Publications by authors named "Kirill Kiselyov"

54 Publications

Using Ligand-Accelerated Catalysis to Repurpose Fluorogenic Reactions for Platinum or Copper.

ACS Cent Sci 2020 Oct 18;6(10):1772-1788. Epub 2020 Sep 18.

Department of Chemistry, University of Pittsburgh, 219 Parkman Avenue, Pittsburgh, Pennsylvania 15260, United States.

The development of a fluorescent probe for a specific metal has required exquisite design, synthesis, and optimization of fluorogenic molecules endowed with chelating moieties with heteroatoms. These probes are generally chelation- or reactivity-based. Catalysis-based fluorescent probes have the potential to be more sensitive; however, catalytic methods with a biocompatible fluorescence turn-on switch are rare. Here, we have exploited ligand-accelerated metal catalysis to repurpose known fluorescent probes for different metals, a new approach in probe development. We used the cleavage of allylic and propargylic ethers as platforms that were previously designed for palladium. After a single experiment that combinatorially examined >800 reactions with two variables (metal and ligand) for each ether, we discovered a platinum- or copper-selective method with the ligand effect of specific phosphines. Both metal-ligand systems were previously unknown and afforded strong signals owing to catalytic turnover. The fluorometric technologies were applied to geological, pharmaceutical, serum, and live cell samples and were used to discover that platinum accumulates in lysosomes in cisplatin-resistant cells in a manner that appears to be independent of copper distribution. The use of ligand-accelerated catalysis may present a new blueprint for engineering metal selectivity in probe development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acscentsci.0c00676DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7596870PMC
October 2020

Early evidence of delayed oligodendrocyte maturation in the mouse model of mucolipidosis type IV.

Dis Model Mech 2020 07 30;13(7). Epub 2020 Jul 30.

Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA

Mucolipidosis type IV (MLIV) is a lysosomal disease caused by mutations in the gene that encodes the endolysosomal transient receptor potential channel mucolipin-1, or TRPML1. MLIV results in developmental delay, motor and cognitive impairments, and vision loss. Brain abnormalities include thinning and malformation of the corpus callosum, white-matter abnormalities, accumulation of undegraded intracellular 'storage' material and cerebellar atrophy in older patients. Identification of the early events in the MLIV course is key to understanding the disease and deploying therapies. The mouse model reproduces all major aspects of the human disease. We have previously reported hypomyelination in the MLIV mouse brain. Here, we investigated the onset of hypomyelination and compared oligodendrocyte maturation between the cortex/forebrain and cerebellum. We found significant delays in expression of mature oligodendrocyte markers , and in the cortex, manifesting as early as 10 days after birth and persisting later in life. Such delays were less pronounced in the cerebellum. Despite our previous finding of diminished accumulation of the ferritin-bound iron in the brain, we report no significant changes in expression of the cytosolic iron reporters, suggesting that iron-handling deficits in MLIV occur in the lysosomes and do not involve broad iron deficiency. These data demonstrate very early deficits of oligodendrocyte maturation and critical regional differences in myelination between the forebrain and cerebellum in the mouse model of MLIV. Furthermore, they establish quantitative readouts of the MLIV impact on early brain development, useful to gauge efficacy in pre-clinical trials.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1242/dmm.044230DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7406328PMC
July 2020

Structural and functional divergence of GDAP1 from the glutathione S-transferase superfamily.

FASEB J 2020 05 10;34(5):7192-7207. Epub 2020 Apr 10.

Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA, USA.

Mutations in ganglioside-induced differentiation-associated protein 1 (GDAP1) alter mitochondrial morphology and result in several subtypes of the inherited peripheral neuropathy Charcot-Marie-Tooth disease; however, the mechanism by which GDAP1 functions has remained elusive. GDAP1 contains primary sequence homology to the GST superfamily; however, the question of whether GDAP1 is an active GST has not been clearly resolved. Here, we present biochemical evidence, suggesting that GDAP1 has lost the ability to bind glutathione without a loss of substrate binding activity. We have revealed that the α-loop, located within the H-site motif is the primary determinant for substrate binding. Using structural data of GDAP1, we have found that critical residues and configurations in the G-site which canonically interact with glutathione are altered in GDAP1, rendering it incapable of binding glutathione. Last, we have found that the overexpression of GDAP1 in HeLa cells results in a mitochondrial phenotype which is distinct from oxidative stress-induced mitochondrial fragmentation. This phenotype is dependent on the presence of the transmembrane domain, as well as a unique hydrophobic domain that is not found in canonical GSTs. Together, we data point toward a non-enzymatic role for GDAP1, such as a sensor or receptor.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1096/fj.202000110RDOI Listing
May 2020

Copper-dependent ATP7B up-regulation drives the resistance of TMEM16A-overexpressing head-and-neck cancer models to platinum toxicity.

Biochem J 2019 12;476(24):3705-3719

Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA, U.S.A.

Platinum-containing drugs such as cisplatin and carboplatin are routinely used for the treatment of many solid tumors including squamous cell carcinoma of the head and neck (SCCHN). However, SCCHN resistance to platinum compounds is well documented. The resistance to platinum has been linked to the activity of divalent transporter ATP7B, which pumps platinum from the cytoplasm into lysosomes, decreasing its concentration in the cytoplasm. Several cancer models show increased expression of ATP7B; however, the reason for such an increase is not known. Here we show a strong positive correlation between mRNA levels of TMEM16A and ATP7B in human SCCHN tumors. TMEM16A overexpression and depletion in SCCHN cell lines caused parallel changes in the ATP7B mRNA levels. The ATP7B increase in TMEM16A-overexpressing cells was reversed by suppression of NADPH oxidase 2 (NOX2), by the antioxidant N-Acetyl-Cysteine (NAC) and by copper chelation using cuprizone and bathocuproine sulphonate (BCS). Pretreatment with either chelator significantly increased cisplatin's sensitivity, particularly in the context of TMEM16A overexpression. We propose that increased oxidative stress in TMEM16A-overexpressing cells liberates the chelated copper in the cytoplasm, leading to the transcriptional activation of ATP7B expression. This, in turn, decreases the efficacy of platinum compounds by promoting their vesicular sequestration. We think that such a new explanation of the mechanism of SCCHN tumors' platinum resistance identifies novel approach to treating these tumors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1042/BCJ20190591DOI Listing
December 2019

Whole-Cell Scale Dynamic Organization of Lysosomes Revealed by Spatial Statistical Analysis.

Cell Rep 2018 06;23(12):3591-3606

Department of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, PA 15213, USA; Department of Computational Biology, Carnegie Mellon University, Pittsburgh, PA 15213, USA. Electronic address:

In eukaryotic cells, lysosomes are distributed in the cytoplasm as individual membrane-bound compartments to degrade macromolecules and to control cellular metabolism. A fundamental yet unanswered question is whether and, if so, how individual lysosomes are organized spatially to coordinate and integrate their functions. To address this question, we analyzed their collective behavior in cultured cells using spatial statistical techniques. We found that in single cells, lysosomes maintain non-random, stable, yet distinct spatial distributions mediated by the cytoskeleton, the endoplasmic reticulum (ER), and lysosomal biogenesis. Throughout the intracellular space, lysosomes form dynamic clusters that significantly increase their interactions with endosomes. Cluster formation is associated with local increases in ER spatial density but does not depend on fusion with endosomes or spatial exclusion by mitochondria. Taken together, our findings reveal whole-cell scale spatial organization of lysosomes and provide insights into how organelle interactions are mediated and regulated across the entire intracellular space.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2018.05.079DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6086371PMC
June 2018

Robust lysosomal calcium signaling through channel TRPML1 is impaired by lysosomal lipid accumulation.

FASEB J 2018 02 4;32(2):782-794. Epub 2018 Jan 4.

Department of Anatomy and Cell Biology, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

The transient receptor potential cation channel mucolipin 1 (TRPML1) channel is a conduit for lysosomal calcium efflux, and channel activity may be affected by lysosomal contents. The lysosomes of retinal pigmented epithelial (RPE) cells are particularly susceptible to build-up of lysosomal waste products because they must degrade the outer segments phagocytosed daily from adjacent photoreceptors; incomplete degradation leads to accumulation of lipid waste in lysosomes. This study asks whether stimulation of TRPML1 can release lysosomal calcium in RPE cells and whether such release is affected by lysosomal accumulations. The TRPML agonist ML-SA1 raised cytoplasmic calcium levels in mouse RPE cells, hesRPE cells, and ARPE-19 cells; this increase was rapid, robust, reversible, and reproducible. The increase was not altered by extracellular calcium removal or by thapsigargin but was eliminated by lysosomal rupture with glycyl-l-phenylalanine-β-naphthylamide. Treatment with desipramine to inhibit acid sphingomyelinase or YM201636 to inhibit PIKfyve also reduced the cytoplasmic calcium increase triggered by ML-SA1, whereas RPE cells from TRPML1 mice showed no response to ML-SA1. Cotreatment with chloroquine and U18666A induced formation of neutral, autofluorescent lipid in RPE lysosomes and decreased lysosomal Ca release. Lysosomal Ca release was also impaired in RPE cells from the ATP-binding cassette, subfamily A, member 4 mouse model of Stargardt's retinal dystrophy. Neither TRPML1 mRNA nor total lysosomal calcium levels were altered in these models, suggesting a more direct effect on the channel. In summary, stimulation of TRPML1 elevates cytoplasmic calcium levels in RPE cells, but this response is reduced by lysosomal accumulation.-Gómez, N. M., Lu, W. Lim, J. C., Kiselyov, K., Campagno, K. E., Grishchuk, Y., Slaugenhaupt, S. A., Pfeffer, B., Fliesler, S. J., Mitchell, C. H. Robust lysosomal calcium signaling through channel TRPML1 is impaired by lysosomal lipid accumulation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1096/fj.201700220RRDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5888396PMC
February 2018

Effects of DHA on Hippocampal Autophagy and Lysosome Function After Traumatic Brain Injury.

Mol Neurobiol 2018 03 1;55(3):2454-2470. Epub 2017 Apr 1.

Department of Neurology, University of Pittsburgh, Pittsburgh, PA, 15213, USA.

Traumatic brain injury (TBI) triggers endoplasmic reticulum (ER) stress and impairs autophagic clearance of damaged organelles and toxic macromolecules. In this study, we investigated the effects of the post-TBI administration of docosahexaenoic acid (DHA) on improving hippocampal autophagy flux and cognitive functions of rats. TBI was induced by cortical contusion injury in Sprague-Dawley rats, which received DHA (16 mg/kg in DMSO, intraperitoneal administration) or vehicle DMSO (1 ml/kg) with an initial dose within 15 min after the injury, followed by a daily dose for 3 or 7 days. First, RT-qPCR reveals that TBI induced a significant elevation in expression of autophagy-related genes in the hippocampus, including SQSTM1/p62 (sequestosome 1), lysosomal-associated membrane proteins 1 and 2 (Lamp1 and Lamp2), and cathepsin D (Ctsd). Upregulation of the corresponding autophagy-related proteins was detected by immunoblotting and immunostaining. In contrast, the DHA-treated rats did not exhibit the TBI-induced autophagy biogenesis and showed restored CTSD protein expression and activity. T-weighted images and diffusion tensor imaging (DTI) of ex vivo brains showed that DHA reduced both gray matter and white matter damages in cortical and hippocampal tissues. DHA-treated animals performed better than the vehicle control group on the Morris water maze test. Taken together, these findings suggest that TBI triggers sustained stimulation of autophagy biogenesis, autophagy flux, and lysosomal functions in the hippocampus. Swift post-injury DHA administration restores hippocampal lysosomal biogenesis and function, demonstrating its therapeutic potential.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s12035-017-0504-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5623615PMC
March 2018

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

Front Biosci (Landmark Ed) 2017 03 1;22:1330-1343. Epub 2017 Mar 1.

Dept. of Biological Sciences, University of Pittsburgh, 519 Langley Hall, 4249 Fifth Avenue, Pittsburgh, PA 15260, USA,

Lysosomes are emerging as important players in cellular zinc ion (Zn) homeostasis. The series of work on Zn2+ accumulation in the neuronal lysosomes and the mounting evidence on the role of lysosomal Zn in cell death during mammary gland involution set a biological precedent for the central role of the lysosomes in cellular Zn 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 Zn 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 Zn uptake by the lysosomes. In this review, we summarize the current knowledge on molecular determinants of the lysosomal Zn handling, uptake, and release pathways, as well as discuss their possible roles in health and disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5517152PMC
http://dx.doi.org/10.2741/4546DOI Listing
March 2017

Biphasic regulation of lysosomal exocytosis by oxidative stress.

Cell Calcium 2016 11 29;60(5):356-362. Epub 2016 Aug 29.

Department of Biological Sciences, Pittsburgh, PA 15260, USA. Electronic address:

Oxidative stress drives cell death in a number of diseases including ischemic stroke and neurodegenerative diseases. A better understanding of how cells recover from oxidative stress is likely to lead to better treatments for stroke and other diseases. The recent evidence obtained in several models ties the process of lysosomal exocytosis to the clearance of protein aggregates and toxic metals. The mechanisms that regulate lysosomal exocytosis, under normal or pathological conditions, are only beginning to emerge. Here we provide evidence for the biphasic effect of oxidative stress on lysosomal exocytosis. Lysosomal exocytosis was measured using the extracellular levels of the lysosomal enzyme beta-hexosaminidase (ß-hex). Low levels or oxidative stress stimulated lysosomal exocytosis, but inhibited it at high levels. Deletion of the lysosomal ion channel TRPML1 eliminated the stimulatory effect of low levels of oxidative stress. The inhibitory effects of oxidative stress appear to target the component of lysosomal exocytosis that is driven by extracellular Ca. We propose that while moderate oxidative stress promotes cellular repair by stimulating lysosomal exocytosis, at high levels oxidative stress has a dual pathological effect: it directly causes cell damage and impairs damage repair by inhibiting lysosomal exocytosis. Harnessing these adaptive mechanisms may point to pharmacological interventions for diseases involving oxidative proteotoxicity or metal toxicity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5077686PMC
http://dx.doi.org/10.1016/j.ceca.2016.08.002DOI Listing
November 2016

ER stress and impaired autophagy flux in neuronal degeneration and brain injury.

Ageing Res Rev 2017 Mar 1;34:3-14. Epub 2016 Sep 1.

Department of Neurology, University of Pittsburgh, Pittsburgh, PA 15213, United States; Veterans Affairs Pittsburgh Health Care System, Geriatric Research, Education and Clinical Center, Pittsburgh, PA 15213, United States. Electronic address:

Autophagy is a highly controlled lysosome-mediated function in eukaryotic cells to eliminate damaged or aged long-lived proteins and organelles. It is required for restoring cellular homeostasis in cell survival under multiple stresses. Autophagy is known to be a double-edged sword because too much activation or inhibition of autophagy can disrupt homeostatic degradation of protein and organelles within the brain and play a role in neuronal cell death. Many factors affect autophagy flux function in the brain, including endoplasmic reticulum (ER) stress, oxidative stress, and aging. Newly emerged research indicates that altered autophagy flux functionality is involved in neurodegeneration of the aged brain, chronic neurological diseases, and after traumatic and ischemic brain injuries. In search to identify neuroprotective agents that may reduce oxidative stress and stimulate autophagy, one particular neuroprotective agent docosahexaenoic acid (DHA) presents unique functions in reducing ER and oxidative stress and modulating autophagy. This review will summarize the recent findings on changes of autophagy in aging, neurodegenerative diseases, and brain injury after trauma or ischemic strokes. Discussion of DHA functions is focused on modulating ER stress and autophagy in regard to its neuroprotection and anti-tumor functions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.arr.2016.08.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5250579PMC
March 2017

ROS and intracellular ion channels.

Cell Calcium 2016 08 11;60(2):108-14. Epub 2016 Mar 11.

Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, United States; Epithelial Signaling and Transport Section, Molecular Physiology and Therapeutics Branch NIH, NIDCR, Bethesda, MD 20892, United States. Electronic address:

Oxidative stress is a well-known driver of numerous pathological processes involving protein and lipid peroxidation and DNA damage. The resulting increase of pro-apoptotic pressure drives tissue damage in a host of conditions, including ischemic stroke and reperfusion injury, diabetes, death in acute pancreatitis and neurodegenerative diseases. Somewhat less frequently discussed, but arguably as important, is the signaling function of oxidative stress stemming from the ability of oxidative stress to modulate ion channel activity. The evidence for the modulation of the intracellular ion channels and transporters by oxidative stress is constantly emerging and such evidence suggests new regulatory and pathological circuits that can be explored towards new treatments for diseases in which oxidative stress is an issue. In this review we summarize the current knowledge on the effects of oxidative stress on the intracellular ion channels and transporters and their role in cell function.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ceca.2016.03.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4996699PMC
August 2016

Impaired myelination and reduced brain ferric iron in the mouse model of mucolipidosis IV.

Dis Model Mech 2015 Dec 17;8(12):1591-601. Epub 2015 Sep 17.

Center for Human Genetic Research and Department of Neurology, Massachusetts General Hospital and Harvard Medical School, 185 Cambridge Street, Boston, MA 02114, USA.

Mucolipidosis type IV (MLIV) is a lysosomal storage disease caused by mutations in the MCOLN1 gene, which encodes the lysosomal transient receptor potential ion channel mucolipin-1 (TRPML1). MLIV causes impaired motor and cognitive development, progressive loss of vision and gastric achlorhydria. How loss of TRPML1 leads to severe psychomotor retardation is currently unknown, and there is no therapy for MLIV. White matter abnormalities and a hypoplastic corpus callosum are the major hallmarks of MLIV brain pathology. Here, we report that loss of TRPML1 in mice results in developmental aberrations of brain myelination as a result of deficient maturation and loss of oligodendrocytes. Defective myelination is evident in Mcoln1(-/-) mice at postnatal day 10, an active stage of postnatal myelination in the mouse brain. Expression of mature oligodendrocyte markers is reduced in Mcoln1(-/-) mice at postnatal day 10 and remains lower throughout the course of the disease. We observed reduced Perls' staining in Mcoln1(-/-) brain, indicating lower levels of ferric iron. Total iron content in unperfused brain is not significantly different between Mcoln1(-/-) and wild-type littermate mice, suggesting that the observed maturation delay or loss of oligodendrocytes might be caused by impaired iron handling, rather than by global iron deficiency. Overall, these data emphasize a developmental rather than a degenerative disease course in MLIV, and suggest that there should be a stronger focus on oligodendrocyte maturation and survival to better understand MLIV pathogenesis and aid treatment development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1242/dmm.021154DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4728313PMC
December 2015

Transition metals activate TFEB in overexpressing cells.

Biochem J 2015 Aug 11;470(1):65-76. Epub 2015 Jun 11.

Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15206, U.S.A.

Transition metal toxicity is an important factor in the pathogenesis of numerous human disorders, including neurodegenerative diseases. Lysosomes have emerged as important factors in transition metal toxicity because they handle transition metals via endocytosis, autophagy, absorption from the cytoplasm and exocytosis. Transcription factor EB (TFEB) regulates lysosomal biogenesis and the expression of lysosomal proteins in response to lysosomal and/or metabolic stresses. Since transition metals cause lysosomal dysfunction, we proposed that TFEB may be activated to drive gene expression in response to transition metal exposure and that such activation may influence transition metal toxicity. We found that transition metals copper (Cu) and iron (Fe) activate recombinant TFEB and stimulate the expression of TFEB-dependent genes in TFEB-overexpressing cells. In cells that show robust lysosomal exocytosis, TFEB was cytoprotective at moderate levels of Cu exposure, decreasing oxidative stress as reported by the expression of heme oxygenase-1 (HMOX1) gene. However, at high levels of Cu exposure, particularly in cells with low levels of lysosomal exocytosis, activation of overexpressed TFEB was toxic, increasing oxidative stress and mitochondrial damage. Based on these data, we conclude that TFEB-driven gene network is a component of the cellular response to transition metals. These data suggest limitations and disadvantages of TFEB overexpression as a therapeutic approach.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1042/BJ20140645DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4613523PMC
August 2015

Brief exposure to copper activates lysosomal exocytosis.

Cell Calcium 2015 Apr 12;57(4):257-62. Epub 2015 Jan 12.

Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, United States. Electronic address:

Copper (Cu) is essential mineral, but its toxicity necessitates existence of powerful machinery responsible for the extraction of excess Cu from the cell. Cu exposure was recently shown to induce the translocation of Cu pump ATP7B to the lysosomes followed by lysosomal exocytosis. Here we sought to investigate the mechanisms underlying the effect of Cu on lysosomal exocytosis. We found that brief exposure to Cu activates lysosomal exocytosis, which was measured as a release of the lysosomal digestive enzyme β-hexosaminidase (β-hex) into the extracellular medium and by the presence lysosomal protein LAMP1 at the plasma membrane. Such release depends on calcium (Ca) and on the lysosomal SNARE VAMP7. ATP7B knockdown using RNAi suppressed the basal lysosomal exocytosis, but did not affect the ability of Cu to activate it. ATP7B knockdown was associated with sustained oxidative stress. The removal of Ca from the extracellular medium suppressed the Cu-dependent component of the lysosomal exocytosis. We propose that Cu promotes lysosomal exocytosis by facilitating a Ca-dependent step of the lysosomal exocytosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ceca.2015.01.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4363267PMC
April 2015

A novel role for 12/15-lipoxygenase in regulating autophagy.

Redox Biol 2015 3;4:40-7. Epub 2014 Dec 3.

Institute of Infection and Immunity, School of Medicine, Cardiff University, Cardiff CF14 4XN, UK. Electronic address:

12/15-Lipoxygenase (LOX) enzymatically generates oxidized phospholipids in monocytes and macrophages. Herein, we show that cells deficient in 12/15-LOX contain defective mitochondria and numerous cytoplasmic vacuoles containing electron dense material, indicating defects in autophagy or membrane processing, However, both LC3 expression and lipidation were normal both basally and on chloroquine treatment. A LOX-derived oxidized phospholipid, 12-hydroxyeicosatetraenoic acid-phosphatidylethanolamine (12-HETE-PE) was found to be a preferred substrate for yeast Atg8 lipidation, versus native PE, while both native and oxidized PE were effective substrates for LC3 lipidation. Last, phospholipidomics demonstrated altered levels of several phospholipid classes. Thus, we show that oxidized phospholipids generated by 12/15-LOX can act as substrates for key proteins required for effective autophagy and that cells deficient in this enzyme show evidence of autophagic dysfunction. The data functionally link phospholipid oxidation with autophagy for the first time.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.redox.2014.11.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4309860PMC
June 2016

Permeation, regulation and control of expression of TRP channels by trace metal ions.

Pflugers Arch 2015 Jun 10;467(6):1143-64. Epub 2014 Aug 10.

CNRS, 38054, Grenoble, France,

Transient receptor potential (TRP) channels form a diverse family of cation channels comprising 28 members in mammals. Although some TRP proteins can only be found on intracellular membranes, most of the TRP protein isoforms reach the plasma membrane where they form ion channels and control a wide number of biological processes. There, their involvement in the transport of cations such as calcium and sodium has been well documented. However, a growing number of studies have started to expand our understanding of these proteins by showing that they also transport other biologically relevant metal ions like zinc, magnesium, manganese and cobalt. In addition to this newly recognized property, the activity and expression of TRP channels can be regulated by metal ions like magnesium, gadolinium, lanthanum or cisplatin. The aim of this review is to highlight the complex relationship between metal ions and TRP channels.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00424-014-1590-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4435931PMC
June 2015

Zn2+ efflux through lysosomal exocytosis prevents Zn2+-induced toxicity.

J Cell Sci 2014 Jul 14;127(Pt 14):3094-103. Epub 2014 May 14.

The Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA

Zn(2+) is an essential micronutrient and an important ionic signal whose excess, as well as scarcity, is detrimental to cells. Free cytoplasmic Zn(2+) is controlled by a network of Zn(2+) transporters and chelating proteins. Recently, lysosomes became the focus of studies in Zn(2+) transport, as they were shown to play a role in Zn(2+)-induced toxicity by serving as Zn(2+) sinks that absorb Zn(2+) from the cytoplasm. Here, we investigated the impact of the lysosomal Zn(2+) sink on the net cellular Zn(2+) distribution and its role in cell death. We found that lysosomes played a cytoprotective role during exposure to extracellular Zn(2+). Such a role required lysosomal acidification and exocytosis. Specifically, we found that the inhibition of lysosomal acidification using Bafilomycin A1 (Baf) led to a redistribution of Zn(2+) pools and increased apoptosis. Additionally, the inhibition of lysosomal exocytosis through knockdown (KD) of the lysosomal SNARE proteins VAMP7 and synaptotagmin VII (SYT7) suppressed Zn(2+) secretion and VAMP7 KD cells had increased apoptosis. These data show that lysosomes play a central role in Zn(2+) handling, suggesting that there is a new Zn(2+) detoxification pathway.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1242/jcs.145318DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4095854PMC
July 2014

The biology of zinc transport in mammary epithelial cells: implications for mammary gland development, lactation, and involution.

J Mammary Gland Biol Neoplasia 2014 Mar 15;19(1):59-71. Epub 2013 Dec 15.

Department of Nutritional Sciences, The Pennsylvania State University, University Park, PA, USA.

Zinc plays a critical role in a vast array of cellular functions including gene transcription, protein translation, cell proliferation, differentiation, bioenergetics, and programmed cell death. The mammary gland depends upon tight coordination of these processes during development and reproduction for optimal expansion, differentiation, and involution. For example, zinc is required for activation of matrix metalloproteinases, intracellular signaling cascades such as MAPK and PKC, and the activation of both mitochondrial-mediated apoptosis and lysosomal-mediated cell death. In addition to functional needs, during lactation the mammary gland must balance providing optimal zinc for cellular requirements with the need to secrete a substantial amount of zinc into milk to meet the requirements of the developing neonate. Finally, the mammary gland exhibits the most profound example of programmed cell death, which is driven by both apoptotic and lysosomal-mediated cell death. Two families of zinc-specific transporters regulate zinc delivery for these diverse functions. Members of the ZIP family of zinc transporters (ZIP1-14) import zinc into the cytoplasm from outside the cell or from subcellular organelles, while members of the ZnT family (ZnT1-10) export zinc from the cytoplasm. Recently, the ion channel transient receptor potential mucolipin 1 (TRPML1) has also been implicated in zinc transport. Herein, we review our current understanding of the molecular mechanisms through which mammary epithelial cells utilize zinc with a focus on the transport of zinc into discrete subcellular organelles for specific cellular functions during mammary gland development, lactation, and involution.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s10911-013-9314-4DOI Listing
March 2014

Altered dynamics of a lipid raft associated protein in a kidney model of Fabry disease.

Mol Genet Metab 2014 Feb 19;111(2):184-92. Epub 2013 Oct 19.

Renal-Electrolyte Division, University of Pittsburgh, Pittsburgh, PA 15261, USA. Electronic address:

Accumulation of globotriaosylceramide (Gb3) and other neutral glycosphingolipids with galactosyl residues is the hallmark of Fabry disease, a lysosomal storage disorder caused by deficiency of the enzyme alpha-galactosidase A (α-gal A). These lipids are incorporated into the plasma membrane and intracellular membranes, with a preference for lipid rafts. Disruption of raft mediated cell processes is implicated in the pathogenesis of several human diseases, but little is known about the effects of the accumulation of glycosphingolipids on raft dynamics in the context of Fabry disease. Using siRNA technology, we have generated a polarized renal epithelial cell model of Fabry disease in Madin-Darby canine kidney cells. These cells present increased levels of Gb3 and enlarged lysosomes, and progressively accumulate zebra bodies. The polarized delivery of both raft-associated and raft-independent proteins was unaffected by α-gal A knockdown, suggesting that accumulation of Gb3 does not disrupt biosynthetic trafficking pathways. To assess the effect of α-gal A silencing on lipid raft dynamics, we employed number and brightness (N&B) analysis to measure the oligomeric status and mobility of the model glycosylphosphatidylinositol (GPI)-anchored protein GFP-GPI. We observed a significant increase in the oligomeric size of antibody-induced clusters of GFP-GPI at the plasma membrane of α-gal A silenced cells compared with control cells. Our results suggest that the interaction of GFP-GPI with lipid rafts may be altered in the presence of accumulated Gb3. The implications of our results with respect to the pathogenesis of Fabry disease are discussed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ymgme.2013.10.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3946758PMC
February 2014

Loss of TRPML1 promotes production of reactive oxygen species: is oxidative damage a factor in mucolipidosis type IV?

Biochem J 2014 Jan;457(2):361-8

*Department of Biological Sciences, University of Pittsburgh, 4249 Fifth Ave, Pittsburgh, PA 15260, U.S.A.

TRPML1 (transient receptor potential mucolipin 1) is a lysosomal ion channel permeable to cations, including Fe2+. Mutations in MCOLN1, the gene coding for TRPML1, cause the LSD (lysosomal storage disease) MLIV (mucolipidosis type IV). The role of TRPML1 in the cell is disputed and the mechanisms of cell deterioration in MLIV are unclear. The demonstration of Fe2+ buildup in MLIV cells raised the possibility that TRPML1 dissipates lysosomal Fe2+ and prevents its accumulation. Since Fe2+ catalyses the production of ROS (reactive oxygen species), we set out to test whether or not the loss of TRPML1 promotes ROS production by Fe2+ trapped in lysosomes. Our data show that RPE1 (retinal pigmented epithelial 1) cells develop a punctate mitochondrial phenotype within 48 h of siRNA-induced TRPML1-KD (knockdown). This mitochondrial fragmentation was aggravated by Fe2+ exposure, but was reversed by incubation with the ROS chelator α-Toc (α-tocopherol). The exposure of TRPML1-KD cells to Fe2+ led to loss of ΔΨm (mitochondrial membrane potential), ROS buildup, lipid peroxidation and increased transcription of genes responsive to cytotoxic oxidative stress in TRPML1-KD cells. These data suggest that TRPML1 redistributes Fe2+ between the lysosomes and the cytoplasm. Fe2+ buildup caused by TRPML1 loss potentiates ROS production and leads to mitochondrial deterioration. Beyond suggesting a new model for MLIV pathogenesis, these data show that TRPML1's role in the cell extends outside lysosomes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1042/BJ20130647DOI Listing
January 2014

Zinc-dependent lysosomal enlargement in TRPML1-deficient cells involves MTF-1 transcription factor and ZnT4 (Slc30a4) transporter.

Biochem J 2013 Apr;451(2):155-63

Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA.

Zinc is critical for a multitude of cellular processes, including gene expression, secretion and enzymatic activities. Cellular zinc is controlled by zinc-chelating proteins and by zinc transporters. The recent identification of zinc permeability of the lysosomal ion channel TRPML1 (transient receptor potential mucolipin 1), and the evidence of abnormal zinc levels in cells deficient in TRPML1, suggested a role for TRPML1 in zinc transport. In the present study we provide new evidence for such a role and identify additional cellular components responsible for it. In agreement with the previously published data, an acute siRNA (small interfering RNA)-driven TRPML1 KD (knockdown) leads to the build-up of large cytoplasmic vesicles positive for LysoTracker™ and zinc staining, when cells are exposed to high concentrations of zinc. We now show that lysosomal enlargement and zinc build-up in TRPML1-KD cells exposed to zinc are ameliorated by KD of the zinc-sensitive transcription factor MTF-1 (metal-regulatory-element-binding transcription factor-1) or the zinc transporter ZnT4. TRPML1 KD is associated with a build-up of cytoplasmic zinc and with enhanced transcriptional response of mRNA for MT2a (metallothionein 2a). TRPML1 KD did not suppress lysosomal secretion, but it did delay zinc leak from the lysosomes into the cytoplasm. These results underscore a role for TRPML1 in zinc metabolism. Furthermore, they suggest that TRPML1 works in concert with ZnT4 to regulate zinc translocation between the cytoplasm and lysosomes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1042/BJ20121506DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3654546PMC
April 2013

Mutant LRRK2 elicits calcium imbalance and depletion of dendritic mitochondria in neurons.

Am J Pathol 2013 Feb 8;182(2):474-84. Epub 2012 Dec 8.

Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, USA.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) have been associated with familial and sporadic cases of Parkinson disease. Mutant LRRK2 causes in vitro and in vivo neurite shortening, mediated in part by autophagy, and a parkinsonian phenotype in transgenic mice; however, the underlying mechanisms remain unclear. Because mitochondrial content/function is essential for dendritic morphogenesis and maintenance, we investigated whether mutant LRRK2 affects mitochondrial homeostasis in neurons. Mouse cortical neurons expressing either LRRK2 G2019S or R1441C mutations exhibited autophagic degradation of mitochondria and dendrite shortening. In addition, mutant LRRK2 altered the ability of the neurons to buffer intracellular calcium levels. Either calcium chelators or inhibitors of voltage-gated L-type calcium channels prevented mitochondrial degradation and dendrite shortening. These data suggest that mutant LRRK2 causes a deficit in calcium homeostasis, leading to enhanced mitophagy and dendrite shortening.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ajpath.2012.10.027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3562730PMC
February 2013

The intracellular Ca²⁺ channels of membrane traffic.

Channels (Austin) 2012 Sep-Oct;6(5):344-51. Epub 2012 Aug 21.

Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA, USA.

Regulation of organellar fusion and fission by Ca ( 2+) has emerged as a central paradigm in intracellular membrane traffic. Originally formulated for Ca ( 2+) -driven SNARE-mediated exocytosis in the presynaptic terminals, it was later expanded to explain membrane traffic in other exocytic events within the endo-lysosomal system. The list of processes and conditions that depend on the intracellular membrane traffic includes aging, antigen and lipid processing, growth factor signaling and enzyme secretion. Characterization of the ion channels that regulate intracellular membrane fusion and fission promises novel pharmacological approaches in these processes when their function becomes aberrant. The recent identification of Ca ( 2+) permeability through the intracellular ion channels comprising the mucolipin (TRPMLs) and the two-pore channels (TPCs) families pinpoints the candidates for the Ca ( 2+) channel that drive intracellular membrane traffic. The present review summarizes the recent developments and the current questions relevant to this topic.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4161/chan.21723DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3508773PMC
May 2013

Loss of lysosomal ion channel transient receptor potential channel mucolipin-1 (TRPML1) leads to cathepsin B-dependent apoptosis.

J Biol Chem 2012 Mar 18;287(11):8082-91. Epub 2012 Jan 18.

Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA.

Mucolipidosis type IV (MLIV) is a lysosomal storage disease caused by mutations in the gene MCOLN1, which codes for the transient receptor potential family ion channel TRPML1. MLIV has an early onset and is characterized by developmental delays, motor and cognitive deficiencies, gastric abnormalities, retinal degeneration, and corneal cloudiness. The degenerative aspects of MLIV have been attributed to cell death, whose mechanisms remain to be delineated in MLIV and in most other storage diseases. Here we report that an acute siRNA-mediated loss of TRPML1 specifically causes a leak of lysosomal protease cathepsin B (CatB) into the cytoplasm. CatB leak is associated with apoptosis, which can be prevented by CatB inhibition. Inhibition of the proapoptotic protein Bax prevents TRPML1 KD-mediated apoptosis but does not prevent cytosolic release of CatB. This is the first evidence of a mechanistic link between acute TRPML1 loss and cell death.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M111.285536DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3318733PMC
March 2012

TRPML: transporters of metals in lysosomes essential for cell survival?

Cell Calcium 2011 Sep 31;50(3):288-94. Epub 2011 May 31.

Department of Biological Science, University of Pittsburgh, Pittsburgh, PA 15260, USA.

Key aspects of lysosomal function are affected by the ionic content of the lysosomal lumen and, therefore, by the ion permeability in the lysosomal membrane. Such functions include regulation of lysosomal acidification, a critical process in delivery and activation of the lysosomal enzymes, release of metals from lysosomes into the cytoplasm and the Ca(2+)-dependent component of membrane fusion events in the endocytic pathway. While the basic mechanisms of lysosomal acidification have been largely defined, the lysosomal metal transport system is not well understood. TRPML1 is a lysosomal ion channel whose malfunction is implicated in the lysosomal storage disease Mucolipidosis Type IV. Recent evidence suggests that TRPML1 is involved in Fe(2+), Ca(2+) and Zn(2+) transport across the lysosomal membrane, ascribing novel physiological roles to this ion channel, and perhaps to its relatives TRPML2 and TRPML3 and illuminating poorly understood aspects of lysosomal function. Further, alterations in metal transport by the TRPMLs due to mutations or environmental factors may contribute to their role in the disease phenotype and cell death.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ceca.2011.04.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3164942PMC
September 2011

TRPML1.

Adv Exp Med Biol 2011 ;704:209-19

Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA.

TRPML1 (or mucolipin 1) is the first member of the TRP family of ion channels that was found to function in the lower portions of the endocytic pathway. Mutations in the gene coding for TRPML1 (MCOLN1) cause the lysosomal storage disease mucolipidosis type IV (MLIV). TRPML1 localization in the lysosomes and the similarity of mucolipidosis type IV phenotype to lysosomal storage diseases whose origin has been directly linked to lysosomal dysfunction, suggest that TRPML1 activity drives some vitally important processes within the endocytic machinery. The specific aspect(s) of TRPML1 activity that make it indispensable for the proper function of the endocytic pathway as well as the specific aspect(s) of the endocytic activity that depend on TRPML1 are currently being discussed. Among the candidates are: membrane fusion within the lower portion of the endocytic pathway possibly mediated by Ca(2+) release through TRPML1, or regulation of lysosomal ion homeostasis (pH or Fe content) by TRPML1. In addition to delineating the mechanisms of MLIV pathogenesis, identifying the role of TRPML1 in the endocytic pathway will lead to important developments in our understanding of the endocytic pathway and, due to the neurodegenerative nature of MLIV, of the integrative function of the cell. Moreover, molecular modulators of TRPML1 function may lead to novel approaches to modulating biological processes that depend on the endocytic pathway such as growth factor signaling. The present review will focus on the recent developments in identifying the TRPML1 function.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-94-007-0265-3_11DOI Listing
July 2011

TRPC channels in pheromone sensing.

Vitam Horm 2010 ;83:197-213

Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.

Pheromone recognition relies on an amplification cascade that is triggered by pheromone binding to G protein-coupled receptors (GPCR). The first step in translation of GPCR activation by pheromones in the vomeronasal organ and main olfactory epithelium (MOE) into a cellular response is the activation of a transient receptor potential (TRP) family member, TRPC2 [Zufall, F., Ukhanov, K., Lucas, P., Liman, E. R., and Leinders-Zufall, T. (2005). Neurobiology of TRPC2: From gene to behavior. Pflugers Arch.451, 61-71; Yildirim, E., and Birnbaumer, L. (2007). TRPC2: Molecular biology and functional importance. Handb. Exp. Pharmacol. 53-75]. The members of the canonical (TRPC) family of TRP channels mediate membrane permeability, specifically, Ca(2+) influx into the cytoplasm in response to activation of GPCR and tyrosine kinase receptors by hormones, neurotransmitters, and growth factors [Nilius, B. (2007). TRP channels in disease. Biochim. Biophys. Acta1772, 805-812; Venkatachalam, K., and Montell, C. (2007). TRP channels. Annu. Rev. Biochem.76, 387-417]. Mechanisms of their activation have been the focus of intense interest during the last decade. The data obtained from studies of TRPC2 have resulted in a better understanding of ion channel physiology and led to novel paradigms in modern cell biology [Lucas, P., Ukhanov, K., Leinders-Zufall, T., and Zufall, F. (2003). A diacylglycerol-gated cation channel in vomeronasal neuron dendrites is impaired in TRPC2 mutant mice: Mechanism of pheromone transduction. Neuron40, 551-561; Stowers, L., Holy, T. E., Meister, M., Dulac, C., and Koentges, G. (2002). Loss of sex discrimination and male-male aggression in mice deficient for TRP2. Science295, 1493-1500; Leypold, B. G., Yu, C. R., Leinders-Zufall, T., Kim, M. M., Zufall, F., and Axel, R. (2002). Altered sexual and social behaviors in trp2 mutant mice. Proc. Natl. Acad. Sci. USA99, 6376-6381]. Although TRPC2 activation by pheromones presents one of the most straightforward examples of physiological function of TRPC channels, the molecular aspects of its activation are not well understood (Yildirim, E., and Birnbaumer, L. (2007). TRPC2: Molecular biology and functional importance. Handb. Exp. Pharmacol. 53-75). It is natural to expect that better understanding of TRPC2 activation mechanisms will lead to breakthroughs in understanding ion channel activation mechanisms, as well as applied behavioral pharmacology. The present review is focused on the current knowledge of TRPC2 physiology with a specific focus on TRPC activation mechanisms.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/S0083-6729(10)83008-0DOI Listing
January 2011

Mitotic slippage in non-cancer cells induced by a microtubule disruptor, disorazole C1.

BMC Chem Biol 2010 Feb 11;10. Epub 2010 Feb 11.

Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA.

Background: Disorazoles are polyene macrodiolides isolated from a myxobacterium fermentation broth. Disorazole C1 was newly synthesized and found to depolymerize microtubules and cause mitotic arrest. Here we examined the cellular responses to disorazole C1 in both non-cancer and cancer cells and compared our results to vinblastine and taxol.

Results: In non-cancer cells, disorazole C1 induced a prolonged mitotic arrest, followed by mitotic slippage, as confirmed by live cell imaging and cell cycle analysis. This mitotic slippage was associated with cyclin B degradation, but did not require p53. Four assays for apoptosis, including western blotting for poly(ADP-ribose) polymerase cleavage, microscopic analyses for cytochrome C release and annexin V staining, and gel electrophoresis examination for DNA laddering, were conducted and demonstrated little induction of apoptosis in non-cancer cells treated with disorazole C1. On the contrary, we observed an activated apoptotic pathway in cancer cells, suggesting that normal and malignant cells respond differently to disorazole C1.

Conclusion: Our studies demonstrate that non-cancer cells undergo mitotic slippage in a cyclin B-dependent and p53-independent manner after prolonged mitotic arrest caused by disorazole C1. In contrast, cancer cells induce the apoptotic pathway after disorazole C1 treatment, indicating a possibly significant therapeutic window for this compound.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/1472-6769-10-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2834648PMC
February 2010

Aberrant Ca2+ handling in lysosomal storage disorders.

Cell Calcium 2010 Feb 6;47(2):103-11. Epub 2010 Jan 6.

Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA.

Lysosomal storage diseases (LSDs) are caused by inability of cells to process the material captured during endocytosis. While they are essentially diseases of cellular "indigestion", LSDs affect large number of cellular activities and, as such, they teach us about the integrative function of the cell, as well as about the gaps in our knowledge of the endocytic pathway and membrane transport. The present review summarizes recent findings on Ca2+ handling in LSDs and attempts to identify the key questions on alterations in Ca2+ signaling and membrane transport in this group of diseases, answers to which may lie in delineating the cellular pathogeneses of LSDs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ceca.2009.12.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2838446PMC
February 2010