Transmembrane 163 (TMEM163) protein interacts with specific mammalian SLC30 zinc efflux transporter family members.

Recently, we reported that TMEM163 is a zinc efflux transporter that likely belongs to the mammalian solute carrier 30 (Slc30/ZnT) subfamily of the cation diffusion facilitator (CDF) protein superfamily. We hypothesized that human TMEM163 forms functional heterodimers with certain ZNT proteins based on their overlapping subcellular localization with TMEM163 and previous reports that some ZNT monomers interact with each other. In this study, we heterologously expressed individual constructs with a unique peptide tag containing TMEM163, ZNT1, ZNT2, ZNT3, and ZNT4 (negative control) or co-expressed TMEM163 with each ZNT in cultured cells for co-immunoprecipitation (co-IP) experiments. We also co-expressed TMEM163 with two different peptide tags as a positive co-IP control. Western blot analyses revealed that TMEM163 dimerizes with itself but that it also heterodimerizes with ZNT1, ZNT2, ZNT3, and ZNT4 proteins. Confocal microscopy revealed that TMEM163 and ZNT proteins partially co-localize in cells, suggesting that they exist as homodimers and heterodimers in their respective subcellular sites. Functional zinc flux assays using Fluozin-3 and Newport Green dyes show that TMEM163/ZNT heterodimers exhibit similar efflux function as TMEM163 homodimers. Cell surface biotinylation revealed that the plasma membrane localization of TMEM163 is not markedly influenced by ZNT co-expression. Overall, our results show that the interaction between TMEM163 and distinct ZNT proteins is physiologically relevant and that their heterodimerization may serve to increase the functional diversity of zinc effluxers within specific tissues or cell types.

Functional Study of TMEM163 Gene Variants Associated with Hypomyelination Leukodystrophy.

Hypomyelinating leukodystrophies (HLDs) are a rare group of heterogeneously genetic disorders characterized by persistent deficit of myelin observed on magnetic resonance imaging (MRI). To identify a new disease-associated gene of HLD, trio-based whole exome sequencing was performed for unexplained patients with HLD. Functional studies were performed to confirm the phenotypic effect of candidate protein variants. Two de novo heterozygous variants, c.227T>G p.(L76R) or c.227T>C p.(L76P) in TMEM163 were identified in two unrelated HLD patients. TMEM163 protein is a zinc efflux transporter localized within the plasma membrane, lysosomes, early endosomes, and other vesicular compartments. It has not been associated with hypomyelination. Functional zinc flux assays in HeLa cells stably-expressing TMEM163 protein variants, L76R and L76P, revealed distinct attenuation or enhancement of zinc efflux, respectively. Experiments using a zebrafish model with knockdown of tmem163a and tmem163b (morphants) showed that loss of tmem163 causes dysplasia of the larvae, locomotor disability and myelin deficit. Expression of human wild type TMEM163 mRNAs in morphants rescues the phenotype, while the TMEM163 L76P and L76R mutants aggravated the condition. Moreover, poor proliferation, elevated apoptosis of oligodendrocytes, and reduced oligodendrocytes and neurons were also observed in zebrafish morphants. Our findings suggest an unappreciated role for TMEM163 protein in myelin development and add TMEM163 to a growing list of genes associated with hypomyelination leukodystrophy.

TMEM176B Regulates AKT/mTOR Signaling and Tumor Growth in Triple-Negative Breast Cancer.

TMEM176B is a member of the membrane spanning 4-domains (MS4) family of transmembrane proteins, and a putative ion channel that is expressed in immune cells and certain cancers. We aimed to understand the role of TMEM176B in cancer cell signaling, gene expression, cell proliferation, and migration in vitro, as well as tumor growth in vivo. We generated breast cancer cell lines with overexpressed and silenced TMEM176B, and a therapeutic antibody targeting TMEM176B. Proliferation and migration assays were performed in vitro, and tumor growth was evaluated in vivo. We performed gene expression and Western blot analyses to identify the most differentially regulated genes and signaling pathways in cells with TMEM176B overexpression and silencing. Silencing TMEM176B or inhibiting it with a therapeutic antibody impaired cell proliferation, while overexpression increased proliferation in vitro. Syngeneic and xenograft tumor studies revealed the attenuated growth of tumors with TMEM176B gene silencing compared with controls. We found that the AKT/mTOR signaling pathway was activated or repressed in cells overexpressing or silenced for TMEM176B, respectively. Overall, our results suggest that TMEM176B expression in breast cancer cells regulates key signaling pathways and genes that contribute to cancer cell growth and progression, and is a potential target for therapeutic antibodies.

Zinc: Multidimensional Effects on Living Organisms.

Zinc is a redox-inert trace element that is second only to iron in abundance in biological systems. In cells, zinc is typically buffered and bound to metalloproteins, but it may also exist in a labile or chelatable (free ion) form. Zinc plays a critical role in prokaryotes and eukaryotes, ranging from structural to catalytic to replication to demise. This review discusses the influential properties of zinc on various mechanisms of bacterial proliferation and synergistic action as an antimicrobial element. We also touch upon the significance of zinc among eukaryotic cells and how it may modulate their survival and death through its inhibitory or modulatory effect on certain receptors, enzymes, and signaling proteins. A brief discussion on zinc chelators is also presented, and chelating agents may be used with or against zinc to affect therapeutics against human diseases. Overall, the multidimensional effects of zinc in cells attest to the growing number of scientific research that reveal the consequential prominence of this remarkable transition metal in human health and disease.

Transmembrane 163 (TMEM163) Protein: A New Member of the Zinc Efflux Transporter Family.

A growing body of evidence continues to demonstrate the vital roles that zinc and its transporters play on human health. The mammalian solute carrier 30 (SLC30) family, with ten current members, controls zinc efflux transport in cells. TMEM163, a recently reported zinc transporter, has similar characteristics in both predicted transmembrane domain structure and function to the cation diffusion facilitator (CDF) protein superfamily. This review discusses past and present data indicating that TMEM163 is a zinc binding protein that transports zinc in cells. We provide a brief background on TMEM163's discovery, transport feature, protein interactome, and similarities, as well as differences, with known SLC30 (ZnT) protein family. We also examine recent reports that implicate TMEM163 directly or indirectly in various human diseases such as Parkinson's disease, Mucolipidosis type IV and diabetes. Overall, the role of TMEM163 protein in zinc metabolism is beginning to be realized, and based on current evidence, we propose that it is likely a new CDF member belonging to mammalian SLC30 (ZnT) zinc efflux transporter proteins.

Protocol for Quantifying Zinc Flux in Cultured Cells using Fluorescent Indicators.

Zinc (Zn2+) plays a vital role in the functioning of the cell. Cells have influx and efflux zinc transporters to regulate the levels of Zn2+ in the cytoplasm and organellar compartments to maintain homeostasis. We present a protocol to measure changes in cellular zinc concentrations using either a low-affinity membrane permeable or a high-affinity membrane impermeable fluorescent dye. Overall, zinc-specific fluorescent indicators using the assay can reliably detect the Zn2+ flux into or out of cultured cells. For complete details on the use and execution of this protocol, please refer to Sanchez et al. (2019).

Transmembrane 163 (TMEM163) protein effluxes zinc.

Recent investigations of rodent Tmem163 suggest that it binds to and transports zinc as a dimer, and that alanine mutagenesis of its two species-conserved aspartate (D123A/D127A) residues proposed to bind zinc, perturbs protein function. Direct corroboration, however, is lacking whether it is an influx or efflux transporter in cells. We hypothesized that human TMEM163 is a zinc effluxer based on its predicted protein characteristics. We used cultured human cell lines that either stably or transiently expressed TMEM163, and pre-loaded the cells with zinc to determine transport activity. We found that TMEM163-expressing cells exhibited significant reduction of intracellular zinc levels as evidenced by two zinc-specific fluorescent dyes and radionuclide zinc-65. The specificity of the fluorescence signal was confirmed upon treatment with TPEN, a high-affinity zinc chelator. Multiple sequence alignment and phylogenetic analyses showed that TMEM163 is related to distinct members of the cation diffusion facilitator (CDF) protein family. To further characterize the efflux function of TMEM163, we substituted alanine in two homologous aspartate residues (D124A/D128A) and performed site-directed mutagenesis of several conserved amino acid residues identified as non-synonymous single nucleotide polymorphism (S61R, S95C, S193P, and E286K). We found a significant reduction of zinc efflux upon cellular expression of D124A/D128A or E286K protein variant when compared with wild-type, suggesting that these particular amino acids are important for normal protein function. Taken together, our findings demonstrate that TMEM163 effluxes zinc, and it should now be designated ZNT11 as a new member of the mammalian CDF family of zinc efflux transporters.

ZnT3 expression levels are down-regulated in the brain of Mcoln1 knockout mice.

AIM: Zinc is a critical divalent cation in mammalian brain, but its concentration must be strictly-controlled. Within certain subsets of glutamatergic neurons, ZnT3 (encoded by the Slc30a3 gene) facilitates the transport and storage of zinc in synaptic vesicles. It has been previously reported that Slc30a3 mRNA levels are perturbed in numerous neurodegenerative disorders. Given the growing evidence of zinc dysregulation in another neurodegenerative disease known as Mucolipidosis IV (MLIV), we hypothesized that abnormal ZnT3 expression would be observed in the brain of MLIV mouse model. Elucidating the link between abnormal ZnT3 and zinc levels could reveal the neuropathological correlates between MLIV and other age-related brain disorders. METHODS: Total RNAs from cortical tissues of Mucolipin-1 knockout (Mcoln1-/- KO) and Mcoln1+/+ wild-type (WT) littermate control mice were analyzed for differential gene expression (DGE) using RNA sequencing (RNA-seq). Real-time quantitative PCR (qPCR) and Western blot techniques were used to validate the data. RESULTS: RNA-seq analysis showed a marked decrease in baseline levels of Slc30a3 mRNA in Mcoln1-/- mice. Real-time qPCR and Western blot analyses confirmed that Slc30a3 transcripts and its protein levels were significantly reduced. Our observations add MLIV to a growing list of neurodegenerative diseases that parallels abnormal ZnT3 expression with zinc dyshomeostasis.

Comparative De Novo Transcriptome Assembly of Notophthalmus viridescens RNA-seq Data using Two Commercial Software Programs.

BACKGROUND AND PURPOSE: The reduction of cost and ease of using core laboratories or commercial sequencing companies have allowed biomedical and health researchers alike to employ reference-based genomic or transcriptomic sequencing (RNA-seq) projects to expand their work. Non-reference based data analysis, in cases of inexperienced researchers, become more challenging despite the availability of many open source and commercial software programs. METHODS: We performed de novo assembly of RNA-seq data obtained from a non-model organism (Eastern Newt skin) to compare data output of two commercially available software workflows. RESULTS: Our results show that the software packages performed satisfactorily albeit with differences in how the annotated and novel transcripts were identified and listed. CONCLUSION: Overall, we conclude that the use of commercial software platforms has a clear advantage to that of open source programs because of convenience with data analysis workflows. One caveat is that users need to know the software's basic algorithm and technical approach, in order to determine the precision and validity of the data output. Thus, it is imperative that researchers fully evaluate the software according to their needs to determine their suitability.

The mucolipin-1 (TRPML1) ion channel, transmembrane-163 (TMEM163) protein, and lysosomal zinc handling.

Lysosomes are emerging as important players in cellular zinc ion (Zn2+) homeostasis. The series of work on Zn2+ accumulation in the neuronal lysosomes and the mounting evidence on the role of lysosomal Zn2+ in cell death during mammary gland involution set a biological precedent for the central role of the lysosomes in cellular Zn2+ handling. Such a role appears to involve cytoprotection on the one hand, and cell death on the other. The recent series of work began to identify the molecular determinants of the lysosomal Zn2+ handling. In addition to zinc transporters (ZnT) of the solute-carrier family type 30A (SLC30A), the lysosomal ion channel TRPML1 and the poorly understood novel transporter TMEM163 have been shown to play a role in the Zn2+ uptake by the lysosomes. In this review, we summarize the current knowledge on molecular determinants of the lysosomal Zn2+ handling, uptake, and release pathways, as well as discuss their possible roles in health and disease.

The mucolipin-2 (TRPML2) ion channel: a tissue-specific protein crucial to normal cell function.

The discovery of the TRPML subfamily of ion channels has created an exciting niche in the fields of membrane trafficking, signal transduction, autophagy, and metal homeostasis. The TRPML protein subfamily consists of three members, TRPML1, TRPML2, and TRPML3, which are encoded by MCOLN1, MCOLN2, and MCOLN3 genes, respectively. They are non-selective cation channels with six predicted transmembrane domains and intracellular amino- and carboxyl-terminus regions. They localize to the plasma membrane, endosomes, and lysosomes of cells. TRPML1 is associated with the human lysosomal storage disease known as mucolipidosis type IV (MLIV), but TRPML2 and TRPML3 have not been linked with a human disease. Although TRPML1 is expressed in many tissues, TRPML3 is expressed in a varied but limited set of tissues, while TRPML2 has a more limited expression pattern where it is mostly detected in lymphoid and myeloid tissues. This review focuses on TRPML2 because it appears to play an important, yet unrecognized role in the immune system. While the evidence has been mostly indirect, we present and discuss relevant data that strengthen the connection of TRPML2 with cellular immunity. We also discuss the functional redundancy between the TRPML proteins, and how such features could be exploited as a potential therapeutic strategy for MLIV disease. We present evidence that TRPML2 expression may complement certain phenotypic alterations in MLIV cells and briefly examine the challenges of functional complementation. In conclusion, the function of TRPML2 still remains obscure, but emerging data show that it may serve a critical role in immune cell development and inflammatory responses.

(p)ppGpp modulates cell size and the initiation of DNA replication in Caulobacter crescentus in response to a block in lipid biosynthesis.

Stress conditions, such as a block in fatty acid synthesis, signal bacterial cells to exit the cell cycle. Caulobacter crescentus FabH is a cell-cycle-regulated ß-ketoacyl-acyl carrier protein synthase that initiates lipid biosynthesis and is essential for growth in rich media. To explore how C. crescentus responds to a block in lipid biosynthesis, we created a FabH-depletion strain. We found that FabH depletion blocks lipid biosynthesis in rich media and causes a cell cycle arrest that requires the alarmone (p)ppGpp for adaptation. Notably, basal levels of (p)ppGpp coordinate both a reduction in cell volume and a block in the over-initiation of DNA replication in response to FabH depletion. The gene ctrA encodes a master transcription factor that directly regulates 95 cell-cycle-controlled genes while also functioning to inhibit the initiation of DNA replication. Here, we demonstrate that ctrA transcription is (p)ppGpp-dependent during fatty acid starvation. CtrA fails to accumulate when FabH is depleted in the absence of (p)ppGpp due to a substantial reduction in ctrA transcription. The (p)ppGpp-dependent maintenance of ctrA transcription during fatty acid starvation initiated from only one of the two ctrA promoters. In the absence of (p)ppGpp, the majority of FabH-depleted cells enter a viable but non-culturable state, with multiple chromosomes, and are unable to recover from the miscoordination of cell cycle events. Thus, basal levels of (p)ppGpp facilitate C. crescentus' re-entry into the cell cycle after termination of fatty acid starvation.

PAX5 is the transcriptional activator of mucolipin-2 (MCOLN2) gene.

Transient receptor potential mucolipin (TRPML) proteins belong to the TRP superfamily of non-selective cation channels. The TRPML1, -2, and -3 proteins are encoded by Mucolipin (MCOLN)-1, -2 and -3 genes, respectively. TRPML1 has been associated with mucolipidosis type IV (MLIV), while no disease phenotype has been linked with TRPML2 or -3 protein. The TRPML proteins share high sequence similarities, form hetero-tetramers, and serve in membrane trafficking, autophagy, and metal homeostasis. Previous studies suggest that TRPML2 serves a role in the immune system; however, the evidence is mostly indirect. We hypothesize that if TRPML2 is involved in immune function its expression would be likely regulated by an immune-associated transcription factor protein. Thus, we set out to identify the core promoter region and the transcription factor responsible for MCOLN2 gene expression. Using dual-luciferase assay and over-expression analyses, we reveal for the first time that B-cell lineage specific activator protein (BSAP), also known as paired box 5 (PAX5), controls MCOLN2 expression. Specifically, heterologous expression of PAX5 in HEK-293 cells significantly increased endogenous MCOLN2 transcript and TRPML2 protein levels, while RNA interference targeting endogenous PAX5 reduced its effect. Site-directed mutagenesis studies showed that the core promoter and PAX5 binding region to be between -79 and -60 base pairs upstream of the transcriptional start site. Thus, our findings add to a growing list of evidence for TRPML2's possible involvement in the immune system. The knowledge gained from this study could be used to further characterize the role of TRPML2 in B-cell development and function.

Cellular zinc levels are modulated by TRPML1-TMEM163 interaction.

Mucolipidosis type IV (MLIV) is caused by loss of function mutations in the TRPML1 ion channel. We previously reported that tissue zinc levels in MLIV were abnormally elevated; however, the mechanism behind this pathologic accumulation remains unknown. Here, we identify transmembrane (TMEM)-163 protein, a putative zinc transporter, as a novel interacting partner for TRPML1. Evidence from yeast two-hybrid, tissue expression pattern, co-immunoprecipitation, mass spectrometry and confocal microscopy studies confirmed the physical association of TMEM163 with TRPML1. This interaction is disrupted when a part of TMEM163's N-terminus was deleted. Further studies to define the relevance of their interaction revealed that the plasma membrane (PM) levels of TMEM163 significantly decrease when TRPML1 is co-expressed in HEK-293 cells, while it mostly localizes within the PM when co-expressed with a mutant TRPML1 that distributes mostly in the PM. Meanwhile, co-expression of TMEM163 does not alter TRPML1 channel activity, but its expression levels in MLIV patient fibroblasts are reduced, which correlate with marked accumulation of zinc in lysosomes when these cells are acutely exposed to exogenous zinc (100 µM). When TMEM163 is knocked down or when TMEM163 and TRPML1 are co-knocked down in HEK-293 cells treated overnight with 100 nm zinc, the cells have significantly higher intracellular zinc levels than untreated control. Overall, these findings suggest that TMEM163 and TRPML1 proteins play a critical role in cellular zinc homeostasis, and thus possibly explain a novel mechanism for the pathological overload of zinc in MLIV disease.

The Caulobacter crescentus ctrA P1 promoter is essential for the coordination of cell cycle events that prevent the overinitiation of DNA replication.

The master regulator CtrA oscillates during the Caulobacter cell cycle due to temporally regulated proteolysis and transcription. It is proteolysed during the G1-S transition and reaccumulates in predivisional cells as a result of transcription from two sequentially activated promoters, P1 and P2. CtrA reinforces its own synthesis by directly mediating the activation of P2 concurrently with repression of P1. To explore the role of P1 in cell cycle control, we engineered a mutation into the native ctrA locus that prevents transcription from P1 but not P2. As expected, the ctrA P1 mutant exhibits striking growth, morphological and DNA replication defects. Unexpectedly, we found CtrA and its antagonist SciP, but not DnaA, GcrA or CcrM accumulation to be dramatically reduced in the ctrA P1 mutant. SciP levels closely paralleled CtrA accumulation, suggesting that CtrA acts as a rheostat to modulate SciP abundance. Furthermore, the reappearance of CtrA and CcrM in predivisional cells was delayed in the P1 mutant by 0.125 cell cycle unit in synchronized cultures. High levels of ccrM transcription despite low levels of CtrA and increased transcription of ctrA P2 in the ctrA P1 mutant are two examples of robustness in the cell cycle. Thus, Caulobacter can adjust regulatory pathways to partially compensate for reduced and delayed CtrA accumulation in the ctrA P1 mutant.

Abnormal accumulation of human transmembrane (TMEM)-176A and 176B proteins is associated with cancer pathology.

Transmembrane (TMEM)-176A and 176B proteins belong to the MS4A family of proteins whose function in the immune system remains unclear. TMEM176A transcripts were previously shown to be elevated in liver cancer or kidney tissue with proteinuria, while marked changes in TMEM176B transcripts have been found in tolerated tissue allografts and neoplastic fibroblasts. To study the functional relationship between human TMEM176A and 176B and their putative link to cancer, we used polymerase chain reaction and biochemical assays. Here, we show that TMEM176A and 176B are widely expressed in all human tissues examined. Co-immunoprecipitation of heterologously expressed TMEM176A and 176B revealed direct physical interaction. To determine the relevance of such interaction to cancer pathology, we analyzed biopsied tissue samples from a variety of normal and cancer tissues. Our data reveal that human TMEM176A and 176B protein levels are significantly elevated in lymphoma, but not in normal tissues. The protein levels of TMEM176A are also significantly increased in lung carcinoma. Finally, analysis of the protein expression ratio of TMEM176A over 176B showed significant differences between normal and cancer tissues of the breast, lymph, skin, and liver, which indicates that both TMEM proteins could be potential useful markers for certain human cancers.

Zinc dyshomeostasis is linked with the loss of mucolipidosis IV-associated TRPML1 ion channel.

Chelatable zinc is important in brain function, and its homeostasis is maintained to prevent cytotoxic overload. However, certain pathologic events result in intracellular zinc accumulation in lysosomes and mitochondria. Abnormal lysosomes and mitochondria are common features of the human lysosomal storage disorder known as mucolipidosis IV (MLIV). MLIV is caused by the loss of TRPML1 ion channel function. MLIV cells develop large hyperacidic lysosomes, membranous vacuoles, mitochondrial fragmentation, and autophagic dysfunction. Here, we observed that RNA interference of mucolipin-1 gene (TRPML1) in HEK-293 cells mimics the MLIV cell phenotype consisting of large lysosomes and membranous vacuoles that accumulate chelatable zinc. To show that abnormal chelatable zinc levels are indeed correlated with MLIV pathology, we quantified its concentration in cultured MLIV patient fibroblast and control cells with a spectrofluorometer using N-(6-methoxy-8-quinolyl)-p-toluene sulfonamide fluorochrome. We found a significant increase of chelatable zinc levels in MLIV cells but not in control cells. Furthermore, we quantified various metal isotopes in whole brain tissue of TRPML1(-/-) null mice and wild-type littermates using inductively coupled plasma mass spectrometry and observed that the zinc-66 isotope is markedly elevated in the brain of TRPML1(-/-) mice when compared with controls. In conclusion, we show for the first time that the loss of TRPML1 function results in intracellular chelatable zinc dyshomeostasis. We propose that chelatable zinc accumulation in large lysosomes and membranous vacuoles may contribute to the pathogenesis of the disease and progressive cell degeneration in MLIV patients.

Small molecule activators of TRPML3.

We conducted a high-throughput screen for small molecule activators of the TRPML3 ion channel, which, when mutated, causes deafness and pigmentation defects. Cheminformatics analyses of the 53 identified and confirmed compounds revealed nine different chemical scaffolds and 20 singletons. We found that agonists strongly potentiated TRPML3 activation with low extracytosolic [Na(+)]. This synergism revealed the existence of distinct and cooperative activation mechanisms and a wide dynamic range of TRPML3 activity. Testing compounds on TRPML3-expressing sensory hair cells revealed the absence of activator-responsive channels. Epidermal melanocytes showed only weak or no responses to the compounds. These results suggest that TRPML3 in native cells might be absent from the plasma membrane or that the protein is a subunit of heteromeric channels that are nonresponsive to the activators identified in this screen.