Adipocytes robustly synthesize fatty acids (FA) from carbohydrate through the de novo lipogenesis (DNL) pathway, yet surprisingly DNL contributes little to their abundant triglyceride stored in lipid droplets. This conundrum raises the hypothesis that adipocyte DNL instead enables membrane expansions to occur in processes like autophagy, which requires an abundant supply of phospholipids. We report here that adipocyte Fasn deficiency in vitro and in vivo markedly impairs autophagy, evident by autophagosome accumulation and severely compromised degradation of the autophagic substrate p62. Our data indicate the impairment occurs at the level of autophagosome-lysosome fusion, and indeed, loss of Fasn decreases certain membrane phosphoinositides necessary for autophagosome and lysosome maturation and fusion. Autophagy dependence on FA produced by Fasn is not fully alleviated by exogenous FA in cultured adipocytes, and interestingly, imaging studies reveal that Fasn colocalizes with nascent autophagosomes. Together, our studies identify DNL as a critical source of FAs to fuel autophagosome and lysosome maturation and fusion in adipocytes.
Bis(monoacylglycero)phosphates (BMPs), a class of lipids highly enriched within endolysosomal organelles, are key components of the lysosomal intraluminal vesicles responsible for activating sphingolipid catabolic enzymes. While BMPs are understudied relative to other phospholipids, recent reports associate BMP dysregulation with a variety of pathological states including neurodegenerative diseases and lysosomal storage disorders. Since the dramatic lysosomal remodeling characteristic of cellular transformation could impact BMP abundance and function, we employed untargeted lipidomics approaches to identify and quantify BMP species in several in vitro and in vivo models of breast cancer and comparative non-transformed cells and tissues. We observed lower BMP levels within transformed cells relative to normal cells, and consistent enrichment of docosahexaenoic acid (22:6) fatty acyl chain-containing BMP species in both human- and mouse-derived mammary tumorigenesis models. Our functional analysis points to a working model whereby 22:6 BMPs serve as reactive oxygen species scavengers in tumor cells, protecting lysosomes from oxidant-induced lysosomal membrane permeabilization. Our findings suggest that breast tumor cells might divert polyunsaturated fatty acids into BMP lipids as part of an adaptive response to protect their lysosomes from elevated reactive oxygen species levels, and raise the possibility that BMP-mediated lysosomal protection is a tumor-specific vulnerability that may be exploited therapeutically.
- MeSH
- fosfáty metabolismus MeSH
- kyseliny dokosahexaenové * MeSH
- lidé MeSH
- lysofosfolipidy metabolismus MeSH
- lyzozomy metabolismus MeSH
- myši MeSH
- nádory prsu * patologie MeSH
- reaktivní formy kyslíku metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Research Support, N.I.H., Extramural MeSH
The lysosome represents a central degradative compartment of eukaryote cells, yet little is known about the biogenesis and function of this organelle in parasitic protists. Whereas the mannose 6-phosphate (M6P)-dependent system is dominant for lysosomal targeting in metazoans, oligosaccharide-independent sorting has been reported in other eukaryotes. In this study, we investigated the phagolysosomal proteome of the human parasite Trichomonas vaginalis, its protein targeting and the involvement of lysosomes in hydrolase secretion. The organelles were purified using Percoll and OptiPrep gradient centrifugation and a novel purification protocol based on the phagocytosis of lactoferrin-covered magnetic nanoparticles. The analysis resulted in a lysosomal proteome of 462 proteins, which were sorted into 21 classes. Hydrolases represented the largest functional class and included proteases, lipases, phosphatases, and glycosidases. Identification of a large set of proteins involved in vesicular trafficking (80) and turnover of actin cytoskeleton rearrangement (29) indicate a dynamic phagolysosomal compartment. Several cysteine proteases such as TvCP2 were previously shown to be secreted. Our experiments showed that secretion of TvCP2 was strongly inhibited by chloroquine, which increases intralysosomal pH, thus indicating that TvCP2 secretion occurs through lysosomes rather than the classical secretory pathway. Unexpectedly, we identified divergent homologues of the M6P receptor TvMPR in the phagolysosomal proteome, although T. vaginalis lacks enzymes for M6P formation. To test whether oligosaccharides are involved in lysosomal targeting, we selected the lysosome-resident cysteine protease CLCP, which possesses two glycosylation sites. Mutation of any of the sites redirected CLCP to the secretory pathway. Similarly, the introduction of glycosylation sites to secreted β-amylase redirected this protein to lysosomes. Thus, unlike other parasitic protists, T. vaginalis seems to utilize glycosylation as a recognition marker for lysosomal hydrolases. Our findings provide the first insight into the complexity of T. vaginalis phagolysosomes, their biogenesis, and role in the unconventional secretion of cysteine peptidases.
- MeSH
- cystein metabolismus MeSH
- cysteinové proteasy * metabolismus MeSH
- fagozomy metabolismus MeSH
- lidé MeSH
- lyzozomy metabolismus MeSH
- proteasy metabolismus MeSH
- proteomika MeSH
- Trichomonas vaginalis * metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Lysosomes, now known to take part in multiple cellular functions, also respond to various stress stimuli. These include biogenesis in response to nanomolar concentrations of hydrophobic weak-base anticancer drugs. However, since lysosomal stress mediated by accumulation of weak-base drugs at such concentrations has never been proven and these drugs have diverse effects on malignant cells, we investigated whether the interpretation of the data was true. We found that lysosomal accumulation of the drugs daunorubicin, doxorubicin, mitoxantrone, symadex, chloroquine, clomipramine and sunitinib alone, was insufficient to induce lysosomal alkalization i.e., lysosomal stress-mediated biogenesis at nanomolar concentrations. Instead, we found that some of the drugs used induced G2 phase arrest and lysosomal biogenesis that is associated with activation of transcription factor EB (TFEB). Similarly, cantharidin, a control compound that does not belong to the weak base drugs, induced cell cycle arrest in the G2 phase associated with TFEB-driven lysosomal biogenesis. Overall none of the tested drugs caused stress-induced lysosomal biogenesis at nanomolar concentrations. However, daunorubicin, doxorubicin, mitoxantrone, symadex and cantharidin induced a massive block in the G2 phase of the cell cycle which is naturally associated with TFEB-driven lysosomal biogenesis.
In the ciliate Tetrahymena thermophila, lysosome-related organelles called mucocysts accumulate at the cell periphery where they secrete their contents in response to extracellular events, a phenomenon called regulated exocytosis. The molecular bases underlying regulated exocytosis have been extensively described in animals but it is not clear whether similar mechanisms exist in ciliates or their sister lineage, the Apicomplexan parasites, which together belong to the ecologically and medically important superphylum Alveolata. Beginning with a T. thermophila mutant in mucocyst exocytosis, we used a forward genetic approach to uncover MDL1 (Mucocyst Discharge with a LamG domain), a novel gene that is essential for regulated exocytosis of mucocysts. Mdl1p is a 40 kDa membrane glycoprotein that localizes to mucocysts, and specifically to a tip domain that contacts the plasma membrane when the mucocyst is docked. This sub-localization of Mdl1p, which occurs prior to docking, underscores a functional asymmetry in mucocysts that is strikingly similar to that of highly polarized secretory organelles in other Alveolates. A mis-sense mutation in the LamG domain results in mucocysts that dock but only undergo inefficient exocytosis. In contrast, complete knockout of MDL1 largely prevents mucocyst docking itself. Mdl1p is physically associated with 9 other proteins, all of them novel and largely restricted to Alveolates, and sedimentation analysis supports the idea that they form a large complex. Analysis of three other members of this putative complex, called MDD (for Mucocyst Docking and Discharge), shows that they also localize to mucocysts. Negative staining of purified MDD complexes revealed distinct particles with a central channel. Our results uncover a novel macromolecular complex whose subunits are conserved within alveolates but not in other lineages, that is essential for regulated exocytosis in T. thermophila.
- MeSH
- exocytóza genetika MeSH
- lyzozomy metabolismus MeSH
- organely metabolismus MeSH
- sekreční vezikuly genetika metabolismus MeSH
- Tetrahymena thermophila * genetika MeSH
- Tetrahymena * MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Information about cholesterol subcellular localization and transport pathways inside cells is essential for understanding and treatment of cholesterol-related diseases. However, there is a lack of reliable tools to monitor it. This work follows the fate of Sterolight, a BODIPY-labelled sterol, within the cell and demonstrates it as a suitable probe for visualization of sterol/lipid trafficking. Sterolight enters cells through an energy-independent process and knockdown experiments suggest caveolin-1 as its potential cellular carrier. Intracellular transport of Sterolight is a rapid process, and transfer from ER and mitochondria to lysosomes and later to lipid droplets requires the participation of active microtubules, as it can be inhibited by the microtubule disruptor nocodazole. Excess of the probe is actively exported from cells, in addition to being stored in lipid droplets, to re-establish the sterol balance. Efflux occurs through a mechanism requiring energy and may be selectively poisoned with verapamil or blocked in cells with mutated cholesterol transporter NPC1. Sterolight is efficiently transferred within and between different cell populations, making it suitable for monitoring numerous aspects of sterol biology, including the live tracking and visualization of intracellular and intercellular transport.
Receptor tyrosine kinases (RTKs) are membrane receptors that regulate many fundamental cellular processes. A tight regulation of RTK signaling is fundamental for development and survival, and an altered signaling by RTKs can cause cancer. RTKs are localized at the plasma membrane (PM) and the major regulatory mechanism of signaling of RTKs is their endocytosis and degradation. In fact, RTKs at the cell surface bind ligands with their extracellular domain, become active, and are rapidly internalized where the temporal extent of signaling, attenuation, and downregulation are modulated. However, other mechanisms of signal attenuation and termination are known. Indeed, inhibition of RTKs' activity may occur through the modulation of the phosphorylation state of RTKs and the interaction with specific proteins, whereas antagonist ligands can inhibit the biological responses mediated by the receptor. Another mechanism concerns the expression of endogenous inactive receptor variants that are deficient in RTK activity and take part to inactive heterodimers or hetero-oligomers. The downregulation of RTK signals is fundamental for several cellular functions and the homeostasis of the cell. Here, we will review the mechanisms of signal attenuation and termination of RTKs, focusing on FGFRs.
- MeSH
- down regulace MeSH
- lidé MeSH
- lyzozomy metabolismus MeSH
- mutace genetika MeSH
- signální transdukce * MeSH
- tyrosinkinasové receptory antagonisté a inhibitory genetika metabolismus MeSH
- ubikvitinace MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
Niemann-Pick disease type C (NPC) is a complex and rare pathology, which is mainly associated to mutations in the NPC1 gene. This disease is phenotypically characterized by the abnormal accumulation of multiple lipid species in the acidic compartments of the cell. Due to the complexity of stored material, a clear molecular mechanism explaining NPC pathophysiology is still not established. Abnormal sphingosine accumulation was suggested as the primary factor involved in the development of NPC, followed by the accumulation of other lipid species. To provide additional mechanistic insight into the role of sphingosine in NPC development, fluorescence spectroscopy and microscopy were used to study the biophysical properties of biological membranes using different cellular models of NPC. Addition of sphingosine to healthy CHO-K1 cells, in conditions where other lipid species are not yet accumulated, caused a rapid decrease in plasma membrane and lysosome membrane fluidity, suggesting a direct effect of sphingosine rather than a downstream event. Changes in membrane fluidity caused by addition of sphingosine were partially sustained upon impaired trafficking and metabolization of cholesterol in these cells, and could recapitulate the decrease in membrane fluidity observed in NPC1 null Chinese Hamster Ovary (CHO) cells (CHO-M12) and in cells with pharmacologically induced NPC phenotype (treated with U18666A). In summary, these results show for the first time that the fluidity of the membranes is altered in models of NPC and that these changes are in part caused by sphingosine, supporting the role of this lipid in the pathophysiology of NPC.
- MeSH
- buněčná membrána metabolismus MeSH
- CHO buňky MeSH
- cholesterol metabolismus MeSH
- Cricetulus MeSH
- endozomy metabolismus MeSH
- fenotyp MeSH
- lyzozomy metabolismus MeSH
- Niemannova-Pickova nemoc typu C metabolismus patologie MeSH
- sfingolipidy metabolismus MeSH
- sfingosin metabolismus MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Lysosomal sequestration of weak base drugs has been identified as one of the stress-related mechanisms that trigger in vitro lysosomal biogenesis controlled by transcription factor EB (TFEB). Whether such mechanism can induce lysosomal biogenesis in vivo is unknown. In this study, we addressed the question whether prolonged treatment with sunitinib (SUN) in patients with advanced renal cell carcinoma (n = 22) and with imatinib (IM) in those with gastrointestinal stromal tumor (n = 6) could induce lysosomal biogenesis in leukocytes. Lysosomal biogenesis was monitored using immunoblotting of three lysosomal membrane proteins: lysosome-associated membrane proteins 1 and 2 (LAMP1 and LAMP2) and vacuolar H+-ATPase, B2 subunit (ATP6V1B2). Present results indicate that prolonged treatment with SUN affects LAMP1 and LAMP2 expression only marginally in most patients. In contrast, changes in ATP6V1B2 expression were marked and resembled irregular oscillations. Very similar changes in the expression of lysosomal membrane proteins were also found in IM-treated patients. Conclusion: prolonged treatment of cancer patients with SUN and IM did not induce leucocyte lysosomal biogenesis but dramatically affected expression of ATP6V1B2.
- MeSH
- gastrointestinální stromální tumory farmakoterapie metabolismus MeSH
- imatinib mesylát terapeutické užití MeSH
- inhibitory proteinkinas terapeutické užití MeSH
- karcinom z renálních buněk farmakoterapie metabolismus MeSH
- leukocyty metabolismus MeSH
- lidé MeSH
- lyzozomy metabolismus MeSH
- membránové glykoproteiny asociované s lyzozomy metabolismus MeSH
- sunitinib terapeutické užití MeSH
- transkripční faktory BHLH-Zip metabolismus MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
To improve the treatment of psoriasiform inflammation, we developed actively targeted nanocarriers loaded with the phosphodiesterase 4 inhibitor AN2728. Methods: Phospholipid-poly(lactic-co-glycolic acid) nanohybrids were prepared and conjugated with monovalent anti-desmoglein 3 antibody to bind keratinocytes. Results: The actively targeted nanohybrids were 229 nm in mean size with a nearly neutral surface charge. Flow cytometry and confocal microscopy showed a 9-fold increase in keratinocyte uptake of targeted nanohybrids relative to non-targeted nanoparticles. The nanoparticles localized mainly in lysosomes after internalization. AN2728-loaded antibody-conjugated nanocarriers inhibited cytokine/chemokine overexpression in activated keratinocytes without affecting cell viability. The targeted nanohybrids also suppressed neutrophil migration by reducing CXCL1 and CXCL2 release from keratinocytes. Following subcutaneous administration in mice, the nanohybrids distributed to the epidermis and hair follicles. In a psoriasis-like skin mouse model, the actively targeted nanoparticles were superior to free drug and non-targeted nanoparticles in mitigating skin inflammation. Intervention with the targeted nanosystem reduced the epidermal thickness of the psoriasiform lesion from 191 to 42 µm, decreased the Psoriasis Area Severity Index by 74%, restored barrier function, and returned chemokine levels to baseline. Conclusions: Our developed nanosystem was safe and demonstrated efficient targeting properties for the treatment of cutaneous inflammation.
- MeSH
- bicyklické sloučeniny heterocyklické aplikace a dávkování farmakologie MeSH
- buněčné linie keratinocytů HaCaT MeSH
- chemokin CXCL1 účinky léků imunologie MeSH
- chemokin CXCL2 účinky léků imunologie MeSH
- chemotaxe účinky léků MeSH
- desmoglein 3 imunologie MeSH
- epidermis MeSH
- fosfolipidy * MeSH
- imunokonjugáty farmakologie MeSH
- inhibitory fosfodiesterasy 4 aplikace a dávkování farmakologie MeSH
- keratinocyty účinky léků imunologie MeSH
- kopolymer kyseliny glykolové a mléčné * MeSH
- lidé MeSH
- lyzozomy metabolismus ultrastruktura MeSH
- modely nemocí na zvířatech MeSH
- myši MeSH
- nanočástice * MeSH
- neutrofily účinky léků MeSH
- nosiče léků MeSH
- protilátky imunologie MeSH
- psoriáza imunologie patologie MeSH
- sloučeniny boru aplikace a dávkování farmakologie MeSH
- systémy cílené aplikace léků MeSH
- vlasový folikul MeSH
- zánět MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
