Lysosome Fission: Planning for an Exit Saffi, Golam T.; Botelho, Roberto J.
Trends in cell biology,
August 2019, 2019-08-00, 20190801, Letnik:
29, Številka:
8
Journal Article
Recenzirano
Lysosomes are acidic and degradative organelles that receive and digest a plethora of molecular and particulate cargo delivered by endocytosis, autophagy, and phagocytosis. The mechanisms responsible ...for sorting, transporting, and ultimately delivering membranes and cargo to lysosomes through fusion have been intensely investigated. Much less is understood about lysosome fission, which is necessary to balance the incessant flow of cargo into lysosomes and maintain steady-state number, size, and function of lysosomes. Here, we review the emerging picture of how lipid signals, coat and adaptor proteins, and motor-cytoskeletal assemblies drive budding, tubulation, splitting, and ‘kiss-and-run’ events that enable fission and exit from lysosomes and related organelles.
Lysosomes undergo constant fusion–fission cycles, which are important to maintain steady-state lysosome number, size, and function.Lysosome fission proceeds through vesicle formation, membrane tubulation, lysosome splitting, and kiss-and-run.Canonical coat proteins, such as clathrin; adaptor proteins (AP1–5); scission machinery, such as dynamin; and novel proteins, such as spastizin and spatacsin, help mediate fission from lysosomes and related organelles.Phosphoinositide species and Ca2+ may help coordinate the types of fission mechanism that act on lysosomes and related organelles.Overall, lysosome fission, including mechanisms of cargo sorting and targeting, remains a relatively ill-defined process.
Lysosomes are terminal, degradative organelles of the endosomal pathway that undergo repeated fusion-fission cycles with themselves, endosomes, phagosomes, and autophagosomes. Lysosome number and ...size depends on balanced fusion and fission rates. Thus, conditions that favour fusion over fission can reduce lysosome numbers while enlarging their size. Conversely, favouring fission over fusion may cause lysosome fragmentation and increase their numbers. PIKfyve is a phosphoinositide kinase that generates phosphatidylinositol-3,5-bisphosphate to modulate lysosomal functions. PIKfyve inhibition causes an increase in lysosome size and reduction in lysosome number, consistent with lysosome coalescence. This is thought to proceed through reduced lysosome reformation and/or fission after fusion with endosomes or other lysosomes. Previously, we observed that photo-damage during live-cell imaging prevented lysosome coalescence during PIKfyve inhibition. Thus, we postulated that lysosome fusion and/or fission dynamics are affected by reactive oxygen species (ROS). Here, we show that ROS generated by various independent mechanisms all impaired lysosome coalescence during PIKfyve inhibition and promoted lysosome fragmentation during PIKfyve re-activation. However, depending on the ROS species or mode of production, lysosome dynamics were affected distinctly. H2O2 impaired lysosome motility and reduced lysosome fusion with phagosomes, suggesting that H2O2 reduces lysosome fusogenecity. In comparison, inhibitors of oxidative phosphorylation, thiol groups, glutathione, or thioredoxin, did not impair lysosome motility but instead promoted clearance of actin puncta on lysosomes formed during PIKfyve inhibition. Additionally, actin depolymerizing agents prevented lysosome coalescence during PIKfyve inhibition. Thus, we discovered that ROS can generally prevent lysosome coalescence during PIKfyve inhibition using distinct mechanisms depending on the type of ROS.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Lysosomes receive and degrade cargo from endocytosis, phagocytosis and autophagy. They also play an important role in sensing and instructing cells on their metabolic state. The lipid kinase PIKfyve ...generates phosphatidylinositol-3,5-bisphosphate to modulate lysosome function. PIKfyve inhibition leads to impaired degradative capacity, ion dysregulation, abated autophagic flux and a massive enlargement of lysosomes. Collectively, this leads to various physiological defects, including embryonic lethality, neurodegeneration and overt inflammation. The reasons for such drastic lysosome enlargement remain unclear. Here, we examined whether biosynthesis and/or fusion-fission dynamics contribute to swelling. First, we show that PIKfyve inhibition activates TFEB, TFE3 and MITF, enhancing lysosome gene expression. However, this did not augment lysosomal protein levels during acute PIKfyve inhibition, and deletion of TFEB and/or related proteins did not impair lysosome swelling. Instead, PIKfyve inhibition led to fewer but enlarged lysosomes, suggesting that an imbalance favouring lysosome fusion over fission causes lysosome enlargement. Indeed, conditions that abated fusion curtailed lysosome swelling in PIKfyve-inhibited cells.
Objective
To assess the effects of periodontal treatment on endothelial function in patients with coronary artery disease.
Materials and Methods
A randomized controlled trial was conducted with 69 ...patients with stable coronary disease and severe periodontitis. The test group received nonsurgical periodontal therapy consisting of personalized oral hygiene instructions, subgingival scaling, and root planing per quadrant, whereas the control group received equal treatment after the study period. Endothelial function was assessed by measurement of brachial artery flow‐mediated dilation, concentrations of sVCAM‐1, sICAM‐1, and P‐selectin in serum before and 3 months after periodontal therapy.
Results
The test group exhibited statistically better periodontal parameters—plaque, probing depth, periodontal attachment loss, and bleeding on probing. No significant improvements were observed in the control (1.37%) and test (1.39%) groups in flow‐mediated dilation, with no significant between‐group difference. sVCAM‐1 concentration increased in the control group (997.6 ± 384.4–1201.8 ± 412.5; p = 0.03), whereas in the test group, no significant changes were observed (915.1 ± 303.8–1050.3 ± 492.3; p = 0.17), resulting in a significant difference between the two groups (p = 0.04). The same pattern was observed for concentrations of sICAM‐1.
Conclusion
Periodontal treatment did not provide better vasodilation in patients with coronary disease in a short‐term follow‐up period, although it maintained blood concentrations of markers of vascular inflammation.
Aim
To assess the effect of periodontal therapy (PT) on cardiovascular blood biomarkers.
Materials and Methods
This single‐blind, parallel‐design, randomized controlled trial included patients with ...stable coronary artery disease and periodontitis. The test group (TG) received non‐surgical PT, whereas the control group (CG) received one session of plaque removal. Plasma levels of C‐reactive protein (CRP), glycated haemoglobin, lipids and cytokines (IL‐1β, IL‐6, IL‐8, IL‐10, IFN‐γ and TNF‐α) were measured at baseline and after 3 months.
Results
Eighty‐two patients (74.4% women, mean age 59.6 years) were analysed. TG had significantly better periodontal parameters than CG after 3 months, but no significant differences in blood markers were observed between them. In a post hoc subgroup analysis in patients with baseline CRP <3 mg/L, a significant increase in CRP was observed in CG (1.44 ± 0.82 mg/L to 4.35 ± 7.85 mg/L, p = 0.01), whereas CRP remained unchanged in TG (1.40 ± 0.96 mg/L to 1.33 ± 1.26 mg/L, p = 0.85), resulting in a significant difference between groups at 3 months. In patients with CRP ≥3 mg/L, a significant reduction in CRP was observed only in TG (11.3 ± 12.8 mg/L to 5.7 ± 4.1 mg/L, p = 0.04). Levels of IL‐6 and IL‐8 were significantly lower in TG than CG at 3 months.
Conclusions
PT leads to lower levels of CRP, IL‐6 and IL‐8 in cardiovascular patients with high CRP levels.
Vacuolar ATPases (V-ATPases) are multi-subunit ATP-dependent proton pumps necessary for cellular functions, including pH regulation and membrane fusion. The evidence suggests that the V-ATPase ...a-subunit's interaction with the membrane signaling lipid phosphatidylinositol (PIPs) regulates the recruitment of V-ATPase complexes to specific membranes. We generated a homology model of the N-terminal domain of the human a4 isoform (a4NT) using Phyre2.0 and propose a lipid binding domain within the distal lobe of the a4NT. We identified a basic motif, K
IKK
, critical for interaction with phosphoinositides (PIP), and found similar basic residue motifs in all four mammalian and both yeast a-isoforms. We tested PIP binding of wildtype and mutant
NT in vitro. In protein lipid overlay assays, the double mutation K234A/K237A and the autosomal recessive distal renal tubular-causing mutation K237del reduced both PIP binding and association with liposomes enriched with PI(4,5)P
, a PIP enriched within plasma membranes. Circular dichroism spectra of the mutant protein were comparable to wildtype, indicating that mutations affected lipid binding, not protein structure. When expressed in HEK293, wildtype a4NT localized to the plasma membrane in fluorescence microscopy and co-purified with the microsomal membrane fraction in cellular fractionation experiments. a4NT mutants showed reduced membrane association and decreased plasma membrane localization. Depletion of PI(4,5)P
by ionomycin caused reduced membrane association of the WT a4NT protein. Our data suggest that information contained within the soluble a4NT is sufficient for membrane association and that PI(4,5)P
binding capacity is involved in a4 V-ATPase plasma membrane retention.
Lysosome membranes contain diverse phosphoinositide (PtdIns) lipids that coordinate lysosome function and dynamics. The PtdIns repertoire on lysosomes is tightly regulated by the actions of diverse ...PtdIns kinases and phosphatases; however, specific roles for PtdIns in lysosomal functions and dynamics are currently unclear and require further investigation. It was previously shown that PIKfyve, a lipid kinase that synthesizes PtdIns(3,5)P2 from PtdIns(3)P, controls lysosome “fusion-fission” cycle dynamics, autophagosome turnover, and endocytic cargo delivery. Furthermore, INPP4B, a PtdIns 4-phosphatase that hydrolyzes PtdIns(3,4)P2 to form PtdIns(3)P, is emerging as a cancer-associated protein with roles in lysosomal biogenesis and other lysosomal functions. Here, we investigated the consequences of disrupting PIKfyve function in Inpp4b-deficient mouse embryonic fibroblasts. Through confocal fluorescence imaging, we observed the formation of massively enlarged lysosomes, accompanied by exacerbated reduction of endocytic trafficking, disrupted lysosome fusion-fission dynamics, and inhibition of autophagy. Finally, HPLC scintillation quantification of 3H-myo-inositol labeled PtdIns and PtdIns immunofluorescence staining, we observed that lysosomal PtdIns(3)P levels were significantly elevated in Inpp4b-deficient cells due to the hyperactivation of phosphatidylinositol 3-kinase catalytic subunit VPS34 enzymatic activity. In conclusion, our study identifies a novel signaling axis that maintains normal lysosomal homeostasis and dynamics, which includes the catalytic functions of Inpp4b, PIKfyve, and VPS34.
PARPs and the DNA damage response SOUSA, Fabricio G; MATUO, Renata; SOARES, Daniele G ...
Carcinogenesis (New York),
08/2012, Letnik:
33, Številka:
8
Journal Article
Recenzirano
Odprti dostop
Adenosine diphosphate (ADP)-ribosylation is an important posttranslational modification catalyzed by a variety of enzymes, including poly (ADP ribose) polymerases (PARPs), which use nicotinamide ...adenine dinucleotide (NAD(+)) as a substrate to synthesize and transfer ADP-ribose units to acceptor proteins. The PARP family members possess a variety of structural domains, span a wide range of functions and localize to various cellular compartments. Among the molecular actions attributed to PARPs, their role in the DNA damage response (DDR) has been widely documented. In particular, PARPs 1-3 are involved in several cellular processes that respond to DNA lesions, which include DNA damage recognition, signaling and repair as well as local transcriptional blockage, chromatin remodeling and cell death induction. However, how these enzymes are able to participate in such numerous and diverse mechanisms in response to DNA damage is not fully understood. Herein, the DDR functions of PARPs 1-3 and the emerging roles of poly (ADP ribose) polymers in DNA damage are reviewed. The development of PARP inhibitors, their applications and mechanisms of action are also discussed in the context of the DDR.
Abstract
Aim
Non-invasive left ventricular (LV) pressure-strain loops provide a novel method for quantifying myocardial work by incorporating LV pressure in measurements of myocardial deformation. ...Early studies suggest that myocardial work parameters such as Global Constructive Work (GCW) could be useful and reliable in arrhythmia prediction particularly in patients undergoing cardiac resynchronization therapy.
The aim of this study was to evaluate whether the magnitude of GCW was associated with occurrence of ventricular arrhythmias in patients after cardiac resynchronization therapy.
Method and results
Patients on guideline-recommended treatment with a cardiac resynchronization therapy defibrillator (CRT-D) were evaluated by 2D speckle-tracking echocardiography including measurements of GCW at least six months after implantation. The primary outcome was a composite of appropriate defibrillator therapy and sustained ventricular arrhythmia under the monitor zone. A total of 162 patients (mean age 66 years (±10), 122 males (75%)), were included. 16 (10%) patients experienced the primary outcome during a median follow-up of 18 months (IQR: 12-25) after the index echocardiography. Patients with below-median GCW (<1,473 mmHg%) had a hazard ratio for the outcome of 8.14 95% CI: 1.83-36.08, P=0.006 compared to patients above the median in a univariate model and remained an independent predictor after multivariate adjustment for eGFR and QRS duration (hazard ratio 4.75 95% CI: 1.01 – 22.28, P<0.05.
Conclusion
In patients treated with CRT-D, GCW below median level was associated with a 5-fold increase in risk of ventricular arrhythmias.
Lesions with great loss of skin and extensive burns are usually treated with heterologous skin grafts, which may lead rejection. Cell therapy with mesenchymal stem cells is arising as a new proposal ...to accelerate the healing process. We tested a new therapy consisting of sodium carboxymethylcellulose (CMC) as a biomaterial, in combination with adipose-derived stem cells (ADSCs), to treat skin lesions in an in vivo rat model. This biomaterial did not affect membrane viability and induced a small and transient genotoxicity, only at the highest concentration tested (40 mg/mL). In a rat wound model, CMC at 10 mg/mL associated with ADSCs increased the rate of cell proliferation of the granulation tissue and epithelium thickness when compared to untreated lesions (Sham), but did not increase collagen fibers nor alter the overall speed of wound closure. Taken together, the results show that the CMC is capable to allow the growth of ADSCs and is safe for this biological application up to the concentration of 20 mg/mL. These findings suggest that CMC is a promising biomaterial to be used in cell therapy.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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