Phospholipid Phosphatidylserine Research Articles

Cofilin-1 Activation in Stored Platelets

BackgroundPlatelets, harvested for transfusion, have a relatively short “shelf life” of 5 days. With increasing storage, platelets develop structural and functional changes called “platelet storage lesions”. These changes result not only in reduced hemostatic response to agonists but also in an accelerated clearance from the circulation in vivo. The precise mechanisms of clearance of platelets aged in vitro (or vivo) are not known. There is an increased exposure of the anionic phospholipid phosphatidylserine (PS) on the cell surface of stored platelets. PS is a well-known tag for clearance of cells from the circulation by the macrophage PS receptors. PS-expressing stored platelets are also rapidly cleared from the circulation, presumably through the similar mechanism. Targeting this pathway could improve the recovery of stored platelets following transfusion by inhibiting storage-induced PS exposureResults and DiscussionActin is a major PS binding proteins in platelets. In resting platelets, actin filaments are at the periphery and in close proximity to the membrane. We hypothesize that the interaction with actin filaments constrains the mobility of PS in the inner leaflet. We generated artificial bilamellar liposomes mimicking the intra (PS) or extra (PC) cellular phospholipid composition of the membrane bilayer. When these liposomes were incubated with platelet lysate, the most striking enrichment was seen in actin, which was increased 5-fold in PS-containing liposome compared to control PC liposomes. These data suggest that actin and actin-associated proteins may, in fact, be the major inner leaflet PS-binding proteins in resting platelets.Cortical actin retracts from the membrane with PS exposure in senescent platelets: We examined the morphological changes in resting fresh platelets, five-day-old platelets and ABT-737 (10 μM) treated platelets by immunofluorescence microscopy. Platelets immobilized on polylysine-coated plates were immunostained with FITC-lactadherin and Alexa- 647-phalloidin. As shown in Figure 1, resting platelets do not express PS (no or minimal FITC-lactadherin staining). Actin filaments (Alexa-647-phalloidin) are distributed in the subcortical region as a thin submembranous network. In 6-day old stored platelets, there is significant increase in platelet surface FITC-lactadherin staining. In addition, in most platelets actin filaments have retracted from the subcortical region towards the center of the platelet (compare figure 1 panels A and BBad phosphorylation is significantly diminished in stored platelets. Phosphorylated Bad is sequestered in the cytosol by binding to the scaffolding protein 14-3-3. Furthermore, phosphorylation of S118 inhibits apoptosis by preventing its association with Bcl-xL. Dephosphorylation of Bad may initiate intrinsic apoptosis by associating with Bcl-xL. We determined the phosphorylation status of Bad in stored platelets by Phosphoproteomic and WB analyses of stored and fresh human platelets ( Figure-2) Both methods showed a drastic decrease in Bad is phosphorylation at serine residues 99 and 118 in stored platelets.Activation of cofilin-1 by dephosphorylation in stored platelets. Like Bad, phosphorylation of cofilin-1 is also markedly decrease in stored platelets as shown by WB analyses (Figure-3). It is unclear how the apoptotic pathways lead to cofilin-1 activation (dephosphorylation). Caspases are known to activate slingshot phosphatase which could then activate cofilina1 by its dephosphorylation. LIM- kinases phosphorylate and, thus, inactivate cofilin-1 whereas slingshot phosphatases dephosphorylate and activate cofilin-1. Our data show that the Lim kinase inhibitor, as expected increased ABT-737-induced PS exposure on platelets (Figure 4A), whereas slingshot phosphatase inhibitor inhibited ABT-737-induced PS exposure at a 1-μM concentration (Figure 4B)ConclusionOur data show that activation of apoptotic pathways is responsible for activation of cofilin and subsequent PS exposure by actin reorganization in stored platelets. Targeting this pathway could improve the recovery of stored platelets following transfusion by inhibiting storage-induced PS exposure. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Tyrosine Kinase Inhibitor Pazopanib Inhibits Platelet Procoagulant Activity in Renal Cell Carcinoma Patients.

Pazopanib is an angiostatic tyrosine kinase inhibitor (TKI) presently used for cancer treatment, particularly in patients with renal cell carcinoma (RCC). This treatment can be accompanied by mild bleeding as an adverse effect. Given the role of protein tyrosine kinases in platelet activation processes, we investigated whether and how pazopanib can affect platelet functions in purified systems and during treatment of advanced RCC patients. In isolated platelets from healthy volunteers, pazopanib dose-dependently reduced collagen-induced integrin activation and secretion, as well as platelet aggregation. Pazopanib addition diminished glycoprotein (GP) VI-dependent tyrosine phosphorylation of multiple platelet proteins, including the tyrosine kinase Syk. Furthermore, pazopanib inhibited GPVI-induced Ca2+ elevation, resulting in reduced exposure of the procoagulant phospholipid phosphatidylserine (PS). Upon perfusion of control blood over a collagen surface, pazopanib inhibited thrombus size as well as PS exposure. Blood samples from 10 RCC patients were also analyzed before and after 14 days of pazopanib treatment as monotherapy. This treatment caused an overall lowering in platelet count, with 3 out of 10 patients experiencing mild bleeding. Platelets isolated from pazopanib-treated patients showed a significant lowering of PS exposure upon activation. In addition, platelet procoagulant activity was inhibited in thrombi formed under flow conditions. Control experiments indicated that higher pazopanib concentrations were required to inhibit GPVI-mediated PS exposure in the presence of plasma. Together, these results indicated that pazopanib suppresses GPVI-induced platelet activation responses in a way partly antagonized by the presence of plasma. In treated cancer patients, pazopanib effects were confined to a reduction in GPVI-dependent PS exposure. Together with the reduced platelet count, this may explain the mild bleeding tendency observed in pazopanib-treated patients.

The Curcumin Analogs 2-Pyridyl Cyclohexanone Induce Apoptosis via Inhibition of the JAK2-STAT3 Pathway in Human Esophageal Squamous Cell Carcinoma Cells.

Multiple modifications to the structure of curcumin have been investigated with an aim to improve its potency and biochemical properties. Previously, we have synthesized a series of curcumin analogs. In the present study, the anticancer effect of 2-pyridyl cyclohexanone, one of the curcumin analogs, on esophageal carcinoma Eca109 and EC9706 cell lines and its molecular mechanisms were investigated. 2-Pyridyl cyclohexanone inhibited the proliferation of Eca109 and EC9706 cells by inducing apoptosis as indicated by morphological changes, membrane phospholipid phosphatidylserine ectropion, caspase 3 activation, and cleavage of poly(ADP-ribose) polymerase. Mechanistic studies indicated that 2-pyridyl cyclohexanone disrupted mitochondrial membrane potential, disturbed the balance of the Bcl-2 family proteins, and triggered apoptosis via the mitochondria-mediated intrinsic pathway. In 2-pyridine cyclohexanone-treated cells, the phosphorylation levels of JAK2 and STAT3 were dose-dependently decreased and p38 and p-ERK signals were notably activated in a dose-dependent manner. Moreover, we found that the addition of S3I-201, a STAT3 inhibitor, led to a decreased expression level of Bcl-2 in Eca109 cells. The chromatin immunoprecipitation assay demonstrated that STAT3 bound to the promoter of Bcl-2 in the Eca109 cells. Furthermore, the mutation of four STAT3 binding sites (−1733/−1723, −1627/−1617, −807/−797, and −134/−124) on the promote of Bcl-2 gene alone attenuated the transcriptional activation of STAT3. In addition, down-regulation of STAT3 resulted in less of transcriptional activity of STAT3 on Bcl-2 expression. These data provide a potential molecular mechanism of the apoptotic induction function of 2-pyridyl cyclohexanone, and emphasize its important roles as a therapeutic agent for esophageal squamous carcinoma.

Shear-induced integrin signaling in platelet phosphatidylserine exposure, microvesicle release, and coagulation

AbstractIt is currently unclear why agonist-stimulated platelets require shear force to efficiently externalize the procoagulant phospholipid phosphatidylserine (PS) and release PS-exposed microvesicles (MVs). We reveal that integrin outside-in signaling is an important mechanism for this requirement. PS exposure and MV release were inhibited in β3−/− platelets or by integrin antagonists. The impaired MV release and PS exposure in β3−/− platelets were rescued by expression of wild-type β3 but not a Gα13 binding–deficient β3 mutant (E733EE to AAA), which blocks outside-in signaling but not ligand binding. Inhibition of Gα13 or Src also diminished agonist/shear-dependent PS exposure and MV release, further indicating a role for integrin outside-in signaling. PS exposure in activated platelets was induced by application of pulling force via an integrin ligand, which was abolished by inhibiting Gα13-integrin interaction, suggesting that Gα13-dependent transmission of mechanical signals by integrins induces PS exposure. Inhibition of Gα13 delayed coagulation in vitro. Furthermore, inhibition or platelet-specific knockout of Gα13 diminished laser-induced intravascular fibrin formation in arterioles in vivo. Thus, β3 integrins serve as a shear sensor activating the Gα13-dependent outside-in signaling pathway to facilitate platelet procoagulant function. Pharmacological targeting of Gα13-integrin interaction prevents occlusive thrombosis in vivo by inhibiting both coagulation and platelet thrombus formation.

Multivalent Cation-Bridged PI(4,5)P2 Clusters Form at Very Low Concentrations

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2 or PIP2), is a key component of the inner leaflet of the plasma membrane in eukaryotic cells. In model membranes, PIP2 has been reported to form clusters, but whether these locally different conditions could give rise to distinct pools of unclustered and clustered PIP2 is unclear. By use of both fluorescence self-quenching and Förster resonance energy transfer assays, we have discovered that PIP2 self-associates at remarkably low concentrations starting below 0.05 mol% of total lipids. Formation of these clusters was dependent on physiological divalent metal ions, such as Ca2+, Mg2+, Zn2+, or trivalent ions Fe3+ and Al3+. Formation of PIP2 clusters was also headgroup-specific, being largely independent of the type of acyl chain. The similarly labeled phospholipids phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol exhibited no such clustering. However, six phosphoinositide species coclustered with PIP2. The degree of PIP2 cation clustering was significantly influenced by the composition of the surrounding lipids, with cholesterol and phosphatidylinositol enhancing this behavior. We propose that PIP2 cation-bridged cluster formation, which might be similar to micelle formation, can be used as a physical model for what could be distinct pools of PIP2 in biological membranes. To our knowledge, this study provides the first evidence of PIP2 forming clusters at such low concentrations. The property of PIP2 to form such clusters at such extremely low concentrations in model membranes reveals, to our knowledge, a new behavior of PIP2 proposed to occur in cells, in which local multivalent metal ions, lipid compositions, and various binding proteins could greatly influence PIP2 properties. In turn, these different pools of PIP2 could further regulate cellular events.

PS, It's Complicated: The Roles of Phosphatidylserine and Phosphatidylethanolamine in the Pathogenesis of Candida albicans and Other Microbial Pathogens.

The phospholipids phosphatidylserine (PS) and phosphatidylethanolamine (PE) play important roles in the virulence of Candida albicans and loss of PS synthesis or synthesis of PE from PS (PS decarboxylase) severely compromises virulence in C. albicans in a mouse model of systemic candidiasis. This review discusses synthesis of PE and PS in C. albicans and mechanisms by which these lipids impact virulence in this fungus. This is further compared to how PS and PE synthesis impact virulence in other fungi, parasites and bacteria. Furthermore, the impact of PS asymmetry on virulence and extracellular vesicle formation in several microbes is reviewed. Finally, the potential for PS and PE synthases as drug targets in these various kingdoms is also examined.

Molecular Imaging of Apoptosis in Ischemia Reperfusion Injury With Radiolabeled Duramycin Targeting Phosphatidylethanolamine: Effective Target Uptake and Reduced Nontarget Organ Radiation Burden

ObjectivesThe purpose of this study was to evaluate the feasibility of imaging apoptosis in experimental ischemia-reperfusion model by technetium-99m (99mTc)-labeled Duramycin, and compare it to an established tracer, 99mTc-labeled Annexin-V, which has a relative disadvantage of high radiation burden to nontarget organs. BackgroundDuring apoptosis, the cell membrane phospholipids-phosphatidylserine (PS) and phosphatidylethanolamine (PE) are exposed and can be targeted by Annexin-V and Duramycin, respectively, for in vivo imaging. Identification of a reversible cell death process should permit therapeutic intervention to help reduce myocyte loss and left ventricle dysfunction. MethodsIn a 40-min left coronary artery ischemia-reperfusion model in 17 rabbits, 7 mCi of 99mTc-labeled Duramycin (n = 10), 99mTc-linear Duramycin (a negative tracer control; n = 3), or 99mTc-Annexin-V (a positive tracer-control; n = 4) were intravenously administered 30 min after reperfusion. Of the 10 Duramycin group animals, 4 animals were treated with an antiapoptotic agent, minocycline at the time of reperfusion. In vivo and ex vivo micro–single-photon emission computed tomography (μSPECT) and micro-computed tomography (μCT) imaging was performed 3 h after reperfusion, followed by quantitative assessment of tracer uptake and pathological characterization. Fluorescent Duramycin and Annexin-V were injected in 4 rats to visualize colocalization in infarct areas in a 40-min left coronary artery occlusion and 30-min reperfusion model. ResultsIntense uptake of Duramycin and Annexin-V was observed in the apical (infarcted) areas. The percent injected dose per gram uptake of Duramycin in apical region (0.751 ± 0.262%) was significantly higher than remote area in same animals (0.045 ± 0.029%; p < 0.01). Duramycin uptake was insignificantly lower than Annexin-V uptake (1.23 ± 0.304%; p > 0.01) but demonstrated substantially lower radiation burden to kidneys (0.358 ± 0.210% vs. 1.58 ± 0.316%, respectively; p < 0.001). Fluorescence studies with Duramycin and Annexin V showed colocalization in infarct areas. Minocycline treatment substantially resolved Duramycin uptake (0.354% ± 0.0624%; p < 0.01). ConclusionsDuramycin is similarly effective in imaging apoptotic cell death as Annexin-V with lower nontarget organ radiation. Clinical feasibility of apoptosis imaging with a PE-seeking tracer should be tested.

Influence of phosphatidylserine and phosphatidylethanolamine on farnesol tolerance in Candida albicans.

Candida albicans is among the most common human fungal pathogens. The ability to undergo the morphological transition from yeast to hyphal growth is critical for its pathogenesis. Farnesol, a precursor in the isoprenoid/sterol pathway, is a quorum-sensing molecule produced by C. albicans that inhibits hyphal growth in this polymorphic fungus. Interestingly, C. albicans can tolerate farnesol concentrations that are toxic to other fungi. We hypothesized that changes in phospholipid composition are one of the factors contributing to farnesol tolerance in C. albicans. In this study, we found that loss of enzymes that synthesize the phospholipids phosphatidylserine (PS) and/or phosphatidylethanolamine (PE) compromise the tolerance of C. albicans to farnesol. Compared with wild type, the phospholipid mutant cho1∆/∆ (loss of PS and decreased PE synthesis) shows greater inhibition of growth, loss of ATP production, increased consumption of oxygen, and increased formation of reactive oxygen species in the presence of farnesol. The cho1∆/∆ mutant also exhibits decreased sensitivity to mitochondrial ATPase inhibition, suggesting that cells lacking PS and/or downstream PE rely less on mitochondrial function for ATP synthesis. These data reveal that PS and PE play roles in farnesol tolerance and maintaining mitochondrial respiratory function.

Methylphenidate disrupts cytoskeletal homeostasis and reduces membrane-associated lipid content in juvenile rat hippocampus.

Although methylphenidate (MPH) is ubiquitously prescribed to children and adolescents, the consequences of chronic utilization of this psychostimulant are poorly understood. In this study, we investigated the effects of MPH on cytoskeletal homeostasis and lipid content in rat hippocampus. Wistar rats received intraperitoneal injections of MPH (2.0mg/kg) or saline solution (controls), once a day, from the 15th to the 44th day of age. Results showed that MPH provoked hypophosphorylation of glial fibrillary acidic protein (GFAP) and reduced its immunocontent. Middle and high molecular weight neurofilament subunits (NF-M, NF-H) were hypophosphorylated by MPH on KSP repeat tail domains, while NFL, NFM and NFH immunocontents were not altered. MPH increased protein phosphatase 1 (PP1) and 2A (PP2A) immunocontents. MPH also decreased the total content of ganglioside and phospholipid, as well as the main brain gangliosides (GM1, GD1a, and GD1b) and the major brain phospholipids (sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine). Total cholesterol content was also reduced in the hippocampi of juvenile rats treated with MPH. These results provide evidence that disruptions of cytoskeletal and lipid homeostasis in hippocampus of juvenile rats are triggers by chronic MPH treatment and present a new basis for understanding the effects and consequences associated with chronic use of this psychostimulant during the development of the central nervous system.

Tafazzin (TAZ) Regulates the Differentiation of AML Cells By Reducing Levels of the Phospholipid Phosphatidylethanolamine

AML cells have unique mitochondrial characteristics with an increased reliance on mitochondrial metabolism and oxidative phosphorylation. To identify new biological vulnerabilities in the mitochondria of AML, we conducted a CRISPR knockout screen. CAS9-overexpressing human OCI-AML2 leukemia cells were transduced with a library of 91,320 sgRNAs in barcoded lentiviral vectors targeting 17,237 nuclear-encoded genes. Cells were harvested, genomic DNA was isolated, and the relative abundance of sgRNAs were determined by sequencing barcodes 14 days after puromycin selection. We focused on the sgRNAs targeting the 1050 mitochondrial proteins to identify targets in the mitochondrial proteome whose knockout reduced AML growth and viability.The cardiolipin remodeling enzyme tafazzin (TAZ) was among the top 1% of mitochondrial hits. Using individual sgRNA, we confirmed that knockout of TAZ reduced the growth of CAS9-OCI-AML2 cells by >70%, thus validating the findings from our screen. We also knocked down TAZ with 2 independent shRNA and demonstrated reductions in growth and viability of a panel of AML cells: OCI-AML2 (>80%), TEX (>50%), K562 (>50%), and U937 (>40%). Moreover, TAZ knockdown significantly reduced the engraftment of TEX leukemia cells in vivo by 80%, indicating that TAZ-knockdown reduces AML growth in vivo and can target leukemia initiating cells. In contrast, knockdown of TAZ in mouse models did not impair normal hematopoiesis nor reduced the abundance of hematopoietic stem cells, although more subtle defects in the hematopoietic stem cells might explain transient episodes of neutropenia seen in Barth's syndrome, a congenital condition associated with X-linked TAZ mutations.TAZ is responsible for the majority of Cardiolipin (CL) remodeling under physiological conditions. As expected the knockdown of TAZ in both AML and normal mouse hematopoietic cells increased the substrate (monlysocardiolipin) to product (CL) ratio of TAZ. CL is required for the proper localization, and efficient function of, respiratory chain enzymes. However, in AML cells, knockdown of TAZ did not alter respiratory chain complex activity, basal oxygen consumption, or respiratory chain reserve capacity.Recent studies have shown that mitochondrial pathways can regulate cell fate and differentiation independent of their effects on oxidative phosphorylation. Therefore, we examined changes in AML cell differentiation after TAZ knockdown. Knockdown of TAZ promoted the differentiation of AML cells as evidenced by increased non-specific esterase staining and increased CD11b expression on the cell surface.In breast cancer cells decreasing phosphatidylethanolamine (PE) levels, induced the differentiation of these cells. As TAZ regulates phospholipid remodeling, therefore we measured levels of PE and phosphatidylserine (PS) after TAZ-knockdown by spot densitometry. Interestingly, knockdown of TAZ in OCI-AML2 cells decreased PE and increased PS lipid levels. To determine whether alterations in PE and PS phospholipids are functionally important for differentiation of AML cells, we treated AML cells with MMV007285, an inhibitor of the phosphatidylserine decarboxylase (PISD), an enzyme that converts PS to PE. MMV007285 mimicked the effects of TAZ-knockdown and increased differentiation of OCI-AML2 and 8227 AML cells.In summary, the cardiolipin remodeling enzyme TAZ regulates the differentiation of AML cells by controlling levels of PS and PE, thereby highlighting a new mechanism by which phospholipids and mitochondrial enzymes regulate AML cell fate and differentiation. Moreover, PISD inhibition may be a novel therapeutic strategy to selectively promote the differentiation of AML. DisclosuresNo relevant conflicts of interest to declare.

Phosphatidylserine save-me signals drive functional recovery of severed axons in Caenorhabditis elegans

Functional regeneration after axonal injury requires transected axons to regrow and reestablish connection with their original target tissue. The spontaneous regenerative mechanism known as axonal fusion provides a highly efficient means of achieving targeted reconnection, as a regrowing axon is able to recognize and fuse with its own detached axon segment, thereby rapidly reestablishing the original axonal tract. Here, we use behavioral assays and fluorescent reporters to show that axonal fusion enables full recovery of function after axotomy of Caenorhabditis elegans mechanosensory neurons. Furthermore, we reveal that the phospholipid phosphatidylserine, which becomes exposed on the damaged axon to function as a "save-me" signal, defines the level of axonal fusion. We also show that successful axonal fusion correlates with the regrowth potential and branching of the proximal fragment and with the retraction length and degeneration of the separated segment. Finally, we identify discrete axonal domains that vary in their propensity to regrow through fusion and show that the level of axonal fusion can be genetically modulated. Taken together, our results reveal that axonal fusion restores full function to injured neurons, is dependent on exposure of phospholipid signals, and is achieved through the balance between regenerative potential and level of degeneration.

Abstract 3219: Targeting lipid metabolism and ER stress in renal cell carcinoma by englerin A

Abstract Renal cell carcinoma (RCC) is among the top ten most common forms of cancer and is the leading malignancy of the kidney. Clear cell renal carcinoma (cc-RCC), the most frequent type of RCC, is one of the most refractory cancers with an incidence that is on the rise. The two-year survival rate for patients with metastatic disease is under 20% despite approved targeted therapies. Hence, there is an urgent need to investigate novel agents. Screening of plant extracts in search of new anti-cancer agents resulted in the discovery of englerin A (EA), a guaiane sesquiterpene with potent cytotoxicity against RCC and a small subset of other cancers. Although a few cellular targets have been identified for EA, in most instances, modulation of these targets required concentrations of EA much higher than that required to kill tumor cells highly sensitive to EA. Hence, it is still not clear what mechanisms account for the cytotoxicity of EA in tumor cells highly sensitive to EA. Unlike any prior study, the current study used a systems biology approach to explore the mechanism(s) of action of EA. Metabolomics analyses were conducted after treatment of human RCC A498 cells with 100 nM EA for 24 and 48 h. Pathway analysis revealed that 88% of the metabolic disturbance associated with englerin A at 24 h was due to changes in lipid metabolism. In particular, among the top metabolites, several ceramides and glucosylceramides increased up to 18- and 178-fold, respectively. On the other hand, all sphingomyelins decreased up to 5.5-fold. Furthermore, EA induced depletion in phosphatidylcholine, phosphatidylglycerol, and phosphatidylethanolamine phospholipids. At 48 h of englerin A treatment, the main findings from pathway analysis were the accumulation of fatty acylcarnitine derivatives, purines, pyrimidines, and phosphatidyl serine phospholipids, as well as the depletion of reduced glutathione indicating dysfunctional mitochondria and oxidative stress. Moreover, microarray analyses determined that EA (100 nM) induced ER stress signaling and an acute inflammatory response including interferon signaling, likely due to the generation of ceramides which were toxic to both A498 and UO-31 renal cancer cells. Quantitative PCR and Western Blot analyses confirmed that EA induced both the unfolded protein and acute inflammatory response. Furthermore, fluorescence confocal microscopy revealed that EA at 25 nM disrupted the morphology of the ER confirming the deleterious effect of EA on the ER. Collectively, our findings suggest that cc-RCC is highly sensitive to disruptions in lipid metabolism and ER stress and that these vulnerabilities can be targeted for the treatment of cc-RCC and possibly other lipid storing cancers. Furthermore, our results also suggest that ceramides may be a mediator of some of the actions of EA. Lastly, the acute inflammatory response induced by EA may mediate anti-tumor immunity. Note: This abstract was not presented at the meeting. Citation Format: Ayse Batova, Diego Altomare, Kim E. Creek, Robert K. Naviaux, Lin Wang, Kefeng Li, Erica Green, Richard Williams, Jane C. Naviaux, Mitchell Diccianni, Alice L. Yu. Targeting lipid metabolism and ER stress in renal cell carcinoma by englerin A [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3219. doi:10.1158/1538-7445.AM2017-3219

Structure of human nSMase2 reveals an interdomain allosteric activation mechanism for ceramide generation

Neutral sphingomyelinase 2 (nSMase2, product of the SMPD3 gene) is a key enzyme for ceramide generation that is involved in regulating cellular stress responses and exosome-mediated intercellular communication. nSMase2 is activated by diverse stimuli, including the anionic phospholipid phosphatidylserine. Phosphatidylserine binds to an integral-membrane N-terminal domain (NTD); however, how the NTD activates the C-terminal catalytic domain is unclear. Here, we identify the complete catalytic domain of nSMase2, which was misannotated because of a large insertion. We find the soluble catalytic domain interacts directly with the membrane-associated NTD, which serves as both a membrane anchor and an allosteric activator. The juxtamembrane region, which links the NTD and the catalytic domain, is necessary and sufficient for activation. Furthermore, we provide a mechanistic basis for this phenomenon using the crystal structure of the human nSMase2 catalytic domain determined at 1.85-Å resolution. The structure reveals a DNase-I-type fold with a hydrophobic track leading to the active site that is blocked by an evolutionarily conserved motif which we term the "DK switch." Structural analysis of nSMase2 and the extended N-SMase family shows that the DK switch can adopt different conformations to reposition a universally conserved Asp (D) residue involved in catalysis. Mutation of this Asp residue in nSMase2 disrupts catalysis, allosteric activation, stimulation by phosphatidylserine, and pharmacological inhibition by the lipid-competitive inhibitor GW4869. Taken together, these results demonstrate that the DK switch regulates ceramide generation by nSMase2 and is governed by an allosteric interdomain interaction at the membrane interface.

Effect of bavituximab in combination with nivolumab on tumor immune response in a 3D ex vivo system of lung cancer patients.

e23091 Background: Bavituximab is a chimeric monoclonal antibody that targets the membrane phospholipid phosphatidylserine (PS) exposed on endothelial cells and cancer cells in solid tumors. Our previous studies showed that bavituximab enhances the activation of CD8+ TILs that correlates with increased cytokine production by lymphoid and myeloid cells in lung cancer with low PD-L1 expression suggesting that the interruption of the PD-1/PD-L1 axis by nivolumab may enhance the bavituximab effect in tumors. Methods: Fresh tumor tissues obtained from consented patients with NSCLC at the time of surgical resection were utilized in a 3D ex vivo tumor miscrosphere assay, where 3D tumor microspheres were treated with bavituximab or nivolumab alone or in combination at 10 mg/ml for 36 hours. At the end of the treatment, a multiplex human cytokine assay was used to simultaneously analyze the differential secretion of cytokines, including human IFNg, in culture media as a surrogate of TIL activation. In addition, a gene expression analysis using a NanoString platform containing probes to quantitate 770 immune function genes. Results: Preliminary results indicate the combination treatment with bavituximab and nivolumab led to increased expression of genes involved in M1 polarization of tumor associated macrophages in a subpopulation of lung tumors that closely correlated with release of cytokines such as MIP1b (CCL4) which is a chemoattractant for natural killer cells, monocytes and a variety of other cells involved in tumor immune response. Conclusions: This lung patient derived ex-vivo approach indicates that bavituximab in combination with nivolumab may enhance immune response. This response likely involves M1 polarization of tumor associated macrophages and suggests potential clinical implications in the treatment of lung cancer.

Lethal H1N1 influenza A virus infection alters the murine alveolar type II cell surfactant lipidome.

Alveolar type II (ATII) epithelial cells are the primary site of influenza virus replication in the distal lung. Development of acute respiratory distress syndrome in influenza-infected mice correlates with significant alterations in ATII cell function. However, the impact of infection on ATII cell surfactant lipid metabolism has not been explored. C57BL/6 mice were inoculated intranasally with influenza A/WSN/33 (H1N1) virus (10,000 plaque-forming units/mouse) or mock-infected with virus diluent. ATII cells were isolated by a standard lung digestion protocol at 2 and 6 days postinfection. Levels of 77 surfactant lipid-related compounds of known identity in each ATII cell sample were measured by ultra-high-performance liquid chromatography-mass spectrometry. In other mice, bronchoalveolar lavage fluid was collected to measure lipid and protein content using commercial assay kits. Relative to mock-infected animals, ATII cells from influenza-infected mice contained reduced levels of major surfactant phospholipids (phosphatidylcholine, phosphatidylglycerol, and phosphatidylethanolamine) but increased levels of minor phospholipids (phosphatidylserine, phosphatidylinositol, and sphingomyelin), cholesterol, and diacylglycerol. These changes were accompanied by reductions in cytidine 5'-diphosphocholine and 5'-diphosphoethanolamine (liponucleotide precursors for ATII cell phosphatidylcholine and phosphatidylethanolamine synthesis, respectively). ATII cell lamellar bodies were ultrastructurally abnormal after infection. Changes in ATII cell phospholipids were reflected in the composition of bronchoalveolar lavage fluid, which contained reduced amounts of phosphatidylcholine and phosphatidylglycerol but increased amounts of sphingomyelin, cholesterol, and protein. Influenza infection significantly alters ATII cell surfactant lipid metabolism, which may contribute to surfactant dysfunction and development of acute respiratory distress syndrome in influenza-infected mice.

Immobilization and detection of platelet-derived extracellular vesicles on functionalized silicon substrate: cytometric and spectrometric approach

Among the various biomarkers that are used to diagnose or monitor disease, extracellular vesicles (EVs) represent one of the most promising targets in the development of new therapeutic strategies and the application of new diagnostic methods. The detection of circulating platelet-derived microvesicles (PMVs) is a considerable challenge for laboratory diagnostics, especially in the preliminary phase of a disease. In this study, we present a multistep approach to immobilizing and detecting PMVs in biological samples (microvesicles generated from activated platelets and human platelet-poor plasma) on functionalized silicon substrate. We describe the application of time-of-flight secondary ion mass spectrometry (TOF-SIMS) and spectroscopic ellipsometry methods to the detection of immobilized PMVs in the context of a novel imaging flow cytometry (ISX) technique and atomic force microscopy (AFM). This novel approach allowed us to confirm the presence of the abundant microvesicle phospholipids phosphatidylserine (PS) and phosphatidylethanolamine (PE) on a surface with immobilized PMVs. Phosphatidylcholine groups (C5H12N+; C5H15PNO4+) were also detected. Moreover, we were able to show that ellipsometry permitted the immobilization of PMVs on a functionalized surface to be evaluated. The sensitivity of the ISX technique depends on the size and refractive index of the analyzed microvesicles.Graphical abstractHuman platelets activated with thrombin (in concentration 1IU/mL) generate population of PMVs (platelet derived microvesicles), which can be detected and enumerated with fluorescent-label method (imaging cytometry). Alternatively, PMVs can be immobilized on the modified silicon substrate which is functionalized with a specific IgM murine monoclonal antibody against human glycoprotein IIb/IIIa complex (PAC-1). Immobilized PMVs can be subjected to label-free analyses by means ellipsometry, atomic force microscopy (AFM) and time-of-flight secondary ion mass spectrometry (TOF-SIMS).

Mammalian Nonmuscle Myosin II Binds to Anionic Phospholipids with Concomitant Dissociation of the Regulatory Light Chain

Mammalian cells express three Class II nonmuscle myosins (NM): NM2A, NM2B, and NM2C. The three NM2s have well established essential roles in cell motility, adhesion, and cytokinesis and less well defined roles in vesicle transport and other processes that would require association of NM2s with cell membranes. Previous evidence for the mechanism of NM2-membrane association includes direct interaction of NM2s with membrane lipids and indirect interaction by association of NM2s with membrane-bound F-actin or peripheral membrane proteins. Direct binding of NM2s to phosphatidylserine-liposomes, but not to phosphatidylcholine-liposomes, has been reported, but the molecular basis of the interaction between NM2s and acidic phospholipids has not been previously investigated. We now show that filamentous, full-length NM2A, NM2B, and NM2C and monomeric, non-filamentous heavy meromyosin bind to liposomes containing one or more acidic phospholipids (phosphatidylserine, phosphatidylinositol 4,5-diphosphate, and phosphatidylinositol 3,4,5-triphosphate) but do not bind to 100% phosphatidylcholine-liposomes. Binding of NM2s to acidic liposomes occurs predominantly through interaction of the liposomes with the regulatory light chain (RLC) binding site in the myosin heavy chain with concomitant dissociation of the RLC. Phosphorylation of myosin-bound RLC by myosin light chain kinase substantially inhibits binding to liposomes of both filamentous NM2 and non-filamentous heavy meromyosin; the addition of excess unbound RLC, but not excess unbound essential light chain, competes with liposome binding. Consistent with the in vitro data, we show that endogenous and expressed NM2A associates with the plasma membrane of HeLa cells and fibrosarcoma cells independently of F-actin.

IN VITRO SELECTION AND BINDING STUDIES OF A DNA APTAMER TARGETING PHOSPHATIDYLSERINE (PS) USING NON-FLUORESCENCE MEASUREMENTS

Phosphatidylserine (PS) is a phospholipid that is an important component of all cell membranes. Normally, phospholipid PS is only found on the cytosolic surface of the plasma membrane. Obligatory translocation of PS from the interior to the exterior side of the plasma membranes is a potential biomarker for apoptotic cell death. The SELEX technology (Systematic Evolution of Ligands by Exponential enrichment) is widely applied as an in vitro selection and amplification method to generate target-specific aptamers. In this study, entropic fragment based approach (EFBA) was used to design a set of DNA aptamers bind specifically with PS based on seed-and-grow strategy. Each member of designed short-sequence DNA aptamers consists of a 6 nucleotides while long-sequence DNA aptamers consists of an 11 nucleotides. The binding properties of the designed DNA aptamers with phospholipid PS have been studied using NanoDrop without fluorescent tags attached to them. This technique has been proposed to measure concentrations of relative PS bound aptamers versus unbound ones and to analyze the phospholipid binding properties of aptamers. The Short-sequence DNA aptamers show stronger binding affinity for phospholipid PS compared with long ones. Also short-sequence DNA aptamer (AAAGAC) has the highest binding affinity compared with the other short ones and also the other two long ones. These results suggest that the phospholipid PS binding of DNA aptamers is very much sequence specific and depends also on the number of nucleotides in the DNA aptamers. The present results thus disclose a novel assay for phospholipid PS recognition in apoptotic cells, which may open up the possibility of discovering aptamer based diagnostic tools to be used in treatment of many diseases including cancer.

Could Bat Cell Temperature and Filovirus Filament Length Explain the Emergence of Ebola Virus in Mammals? Predictions of a Thermodynamic Model.

The host reservoir of Zaire ebolavirus (EBOV) remains elusive. One suggestion is that EBOV emerges in mammals when the precursor virus jumps from mayflies (or other riverine insects) to insectivorous bats. However, this does not fit with the current view that filoviruses cannot infect arthropods. Here, it is first argued that the evidence that arthropods are refractory is not definitive. Second, it is proposed that a combination of filovirus filament length and the high temperature (~42°C) experienced by an insect virus ingested by a flying bat, together with the large number of insects eaten by bats (e.g. during an ephemeral mass emergence of mayflies), facilitate jumping the species barrier. The length of a filovirus filament is related to the number of genome copies (GC). Predictions from a preliminary thermodynamic model developed here suggest that filament length could greatly affect EBOV infectivity to mammalian cells with infectivity peaking for filaments of a certain length. Importantly, the infectivity to mammals of even short filaments may be more than one million-fold higher than that for the single GC virion. Third, it is proposed that at the high temperature within the bat, the phospholipid phosphatidylserine in the virus envelope promotes filament formation through fusion of single GC particles within the ingested insect, thus hugely increasing their infectivity to bats. Forth, according to the thermodynamic model, increasing the temperature from 27°C (insect cell temperature at average air temperature in Guinea, West Africa) to 42°C (bat) could increase the affinity of the filaments for bat cells by 1-2 orders of magnitude, while having no effect on the binding affinity of the single GC virions. The thermodynamic model developed here is supported by the counterintuitive observation that high glycoprotein densities on the EBOV surface reduce its infectivity in contrast to other viruses such as HIV.

How membrane asymmetry regulates ADAM17 sheddase function

Asymmetrical distribution of lipids in the cell membrane is a fundamental feature of all eukaryotic cells. The most important negatively charged phospholipids phosphatidylserine (PS) and phosphatidylinositol species reside almost exclusively in the cytoplasmic leaflet, where they serve as essential co-factors for many membrane-bound enzymes including protein kinase C, phosphatase PTEN, tyrosine kinase c-Src, and MARCKS.1 Clusters of basic amino acid residues draw the proteins via electrostatic interactions to the lipid, enabling them to exert their function at the cytosolic membrane face.