Phosphatidylserine-binding Protein Research Articles

Phosphatidic acid binding proteins display differential binding as a function of membrane curvature stress and chemical properties

Phosphatidic acid (PA) is a crucial membrane phospholipid involved in de novo lipid synthesis and numerous intracellular signaling cascades. The signaling function of PA is mediated by peripheral membrane proteins that specifically recognize PA. While numerous PA-binding proteins are known, much less is known about what drives specificity of PA-protein binding. Previously, we have described the ionization properties of PA, summarized in the electrostatic-hydrogen bond switch, as one aspect that drives the specific binding of PA by PA-binding proteins. Here we focus on membrane curvature stress induced by phosphatidylethanolamine and show that many PA-binding proteins display enhanced binding as a function of negative curvature stress. This result is corroborated by the observation that positive curvature stress, induced by lyso phosphatidylcholine, abolishes PA binding of target proteins. We show, for the first time, that a novel plant PA-binding protein, Arabidopsis Epsin-like Clathrin Adaptor 1 (ECA1) displays curvature-dependence in its binding to PA. Other established PA targets examined in this study include, the plant proteins TGD2, and PDK1, the yeast proteins Opi1 and Spo20, and, the mammalian protein Raf-1 kinase and the C2 domain of the mammalian phosphatidylserine binding protein Lact as control. Based on our observations, we propose that liposome binding assays are the preferred method to investigate lipid binding compared to the popular lipid overlay assays where membrane environment is lost. The use of complex lipid mixtures is important to elucidate further aspects of PA binding proteins.

Proteinase 3 Is a Phosphatidylserine-binding Protein That Affects the Production and Function of Microvesicles

Proteinase 3 (PR3), the autoantigen in granulomatosis with polyangiitis, is expressed at the plasma membrane of resting neutrophils, and this membrane expression increases during both activation and apoptosis. Using surface plasmon resonance and protein-lipid overlay assays, this study demonstrates that PR3 is a phosphatidylserine-binding protein and this interaction is dependent on the hydrophobic patch responsible for membrane anchorage. Molecular simulations suggest that PR3 interacts with phosphatidylserine via a small number of amino acids, which engage in long lasting interactions with the lipid heads. As phosphatidylserine is a major component of microvesicles (MVs), this study also examined the consequences of this interaction on MV production and function. PR3-expressing cells produced significantly fewer MVs during both activation and apoptosis, and this reduction was dependent on the ability of PR3 to associate with the membrane as mutating the hydrophobic patch restored MV production. Functionally, activation-evoked MVs from PR3-expressing cells induced a significantly larger respiratory burst in human neutrophils compared with control MVs. Conversely, MVs generated during apoptosis inhibited the basal respiratory burst in human neutrophils, and those generated from PR3-expressing cells hampered this inhibition. Given that membrane expression of PR3 is increased in patients with granulomatosis with polyangiitis, MVs generated from neutrophils expressing membrane PR3 may potentiate oxidative damage of endothelial cells and promote the systemic inflammation observed in this disease.

Reverse engineering B. Schlosseri developmental-regulated cell death programs for cancer immunotherapy

Abstract B. schlosseri, a colonial marine species, offers a unique platform to study mechanisms underlying cellular recognition. We used adoptive transfer studies, FACs analysis, and RNA-sequencing to study the progression by which fused-colonies eliminate chimeric partners. We show the principle cell type mediating elimination of partners in a chimera is a cytotoxic Morula cell (MC), which utilizes developmental-regulated programmed cell death pathways, initiated during the ‘takeover’ phase of colony life, to eliminate partners. Adoptive transfer of FACs purified cytotoxic MCs from third party donors into recipients recapitulates events of chimeric partner elimination. Transfer of 1×10^5 allogeneic MCs eliminated 33 of 78 (42%) of recipient palleal buds and 20 of 76 (20.5%) adult zooids by day 14 post-transfer. Transfer of phagocytic populations had only minimal effects on recipient colonies. We identified 275 genes (FDR < 0.05) from global RNA-seq expression profiling that increased by expression of at least 50 fold associated with elimination of chimeric partners. These include an IL-17 family member (IL-17C), the phagocytic receptors (MerTK, AXL), and programmed cell removal factors (CALR, MFGE-8). To probe the role of specific receptors we used in vitro studies. Recombinant Il-17c promoted allogeneic cytotoxicity while MFGE-8, a phosphatidylserine binding protein, induced engulfment of apoptotic cells. Studies using chimeric phosphatidylserine binding-cytokine fusion proteins are underway. Preliminary data suggests these fusion proteins promote inflammatory turnover of apoptotic cells. These studies provide new insights by which reverse engineering cell death programs can be utilized to enhance anti-tumor immunity.

Protection of Insects against Viral Infection by Apoptosis-Dependent Phagocytosis.

We investigated whether phagocytosis participates in the protection of insects from viral infection using the natural host-virus interaction between Drosophila melanogaster and Drosophila C virus (DCV). Drosophila S2 cells were induced to undergo apoptotic cell death upon DCV infection. However, UV-inactivated virus was unable to cause apoptosis, indicating the need for productive infection for apoptosis induction. S2 cells became susceptible to phagocytosis by hemocyte-derived l(2)mbn cells after viral infection, and the presence of phagocytes in S2 cell cultures reduced viral proliferation. Phagocytosis depended, in part, on caspase activity in S2 cells, as well as the engulfment receptors Draper and integrin βν in phagocytes. To validate the in vivo situation, adult flies were abdominally infected with DCV, followed by the analysis of fly death and viral growth. DCV infection killed flies in a dose-responding manner, and the activation of effector caspases was evident, as revealed by the cleavage of a target protein ectopically expressed in flies. Furthermore, hemocytes isolated from infected flies contained DCV-infected cells, and preinjection of latex beads to inhibit the phagocytic activity of hemocytes accelerated fly death after viral infection. Likewise, viral virulence was exaggerated in flies lacking the engulfment receptors, and was accompanied by the augmented proliferation of virus. Finally, phagocytosis of DCV-infected cells in vitro was inhibited by phosphatidylserine-containing liposome, and virus-infected flies died early when a phosphatidylserine-binding protein was ectopically expressed. Collectively, our study demonstrates that the apoptosis-dependent, phosphatidylserine-mediated phagocytosis of virus-infected cells plays an important role in innate immune responses against viral infection in Drosophila.

MARCKS ED Inhibits Fibrin Formation By Blocking Coagulation Protein Complex Assembly on Phosphatidylserine

Introduction: Prothrombinase is an essential enzyme of the coagulation cascade that is made up of a heterodimer of coagulation factor Xa and Va. The complex most readily forms in the presence of lipid membranes containing the anionic lipid phosphatidylserine. The phosphatidylserine lipid head groups provide a surface for the prothrombinase complex to assemble and significantly increase the enzyme's turnover number. Here, we investigate how truncation and modification of the naturally occurring myristoylated alanine-rich C-kinase substrate (MARCKS) protein into small peptide and peptidomimetic fragments (MARCKS ED) forms lipid probes that bind to phosphatidylserine, and block the assembly and enzymatic activity of the prothrombinase complex. Compared to known phosphatidylserine binding proteins such as annexin V or lactadherin, the MARCKS ED probes are less than a tenth of the molecular weight and do not require calcium to bind to phosphatidylserine.Methods: Using biophysical assays, we examine how the MARCKS ED probes assemble on lipid membranes containing phosphatidylserine and how this assembly alters the binding of factor Xa to these surfaces. Effects on the enzymatic activity of the prothrombinase complex in the presence of activated platelets and other phosphatidylserine exposing membranes are explored using a modified prothrombinase assay. The binding and anticoagulant activity of the MARCKS ED probes during whole blood coagulation are examined by quantifying fluorescently labeled MARCKS ED probe localization, platelet accumulation, and fibrin formation in microfluidic flow assays.Results: We find that the MARCKS ED probes can antagonize factor Xa from binding phosphatidylserine by measuring reduced association of factor Xa to lipid membranes treated with the MARCKS ED probes. Pre-treatment of activated platelets with the MARCKS ED probes significantly reduced the assembly and enzymatic activity of prothrombinase (100% activity with vehicle control vs. 51% with 1 μM MARCKS ED peptide treatment, P < 0.05). Similar inhibitory activity was also observed when activated platelets were replaced with other phosphatidylserine exposing lipid membranes, such as synthetic liposomes or biologic microparticles. In the whole blood microfluidic flow assay, the MARCKS ED probes colocalized with adherent platelets and inhibited fibrin formation (100% peak fibrin intensity with vehicle control vs. 17% peak fibrin intensity with 1 μM MARCKS ED peptide treatment, P < 0.05). The MARCKS ED probes did not alter platelet surface area coverage. Electron microscopy images of the final clots formed in the microfluidic flow assay show an absence of fibrin formation with MARCKS ED probe treatment.Conclusions: We demonstrate that the MARCKS ED probes can inhibit the assembly and enzymatic activity of prothrombinase, and that this activity translates to significantly reduced fibrin formation in whole blood. The MARCKS ED peptide and peptidomimetic probes may provide a new therapeutic tool for inhibiting the assembly of phosphatidylserine dependent coagulation factors, as well as a research tool for identifying platelet phosphatidylserine exposure. DisclosuresKastelowitz:University of Colorado Boulder: Patents & Royalties. Yin:University of Colorado Boulder: Patents & Royalties.

Abstract IA25: Mechanisms of protective tumor immunity

Abstract Efficacious cancer immunotherapies will likely require combinations of strategies that enhance tumor antigen presentation and antagonize negative immune regulatory circuits. We demonstrated that vaccination with irradiated, autologous melanoma cells engineered to secrete GM-CSF followed by antibody blockade of CTLA-4 accomplishes clinically significant tumor destruction with minimal toxicity in a majority of stage IV metastatic melanoma and some advanced ovarian carcinoma patients. The extent of tumor necrosis in post-treatment biopsies was linearly related to the natural logarithm of the ratio of infiltrating CD8+ effector T cells to FoxP3+ Tregs, suggesting that further Treg inhibition might increase the frequency of clinical responses. Through an analysis of cytokine deficient mice, we delineated a critical role for GM-CSF in Treg homeostasis. GM-CSF is required for the expression of the phosphatidylserine binding protein MFG-E8 in antigen presenting cells, whereas the uptake of apoptotic cells by phagocyte-derived MFG-E8 maintains peripheral Treg activity. The pharmacologic inhibition of MFG-E8 function through genetic or engineering-based approaches blocks Treg induction, which intensifies vaccine-induced responses, leading to the regression of established tumors in mice. The clinical translation of these therapeutic strategies to Phase I testing in humans is underway. The detailed analysis of patients achieving sustained clinical benefits from irradiated, autologous GM-CSF secreting tumor cell vaccines and CTLA-4 antibody blockade also affords a rich opportunity to identify antigens associated with immune-mediated tumor destruction and to delineate mechanisms of therapeutic immunity. In this regard, we have identified antibodies against a panel of secreted and cell surface proteins that play important roles in tumor development and immune evasion. Isolation of the rare, antigen-specific memory B cells directed towards these important targets provides new opportunities for developing human monoclonal antibodies for cancer immunotherapy. Citation Format: Glenn Dranoff. Mechanisms of protective tumor immunity. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr IA25.

Molecular imaging of cell death in tumors. Increasing annexin A5 size reduces contribution of phosphatidylserine-targeting function to tumor uptake.

ObjectiveAnnexin A5 is a phosphatidylserine binding protein that binds dying cells in vivo. Annexin A5 is a potential molecular imaging agent to determine efficacy of anti-cancer therapy in patients. Its rapid clearance from circulation limits tumor uptake and, hence, its sensitivity. The aim of this study is to determine if non-invasive imaging of cell death in tumors will benefit from increasing circulation time of annexin A5 by increasing its size.ProceduresAnnexin A5 size was increased by complexation of biotinylated annexin A5 with Alexa-Fluor680-labeled streptavidin. The non-binding variant of annexin A5, M1234, was used as negative control. The HT29 colon carcinoma xenograft model in NMRI nude mice was used to measure tumor uptake in vivo. Tumor uptake of fluorescent annexin A5-variants was measured using non-invasive optical imaging.ResultsThe annexin A5-streptavidin complex (4∶1, moles:moles, Mw ∼200 kDa) binds phosphatidylserine-expressing membranes with a Hill-coefficient of 5.7±0.5 for Ca2+-binding and an EC50 of 0.9±0.1 mM Ca2+ (EC50 is the Ca2+ concentration required for half maximal binding)(annexin A5: Hill-coefficient 3.9±0.2, EC50 1.5±0.2 mM Ca2+). Circulation half-life of annexin A5-streptavidin is ±21 minutes (circulation half-life of annexin A5 is ±4 min.). Tumor uptake of annexin A5-streptavidin was higher and persisted longer than annexin A5-uptake but depended less on phosphatidylserine binding.ConclusionIncreasing annexin A5 size prolongs circulation times and increases tumor uptake, but decreases contribution of PS-targeting to tumor uptake and abolishes power to report efficacy of therapy.

Role of Phosphatidylserine Receptors in Enveloped Virus Infection

We recently demonstrated that a soluble protein, Gas6, can facilitate viral entry by bridging viral envelope phosphatidylserine to Axl, a receptor tyrosine kinase expressed on target cells. The interaction between phosphatidylserine, Gas6, and Axl was originally shown to be a molecular mechanism through which phagocytes recognize phosphatidylserine exposed on dead cells. Since our initial report, several groups have confirmed that Axl/Gas6, as well as other phosphatidylserine receptors, facilitate entry of dengue, West Nile, and Ebola viruses. Virus binding by viral envelope phosphatidylserine is now a viral entry mechanism generalized to many families of viruses. In addition to Axl/Gas6, various molecules are known to recognize phosphatidylserine; however, the effects of these molecules on virus binding and entry have not been comprehensively evaluated and compared. In this study, we examined most of the known human phosphatidylserine-recognizing molecules, including MFG-E8, TIM-1, -3, and -4, CD300a, BAI1, and stabilin-1 and -2, for their abilities to facilitate virus binding and infection. Using pseudotyped lentiviral vectors, we found that a soluble phosphatidylserine-binding protein, MFG-E8, enhances transduction. Cell surface receptors TIM-1 and -4 also enhance virus binding/transduction. The extent of enhancement by these molecules varies, depending on the type of pseudotyping envelope proteins. Mutated MFG-E8, which binds viral envelope phosphatidylserine without bridging virus to cells, but, surprisingly, not annexin V, which has been used to block phagocytosis of dead cells by concealing phosphatidylserine, efficiently blocks these phosphatidylserine-dependent viral entry mechanisms. These results provide insight into understanding the role of viral envelope phosphatidylserine in viral infection. Envelope phosphatidylserine has previously been shown to be important for replication of various envelope viruses, but details of this mechanism(s) were unclear. We were the first to report that a bifunctional serum protein, Gas6, bridges envelope phosphatidylserine to a cell surface receptor, Axl. Recent studies demonstrated that many envelope viruses, including vaccinia, dengue, West Nile, and Ebola viruses, utilize Axl/Gas6 to facilitate their entry, suggesting that the phosphatidylserine-mediated viral entry mechanism can be shared by various enveloped viruses. In addition to Axl/Gas6, various molecules are known to recognize phosphatidylserine; however, the effects of these molecules on virus binding and entry have not been comprehensively evaluated and compared. In this study, we examined most human phosphatidylserine-recognizing molecules for their abilities to facilitate viral infection. The results provide insights into the role(s) of envelope phosphatidylserine in viral infection, which can be applicable to the development of novel antiviral reagents that block phosphatidylserine-mediated viral entry.

Dobutamine Stress–Induced Microparticle Release

Despite numerous advances in the diagnosis and treatment of cardiovascular disease (CVD), it remains a leading cause of morbidity and mortality in the developed world. CVD is the leading cause of death in the United States with total costs associated with CVD treatment reaching 17% of the national health expenditures.1 A substantial portion of the cost and time associated with CVD is in the administration of stress tests, including dobutamine stress echocardiogram (DSE), for noninvasive risk assessment of patients presenting with symptoms of obstructive coronary artery disease (CAD). Article, see p 109 Microparticles (MPs) are subcellular vesicles between 100 nm and 1 μm in diameter which are shed from the plasma membrane of activated or dying cells.2 Microparticles possess the procoagulant lipid phosphatidyl serine (PS) and cell surface proteins of their parent cells, which allow for efficient identification of cellular source via flow cytometry using fluorescently labeled annexin V, a PS binding protein, and receptor-specific antibodies.3 Microparticles contain cytokines and miRNA and act as local and systemic messengers. In addition, microparticle production is vital for removal of stressed-induced cellular by-products.4 Numerous studies have shown that microparticles activate and regulate many physiological and pathological processes.2,5 Microparticles likely play a significant role in CVD and CVD risk factors.3 Studies have shown increased microparticle levels in patients with diabetes mellitus and hypertension.6,7 Elevated microparticle levels have been correlated with a higher calculated 10-year Framingham CAD risk.8 Increases in microparticle levels have also been observed in patients with high risk coronary lesions and correlate …

Cooperative binding of Annexin A5 to phosphatidylserine on apoptotic cell membranes

Healthy cells exhibit an asymmetric plasma membrane with phosphatidylserine (PS) located on the cytoplasmic leaflet of the plasma membrane bilayer. Annexin A5-FITC, a PS binding protein, is commonly used to evaluate apoptosis in flow cytometry. PS exposed by apoptotic cells serves as a major ‘eat-me’ signal for phagocytes. Although exposition of PS has been observed after alternative stimuli, no clearance of viable, PS exposing cells has been detected. Thus, besides PS exposure, membranes of viable and apoptotic cells might exhibit specific characteristics. Here, we show that Annexin A5 binds in a cooperative manner to different types of dead cells. Shrunken apoptotic cells thereby showed the highest Hill coefficient values. Contrarily, parafomaldehyde fixation of apoptotic cells completely abrogates the cooperativity effect seen with dead and dying cells. We tend to speculate that the cooperative binding of Annexin A5 to the membranes of apoptotic cells reflects higher fluidity of the exposed membranes facilitating PS clustering.

Abstract PL07-03: Mechanisms of protective tumor immunity.

Abstract Efficacious cancer immunotherapies will likely require combinations of strategies that enhance tumor antigen presentation and antagonize negative immune regulatory circuits. We demonstrated that vaccination with irradiated, autologous melanoma cells engineered to secrete GM-CSF followed by antibody blockade of CTLA-4 accomplishes clinically significant tumor destruction with minimal toxicity in a majority of stage IV metastatic melanoma and some advanced ovarian carcinoma patients. The extent of tumor necrosis in post-treatment biopsies was linearly related to the natural logarithm of the ratio of infiltrating CD8+ effector T cells to FoxP3+ Tregs, suggesting that further Treg inhibition might increase the frequency of clinical responses. Through an analysis of cytokine deficient mice, we delineated a critical role for GM-CSF in Treg homeostasis. GM-CSF is required for the expression of the phosphatidylserine binding protein MFG-E8 in antigen presenting cells, whereas the uptake of apoptotic cells by phagocyte-derived MFG-E8 maintains peripheral Treg activity. The pharmacologic inhibition of MFG-E8 function through genetic or engineering-based approaches blocks Treg induction, which intensifies vaccine-induced responses, leading to the regression of established tumors in mice. The clinical translation of these therapeutic strategies to Phase I testing in humans is underway. The detailed analysis of patients achieving sustained clinical benefits from irradiated, autologous GM-CSF secreting tumor cell vaccines and CTLA-4 antibody blockade also affords a rich opportunity to identify antigens associated with immune-mediated tumor destruction and to delineate mechanisms of therapeutic immunity. We elucidated several of the molecular pathways that underlie these antitumor effects, including the NKG2D system and multiple secreted/or cell surface proteins that contribute to tumor promoting inflammation. The therapy-induced antibodies manifest functional activity in vitro, antagonizing tumor cell survival, invasive potential, and angiogenesis. These findings support a key role for humoral immunity in tumor destruction, and highlight interest in more detailed characterization of the antitumor B cell repertoire in vaccinated patients. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):PL07-03. Citation Format: Glenn Dranoff. Mechanisms of protective tumor immunity. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr PL07-03.

A putative link between phagocytosis-induced apoptosis and hemocyanin-derived phenoloxidase activation

Apoptosis and phagocytosis are crucial processes required for developmental morphogenesis, pathogen deterrence and immunomodulation in metazoans. We present data showing that amebocytes of the chelicerate, Limulus polyphemus, undergo phagocytosis-induced cell death after ingesting spores of the fungus, Beauveria bassiana, in vitro. The observed biochemical and morphological modifications associated with dying amebocytes are congruent with the hallmarks of apoptosis, including: extracellularisation of phosphatidylserine, intranucleosomal DNA fragmentation and an increase in caspase 3/7-like activities. Previous studies have demonstrated that phosphatidylserine is a putative endogenous activator of hemocyanin-derived phenoloxidase, inducing conformational changes that permit phenolic substrate access to the active site. Here, we observed extracellular hemocyanin-derived phenoloxidase activity levels increase in the presence of apoptotic amebocytes. Enzyme activity induced by phosphatidylserine or apoptotic amebocytes was reduced completely upon incubation with the phosphatidylserine binding protein, annexin V. We propose that phosphatidylserine redistributed to the outer plasma membrane of amebocytes undergoing phagocytosis-induced apoptosis could interact with hemocyanin, thus facilitating its conversion into a phenoloxidase-like enzyme, during immune challenge.

Milk fat globule-EGF factor 8 mediates the enhancement of apoptotic cell clearance by glucocorticoids

The phagocytic clearance of apoptotic cells is essential to prevent chronic inflammation and autoimmunity. The phosphatidylserine-binding protein milk fat globule-EGF factor 8 (MFG-E8) is a major opsonin for apoptotic cells, and MFG-E8(-/-) mice spontaneously develop a lupus-like disease. Similar to human systemic lupus erythematosus (SLE), the murine disease is associated with an impaired clearance of apoptotic cells. SLE is routinely treated with glucocorticoids (GCs), whose anti-inflammatory effects are consentaneously attributed to the transrepression of pro-inflammatory cytokines. Here, we show that the GC-mediated transactivation of MFG-E8 expression and the concomitantly enhanced elimination of apoptotic cells constitute a novel aspect in this context. Patients with chronic inflammation receiving high-dose prednisone therapy displayed substantially increased MFG-E8 mRNA levels in circulating monocytes. MFG-E8 induction was dependent on the GC receptor and several GC response elements within the MFG-E8 promoter. Most intriguingly, the inhibition of MFG-E8 induction by RNA interference or genetic knockout strongly reduced or completely abolished the phagocytosis-enhancing effect of GCs in vitro and in vivo. Thus, MFG-E8-dependent promotion of apoptotic cell clearance is a novel anti-inflammatory facet of GC treatment and renders MFG-E8 a prospective target for future therapeutic interventions in SLE.

Abstract SY27-04: Engineering improved cancer vaccines.

Abstract Efficacious cancer immunotherapies will likely require combinations of strategies that enhance tumor antigen presentation and antagonize negative immune regulatory circuits. We demonstrated that vaccination with irradiated, autologous melanoma cells engineered to secrete GM-CSF followed by antibody blockade of CTLA-4 accomplishes clinically significant tumor destruction with minimal toxicity in a majority of stage IV metastatic melanoma and some advanced ovarian carcinoma patients. The extent of tumor necrosis in post-treatment biopsies was linearly related to the natural logarithm of the ratio of infiltrating CD8+ effector T cells to FoxP3+ Tregs, suggesting that further Treg inhibition might increase the frequency of clinical responses. Through an analysis of cytokine deficient mice, we delineated a critical role for GM-CSF in Treg homeostasis. GM-CSF is required for the expression of the phosphatidylserine binding protein MFG-E8 in antigen presenting cells, whereas the uptake of apoptotic cells by phagocyte-derived MFG-E8 maintains peripheral Treg activity. The pharmacologic inhibition of MFG-E8 function through genetic or engineering-based approaches blocks Treg induction, which intensifies vaccine-induced responses, leading to the regression of established tumors in mice. The clinical translation of these therapeutic strategies to Phase I testing in humans is underway. The detailed analysis of patients achieving sustained clinical benefits from irradiated, autologous GM-CSF secreting tumor cell vaccines and CTLA-4 antibody blockade also affords a rich opportunity to identify antigens associated with immune-mediated tumor destruction and to delineate mechanisms of therapeutic immunity. We elucidated several of the molecular pathways that underlie these anti-tumor effects, including the NKG2D system and multiple secreted/or cell surface proteins that contribute to tumor promoting inflammation. The therapy-induced antibodies manifest functional activity in vitro, antagonizing tumor cell survival, invasive potential, and angiogenesis. These findings support a key role for humoral immunity in tumor destruction, and highlight interest in more detailed characterization of the anti-tumor B cell repertoire in vaccinated patients. Citation Format: Glenn Dranoff. Engineering improved cancer vaccines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr SY27-04. doi:10.1158/1538-7445.AM2013-SY27-04

Phosphatidylserine-binding protein lactadherin inhibits protein translocation across the ER membrane

Secretory and membrane proteins are translocated across and inserted into the endoplasmic reticulum membrane via translocon channels. To investigate the effect of the negatively-charged phospholipid phosphatidylserine on the translocation of nascent polypeptide chains through the translocon, we used the phosphatidylserine-binding protein lactadherin C2-domain. Lactadherin inhibited targeting of nascent chain to the translocon by signal sequence and the initiation of translocation. Moreover, lactadherin inhibited the movement of the translocating polypeptide chain regardless of the presence or absence of positively-charged residues. Phosphatidylserine might be critically involved in translocon function, but it is not a major determinant for translocation arrest of positively-charged residues.

Proteinase 3 (PR3) is a phosphatidylserine-binding protein that can bind microparticles: Relevance in the context of granulomatosis with polyangiitis (GPA)

Proteinase 3 (PR3) is a phosphatidylserine-binding protein that can bind microparticles: Relevance in the context of granulomatosis with polyangiitis (GPA)

Pyroptotic cells externalize eat-me and release find-me signals and are efficiently engulfed by macrophages

Pathogenic intracellular bacteria often hijack macrophages for their propagation. The infected macrophages release IL-1β and IL-18 and simultaneously commit suicide, which is called pyroptosis; both responses require caspase-1. Here, we found that pyroptotic cells induced by microbial infection were efficiently engulfed by human monocytic THP-1-cell-derived macrophages or mouse peritoneal macrophages. This engulfment was inhibited by the D89E mutant of milk fat globule (MFG) epidermal growth factor (EGF) factor 8 (MFG-E8; a phosphatidylserine-binding protein) that has been shown previously to inhibit phosphatidylserine-dependent engulfment of apoptotic cells by macrophages, suggesting that the engulfment of pyroptotic cells by macrophages was also phosphatidylserine dependent. Using a pair of cell lines that respectively exhibited pyroptosis or apoptosis after muramyl dipeptide treatment, we showed that both pyroptotic and apoptotic cells bound to a T-cell immunoglobulin and mucin domain-containing 4 (Tim4; another phosphatidylserine-binding protein)-coated plate, whereas heat-killed necrotic cells did not, indicating that phosphatidylserine was externalized in pyroptosis and apoptosis but not in accidental necrosis. Macrophages engulfed apoptotic cells most efficiently, followed by pyroptotic and then heat-killed necrotic cells. Pyroptotic cells also released a macrophage attractant(s), 'find-me' signal, whose activity was diminished by apyrase that degrades nucleoside triphosphate to nucleoside monophosphate. Heat-killed necrotic cells and pyroptotic cells released ATP much more efficiently than apoptotic cells. These results suggest that pyroptotic cells, like apoptotic cells, actively induce phagocytosis by macrophages using 'eat-me' and find-me signals. Based on these results, a possible role of coordinated induction of pyroptosis and inflammatory cytokine production is discussed.

Phosphatidylserine recognition and induction of apoptotic cell clearance by Drosophila engulfment receptor Draper

The membrane phospholipid phosphatidylserine is exposed on the cell surface during apoptosis and acts as an eat-me signal in the phagocytosis of apoptotic cells in mammals and nematodes. However, whether this is also true in insects was unclear. When milk fat globule-epidermal growth factor 8, a phosphatidylserine-binding protein of mammals, was ectopically expressed in Drosophila, the level of phagocytosis was reduced, whereas this was not the case for the same protein lacking a domain responsible for the binding to phosphatidylserine. We found that the extracellular region of Draper, an engulfment receptor of Drosophila, binds to phosphatidylserine in an enzyme-linked immunosorbent assay-like solid-phase assay and in an assay for surface plasmon resonance. A portion of Draper containing domains EMI and NIM located close to the N-terminus was required for binding to phosphatidylserine, and a Draper protein lacking this region was not active in Drosophila. Finally, the level of tyrosine-phosphorylated Draper, indicative of the activation of Draper, in a hemocyte-derived cell line was increased after treatment with phosphatidylserine-containing liposome. These results indicated that phosphatidylserine serves as an eat-me signal in the phagocytic removal of apoptotic cells in Drosophila and that Draper is a phosphatidylserine-binding receptor for phagocytosis.

Abstract IA8: The interplay of inflammation and cancer.

Abstract Efficacious cancer immunotherapies will likely require combinations of strategies that enhance tumor antigen presentation and antagonize negative immune regulatory circuits. We demonstrated that vaccination with irradiated, autologous melanoma cells engineered to secrete GM-CSF followed by antibody blockade of CTLA-4 accomplishes clinically significant tumor destruction with minimal toxicity in a majority of stage IV metastatic melanoma and some advanced ovarian carcinoma patients. The detailed analysis of patients achieving sustained clinical benefits from these therapies illustrated the importance of a coordinated cellular and humoral anti-tumor immune response. Indeed, the extent of tumor necrosis in post-treatment biopsies was linearly related to the natural logarithm of the ratio of infiltrating CD8+ effector T cells to FoxP3+ Tregs. Moreover, therapy induced a potent antibody response to several key tumor cell surface and secreted proteins; these antibodies manifest functional activity in vitro, antagonizing tumor cell survival, invasive potential, and angiogenesis. Through an analysis of cytokine deficient mice, we delineated a critical role for GM-CSF in Treg homeostasis. GM-CSF is required for the expression of the phosphatidylserine binding protein MFG-E8 in antigen presenting cells, whereas the uptake of apoptotic cells by phagocyte-derived MFG-E8 maintains peripheral Treg activity. While blockade of MFG-E8 can enhance therapeutic immunity in the context of cancer vaccines, loss of Treg homeostasis with MFG-E8 deficiency can lead to chronic inflammation. Mice deficient in GM-CSF and IFN-γ develop at high frequency germinal center derived lymphomas and nonsmall cell lung carcinomas. The pathogenesis of these tumors reflects a combination of tumor-promoting inflammation and a loss of protective tumor immunity. Taken together, these findings highlight dual roles for immunity in cancer pathogenesis and therapy. Citation Format: Glenn Dranoff. The interplay of inflammation and cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr IA8.

LRRK2 kinase inhibition prevents pathological microglial phagocytosis in response to HIV-1 Tat protein

BackgroundHuman Immunodeficiency Virus-1 (HIV-1) associated neurocognitive disorders (HANDs) are accompanied by significant morbidity, which persists despite the use of combined antiretroviral therapy (cART). While activated microglia play a role in pathogenesis, changes in their immune effector functions, including phagocytosis and proinflammatory signaling pathways, are not well understood. We have identified leucine-rich repeat kinase 2 (LRRK2) as a novel regulator of microglial phagocytosis and activation in an in vitro model of HANDs, and hypothesize that LRRK2 kinase inhibition will attenuate microglial activation during HANDs.MethodsWe treated BV-2 immortalized mouse microglia cells with the HIV-1 trans activator of transcription (Tat) protein in the absence or presence of LRRK2 kinase inhibitor (LRRK2i). We used Western blot, qRT-PCR, immunocytochemistry and latex bead engulfment assays to analyze LRRK2 protein levels, proinflammatory cytokine and phagocytosis receptor expression, LRRK2 cellular distribution and phagocytosis, respectively. Finally, we utilized ex vivo microfluidic chambers containing primary hippocampal neurons and BV-2 microglia cells to investigate microglial phagocytosis of neuronal axons.ResultsWe found that Tat-treatment of BV-2 cells induced kinase activity associated phosphorylation of serine 935 on LRRK2 and caused the formation of cytoplasmic LRRK2 inclusions. LRRK2i decreased Tat-induced phosphorylation of serine 935 on LRRK2 and inhibited the formation of Tat-induced cytoplasmic LRRK2 inclusions. LRRK2i also decreased Tat-induced process extension in BV-2 cells. Furthermore, LRRK2i attenuated Tat-induced cytokine expression and latex bead engulfment. We examined relevant cellular targets in microfluidic chambers and found that Tat-treated BV-2 microglia cells cleared axonal arbor and engulfed neuronal elements, whereas saline treated controls did not. LRRK2i was found to protect axons in the presence of Tat-activated microglia, as well as AnnexinV, a phosphatidylserine-binding protein. In addition, LRRK2i decreased brain-specific angiogenesis inhibitor 1 (BAI1) receptor expression on BV-2 cells after Tat-treatment, a key receptor in phosphatidylserine-mediated phagocytosis.ConclusionTaken together, these results implicate LRRK2 as a key player in microglial inflammation and, in particular, in the phagocytosis of neuronal elements. These studies show that LRRK2 kinase inhibition may prove an effective therapeutic strategy for HANDs, as well as other neuroinflammatory conditions.