistent γH2A.X detection. Together, these findings describe in TNBC a novel lysosomal independent function for TFEB in responding to DNA damage.

The Role of Mitochondrial Calcium Signaling in the Pathophysiology of Cancer Cells.

The pioneering work of Richard Altman on the presence of mitochondria in cells set in motion a field of research dedicated to uncovering the secrets of the mitochondria. Despite limitations in studying the structure and function of the mitochondria, advances in our understanding of this organelle prompted the development of potential treatments for various diseases, from neurodegenerative conditions to muscular dystrophy and cancer. As the powerhouses of the cell, the mitochondria represent the essence of cellular life and as such, a selective advantage for cancer cells. Much of the function of the mitochondria relies on Ca2+ homeostasis and the presence of effective Ca2+ signaling to maintain the balance between mitochondrial function and dysfunction and subsequently, cell survival. Ca2+ regulates the mitochondrial respiration rate which in turn increases ATP synthesis, but too much Ca2+ can also trigger the mitochondrial apoptosis pathway; however, cancer cells have evolved mechanisms to modulate mitochondrial Ca2+ influx and efflux in order to sustain their metabolic demand and ensure their survival. Therefore, targeting the mitochondrial Ca2+ signaling involved in the bioenergetic and apoptotic pathways could serve as potential approaches to treat cancer patients. This chapter will review the role of Ca2+ signaling in mediating the function of the mitochondria and its involvement in health and disease with special focus on the pathophysiology of cancer.

Localization of keyhole limpet hemocyanin-like immunoreactivity in the nervous system of Biomphalaria alexandrina.

Recent years have led to increased effort to describe and understand the peripheral nervous system and its influence on central mechanisms and behavior in gastropod molluscs. This study revealed that an antibody raised against keyhole limpet hemocyanin (KLH) cross-reacts with an antigen(s) found extensively in both the central and the peripheral nervous systems of Biomphalaria alexandrina. The results revealed KLH-like immunoreactive (LIR) neurons in the cerebral, pedal, buccal, left pleural, right parietal, and visceral ganglion within the CNS with fibers projecting throughout all the peripheral nerves. Numerous KLH-LIR peripheral sensory neurons located in the foot, lips, tentacles, mantle, esophagus, and penis exhibited a bipolar morphology with long tortuous dendrites. KLH-LIR cells were also present in the eye and statocyst, thus suggesting the labeling of multiple sensory modalities/cell types. KLH-LIR cells did not co-localize with tyrosine hydroxylase (TH)-LIR cells, which have previously been described in this and other gastropods. The results thus provide descriptions of thousands of peripheral sensory neurons, not previously described in detail. Future research should seek to pair sensory modalities with peripheral cell type and attempt to further elucidate the nature of KLH-like reactivity. These findings also emphasize the need for caution when analyzing results obtained through use of antibodies raised against haptens conjugated to carrier proteins, suggesting the need for stringent controls to help limit potential confounds caused by cross-reactivity. In addition, this study is the first to describe neuronal cross-reactivity with KLH in Biomphalaria, which could provide a substrate for host-parasite interactions with a parasitic trematode, Schistosoma.

TRP channels in gastric cancer: New hopes and clinical perspectives.

Gastric cancer is a multifactorial disease associated with a combination of and environmental factors. Each year, one million new gastric cancer cases are diagnosed worldwide and two-thirds end up losing the battle with this devastating disease. Currently, surgery represents the only effective treatment option for patients with early stage tumors. However, the asymptomatic phenotype of this disease during the early stages poses as a significant limiting factor to diagnosis and often renders treatments ineffective. To address these issues, scientists are focusing on personalized medicine and discovering new ways to treat cancer patients. Emerging therapeutic options include the transient receptor potential (TRP) channels. Since their discovery, TRP channels have been shown to contribute significantly to the pathophysiology of various cancers, including gastric cancer. This review will summarize the current knowledge about gastric cancer and provide a synopsis of recent advancements on the role and involvement of TRP channels in gastric cancer as well as a discussion of the benefits of targeting TPR channel in the clinical management of gastric cancer.

TRPM2 Silencing Causes G2/M Arrest and Apoptosis in Lung Cancer Cells via Increasing Intracellular ROS and RNS Levels and Activating the JNK Pathway.

BACKGROUND/AIMS: The oxidative stress sensor transient receptor potential melastatin-2 (TRPM2) ion channel has recently gained attention in many types of cancer. The lung tissue is highly susceptible to oxidative stress-mediated injury and diseases; therefore, we aimed to determine whether TRPM2 plays an essential role in protecting lung cancer cells from oxidative damage while promoting cancer cell survival and metastasis. METHODS: We used two non-small cell lung (NSCLC) cell lines A549 and H1299 as a lung cancer model. We investigated the functional expression of TRPM2 using electrophysiology, qRT-PCR and Western blots. CFSE and flow cytometry were used to study TRPM2 role in proliferation, cell cycle and apoptosis. Gap closure chambers and Three-Tiered Chemotaxis Chamber were used to study the role of TRPM2 in metastasis. SCID mice were used to study the role of TRPM2 in lung tumor growth and metastasis. RESULTS: we demonstrate that TRPM2 is functionally expressed in NSCLC cells and that its downregulation significantly inhibits cell proliferation and promotes apoptosis. These results were concomitant with an induction in DNA damage and G2/M cell cycle arrest. TRPM2 silencing inhibits also lung cancer cells invasion ability and alters EMT processes. Mechanistically, TRPM2 downregulation causes an increase in the intracellular levels of reactive oxygen (ROS) and nitrogen (RNS) species, which in turn causes DNA damage and JNK activation leading to G2/M arrest, and an ultimate cell death. Finally, TRPM2 downregulation suppresses the growth of human lung tumour xenograft in SCID mice and TRPM2 depleted tumours exhibited a significant reduction in the mRNA expression level of EMT markers compared to the control tumors. CONCLUSION: Our data provide new insights on the functional expression of TRPM2 in lung cancer, its essential role in tumour growth and metastasis through the control of JNK signaling pathway, and that TRPM2 could be exploited for targeted lung cancer therapies.

The lysosomal TRPML1 channel regulates triple negative breast cancer development by promoting mTORC1 and purinergic signaling pathways.

The triple-negative breast cancer (TNBC) that comprises approximately 10%-20% of breast cancers is an aggressive subtype lacking effective therapeutics. Among various signaling pathways, mTORC1 and purinergic signals have emerged as potentially fruitful targets for clinical therapy of TNBC. Unfortunately, drugs targeting these signaling pathways do not successfully inhibit the progression of TNBC, partially due to the fact that these signaling pathways are essential for the function of all types of cells. In this study, we report that TRPML1 is specifically upregulated in TNBCs and that its genetic downregulation and pharmacological inhibition suppress the growth of TNBC. Mechanistically, we demonstrate that TRPML1 regulates TNBC development, at least partially, through controlling mTORC1 activity and the release of lysosomal ATP. Because TRPML1 is specifically activated by cellular stresses found in tumor microenvironments, antagonists of TRPML1 could represent anticancer drugs with enhanced specificity and potency. Our findings are expected to have a major impact on drug targeting of TNBCs.

TRPM2 ion channel promotes gastric cancer migration, invasion and tumor growth through the AKT signaling pathway.

Transient Receptor Potential Melastatin-2 (TRPM2) ion channel is emerging as a great therapeutic target in many types of cancer, including gastric cancer - a major health threat of cancer related-death worldwide. Our previous study demonstrated the critical role of TRPM2 in gastric cancer cells bioenergetics and survival; however, its role in gastric cancer metastasis, the major cause of patient death, remains unknown. Here, using molecular and functional assays, we demonstrate that TRPM2 downregulation significantly inhibits the migration and invasion abilities of gastric cancer cells, with a significant reversion in the expression level of metastatic markers. These effects were concomitant with decreased Akt and increased PTEN activities. Finally, TRPM2 silencing resulted in deregulation of metastatic markers and abolished the tumor growth ability of AGS gastric cancer cells in NOD/SCID mice. Taken together, our results provide compelling evidence on the important function of TRPM2 in the modulation of gastric cancer cell invasion likely through controlling the PTEN/Akt pathway.

Evolving use of natriuretic peptide receptor type-C as part of strategies for the treatment of pulmonary hypertension due to left ventricle heart failure.

Pulmonary hypertension (PH) due to left ventricular heart failure (LV-HF) is a disabling and life-threatening disease for which there is currently no single marketed pharmacological agent approved. Despite recent advances in the pathophysiological understanding, there is as yet no prospect of cure, and the majority of patients continue to progress to right ventricular failure and die. There is, therefore an urgent unmet need to identify novel pharmacological agents that will prevent or reverse the increase in pulmonary artery pressures while enhancing cardiac performance in PH due to LV-HF. In the present article, we first focused on the Natriuretic Peptide Receptor type C (NPR-C) based therapeutic strategies aimed at lowering pulmonary artery pressure. Second, we reviewed potential NPR-C therapeutic strategies to reverse or least halt the detrimental effects of diastolic dysfunction and impaired nitic oxide signalling pathways, as well as possibilities for neurohumoral modulation.

mTOR signalling: jack-of-all-trades 1.

The mechanistic target of rapamycin (mTOR) is an evolutionarily conserved serine/threonine kinase that senses and integrates environmental information into cellular regulation and homeostasis. Accumulating evidence has suggested a master role of mTOR signalling in many fundamental aspects of cell biology and organismal development. mTOR deregulation is implicated in a broad range of pathological conditions, including diabetes, cancer, neurodegenerative diseases, myopathies, inflammatory, infectious, and autoimmune conditions. Here, we review recent advances in our knowledge of mTOR signalling in mammalian physiology. We also discuss the impact of mTOR alteration in human diseases and how targeting mTOR function can treat human diseases.

The hidden potential of lysosomal ion channels: A new era of oncogenes.

Lysosomes serve as the control centre for cellular clearance. These membrane-bound organelles receive biomolecules destined for degradation from intracellular and extracellular pathways; thus, facilitating the production of energy and shaping the fate of the cell. At the base of their functionality are the lysosomal ion channels which mediate the function of the lysosome through the modulation of ion influx and efflux. Ion channels form pores in the membrane of lysosomes and allow the passage of ions, a seemingly simple task which harbours the potential of overthrowing the cell's stability. Considered the master regulators of ion homeostasis, these integral membrane proteins enable the proper operation of the lysosome. Defects in the structure or function of these ion channels lead to the development of lysosomal storage diseases, neurodegenerative diseases and cancer. Although more than 50 years have passed since their discovery, lysosomes are not yet fully understood, with their ion channels being even less well characterized. However, significant improvements have been made in the development of drugs targeted against these ion channels as a means of combating diseases. In this review, we will examine how Ca2+, K+, Na+ and Cl- ion channels affect the function of the lysosome, their involvement in hereditary and spontaneous diseases, and current ion channel-based therapies.

Conformational change of the extracellular parts of the CFTR protein during channel gating.

Cystic fibrosis can be treated by potentiators, drugs that interact directly with the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel to increase its open probability. These substances likely target key conformational changes occurring during channel opening and closing, however, the molecular bases of these conformational changes, and their susceptibility to manipulation are poorly understood. We have used patch clamp recording to identify changes in the three-dimensional organization of the extracellularly accessible parts of the CFTR protein during channel opening and closing. State-dependent formation of both disulfide bonds and Cd2+ bridges occurred for pairs of cysteine side-chains introduced into the extreme extracellular ends of transmembrane helices (TMs) 1, 6, and 12. Between each of these three TMs, we found that both disulfide bonds and metal bridges formed preferentially or exclusively in the closed state and that these inter-TM cross-links stabilized the closed state. These results indicate that the extracellular ends of these TMs are close together when the channel is closed and that they separate from each other when the channel opens. These findings identify for the first time key conformational changes in the extracellular parts of the CFTR protein that can potentially be manipulated to control channel activity.