TREML4 receptor regulates inflammation and innate immune cell death during polymicrobial sepsis.

Sepsis is a biphasic disease characterized by an acute inflammatory response, followed by a prolonged immunosuppressive phase. Therapies aimed at controlling inflammation help to reduce the time patients with sepsis spend in intensive care units, but they do not lead to a reduction in overall mortality. Recently, the focus has been on addressing the immunosuppressive phase, often caused by apoptosis of immune cells. However, molecular triggers of these events are not yet known. Using whole-genome CRISPR screening in mice, we identified a triggering receptor expressed on myeloid cells (TREM) family receptor, TREML4, as a key regulator of inflammation and immune cell death in sepsis. Genetic ablation of Treml4 in mice demonstrated that TREML4 regulates calcium homeostasis, the inflammatory cytokine response, myeloperoxidase activation, the endoplasmic reticulum stress response and apoptotic cell death in innate immune cells, leading to an overall increase in survival rate, both during the acute and chronic phases of polymicrobial sepsis.

An Ultrasound-Responsive Theranostic Cyclodextrin-Loaded Nanoparticle for Multimodal Imaging and Therapy for Atherosclerosis.

Atherosclerosis is a major cause of mortality and morbidity worldwide. Left undiagnosed and untreated, atherosclerotic plaques can rupture and cause cardiovascular complications such as myocardial infarction and stroke. Atherosclerotic plaques are composed of lipids, including oxidized low-density lipoproteins and cholesterol crystals, and immune cells, including macrophages. 2-Hydroxypropyl-beta-cyclodextrin (CD) is FDA-approved for capturing, solubilizing, and delivering lipophilic drugs in humans. It is also known to dissolve cholesterol crystals and decrease atherosclerotic plaque size. However, its low retention time necessitates high dosages for successful therapy. This study reports CD delivery via air-trapped polybutylcyanoacrylate nanoparticles (with diameters of 388 ± 34 nm) loaded with CD (CDNPs). The multimodal contrast ability of these nanoparticles after being loaded with IR780 dye in mice is demonstrated using ultrasound and near-infrared imaging. It is shown that CDNPs enhance the cellular uptake of CD in murine cells. In an ApoE-/- mouse model of atherosclerosis, treatment with CDNPs significantly improves the anti-atherosclerotic efficacy of CD. Ultrasound triggering further improves CD uptake, highlighting that CDNPs can be used for ultrasound imaging and ultrasound-responsive CD delivery. Thus, CDNPs represent a theranostic nanocarrier for potential application in patients with atherosclerosis.

Defensin-lipid interactions in membrane targeting: mechanisms of action and opportunities for the development of antimicrobial and anticancer therapeutics.

Defensins are a class of host defence peptides (HDPs) that often harbour antimicrobial and anticancer activities, making them attractive candidates as novel therapeutics. In comparison with current antimicrobial and cancer treatments, defensins uniquely target specific membrane lipids via mechanisms distinct from other HDPs. Therefore, defensins could be potentially developed as therapeutics with increased selectivity and reduced susceptibility to the resistance mechanisms of tumour cells and infectious pathogens. In this review, we highlight recent advances in defensin research with a particular focus on membrane lipid-targeting in cancer and infection settings. In doing so, we discuss strategies to harness lipid-binding defensins for anticancer and anti-infective therapies.

Novel Formulation of Undecylenic Acid induces Tumor Cell Apoptosis.

Undecylenic acid, a monounsaturated fatty acid, is currently in clinical use as a topical antifungal agent, however the potential for therapeutic application in other disease settings has not been investigated. In this study, we describe a novel platform for the solubilization of fatty acids using amino acids and utilize this approach to define a tumoricidal activity and underlying mechanism for undecylenic acid. We examined a novel formulation of undecylenic acid compounded with L-Arginine, called GS-1, that induced concentration-dependent tumor cell death, with undecylenic acid being the cytotoxic component. Further investigation revealed that GS-1-mediated cell death was caspase-dependent with a reduction in mitochondrial membrane potential, suggesting a pro-apoptotic mechanism of action. Additionally, GS-1 was found to localize intracellularly to lipid droplets. In contrast to previous studies where lipid droplets have been shown to be protective against fatty acid-induced cell death, we showed that lipid droplets could not protect against GS-1-induced cytotoxicity. We also found a role for Fatty Acid Transport Protein 2 (FATP2) in the uptake of this compound. Collectively, this study demonstrates that GS-1 has effective pro-apoptotic antitumor activity in vitro and, together with the novel platform of fatty acid solubilization, contributes to the re-emerging field of fatty acids as potential anti-cancer therapeutics.

The Heparanase Regulatory Network in Health and Disease.

The extracellular matrix (ECM) is a structural framework that has many important physiological functions which include maintaining tissue structure and integrity, serving as a barrier to invading pathogens, and acting as a reservoir for bioactive molecules. This cellular scaffold is made up of various types of macromolecules including heparan sulfate proteoglycans (HSPGs). HSPGs comprise a protein core linked to the complex glycosaminoglycan heparan sulfate (HS), the remodeling of which is important for many physiological processes such as wound healing as well as pathological processes including cancer metastasis. Turnover of HS is tightly regulated by a single enzyme capable of cleaving HS side chains: heparanase. Heparanase upregulation has been identified in many inflammatory diseases including atherosclerosis, fibrosis, and cancer, where it has been shown to play multiple roles in processes such as epithelial-mesenchymal transition, angiogenesis, and cancer metastasis. Heparanase expression and activity are tightly regulated. Understanding the regulation of heparanase and its downstream targets is attractive for the development of treatments for these diseases. This review provides a comprehensive overview of the regulators of heparanase as well as the enzyme's downstream gene and protein targets, and implications for the development of new therapeutic strategies.

Heparanase and the hallmarks of cancer.

Heparanase is the only mammalian enzyme that cleaves heparan sulphate, an important component of the extracellular matrix. This leads to the remodelling of the extracellular matrix, whilst liberating growth factors and cytokines bound to heparan sulphate. This in turn promotes both physiological and pathological processes such as angiogenesis, immune cell migration, inflammation, wound healing and metastasis. Furthermore, heparanase exhibits non-enzymatic actions in cell signalling and in regulating gene expression. Cancer is underpinned by key characteristic features that promote malignant growth and disease progression, collectively termed the 'hallmarks of cancer'. Essentially, all cancers examined to date have been reported to overexpress heparanase, leading to enhanced tumour growth and metastasis with concomitant poor patient survival. With its multiple roles within the tumour microenvironment, heparanase has been demonstrated to regulate each of these hallmark features, in turn highlighting the need for heparanase-targeted therapies. However, recent discoveries which demonstrated that heparanase can also regulate vital anti-tumour mechanisms have cast doubt on this approach. This review will explore the myriad ways by which heparanase functions as a key regulator of the hallmarks of cancer and will highlight its role as a major component within the tumour microenvironment. The dual role of heparanase within the tumour microenvironment, however, emphasises the need for further investigation into defining its precise mechanism of action in different cancer settings.

Structural and functional characterization of the membrane-permeabilizing activity of Nicotiana occidentalis defensin NoD173 and protein engineering to enhance oncolysis.

Defensins are an extensive family of host defense peptides found ubiquitously across plant and animal species. In addition to protecting against infection by pathogenic microorganisms, some defensins are selectively cytotoxic toward tumor cells. As such, defensins have attracted interest as potential antimicrobial and anticancer therapeutics. The mechanism of defensin action against microbes and tumor cells appears to be conserved and involves the targeting and disruption of cellular membranes. This has been best defined for plant defensins, which upon binding specific phospholipids, such as phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidic acid, form defensin-lipid oligomeric complexes that destabilize membranes, leading to cell lysis. In this study, to further define the anticancer and therapeutic properties of plant defensins, we have characterized a novel plant defensin, Nicotiana occidentalis defensin 173 (NoD173), from N. occidentalis. NoD173 at low micromolar concentrations selectively killed a panel of tumor cell lines over normal primary cells. To improve the anticancer activity of NoD173, we explored increasing cationicity by mutation, with NoD173 with the substitution of Q22 with lysine [NoD173(Q22K)], increasing the antitumor cell activity by 2-fold. NoD173 and the NoD173(Q22K) mutant exhibited only low levels of hemolytic activity, and both maintained activity against tumor cells in serum. The ability of NoD173 to inhibit solid tumor growth in vivo was tested in a mouse B16-F1 model, whereby injection of NoD173 into established subcutaneous tumors significantly inhibited tumor growth. Finally, we showed that NoD173 specifically targets PIP2 and determined by X-ray crystallography that a high-resolution structure of NoD173, which forms a conserved family-defining cysteine-stabilized-αß motif with a dimeric lipid-binding conformation, configured into an arch-shaped oligomer of 4 dimers. These data provide insights into the mechanism of how defensins target membranes to kill tumor cells and provide proof of concept that defensins are able to inhibit tumor growth in vivo.-Lay, F. T., Ryan, G. F., Caria, S., Phan, T. K., Veneer, P. K., White, J. A., Kvansakul, M., Hulett M. D. Structural and functional characterization of the membrane-permeabilizing activity of Nicotiana occidentalis defensin NoD173 and protein engineering to enhance oncolysis.

Endothelial cell apoptosis and the role of endothelial cell-derived extracellular vesicles in the progression of atherosclerosis.

To maintain physiological homeostasis, cell turnover occurs every day in the body via a form of programmed cell death called apoptosis. During apoptosis, cells undergo distinct morphological changes culminating in the disassembly of the dying cell into smaller fragments known as apoptotic bodies (ApoBDs). Dysregulation of apoptosis is associated with diseases including infection, cancer and atherosclerosis. Although the development of atherosclerosis is largely attributed to the accumulation of lipids and inflammatory debris in vessel walls, it is also associated with apoptosis of macrophages, smooth muscle cells (SMCs) and endothelial cells. During cellular activation and apoptosis, endothelial cells can release several types of membrane-bound extracellular vesicles (EVs) including exosomes, microvesicles (MVs)/microparticles and ApoBDs. Emerging evidence in the field suggests that these endothelial cell-derived EVs (EndoEVs) can contribute to intercellular communication during the development of atherosclerosis via the transfer of cellular contents such as protein and microRNA, which may prevent or promote disease progression depending on the context. This review provides an up-to-date overview of the known causes and consequences of endothelial cell death during atherosclerosis along with highlighting current methodological approaches to studying EndoEVs and the potential roles of EndoEVs in atherosclerosis development.

Heparanase: Cloning, Function and Regulation.

In 2019, we mark the 20th anniversary of the cloning of the human heparanase gene. Heparanase remains the only known enzyme to cleave heparan sulfate, which is an abundant component of the extracellular matrix. Thus, elucidating the mechanisms underlying heparanase expression and activity is critical to understanding its role in healthy and pathological settings. This chapter provides a historical account of the race to clone the human heparanase gene, describes the intracellular and extracellular function of the enzyme, and explores the various mechanisms regulating heparanase expression and activity at the gene, transcript, and protein level.

Correction to: Defining the role of cytoskeletal components in the formation of apoptopodia and apoptotic bodies during apoptosis.

The original version of the article unfortunately contained a typo in the fourth author name. The author name was incorrectly listed as Rochelle Tixeria. The correct name should be Rochelle Tixeira. The original article has been corrected.

Defining the role of cytoskeletal components in the formation of apoptopodia and apoptotic bodies during apoptosis.

During apoptosis, dying cells undergo dynamic morphological changes that ultimately lead to their disassembly into fragments called apoptotic bodies (ApoBDs). Reorganisation of the cytoskeletal structures is key in driving various apoptotic morphologies, including the loss of cell adhesion and membrane bleb formation. However, whether cytoskeletal components are also involved in morphological changes that occur later during apoptosis, such as the recently described generation of thin apoptotic membrane protrusions called apoptopodia and subsequent ApoBD formation, is not well defined. Through monitoring the progression of apoptosis by confocal microscopy, specifically focusing on the apoptopodia formation step, we characterised the presence of F-actin and microtubules in a subset of apoptopodia generated by T cells and monocytes. Interestingly, targeting actin polymerisation and microtubule assembly pharmacologically had no major effect on apoptopodia formation. These data demonstrate apoptopodia as a novel type of membrane protrusion that could be formed in the absence of actin polymerisation and microtubule assembly.

Binding of phosphatidic acid by NsD7 mediates the formation of helical defensin-lipid oligomeric assemblies and membrane permeabilization.

Defensins are cationic antimicrobial peptides that serve as important components of host innate immune defenses, often by targeting cell membranes of pathogens. Oligomerization of defensins has been linked to their antimicrobial activity; however, the molecular basis underpinning this process remains largely unclear. Here we show that the plant defensin NsD7 targets the phospholipid phosphatidic acid (PA) to form oligomeric complexes that permeabilize PA-containing membranes. The crystal structure of the NsD7-PA complex reveals a striking double helix of two right-handed coiled oligomeric defensin fibrils, the assembly of which is dependent upon the interaction with PA at the interface between NsD7 dimers. Using site-directed mutagenesis, we demonstrate that key residues in this PA-binding site are required for PA-mediated NsD7 oligomerization and coil formation, as well as permeabilization of PA-containing liposomes. These data suggest that multiple lipids can be targeted to induce oligomerization of defensins during membrane permeabilization and demonstrate the existence of a "phospholipid code" that identifies target membranes for defensin-mediated attack as part of a first line of defense across multiple species.

Importance of phosphoinositide binding by human ß-defensin 3 for Akt-dependent cytokine induction.

Host defense peptides (HDPs) are well-characterized for their antimicrobial activities but also variously display potent immunomodulatory effects. Human ß-defensin 3 (HBD-3) belongs to a well-known HDP family known as defensins and is able to induce leukocyte chemotactic recruitment, leukocyte activation/maturation, proinflammatory cytokine release, and co-stimulatory marker expression. HBD-3-stimulated cytokine induction is NF-κB-dependent and was initially suggested to act via G protein-coupled C-C chemokine receptor phospholipase C (PLC) and/or Toll-like receptor signaling. Subsequent pharmacological inhibition, however, revealed that NF-κB activation by HBD-3 is receptor-independent and instead involves the phosphoinositide 3-kinase (PI3K)-protein kinase B (Akt) pathway, the mechanism of which remains undetermined. Recently, we have shown that HBD-3 can enter mammalian cells and bind to inner membrane phosphoinositide 4,5-bisphosphate [PI(4,5)P2], an important second lipid messenger of PLC and PI3K-Akt pathways. In this study, we report that the interaction of HBD-3 with PI(4,5)P2 is important for PI3K-Akt-NF-κΒ-mediated induction of tumor necrosis factor and interleukin-6. These data provide insights into the mechanism of immunomodulation by HBD-3, and more generally, highlight the complex multifaceted signaling roles of HDPs in innate defense. Furthermore, it is suggested that the proposed mode of action may be conserved in other HDPs.

Tumor cell membrane-targeting cationic antimicrobial peptides: novel insights into mechanisms of action and therapeutic prospects.

There is an ongoing need for effective and targeted cancer treatments that can overcome the detrimental side effects presented by current treatment options. One class of novel anticancer molecules with therapeutic potential currently under investigation are cationic antimicrobial peptides (CAPs). CAPs are small innate immunity peptides found ubiquitously throughout nature that are typically membrane-active against a wide range of pathogenic microbes. A number of CAPs can also target mammalian cells and often display selective activity towards tumor cells, making them attractive candidates as novel anticancer agents warranting further investigation. This current and comprehensive review describes key examples of naturally occurring membrane-targeting CAPs and their modified derivatives that have demonstrated anticancer activity, across multiple species of origin and structural subfamilies. In addition, we address recent advances made in the field and the ongoing challenges faced in translating experimental findings into clinically relevant treatments.

Convergent evolution of defensin sequence, structure and function.

Defensins are a well-characterised group of small, disulphide-rich, cationic peptides that are produced by essentially all eukaryotes and are highly diverse in their sequences and structures. Most display broad range antimicrobial activity at low micromolar concentrations, whereas others have other diverse roles, including cell signalling (e.g. immune cell recruitment, self/non-self-recognition), ion channel perturbation, toxic functions, and enzyme inhibition. The defensins consist of two superfamilies, each derived from an independent evolutionary origin, which have subsequently undergone extensive divergent evolution in their sequence, structure and function. Referred to as the cis- and trans-defensin superfamilies, they are classified based on their secondary structure orientation, cysteine motifs and disulphide bond connectivities, tertiary structure similarities and precursor gene sequence. The utility of displaying loops on a stable, compact, disulphide-rich core has been exploited by evolution on multiple occasions. The defensin superfamilies represent a case where the ensuing convergent evolution of sequence, structure and function has been particularly extreme. Here, we discuss the extent, causes and significance of these convergent features, drawing examples from across the eukaryotes.

The plant defensin NaD1 introduces membrane disorder through a specific interaction with the lipid, phosphatidylinositol 4,5 bisphosphate.

Plant defensins interact with phospholipids in bilayers as part of their cytotoxic activity. Solanaceous class II defensins with the loop 5 sequence pattern "S-[KR]-[ILVQ]-[ILVQ]-[KR]-[KR]" interact with PI(4,5)P2. Here, the prototypical defensin of this class, NaD1, is used to characterise the biophysical interactions between these defensins and phospholipid bilayers. Binding of NaD1 to bilayers containing PI(4,5)P2 occurs rapidly and the interaction is very strong. Dual polarisation interferometry revealed that NaD1 does not dissociate from bilayers containing PI(4,5)P2. Binding of NaD1 to bilayers with or without PI(4,5)P2 induced disorder in the bilayer. However, permeabilisation assays revealed that NaD1 only permeabilised liposomes with PI(4,5)P2 in the bilayer, suggesting a role for this protein-lipid interaction in the plasma membrane permeabilising activity of this defensin. No defensins in the available databases have the PI(4,5)P2 binding sequence outside the solanaceous class II defensins, leading to the hypothesis that PI(4,5)P2 binding co-evolved with the C-terminal propeptide to protect the host cell against the effects of the tight binding of these defensins to their cognate lipid as they travel along the secretory pathway. This data has allowed us to develop a new model to explain how this class of defensins permeabilises plasma membranes to kill target cells.

The Defensins Consist of Two Independent, Convergent Protein Superfamilies.

The defensin and defensin-like proteins are an extensive group of small, cationic, disulfide-rich proteins found in animals, plants, and fungi and mostly perform roles in host defense. The term defensin was originally used for small mammalian proteins found in neutrophils and was subsequently applied to insect proteins and plant γ-thionins based on their perceived sequence and structural similarity. Defensins are often described as ancient innate immunity molecules and classified as a single superfamily and both sequence alignments and phylogenies have been constructed. Here, we present evidence that the defensins have not all evolved from a single ancestor. Instead, they consist of two analogous superfamilies, and extensive convergent evolution is the source of their similarities. Evidence of common origin necessarily gets weaker for distantly related genes, as is the case for defensins, which are both divergent and small. We show that similarities that have been used as evidence for common origin are all expected by chance in short, constrained, disulfide-rich proteins. Differences in tertiary structure, secondary structure order, and disulfide bond connectivity indicate convergence as the likely source of the similarity. We refer to the two evolutionarily independent groups as the cis-defensins and trans-defensins based on the orientation of the most conserved pair of disulfides.

Nicotiana alata Defensin Chimeras Reveal Differences in the Mechanism of Fungal and Tumor Cell Killing and an Enhanced Antifungal Variant.

The plant defensin NaD1 is a potent antifungal molecule that also targets tumor cells with a high efficiency. We examined the features of NaD1 that contribute to these two activities by producing a series of chimeras with NaD2, a defensin that has relatively poor activity against fungi and no activity against tumor cells. All plant defensins have a common tertiary structure known as a cysteine-stabilized α-ß motif which consists of an α helix and a triple-stranded ß-sheet stabilized by four disulfide bonds. The chimeras were produced by replacing loops 1 to 7, the sequences between each of the conserved cysteine residues on NaD1, with the corresponding loops from NaD2. The loop 5 swap replaced the sequence motif (SKILRR) that mediates tight binding with phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and is essential for the potent cytotoxic effect of NaD1 on tumor cells. Consistent with previous reports, there was a strong correlation between PI(4,5)P2 binding and the tumor cell killing activity of all of the chimeras. However, this correlation did not extend to antifungal activity. Some of the loop swap chimeras were efficient antifungal molecules, even though they bound poorly to PI(4,5)P2, suggesting that additional mechanisms operate against fungal cells. Unexpectedly, the loop 1B swap chimera was 10 times more active than NaD1 against filamentous fungi. This led to the conclusion that defensin loops have evolved as modular components that combine to make antifungal molecules with variable mechanisms of action and that artificial combinations of loops can increase antifungal activity compared to that of the natural variants.

The MS4A family: counting past 1, 2 and 3.

The MS4A (membrane-spanning 4-domain family, subfamily A) family of proteins contains some well-known members including MS4A1 (CD20), MS4A2 (FcɛRIß) and MS4A3 (HTm4). These three MS4A family members are expressed on the cell surface of specific leukocyte subsets and have been well characterized as having key roles in regulating cell activation, growth and development. However, beyond MS4A1-3 there are a large number of related molecules (18 to date in humans) where our understanding of their biological roles is at a relatively nascent stage. This review examines the larger MS4A family focusing on their structure, expression, regulation and characterized and/or emerging biological roles. Our own work on one family member MS4A8B, and its possible role in epithelial cell regulation, is also highlighted.

Access to the Parent Tetrakis(pyridine)gold(III) Trication, Facile Formation of Rare Au(III) Terminal Hydroxides, and Preliminary Studies of Biological Properties.

In this paper we report on the use of [NO][BF4] to access tricationic tetrakis(pyridine)gold(III) from Au powder, a species inaccessible using the more traditional (tetrahydrothiophene)AuCl route. It is then demonstrated that this family of compounds can be used to access new terminal Au(III) hydroxides, a challenging class of compounds, and the first crystallographically characterized examples employing bidentate ligands. Finally, preliminary biological studies indicate good activity for derivatives featuring polydentate ligands against the HeLa and PC3 cell lines but also strong inhibition of primary HUVEC cells.