scholarly journals Ablation of Acid Ceramidase Impairs Autophagy and Mitochondria Activity in Melanoma Cells

2021 ◽  
Vol 22 (6) ◽  
pp. 3247
Author(s):  
Michele Lai ◽  
Veronica La Rocca ◽  
Rachele Amato ◽  
Giulia Freer ◽  
Mario Costa ◽  
...  
Keyword(s):  
Melanoma Cells ◽  
Mitochondrial Activity ◽  
High Plasticity ◽  
Wild Type ◽  
Acid Ceramidase ◽  
Over Expression ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
Total Starvation

Cutaneous melanoma is often resistant to therapy due to its high plasticity, as well as its ability to metabolise chemotherapeutic drugs. Sphingolipid signalling plays a pivotal role in its progression and metastasis. One of the ways melanoma alters sphingolipid rheostat is via over-expression of lysosomal acid ceramidase (AC), which catalyses the hydrolysis of pro-apoptotic long-chain ceramides into sphingosine and fatty acid. In this report, we examine the role of acid ceramidase in maintaining cellular homeostasis through the regulation of autophagy and mitochondrial activity in melanoma cell lines. We show that under baseline conditions, wild-type melanoma cells had 3-fold higher levels of the autophagy marker, microtubule-associated proteins 1A/1B light chain 3B (LC3 II), compared to AC-null cells. This difference was further magnified after cell starvation. Moreover, we noticed autophagy impairment in A375 AC-null cells, possibly due to local accumulation of non-metabolized ceramides. Nonetheless, we observed that AC-null cells exhibited a significant increase in mitochondrial membrane potential compared to control cells. Consistent with this observation, we found that, after total starvation, ~30% of AC-null cells undergo apoptosis compared to ~6% of wild-type cells. As expected, AC transfection restored viability in A375 AC-null cells. Together, these findings suggest that AC-null melanoma cells change and adapt their metabolism to survive in the absence of AC, although in a way that does not allow them to cope with the stress of nutrient deprivation.

scholarly journals KIF18A's neck linker permits navigation of microtubule-bound obstacles within the mitotic spindle

2019 ◽  
Vol 2 (1) ◽  
pp. e201800169 ◽  
Author(s):  
Heidi LH Malaby ◽  
Dominique V Lessard ◽  
Christopher L Berger ◽  
Jason Stumpff
Keyword(s):  
Single Molecule ◽  
Mitotic Spindle ◽  
Binding Proteins ◽  
Mitotic Chromosome ◽  
Chromosome Movement ◽  
Wild Type ◽  
Microtubule Associated Proteins ◽  
Chromosome Alignment ◽  
Associated Proteins ◽  
Fiber Dynamics

KIF18A (kinesin-8) is required for mammalian mitotic chromosome alignment. KIF18A confines chromosome movement to the mitotic spindle equator by accumulating at the plus-ends of kinetochore microtubule bundles (K-fibers), where it functions to suppress K-fiber dynamics. It is not understood how the motor accumulates at K-fiber plus-ends, a difficult feat requiring the motor to navigate protein dense microtubule tracks. Our data indicate that KIF18A's relatively long neck linker is required for the motor's accumulation at K-fiber plus-ends. Shorter neck linker (sNL) variants of KIF18A display a deficiency in accumulation at the ends of K-fibers at the center of the spindle. Depletion of K-fiber–binding proteins reduces the KIF18A sNL localization defect, whereas their overexpression reduces wild-type KIF18A's ability to accumulate on this same K-fiber subset. Furthermore, single-molecule assays indicate that KIF18A sNL motors are less proficient in navigating microtubules coated with microtubule-associated proteins. Taken together, these results support a model in which KIF18A's neck linker length permits efficient navigation of obstacles to reach K-fiber ends during mitosis.

Role of HFE in Hepatic Iron Overload: Novel Findings Demonstrate Direct Effects of Mutant C282Y on Hepcidin and Calreticulin mRNA Expression.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1549-1549
Author(s):  
Jorge P. Pinto ◽  
Pedro Ramos ◽  
Sergio de Almeida ◽  
Susana Oliveira ◽  
Laura Breda ◽  
...  
Keyword(s):  
Protective Effect ◽  
Cell Line ◽  
Mononuclear Cells ◽  
Hepatic Cell ◽  
Wild Type ◽  
Peripheral Blood Mononuclear ◽  
Mutant Proteins ◽  
Over Expression ◽  
Blood Mononuclear Cells

Abstract Studies done in non-hepatic cell lines, focusing on the interaction between HFE with TFR1 and β-2M proved insufficient to explain the discrepancies found in the clinical penetrance of hemochromatosis in subjects carrying the C282Y mutation. Our first goal was to investigate the role of HFE wild type (wt) and mutant proteins (C282Y and H63D) in a human hepatic cell line, focusing on the cellular localization and interaction of HFE with the expression of other iron related proteins. HFE mutant C282Y was found to be retained in the endoplasmic reticulum (ER). Thus, in addition, we investigated the effect of HFE wt and mutant proteins on Calreticulin, which is a chaperon protein that responds to ER stress and has a protective effect on oxidative damage in some cell lines. Here we report setting up a stable transfection of wt- and mutant-HFE in a hepatic cell line (HepG2) and examine the intracellular distribution of wt- and HFE mutants, their effect on iron intake independently of TFR1 and on the expression of other iron and ER stress response genes, namely Hepcidin and Calreticulin. In addition, we validated some of the novel effects of HFE on Calreticulin using peripheral blood mononuclear cells from HFE patients. The localization of the HFE variants was analyzed using KDEL and Golgin-97 as ER and the Golgi complex markers, respectively. HFE C282Y shows a high degree of overlap with the ER markers, confirming a retention of this variant in this organelle. Over-expression of the HFE wt impaired the intake of 55Fe relatively to transfected control cells (P<0.008) independently of TFR1, as demonstrated by RNAi silencing. Hamp RNA expression was decreased in cells over expressing C282Y in comparison to HFE wt cells (P<0.011). Finally over-expression of HFE wt decreases Calreticulin mRNA, whereas the C282Y had an opposite effect, compared to the control cell line. A similar result was observed in peripheral blood mononuclear cells (PMBC) of C282Y homozygous HFE patients, compared to wild type blood donors (P<0.006). Interestingly, this data suggest that synthesis of the HFE mutant C282Y triggers a protective effect on oxidative damage mediated by Calreticulin. In fact, HepG2 cells over-expressing C282Y showed lower levels of ROS than HFE wt (P<0.004). This observation might contribute to explain some of the discrepancies seen in the clinical penetrance of the disease in C282Y carrying subjects. The direct effect of the mutant HFE C282Y on mRNA expression of hepcidin also demonstrated here for the first time corroborates and provides a molecular basis for earlier reports of low hepcidin levels in HH patients and in Hfe-KO mice.

Tumor Suppressor Microrna-29a/b and Microrna-34a Mediate Small Molecule PRIMA-1Met-Induced Aoptosis In Multiple Myeloma Cells By Targeting c-Myc

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1919-1919
Author(s):  
Manujendra N. Saha ◽  
Yijun Yang ◽  
Hong Chang
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Mutant P53 ◽  
Pcr Array ◽  
Wild Type ◽  
Down Regulation ◽  
Over Expression ◽  
P53 Status ◽  
Low Expression

Abstract PRIMA-1Met/APR246 (p53 reactivation and induction of massive apoptosis), is a small molecule with remarkable anti-tumor activities in various human tumor cells, and is currently under phase I/II clinical trial. We have previously demonstrated anti-tumor activity of PRIMA-1Met in multiple myeloma (MM) cells irrespective of p53 status. In addition, we have shown that PRIMA-1Met alone or in combination with dexamethasone triggers significant tumor growth inhibition in vivo in a murine xenograft model of human MM. However, the molecular mechanism underlying anti-myeloma activity of PRIMA-1Met has not been fully elucidated. MicroRNAs (miRNAs) are non-coding small RNA molecules that regulate post-transcriptional gene expression and play a critical role in tumor pathogenesis. Since the role of miRNAs and their regulation in response to PRIMA-1Met in MM is not known, here we investigated the relationship between PRIMA-1Met-induced apoptosis and miRNA expression in MM cells. Using a miRNA PCR array platform (Human Cancer Pathway Finder miScript miRNA PCR array, MIHS-102Z, Qiagen Inc), we analyzed the miRNA profiles in two MM cell lines of different p53 status (MM.1S with wild type p53 and 8226 with mutant p53) treated with either PRIMA-1Met or DMSO control. After normalization to a set of housekeeping genes, differential expressions of the miRNAs were analysed. miRNA-29a, miRNA-29b, and miRNA-34a were found significantly up-regulated (more than 2 fold, p<0.05) in cells treated with PRIMA-1Met compared to DMSO-treated cells. To evaluate the effect of over-expression of these miRNAs, we transfected two MM cell lines (MM.1S and 8226) with either miR-29a/b or miR-34a. Cells transfected with scramble miRNA were used as control. Over-expression of the miRNAs resulted in a dose-dependent inhibition of viability and increase in apoptosis of MM.1S or 8226 cells. Next, we examined the endogenous expression of these miRNAs in 5 primary MM samples by qPCR. Results showed a significant low expression of miR-29a/b and miR-34a in 3 of the 5 samples. Treatment of the two primary MM samples with low expression for miR-29a/b and miR-34a with PRIMA-1Met resulted in up-regulation of these miRNAs leading to inhibition of the viability and induction of apoptosis. To identify the possible targets of these miRNAs, we performed bioinformatics analysis. Results obtained from different searches by miRanda and TargetScan algorithm predicted c-Myc as a potential target for miRNA-29a/b and miRNA-34a. c-Myc is an oncogene whose over-expression has been associated with resistance to current chemotherapy in MM. Global gene expression profiling by microarray showed significant down-regulation of c-Myc in two MM cell lines with either wild type or mutant p53 treated with PRIMA-1Met compare to cells treated with DMSO. Importantly, down-regulation of c-Myc (∼2.6-fold) by PRIMA-1Met was also observed in a MM cell line (8226R5) lacking p53 expression suggesting an important role of c-Myc in p53-independent apoptosis of MM cells induced by PRIMA-1Met. By qPCR and Western blot analysis, we confirmed significant down-regulation of c-Myc in PRIMA-1Met-treated MM cells. These data provided the evidence for an inverse correlation between the expression of these miRNAs and c-Myc indicating that apoptosis of MM cells induced by PRIMA-1Met is regulated by miRNAs29a/b or miRNA34a targeting c-Myc. Our results suggest a novel mechanism for PRIMA-1Met-induced apoptotic signaling in MM cells mediated by up-regulation of miR-29a/b and miR-34a targeting c-Myc. Our findings also provide a preclinical framework for development of therapeutic strategies in combination of PRIMA-1Met and miRNA (miR-29a/b or miR-34a) mimics for the treatment of MM patients, especially for those with high c-Myc expressions. Disclosures: No relevant conflicts of interest to declare.

scholarly journals RYR2 Proteins Contribute to the Formation of Ca2+ Sparks in Smooth Muscle

2004 ◽  
Vol 123 (4) ◽  
pp. 377-386 ◽  
Author(s):  
Guangju Ji ◽  
Morris E. Feldman ◽  
Kai Su Greene ◽  
Vincenzo Sorrentino ◽  
Hong-Bo Xin ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Calcium Release ◽  
Ryanodine Receptors ◽  
Wild Type ◽  
Calcium Dependent ◽  
Outward Currents ◽  
Associated Proteins ◽  
Kinetics Of ◽  
Spontaneous Transient Outward Currents

Calcium release through ryanodine receptors (RYR) activates calcium-dependent membrane conductances and plays an important role in excitation-contraction coupling in smooth muscle. The specific RYR isoforms associated with this release in smooth muscle, and the role of RYR-associated proteins such as FK506 binding proteins (FKBPs), has not been clearly established, however. FKBP12.6 proteins interact with RYR2 Ca2+ release channels and the absence of these proteins predictably alters the amplitude and kinetics of RYR2 unitary Ca2+ release events (Ca2+ sparks). To evaluate the role of specific RYR2 and FBKP12.6 proteins in Ca2+ release processes in smooth muscle, we compared spontaneous transient outward currents (STOCs), Ca2+ sparks, Ca2+-induced Ca2+ release, and Ca2+ waves in smooth muscle cells freshly isolated from wild-type, FKBP12.6−/−, and RYR3−/− mouse bladders. Consistent with a role of FKBP12.6 and RYR2 proteins in spontaneous Ca2+ sparks, we show that the frequency, amplitude, and kinetics of spontaneous, transient outward currents (STOCs) and spontaneous Ca2+ sparks are altered in FKBP12.6 deficient myocytes relative to wild-type and RYR3 null cells, which were not significantly different from each other. Ca2+ -induced Ca2+ release was similarly augmented in FKBP12.6−/−, but not in RYR3 null cells relative to wild-type. Finally, Ca2+ wave speed evoked by CICR was not different in RYR3 cells relative to control, indicating that these proteins are not necessary for normal Ca2+ wave propagation. The effect of FKBP12.6 deletion on the frequency, amplitude, and kinetics of spontaneous and evoked Ca2+ sparks in smooth muscle, and the finding of normal Ca2+ sparks and CICR in RYR3 null mice, indicate that Ca2+ release through RYR2 molecules contributes to the formation of spontaneous and evoked Ca2+ sparks, and associated STOCs, in smooth muscle.

scholarly journals A Crucial Role of Mitochondrial Dynamics in Dehydration Resistance in Saccharomyces cerevisiae

2021 ◽  
Vol 22 (9) ◽  
pp. 4607
Author(s):  
Chang-Lin Chen ◽  
Ying-Chieh Chen ◽  
Wei-Ling Huang ◽  
Steven Lin ◽  
Rimantas Daugelavičius ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Survival Rate ◽  
Mitochondrial Dynamics ◽  
Growth Conditions ◽  
Stationary Growth Phase ◽  
Yeast Cells ◽  
Mitochondrial Activity ◽  
Wild Type ◽  
Stationary Growth

Mitochondria are dynamic organelles as they continuously undergo fission and fusion. These dynamic processes conduct not only mitochondrial network morphology but also activity regulation and quality control. Saccharomyces cerevisiae has a remarkable capacity to resist stress from dehydration/rehydration. Although mitochondria are noted for their role in desiccation tolerance, the mechanisms underlying these processes remains obscure. Here, we report that yeast cells that went through stationary growth phase have a better survival rate after dehydration/rehydration. Dynamic defective yeast cells with reduced mitochondrial genome cannot maintain the mitochondrial activity and survival rate of wild type cells. Our results demonstrate that yeast cells balance mitochondrial fusion and fission according to growth conditions, and the ability to adjust dynamic behavior aids the dehydration resistance by preserving mitochondria.

scholarly journals Cardiac-Specific PID1 Overexpression Enhances Pressure Overload-Induced Cardiac Hypertrophy in Mice

2015 ◽  
Vol 35 (5) ◽  
pp. 1975-1985 ◽  
Author(s):  
Yaoqiu Liu ◽  
Yahui Shen ◽  
Jingai Zhu ◽  
Ming Liu ◽  
Xing Li ◽  
...  
Keyword(s):  
Cardiac Hypertrophy ◽  
Mapk Pathway ◽  
Pressure Overload ◽  
Wild Type Mouse ◽  
Heart Tissue ◽  
Wild Type ◽  
Heart Transplants ◽  
Over Expression

Background/Aims: PID1 was originally described as an insulin sensitivity relevance protein, which is also highly expressed in heart tissue. However, its function in the heart is still to be elucidated. Thus this study aimed to investigate the role of PID1 in the heart in response to hypertrophic stimuli. Methods: Samples of human failing hearts from the left ventricles of dilated cardiomyopathy (DCM) patients undergoing heart transplants were collected. Transgenic mice with cardiomyocyte-specific overexpression of PID1 were generated, and cardiac hypertrophy was induced by transverse aortic constriction (TAC). The extent of cardiac hypertrophy was evaluated by echocardiography as well as pathological and molecular analyses of heart samples. Results: A significant increase in PID1 expression was observed in failing human hearts and TAC-treated wild-type mouse hearts. When compared with TAC-treated wild-type mouse hearts, PID1-TG mouse showed a significant exacerbation of cardiac hypertrophy, fibrosis, and dysfunction. Further analysis of the signaling pathway in vivo suggested that these adverse effects of PID1 were associated with the inhibition of AKT, and activation of MAPK pathway. Conclusion: Under pathological conditions, over-expression of PID1 promotes cardiac hypertrophy by regulating the Akt and MAPK pathway.

scholarly journals Role of non-motile microtubule-associated proteins in virus trafficking

2016 ◽  
Vol 7 (5-6) ◽  
pp. 283-292 ◽  
Author(s):  
Débora M. Portilho ◽  
Roger Persson ◽  
Nathalie Arhel
Keyword(s):  
Motor Proteins ◽  
Current Knowledge ◽  
Cell Entry ◽  
Cellular Transport ◽  
Microtubule Associated Proteins ◽  
Virus Trafficking ◽  
Cytoskeletal Dynamics ◽  
Associated Proteins ◽  
Infected Cells

AbstractViruses are entirely dependent on their ability to infect a host cell in order to replicate. To reach their site of replication as rapidly and efficiently as possible following cell entry, many have evolved elaborate mechanisms to hijack the cellular transport machinery to propel themselves across the cytoplasm. Long-range movements have been shown to involve motor proteins along microtubules (MTs) and direct interactions between viral proteins and dynein and/or kinesin motors have been well described. Although less well-characterized, it is also becoming increasingly clear that non-motile microtubule-associated proteins (MAPs), including structural MAPs of the MAP1 and MAP2 families, and microtubule plus-end tracking proteins (+TIPs), can also promote viral trafficking in infected cells, by mediating interaction of viruses with filaments and/or motor proteins, and modulating filament stability. Here we review our current knowledge on non-motile MAPs, their role in the regulation of cytoskeletal dynamics and in viral trafficking during the early steps of infection.

scholarly journals Claisened Hexafluoro Inhibits Metastatic Spreading of Amoeboid Melanoma Cells

Cancers ◽  
2021 ◽  
Vol 13 (14) ◽  
pp. 3551
Author(s):  
Angela Leo ◽  
Erica Pranzini ◽  
Laura Pietrovito ◽  
Elisa Pardella ◽  
Matteo Parri ◽  
...  
Keyword(s):  
Metastatic Melanoma ◽  
Melanoma Cells ◽  
Mitochondrial Activity ◽  
High Plasticity ◽  
Free Survival ◽  
Ampk Signaling ◽  
Amoeboid Motility ◽  
Metastatic Process ◽  
Reported Data

Metastatic melanoma is characterized by poor prognosis and a low free-survival rate. Thanks to their high plasticity, melanoma cells are able to migrate exploiting different cell motility strategies, such as the rounded/amoeboid-type motility and the elongated/mesenchymal-type motility. In particular, the amoeboid motility strongly contributes to the dissemination of highly invasive melanoma cells and no treatment targeting this process is currently available for clinical application. Here, we tested Claisened Hexafluoro as a novel inhibitor of the amoeboid motility. Reported data demonstrate that Claisened Hexafluoro specifically inhibits melanoma cells moving through amoeboid motility by deregulating mitochondrial activity and activating the AMPK signaling. Moreover, Claisened Hexafluoro is able to interfere with the adhesion abilities and the stemness features of melanoma cells, thus decreasing the in vivo metastatic process. This evidence may contribute to pave the way for future possible therapeutic applications of Claisened Hexafluoro to counteract metastatic melanoma dissemination.

scholarly journals MAP7 recruits kinesin-1 to microtubules to direct organelle transport

2019 ◽  
Author(s):  
Abdullah R. Chaudhary ◽  
Hailong Lu ◽  
Elena B. Krementsova ◽  
Carol S. Bookwalter ◽  
Kathleen M. Trybus ◽  
...  
Keyword(s):  
Single Molecule ◽  
Organelle Transport ◽  
Microtubule Associated Proteins ◽  
Microtubule Polymerization ◽  
Binding Rate ◽  
Associated Proteins ◽  
Bidirectional Transport ◽  
Directed Motility ◽  
Clear Shift

Microtubule-associated proteins (MAPs) play well-characterized roles in regulating microtubule polymerization, dynamics, and organization. In addition, MAPs control trans-port along microtubules by regulating the motility of kinesin and dynein. MAP7 (ensconsin, E-MAP-115) is a ubiquitous MAP that organizes the microtubule cytoskeleton in mitosis and neuronal branching. MAP7 also promotes the interaction of kinesin-1 with microtubules. We expressed and purified full-length kinesin-1 and MAP7 in Sf9 cells. In single-molecule motiity assays, MAP7 recruits kinesin-1 to microtubules, increasing the frequency of both diffusive and processive runs. Optical trapping assays on beads transported by single and teams of kinesin-1 motors indicate that MAP7 increases the relative binding rate of kinesin-1 and the number of motors simultaneously engaged in ensembles. To examine the role of MAP7 in regulating bidirectional transport, we isolated late phagosomes along with their native set of kinesin-1, kinesin-2, and dynein motors. Bidirectional cargoes exhibit a clear shift towards plus-end directed motility on MAP7-decorated microtubules due to increased forces exerted by kinesin teams. Collectively, our results indicate that MAP7 enhances kinesin-1 recruitment to microtubules and targets organelle transport to the plus end.

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