scholarly journals Acetate and hypertonic stress stimulate organelle membrane fission using distinct phosphatidylinositol signals

2018 ◽  
Author(s):  
Dipti Patel ◽  
Christopher Leonard Brett
Keyword(s):  
Molecular Mechanisms ◽  
Catalytic Domain ◽  
Kinase Inhibitor ◽  
Rab Gtpase ◽  
Hypertonic Stress ◽  
Membrane Fission ◽  
Vacuole Fusion ◽  
Intermediary Metabolite ◽  
Organelle Morphology

ABSTRACTOrganelle morphology reflects an equilibrium between membrane fusion and fission that determines size, shape and copy number. By studying the yeast vacuole as a model, the conserved molecular mechanisms responsible for organelle fusion have been revealed. However, a detailed understanding of vacuole fission and how these opposing processes respond to the cell cycle, osmoregulation or metabolism to change morphology remain elusive. Thus, herein we describe a new fluorometric assay to measure vacuole fission in vitro. For proof-of-concept, we use this assay to confirm that acetate, a key intermediary metabolite, triggers vacuole fission in vitro and show that it also blocks homotypic vacuole fusion. The basis of this effect is distinct from hypertonic stress, a known trigger of fission and inhibitor of fusion that inactivates the Rab-GTPase Ypt7: Treatment with the phosphatidylinositol-kinase inhibitor wortmannin or the catalytic domain of the Rab-GAP (GTPase Activating Protein) Gyp1 reveal that fission can be triggered by Ypt7 inactivation alone in absence of hypertonic stress, placing it upstream of PI-3,5-P2 synthesis and osmosis required for membrane scission. Whereas acetate seems to block PI-4-kinase to possibly increase the pool of PI on vacuole membranes needed to synthesize sufficient PI-3,5-P2 for fission. Thus, we speculate that both PI-4-P and PI-3-P arms of PI-P signaling drive changes in membrane fission and fusion responsible altering vacuole morphology in response to cellular metabolism or osmoregulation.GRAPHICAL ABSTRACT

scholarly journals Flavonoids and Their Anti-Diabetic Effects: Cellular Mechanisms and Effects to Improve Blood Sugar Levels

Biomolecules ◽  
2019 ◽  
Vol 9 (9) ◽  
pp. 430 ◽  
Author(s):  
AL-Ishaq ◽  
Abotaleb ◽  
Kubatka ◽  
Kajo ◽  
Büsselberg
Keyword(s):  
Health Care Providers ◽  
Molecular Mechanisms ◽  
Glucose Transporter ◽  
Kinase Inhibitor ◽  
Current Knowledge ◽  
Care Providers ◽  
Hepatic Enzymes ◽  
Positive Role ◽  
Dietary Flavonoids

Diabetes mellitus (DM) is a prevailing global health metabolic disorder, with an alarming incidence rate and a huge burden on health care providers. DM is characterized by the elevation of blood glucose due either to a defect in insulin synthesis, secretion, binding to receptor, or an increase of insulin resistance. The internal and external factors such as obesity, urbanizations, and genetic mutations could increase the risk of developing DM. Flavonoids are phenolic compounds existing as secondary metabolites in fruits and vegetables as well as fungi. Their structure consists of 15 carbon skeletons and two aromatic rings (A and B) connected by three carbon chains. Flavonoids are furtherly classified into 6 subclasses: flavonols, flavones, flavanones, isoflavones, flavanols, and anthocyanidins. Naturally occurring flavonoids possess anti-diabetic effects. As in vitro and animal model’s studies demonstrate, they have the ability to prevent diabetes and its complications. The aim of this review is to summarize the current knowledge addressing the antidiabetic effects of dietary flavonoids and their underlying molecular mechanisms on selected pathways: Glucose transporter, hepatic enzymes, tyrosine kinase inhibitor, AMPK, PPAR, and NF-κB. Flavonoids improve the pathogenesis of diabetes and its complications through the regulation of glucose metabolism, hepatic enzymes activities, and a lipid profile. Most studies illustrate a positive role of specific dietary flavonoids on diabetes, but the mechanisms of action and the side effects need more clarification. Overall, more research is needed to provide a better understanding of the mechanisms of diabetes treatment using flavonoids.

scholarly journals Proliferating cell nuclear antigen acts as a cytoplasmic platform controlling human neutrophil survival

2010 ◽  
Vol 207 (12) ◽  
pp. 2631-2645 ◽  
Author(s):  
Véronique Witko-Sarsat ◽  
Julie Mocek ◽  
Dikra Bouayad ◽  
Nicola Tamassia ◽  
Jean-Antoine Ribeil ◽  
...  
Keyword(s):  
Proliferating Cell Nuclear Antigen ◽  
Molecular Mechanisms ◽  
Kinase Inhibitor ◽  
Nuclear Antigen ◽  
Neutrophil Apoptosis ◽  
Proliferating Cells ◽  
Rna Transfection ◽  
Neutrophil Survival ◽  
Proliferating Cell

Neutrophil apoptosis is a highly regulated process essential for inflammation resolution, the molecular mechanisms of which are only partially elucidated. In this study, we describe a survival pathway controlled by proliferating cell nuclear antigen (PCNA), a nuclear factor involved in DNA replication and repairing of proliferating cells. We show that mature neutrophils, despite their inability to proliferate, express high levels of PCNA exclusively in their cytosol and constitutively associated with procaspases, presumably to prevent their activation. Notably, cytosolic PCNA abundance decreased during apoptosis, and increased during in vitro and in vivo exposure to the survival factor granulocyte colony-stimulating factor (G-CSF). Peptides derived from the cyclin-dependent kinase inhibitor p21, which compete with procaspases to bind PCNA, triggered neutrophil apoptosis thus demonstrating that specific modification of PCNA protein interactions affects neutrophil survival. Furthermore, PCNA overexpression rendered neutrophil-differentiated PLB985 myeloid cells significantly more resistant to TNF-related apoptosis-inducing ligand– or gliotoxin-induced apoptosis. Conversely, a decrease in PCNA expression after PCNA small interfering RNA transfection sensitized these cells to apoptosis. Finally, a mutation in the PCNA interdomain-connecting loop, the binding site for many partners, significantly decreased the PCNA-mediated antiapoptotic effect. These results identify PCNA as a regulator of neutrophil lifespan, thereby highlighting a novel target to potentially modulate pathological inflammation.

scholarly journals Improved reconstitution of yeast vacuole fusion with physiological SNARE concentrations reveals an asymmetric Rab(GTP) requirement

2016 ◽  
Vol 27 (16) ◽  
pp. 2590-2597 ◽  
Author(s):  
Michael Zick ◽  
William Wickner
Keyword(s):  
Acyl Chain ◽  
Fatty Acyl ◽  
Fatty Acyl Chain ◽  
Rab Gtpase ◽  
Lower Phase ◽  
Yeast Vacuole ◽  
Vacuole Fusion ◽  
In Vitro Reconstitution

In vitro reconstitution of homotypic yeast vacuole fusion from purified components enables detailed study of membrane fusion mechanisms. Current reconstitutions have yet to faithfully replicate the fusion process in at least three respects: 1) The density of SNARE proteins required for fusion in vitro is substantially higher than on the organelle. 2) Substantial lysis accompanies reconstituted fusion. 3) The Rab GTPase Ypt7 is essential in vivo but often dispensable in vitro. Here we report that changes in fatty acyl chain composition dramatically lower the density of SNAREs that are required for fusion. By providing more physiological lipids with a lower phase transition temperature, we achieved efficient fusion with SNARE concentrations as low as on the native organelle. Although fused proteoliposomes became unstable at elevated SNARE concentrations, releasing their content after fusion had occurred, reconstituted proteoliposomes with substantially reduced SNARE concentrations fused without concomitant lysis. The Rab GTPase Ypt7 is essential on both membranes for proteoliposome fusion to occur at these SNARE concentrations. Strikingly, it was only critical for Ypt7 to be GTP loaded on membranes bearing the R-SNARE Nyv1, whereas the bound nucleotide of Ypt7 was irrelevant on membranes bearing the Q-SNAREs Vam3 and Vti1.

scholarly journals The vacuolar kinase Yck3 maintains organelle fragmentation by regulating the HOPS tethering complex

2005 ◽  
Vol 168 (3) ◽  
pp. 401-414 ◽  
Author(s):  
Tracy J. LaGrassa ◽  
Christian Ungermann
Keyword(s):  
Cellular Membrane ◽  
Membrane Dynamics ◽  
Dependent Manner ◽  
Casein Kinase I ◽  
Functional Protein ◽  
Hypertonic Stress ◽  
Functional Studies ◽  
Vacuole Fusion

The regulation of cellular membrane flux is poorly understood. Yeast respond to hypertonic stress by fragmentation of the normally large, low copy vacuole. We used this phenomenon as the basis for an in vivo screen to identify regulators of vacuole membrane dynamics. We report here that maintenance of the fragmented phenotype requires the vacuolar casein kinase I Yck3: when Yck3 is absent, salt-stressed vacuoles undergo fission, but reassemble in a SNARE-dependent manner, suggesting that vacuole fusion is disregulated. Accordingly, when Yck3 is deleted, in vitro vacuole fusion is increased, and Yck3 overexpression blocks fusion. Morphological and functional studies show that Yck3 modulates the Rab/homotypic fusion and vacuole protein sorting complex (HOPS)-dependent tethering stage of vacuole fusion. Intriguingly, Yck3 mediates phosphorylation of the HOPS subunit Vps41, a bi-functional protein involved in both budding and fusion during vacuole biogenesis. Because Yck3 also promotes efficient vacuole inheritance, we propose that tethering complex phosphorylation is a part of a general, switch-like mechanism for driving changes in organelle architecture.

scholarly journals Programmable N6-methyladenosine modification of CDCP1 mRNA by RCas9-methyltransferase like 3 conjugates promotes bladder cancer development

2020 ◽  
Vol 19 (1) ◽  
Author(s):  
Xiaoling Ying ◽  
Xu Jiang ◽  
Haiqing Zhang ◽  
Bixia Liu ◽  
Yapeng Huang ◽  
...  
Keyword(s):  
Bladder Cancer ◽  
Molecular Mechanisms ◽  
Catalytic Domain ◽  
Mrna Translation ◽  
Guide Rna ◽  
Cub Domain ◽  
Potential Strategy ◽  
Development In Vitro

AbstractAccumulating evidence has revealed significant roles for N6-methyladenosine (m 6 A) modification in the development of various cancers. We previously demonstrated an oncogenic role of m 6 A-modified CUB domain containing protein 1 (CDCP1) in bladder cancer (BC) progression. However, the biological functions and underlying molecular mechanisms of engineered programmable m 6 A modification of CDCP1 mRNA in BC remain obscure. Here, we established a targeted m 6 A RNA methylation system by fusing the catalytic domain of methyltransferase like 3 (METTL3CD) to RCas9 as the RNA-targeting module. The constructed RCas9- METTL3 retained methylation activity and mediated efficient site-specific m 6 A installation in the presence of a cognate single guide RNA and short protospacer adjacent motif-containing ssDNA molecule . Subsequently, targeting m 6 A installation onto the 3′ untranslated region of CDCP1 promoted CDCP1 mRNA translation and facilitated BC development in vitro and in vivo. Our findings demonstrate that the RCas9-METTL3 system mediates efficient sitespecific m 6 A installation on CDCP1 mRNA and promotes BC development. Thus, the RCas9-METTL3 system provides a new tool for studying m 6 A function and a potential strategy for BC epitranscriptome-modulating therapies.

Lycorine Modulates the Expression of p21 Via a p53-Independent Pathway in HL-60 Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4297-4297
Author(s):  
Jing Liu ◽  
Shu-Ling Wang ◽  
Lin Fang ◽  
Mao Ye ◽  
Zhi-Wei Sun ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
Fold Increase ◽  
Alternative Pathways ◽  
Cyclin Dependent Kinase Inhibitor ◽  
M Phase ◽  
Induced Apoptosis

Abstract Abstract 4297 Leukemia is one of the most life-threatening cancers today, and acute promyelogenous leukemia is a common type of leukemia. We have previously shown that lycorine, a natural alkaloid extract from Amaryllidaceae, exhibited anti-leukemia effects in vitro and in vivo. Lycorine treatment of HL-60 cell arrested cell cycle at G2/M phase and induced apoptosis. In the present study, we sought to explore the molecular mechanisms for the anti-leukemia action of lycorine. Gene chip analysis revealed that lycorine treatment of HL-60 cells induced more than 9 fold increase of p21, a cyclin-dependent kinase inhibitor, whose expression is mainly regulated by p53. Since HL-60 cells are p53 null, the above findings suggest that lycorine activates p21 expression through p53-independent pathway. To further explore the alternative pathways for the activation of p21 induced by lycorine, we examined the effect of lycorine on the expression of Rb, pRb, E2F, c-Myc and HDACs which have shown to regulate p21 expression. We show that expression of pRb (ser780) and c-Myc was down-regulated, Rb and E2F were up-regulated, while the expression of HDAC1 and HDAC3 was not changed. Together these findings suggest that lycorine exerts its anti-leukemia effect by activating p21 expression via pRb/E2F and c-Myc pathways. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Stem cell factor affects fate determination of human gonocytes in vitro

Reproduction ◽  
2007 ◽  
Vol 134 (6) ◽  
pp. 757-765 ◽  
Author(s):  
Jiongjiong Tu ◽  
Liqing Fan ◽  
Ke Tao ◽  
Wenbing Zhu ◽  
Jianjun Li ◽  
...  
Keyword(s):  
Stem Cell ◽  
Stem Cell Factor ◽  
Molecular Mechanisms ◽  
Kinase Inhibitor ◽  
Mitogen Activated Protein Kinase ◽  
Tyrosine Kinase Receptor ◽  
Dependent Manner ◽  
Fate Determination

The stem cell factor (SCF), binding its tyrosine kinase receptor c-Kit, has been shown to play essential roles in the proliferation, differentiation, and survival of germline cells. However, few reports are available about the effect of SCF on the development of human gonocytes within the fetal testis. The objective of this study was to investigate whether SCF affects the biological behaviors of human gonocytes before or after they enter the mitotic arrest stage. Employing an organ culture system, we observed that addition of exogenous SCF could influence the morphology of human gonocytesin vitro. Moreover, SCF was able to trigger the colony formation of round gonocytes, which were characterized positive for alkaline phosphatase activity, Oct-4, SSEA-4, and c-Kit as well. We found that SCF exerted actions in a dose- and age-dependent manner, although the stimulatory effect lasted no more than 14 days. We also showed that SCF played a role in suppressing the apoptosis of human gonocytes. Blocking of SCF signaling with either phosphatidylinositol 3-kinase or mitogen-activated protein kinase inhibitor resulted in similar apoptotic features as well as the SCF-withdrawal cultures. Taken together, we report that SCF acts as a potent regulator in the fate determination of human gonocytes. Our studies should form the basis forin vitrostudies and facilitate investigation of the molecular mechanisms underlying this unique stage.

scholarly journals Cytomegalovirus UL97 Kinase Catalytic Domain Mutations That Confer Multidrug Resistance

2013 ◽  
Vol 57 (7) ◽  
pp. 3375-3379 ◽  
Author(s):  
Sunwen Chou ◽  
Ronald J. Ercolani ◽  
Gail Marousek ◽  
Terry L. Bowlin
Keyword(s):  
Multidrug Resistance ◽  
Catalytic Domain ◽  
Kinase Inhibitor ◽  
Growth Defect ◽  
Clinical Specimens ◽  
Kinase Catalytic Domain ◽  
Severe Growth Defect ◽  
Ganciclovir Resistance ◽  
High Level

ABSTRACTHuman cytomegalovirus UL97 kinase mutations that commonly confer ganciclovir resistance cluster in different parts of the gene than those conferring resistance to maribavir, an experimental UL97 kinase inhibitor. The drug resistance, growth, and autophosphorylation phenotypes of several unusual UL97 mutations in the kinase catalytic domain were characterized. Mutations V466G and P521L, described in clinical specimens from ganciclovir-treated subjects, conferred a UL97 kinase knockout phenotype with no autophosphorylation, a severe growth defect, and high-level ganciclovir, cyclopropavir, and maribavir resistance, similar to mutations at the catalytic lysine residue K355. Mutations F342S and V356G, observed after propagation under cyclopropavirin vitro, showed much less growth attenuation and moderate- to high-level resistance to all three drugs while maintaining UL97 autophosphorylation competence and normal cytopathic effect in cell culture, a novel phenotype. F342S is located in the ATP-binding P-loop and is homologous to a c-Abl kinase mutation conferring resistance to imatinib. UL97 mutants with relatively preserved growth fitness and multidrug resistance are of greater concern in antiviral therapy than the severely growth-impaired UL97 knockout mutants. Current diagnostic genotyping assays are unlikely to detect F342S and V356G, and the frequency of their appearance in clinical specimens remains undefined.

scholarly journals Inhibition of MELK Protooncogene as an Innovative Treatment for Intrahepatic Cholangiocarcinoma

Medicina ◽  
2019 ◽  
Vol 56 (1) ◽  
pp. 1 ◽  
Author(s):  
Antonio Cigliano ◽  
Maria Giulia Pilo ◽  
Marta Mela ◽  
Silvia Ribback ◽  
Frank Dombrowski ◽  
...  
Keyword(s):  
Cell Lines ◽  
Intrahepatic Cholangiocarcinoma ◽  
Leucine Zipper ◽  
Molecular Mechanisms ◽  
Kinase Inhibitor ◽  
Prognostic Indicator ◽  
Dependent Manner ◽  
Downstream Target ◽  
Forkhead Box M1

Background and Objectives: Intrahepatic cholangiocarcinoma (iCCA) is a pernicious tumor characterized by a dismal outcome and scarce therapeutic options. To substantially improve the prognosis of iCCA patients, a better understanding of the molecular mechanisms responsible for development and progression of this disease is imperative. In the present study, we aimed at elucidating the role of the maternal embryonic leucine zipper kinase (MELK) protooncogene in iCCA. Materials and Methods: We analyzed the expression of MELK and two putative targets, Forkhead Box M1 (FOXM1) and Enhancer of Zeste Homolog 2 (EZH2), in a collection of human iCCA by real-time RT-PCR and immunohistochemistry (IHC). The effects on iCCA growth of both the multi-kinase inhibitor OTSSP167 and specific small-interfering RNA (siRNA) against MELK were investigated in iCCA cell lines. Results: Expression of MELK was significantly higher in tumors than in corresponding non-neoplastic liver counterparts, with highest levels of MELK being associated with patients’ shorter survival length. In vitro, OTSSP167 suppressed the growth of iCCA cell lines in a dose-dependent manner by reducing proliferation and inducing apoptosis. These effects were amplified when OTSSP167 administration was coupled to the DNA-damaging agent doxorubicin. Similar results, but less remarkable, were obtained when MELK was silenced by specific siRNA in the same cells. At the molecular level, siRNA against MELK triggered downregulation of MELK and its targets. Finally, we found that MELK is a downstream target of the E2F1 transcription factor. Conclusion: Our results indicate that MELK is ubiquitously overexpressed in iCCA, where it may represent a prognostic indicator and a therapeutic target. In particular, the combination of OTSSP167 (or other, more specific MELK inhibitors) with DNA-damaging agents might be a potentially effective therapy for human iCCA.

scholarly journals Expression of Leptin Receptor and Effects of Leptin on Papillary Thyroid Carcinoma Cells

2019 ◽  
Vol 2019 ◽  
pp. 1-6 ◽  
Author(s):  
Marilena Celano ◽  
Valentina Maggisano ◽  
Saverio Massimo Lepore ◽  
Marialuisa Sponziello ◽  
Valeria Pecce ◽  
...  
Keyword(s):  
Thyroid Cancer ◽  
Cell Lines ◽  
Molecular Mechanisms ◽  
Leptin Receptor ◽  
Kinase Inhibitor ◽  
Immunoblot Analysis ◽  
Prolonged Treatment ◽  
Papillary Thyroid

Background. Obesity has been hypothesized to contribute to the aggressiveness of thyroid cancer through the production of abnormal levels of serum adipokines. Leptin receptor (OB-R) expression has also been documented in papillary thyroid cancer (PTC). Aim. In this translational study, we analyzed in vitro the effects of leptin on the growth and migration of thyroid cancer cells (TPC-1 and K1), the molecular mechanisms underlying leptin’s action, and the influence of prolonged leptin exposure on cell response to a protein kinase inhibitor lenvatinib. The expression levels of OB-R mRNA and protein were also investigated in vivo in a series of aggressive PTCs divided into two groups based on the presence of the BRAF mutation. Results. In TPC-1 and K1 cells, prolonged treatment with leptin (500 ng/ml for 96 h) resulted in a mild increase in the proliferation (about 20% over control only in K1 cells, p<0.05) and in the migration of both cancer cell lines. Immunoblot analysis revealed a slight increase in the phosphorylation of AKT, but no effect on β-catenin and phospho-ERK expressions. The inhibitory effects of lenvatinib on the viability of both cell lines were not influenced by the leptin treatment. OB-R transcript (in fresh tissues) and proteins (in formalin-fixed and paraffin-embedded specimens) were expressed in all PTC tissues examined, with no significant differences between BRAF-mutated and BRAF-wild-type tumors. Conclusions. These results demonstrate leptin’s role in mildly increasing the aggressive phenotype of PTC cells but without influencing the action of lenvatinib. Further studies will clarify whether it is possible to target OB-R, expressed in all aggressive PTCs, as an adjuvant treatment approach for these malignancies.

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