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Here, we describe GS-9, a novel water-soluble fatty acid-based formulation comprising L-lysine and arachidonic acid, that we have shown to induce ferroptosis. GS-9 forms vesicle-like structures in solution and mediates lipid peroxidation, as evidenced by increased C11-BODIPY fluorescence and an accumulation of toxic malondialdehyde, a downstream product of lipid peroxidation. Ferroptosis inhibitors counteracted GS-9-induced cell death, whereas caspase 3 and 7 or MLKL knock-out cell lines are resistant to GS-9-induced cell death, eliminating other cell death processes such as apoptosis and necroptosis as the mechanism of action of GS-9. We also demonstrate that through their role of sequestering fatty acids, lipid droplets play a protective role against GS-9-induced ferroptosis, as inhibition of lipid droplet biogenesis enhanced GS-9 cytotoxicity. In addition, Fatty Acid Transport Protein 2 was implicated in GS-9 uptake. Overall, this study identifies and characterises the mechanism of GS-9 as a ferroptosis inducer. This formulation of arachidonic acid offers a novel tool for investigating and manipulating ferroptosis in various cellular and anti-cancer contexts. [ABSTRACT FROM AUTHOR]

Simple Summary: Heparanase (HPSE) has been demonstrated to enhance the progression and metastasis of solid tumours, leading to a poor clinical prognosis. However, robust genetic ablation animal models to investigate the role of HPSE in breast cancer have not been described. The aim of this study was to utilise an HPSE-deficient strain of the well-established MMTV-PyMT murine mammary carcinoma model (MMTV-PyMTxHPSE−/− mice) to investigate the role of HPSE in early establishment, progression, and metastasis of mammary tumours. Contrary to our current understanding, we observed that even though HPSE regulated tumour angiogenesis, the establishment, progression, and metastasis of mammary tumours in MMTV-PyMT animals were HPSE-independent. We further observed no compensation by matrix metalloproteinases in response to the lack of HPSE in MMTV-PyMTxHPSE−/− animals. These findings may have significant implications in the development and clinical utility of HPSE inhibitors. Breast cancer is the second most common human malignancy and is a major global health burden. Heparanase (HPSE) has been widely implicated in enhancing the development and progression of solid tumours, including breast cancer. In this study, the well-established spontaneous mammary tumour-developing MMTV-PyMT murine model was utilised to examine the role of HPSE in breast cancer establishment, progression, and metastasis. The use of HPSE-deficient MMTV-PyMT (MMTV-PyMTxHPSE−/−) mice addressed the lack of genetic ablation models to investigate the role of HPSE in mammary tumours. It was demonstrated that even though HPSE regulated mammary tumour angiogenesis, mammary tumour progression and metastasis were HPSE-independent. Furthermore, there was no evidence of compensatory action by matrix metalloproteinases (MMPs) in response to the lack of HPSE expression in the mammary tumours. These findings suggest that HPSE may not play a significant role in the mammary tumour development of MMTV-PyMT animals. Collectively, these observations may have implications in the clinical setting of breast cancer and therapy using HPSE inhibitors. [ABSTRACT FROM AUTHOR]

Herbicide resistance represents one of the biggest threats to our natural environment and agricultural sector. Thus, new herbicides are urgently needed to tackle the rise in herbicide-resistant weeds. Here, we employed a novel strategy to repurpose a 'failed' antibiotic into a new and target-specific herbicidal compound. Specifically, we identified an inhibitor of bacterial dihydrodipicolinate reductase (DHDPR), an enzyme involved in lysine biosynthesis in plants and bacteria, that exhibited no antibacterial activity but severely attenuated germination of the plant Arabidopsis thaliana. We confirmed that the inhibitor targets plant DHDPR orthologues in vitro, and exhibits no toxic effects against human cell lines. A series of analogues were then synthesised with improved efficacy in germination assays and against soil-grown A. thaliana. We also showed that our lead compound is the first lysine biosynthesis inhibitor with activity against both monocotyledonous and dicotyledonous weed species, by demonstrating its effectiveness at reducing the germination and growth of Lolium rigidum (rigid ryegrass) and Raphanus raphanistrum (wild radish). These results provide proof-of-concept that DHDPR inhibition may represent a much-needed new herbicide mode of action. Furthermore, this study exemplifies the untapped potential of repurposing 'failed' antibiotic scaffolds to fast-track the development of herbicide candidates targeting the respective plant enzymes. A repurposed 'failed' antibiotic, an inhibitor against bacterial dihydrodipicolinate reductase, exhibits effective herbicidal action against both monocotyledonous and dicotyledonous weed species. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Cardiovascular disease (CVD) is the leading cause of death and disability worldwide, and its management places a huge burden on healthcare systems through hospitalisation and treatment. Atherosclerosis is a chronic inflammatory disease of the arterial wall resulting in the formation of lipid-rich, fibrotic plaques under the subendothelium and is a key contributor to the development of CVD. As such, a detailed understanding of the mechanisms involved in the development of atherosclerosis is urgently required for more effective disease treatment and prevention strategies. Heparanase is the only mammalian enzyme known to cleave heparan sulfate of heparan sulfate proteoglycans, which is a key component of the extracellular matrix and basement membrane. By cleaving heparan sulfate, heparanase contributes to the regulation of numerous physiological and pathological processes such as wound healing, inflammation, tumour angiogenesis, and cell migration. Recent evidence suggests a multifactorial role for heparanase in atherosclerosis by promoting underlying inflammatory processes giving rise to plaque formation, as well as regulating lesion stability. This review provides an up-to-date overview of the role of heparanase in physiological and pathological processes with a focus on the emerging role of the enzyme in atherosclerosis. [ABSTRACT FROM AUTHOR]

The environmental control of microbial pathogens currently relies on compounds that do not exert long-lasting activity on surfaces, are impaired by soil, and contribute to the growing problem of antimicrobial resistance. This study presents the scientific development and characterization of GS-2, a novel, water-soluble ammonium carboxylate salt of capric acid and L-arginine that demonstrates activity against a range of bacteria (particularly Gram-negative bacteria), fungi, and viruses. In real-world surface testing, GS-2 was more effective than a benzalkonium chloride disinfectant at reducing the bacterial load on common touch-point surfaces in a high-traffic building (average 1.6 vs. 32.6 CFUs recovered from surfaces 90 min after application, respectively). Toxicology testing in rats confirmed GS-2 ingredients were rapidly cleared and posed no toxicities to humans or animals. To enhance the time-kill against Gram-positive bacteria, GS-2 was compounded at a specific ratio with a naturally occurring monoterpenoid, thymol, to produce a water-based antimicrobial solution. This GS-2 with thymol formulation could generate a bactericidal effect after five minutes of exposure and a viricidal effect after 10 min of exposure. Further testing of the GS-2 and thymol combination on glass slides demonstrated that the compound retained bactericidal activity for up to 60 days. Based on these results, GS-2 and GS-2 with thymol represent a novel antimicrobial solution that may have significant utility in the long-term reduction of environmental microbial pathogens in a variety of settings. [ABSTRACT FROM AUTHOR]

COVID-19 is primarily known as a respiratory disease caused by SARS-CoV-2. However, neurological symptoms such as memory loss, sensory confusion, severe headaches, and even stroke are reported in up to 30% of cases and can persist even after the infection is over (long COVID). These neurological symptoms are thought to be produced by the virus infecting the central nervous system, however we don't understand the molecular mechanisms triggering them. The neurological effects of COVID-19 share similarities to neurodegenerative diseases in which the presence of cytotoxic aggregated amyloid protein or peptides is a common feature. Following the hypothesis that some neurological symptoms of COVID-19 may also follow an amyloid etiology we identified two peptides from the SARS-CoV-2 proteome that self-assemble into amyloid assemblies. Furthermore, these amyloids were shown to be highly toxic to neuronal cells. We suggest that cytotoxic aggregates of SARS-CoV-2 proteins may trigger neurological symptoms in COVID-19. Here the authors report the formation of toxic clumps of protein, similar to amyloid assemblies found in Alzheimer's disease and suggest their possible role for some of the neurological symptoms of long-COVID. [ABSTRACT FROM AUTHOR]

Heparanase is the only mammalian enzyme capable of cleaving heparan sulfate, a glycosaminoglycan of the extracellular matrix and cell surfaces. Most immune cells express heparanase that contributes to a range of functions including cell migration and cytokine expression. Heparanase also promotes natural killer (NK) cell migration; however, its role in other NK cell functions remains to be defined. In this study, heparanase‐deficient (Hpse–/–) mice were used to assess the role of heparanase in NK cell cytotoxicity, activation, and cytokine production. Upon challenge with the immunostimulant polyinosinic:polycytidylic acid (poly(I:C)), NK cells isolated from Hpse–/– mice displayed impaired cytotoxicity against EO771.LMB cells and reduced levels of activation markers CD69 and NKG2D. However, in vitro cytokine stimulation of wild‐type and Hpse–/– NK cells resulted in similar CD69 and NKG2D expression, suggesting the impaired NK cell activation in Hpse–/– mice results from elements within the in vivo niche. NK cells are activated in vivo by dendritic cells (DCs) in response to poly(I:C). Poly(I:C)‐stimulated Hpse–/– bone marrow DCs (BMDCs) expressed less IL‐12, and when cultured with Hpse–/– NK cells, less MCP‐1 mRNA and protein was detected. Although cell‐cell contact is important for DC‐mediated NK cell activation, co‐cultures of Hpse–/– BMDCs and NK cells showed similar levels of contact to wild‐type cells, suggesting heparanase contributes to NK cell activation independently of cell‐cell contact with DCs. These observations define a role for heparanase in NK cell cytotoxicity and activation and have important implications for how heparanase inhibitors currently in clinical trials for metastatic cancer may impact NK cell immunosurveillance. [ABSTRACT FROM AUTHOR]

A family of eight rhenium(I) tricarbonyl complexes bearing pyridyl-imidazolylidene or bis-imidazolylidene ligands in combination with a series of N-acetyl amino acids ligands (glycine, isoleucine, and proline) and an acetate have been synthesised and characterised. These complexes are of interest as potential anticancer agents, where the oxygen bound carboxylate ligand can exchange with water giving rise to cytotoxic cationic complexes. The pseudo-first-order aquation rate constants for the complexes were evaluated using 1H NMR time-course experiments and for the complexes of the bis-imidazolylidene ligand the average k1 value was 6.22 × 10−5 s−1 while for the pyridyl-imidazolylidene ligand the aquation rate was slower with the average k1 value being 3.00 × 10−5 s−1. Cytotoxicity studies in three cancer cell lines (MDA-MB-231, PC3 and HEPG2) showed that the Re(I) complexes of the bis-imidazolylidene ligand were significantly more toxic than those of the pyridyl-imidazolylidene ligand. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Undoubtedly, machine learning will help revolutionize future HDP discovery, which should not only shed light upon HDP biology in understudied organisms (such as plant HDPs as argued by Petre), but also expand the HDP arsenal for therapeutic exploitation. Keywords: immunomodulation; antimicrobial peptides; host defense peptides; antibiotic resistance; innate immunity EN immunomodulation antimicrobial peptides host defense peptides antibiotic resistance innate immunity N.PAG N.PAG 4 03/03/21 20210225 NES 210225 Introduction The term "antimicrobial peptides" (AMPs) classically refers to naturally occurring molecules that are made by most living organisms as key components of innate immunity that help shape the composition of colonizing micrbiota and provide a first line of defense against invading microbial pathogens. Forsaking the classical term "antimicrobial peptides" and embracing "host defense peptides" can not only properly acknowledge the multi-functionality and complexity of HDPs, but may also imprint the currently underexplored activity landscapes and enshrouded therapeutic potential of HDPs. [Extracted from the article]

The innate immune system in mammals include pattern recognition receptors (PRRs), which initiate immune responses to microbial infection via several mechanisms. These PRRs include cell surface Toll-like receptors (TLRs) and cytosolic Nod-like receptors (NLRs) that recognizes extracellular and intracellular danger signals respectively. NLRs are poised to respond specifically to pathogens that access the host cell cytosol. The molecular mechanisms by which NLRs are activated to form inflammasomes and exert downstream inflammatory responses remain poorly understood. Additionally, very little is known about the regulation of cytosolic pathogen sensory NLR family members, except for NLRP3. Recently a deubiquitinase known as STAMBP has been implicated as a regulator of NLRP7 inflammasome assembly. We have investigated the role of STAMBP in regulation of other inflammasome components and its broader role in inflammation using genetic removal of STAMBP protein from cells using CRISPR/Cas9 gene editing and challenging these gene edited cells with an inflammatory stimuli. Our study demonstrated that STAMBP has a critical role in inflammation both in the context of NLR pathway, through NLRP stabilization and TLR pathway, through JNK signaling and downstream cytokine production. The findings indicate that STAMBP has a wider role in inflammation than previously thought to be the case. [ABSTRACT FROM AUTHOR]

The disassembly of apoptotic cells into small membrane-bound vesicles termed apoptotic bodies (ApoBDs) is a hallmark of apoptosis; however, the functional significance of this process is not well defined. We recently discovered a new membrane protrusion (termed beaded apoptopodia) generated by apoptotic monocytes which fragments to release an abundance of ApoBDs. To investigate the function of apoptotic monocyte disassembly, we used influenza A virus (IAV) infection as a proof-of-concept model, as IAV commonly infects monocytes in physiological settings. We show that ApoBDs generated from IAV-infected monocytes contained IAV mRNA, protein and virions and consequently, could facilitate viral propagation in vitro and in vivo, and induce a robust antiviral immune response. We also identified an antipsychotic, Haloperidol, as an unexpected inhibitor of monocyte cell disassembly which could impair ApoBD-mediated viral propagation under in vitro conditions. Together, this study reveals a previously unrecognised function of apoptotic monocyte disassembly in the pathogenesis of IAV infections. Atkin-Smith et al. study apoptotic bodies formed when Influenza A (IAV)-infected monocytes undergo apoptosis. They find that apoptotic bodies contain components of IAV and can contribute to IAV propagation in vitro and in mice, and stimulate an innate and adaptive immune response. This study suggests that apoptotic bodies may take part in virus propagation and/or clearance. [ABSTRACT FROM AUTHOR]

The innate immune system in mammals include pattern recognition receptors (PRRs), which initiate immune responses to microbial infection via several mechanisms. These PRRs include cell surface Toll-like receptors (TLRs) and cytosolic Nod-like receptors (NLRs) that recognizes extracellular and intracellular danger signals respectively. NLRs are poised to respond specifically to pathogens that access the host cell cytosol. The molecular mechanisms by which NLRs are activated to form inflammasomes and exert downstream inflammatory responses remain poorly understood. Additionally, very little is known about the regulation of cytosolic pathogen sensory NLR family members, except for NLRP3. Recently a deubiquitinase known as STAMBP has been implicated as a regulator of NLRP7 inflammasome assembly. We have investigated the role of STAMBP in regulation of other inflammasome components and its broader role in inflammation using genetic removal of STAMBP protein from cells using CRISPR/Cas9 gene editing and challenging these gene edited cells with an inflammatory stimuli. Our study demonstrated that STAMBP has a critical role in inflammation both in the context of NLR pathway, through NLRP stabilization and TLR pathway, through JNK signaling and downstream cytokine production. The findings indicate that STAMBP has a wider role in inflammation than previously thought to be the case. [ABSTRACT FROM AUTHOR]

Apoptosis is a form of programmed cell death that occurs throughout life as part of normal development as well as pathologic processes including chronic inflammation and infection. Although the death of a cell is often considered as the only biological outcome of a cell committed to apoptosis, it is becoming increasingly clear that the dying cell can actively communicate with other cells via soluble factors as well as membrane-bound extracellular vesicles (EVs) to regulate processes including cell clearance, immunity and tissue repair. Compared to EVs generated from viable cells such as exosomes and microvesicles, apoptotic cell-derived EVs (ApoEVs) are less well defined and the basic criteria for ApoEV characterization have not been established in the field. In this study, we will examine the current understanding of ApoEVs, in particular, the ApoEV subtype called apoptotic bodies (ApoBDs). We described that a subset of ApoBDs can be larger than 5 μm and smaller than 1 μm based on flow cytometry and live time-lapse microscopy analysis, respectively. We also described that a subset of ApoBDs can expose a relatively low level of phosphatidylserine on its surface based on annexin A5 staining. Furthermore, we characterized the presence of caspase-cleaved proteins (in particular plasma membrane-associated or cytoplasmic proteins) in samples enriched in ApoBDs. Lastly, using a combination of biochemical-, live imaging- and flow cytometry-based approaches, we characterized the progressive lysis of ApoBDs. Taken together, these results extended our understanding of ApoBDs. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

A series of imidazolium salt, N-heterocyclic carbene (NHC) ligand precursors, combined with 1,2,3-triazoles were synthesized using the Cu(i) catalyzed azide–alkyne cycloaddition ‘click reaction’. These pro-ligands were prepared from imidazolium molecules that were functionalized with either a terminal alkyne or azide group. Methylation of the triazole unit with methyl iodide produced a series of imidazolium/triazolium NHC pro-ligands. From these pro-ligands a family of luminescent Ir(iii) complexes of the general form [Ir(ppy)2(C^N)]+ or [Ir(ppy)2(C^C)]+ (where ppy is 2-phenylpyridine and C^N represents a bidentate imidazolylidene/triazole ligand and C^C represents a bidentate imidazolylidene/triazolylidene ligand) were prepared. The electrochemical, photophysical and electrochemiluminescence properties of the complexes have been evaluated and a preliminary study of two of these compounds as luminescent probes for cell imaging studies was conducted in A549 human lung adenocarcinoma basal epithelial cells. Co-localisation studies with the commercial dye MitoTracker CMXRos were consistent with mitochondrial uptake for these compounds. [ABSTRACT FROM AUTHOR]

Heparanase is a β-D-endoglucuronidase that cleaves heparan sulfate, a complex glycosaminoglycan found ubiquitously throughout mammalian cells and tissues. Heparanase has been strongly associated with important pathological processes including inflammatory disease and tumor metastasis, through its ability to promote various cellular functions such as cell migration, invasion, adhesion, and cytokine release. A number of cell types express heparanase including leukocytes, cells of the vasculature as well as tumor cells. However, the relative contribution of heparanase from these different cell sources to these processes is poorly defined. It is now well-established that the immune system plays a critical role in shaping tumor progression. Intriguingly, leukocyte-derived heparanase has been shown to either assist or impede tumor progression, depending on the setting. This review covers our current knowledge of heparanase in immune regulation of tumor progression, as well as the potential applications and implications of exploiting or inhibiting heparanase in cancer therapy. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Defensins are cationic antimicrobial peptides expressed throughout the plant and animal kingdoms as a first line of defense against pathogens. Membrane targeting and disruption is a crucial function of many defensins, however the precise mechanism remains unclear. Certain plant defensins form dimers that specifically bind the membrane phospholipids phosphatidic acid (PA) and phosphatidylinositol 4,5-bisphosphate, thereby triggering the assembly of defensin-lipid oligomers that permeabilize cell membranes. To understand this permeabilization mechanism, here we determine the crystal structure of the plant defensin NaD1 bound to PA. The structure reveals a 20-mer that adopts a concave sheet- or carpet-like topology where NaD1 dimers form one face and PA acyl chains form the other face of the sheet. Furthermore, we show that Arg39 is critical for PA binding, oligomerization and fungal cell killing. These findings identify a putative defensin-phospholipid membrane attack configuration that supports a longstanding proposed carpet mode of membrane disruption. [ABSTRACT FROM AUTHOR]

Abstract: Host defense peptides (HDPs) are well‐characterized for their antimicrobial activities but also variously display potent immunomodulatory effects. Human β‐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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Programmed cell death or apoptosis is an important component of host defense systems against viral infection. The B-cell lymphoma 2 (Bcl-2) proteins family is the main arbiter of mitochondrially mediated apoptosis, and viruses have evolved sequence and structural mimics of Bcl-2 to subvert premature host cell apoptosis in response to viral infection. The sequencing of the canarypox virus genome identified a putative pro-survival Bcl-2 protein, CNP058. However, a role in apoptosis inhibition for CNP058 has not been identified to date. Here, we report that CNP058 is able to bind several host cell pro-death Bcl-2 proteins, including Bak and Bax, as well as several BH3 only-proteins including Bim, Bid, Bmf, Noxa, Puma, and Hrk with high to moderate affinities. We then defined the structural basis for CNP058 binding to pro-death Bcl-2 proteins by determining the crystal structure of CNP058 bound to Bim BH3. CNP058 adopts the conserved Bcl-2 like fold observed in cellular pro-survival Bcl-2 proteins, and utilizes the canonical ligand binding groove to bind Bim BH3. We then demonstrate that CNP058 is a potent inhibitor of ultraviolet (UV) induced apoptosis in a cell culture model. Our findings suggest that CNP058 is a potent inhibitor of apoptosis that is able to bind to BH3 domain peptides from a broad range of pro-death Bcl-2 proteins, and may play a key role in countering premature host apoptosis. [ABSTRACT FROM AUTHOR]

Defensins are innate immune molecules that upon recognition of specific phospholipids can disrupt microbial membranes by forming oligomeric assemblies. Structures of two related plant defensins, NaD1 and NsD7, bound to phosphatidylinositol 4,5-bisphosphate ( PIP2) and phosphatidic acid ( PA), respectively, revealed striking differences in their oligomeric topologies. To understand how NsD7 binds different phospholipids and rationalize the different topologies, we determined the structure of an NsD7- PIP2 complex. This structure reveals fundamental differences in phospholipid binding compared to NsD7- PA, and an oligomeric topology nearly identical to the previously determined NaD1- PIP2 complex, establishing that the PIP2 fibril topology is conserved between NaD1 and NsD7. Our findings highlight the remarkable ability of defensins to bind different types of phospholipids to form oligomeric fibrils with diverse topologies. [ABSTRACT FROM AUTHOR]

Divalent metal ions are essential nutrients for all living organisms and are commonly protein-bound where they perform important roles in protein structure and function. This regulatory control from metals is observed in the relatively abundant plasma protein histidine-rich glycoprotein (HRG), which displays preferential binding to the second most abundant transition element in human systems, Zinc (Zn2+). HRG has been proposed to interact with a large number of protein ligands and has been implicated in the regulation of various physiological and pathological processes including the formation of immune complexes, apoptotic/necrotic and pathogen clearance, cell adhesion, antimicrobial activity, angiogenesis, coagulation and fibrinolysis. Interestingly, these processes are often associated with sites of tissue injury or tumour growth, where the concentration and distribution of Zn2+ is known to vary. Changes in Zn2+ levels have been shown to modify HRG function by altering its affinity for certain ligands and/or providing protection against proteolytic disassembly by serine proteases. This review focuses on the molecular interplay between HRG and Zn2+, and how Zn2+ binding modifies HRG-ligand interactions to regulate function in different settings of tissue injury. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The serum protein histidine-rich glycoprotein ( HRG) has been implicated in tissue injury and tumour growth. Several HRG functions are regulated by the divalent metal Zn2+, including ligand binding and proteolytic processing that releases active HRG fragments. Although HRG can bind divalent metals other than Zn2+, the impact of these divalent metals on the biophysical properties of HRG remains poorly understood. We now show that HRG binds Zn2+, Ni2+, Cu2+ and Co2+ with micromolar affinities, but differing stoichiometries, and regulate the release of specific HRG fragments during proteolysis. Furthermore, HRG binding to Zn2+ promotes HRG dimer formation in a Zn2+-concentration- and pH-dependent manner. Our data highlight the complex divalent metal-dependent regulatory mechanisms that govern HRG function. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 c-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, disulfiderich 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 cisdefensins and trans-defensins based on the orientation of the most conserved pair of disulfides. [ABSTRACT FROM AUTHOR]

The MS4A (membrane-spanning 4-domain family, subfamily A) family of proteins contains some well-known members including MS4A1 (CD20), MS4A2 (FcεRIβ) 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. [ABSTRACT FROM AUTHOR]

Defensins are a class of ubiquitously expressed cationic antimicrobial peptides (CAPs) that play an important role in innate defense. Plant defensins are active against a broad range of microbial pathogens and act via multiple mechanisms, including cell membrane permeabilization. The cytolytic activity of defensins has been proposed to involve interaction with specific lipid components in the target cell wall or membrane and defensin oligomerization. Indeed, the defensin Nicotiana alata defensin 1 (NaD1) binds to a broad range of membrane phosphatidylinositol phosphates and forms an oligomeric complex with phosphatidylinositol (4,5)-bisphosphate (PIP2) that facilitates membrane lysis of both mammalian tumor and fungal cells. Here, we report that the tomato defensin TPP3 has a unique lipid binding profile that is specific for PIP2 with which it forms an oligomeric complex that is critical for cytolytic activity. Structural characterization of TPP3 by X-ray crystallography and site-directed mutagenesis demonstrated that it forms a dimer in a "cationic grip" conformation that specifically accommodates the head group of PIP2 to mediate cooperative higher-order oligomerization and subsequent membrane permeabilization. These findings suggest that certain plant defensins are innate immune receptors for phospholipids and adopt conserved dimeric configurations to mediate PIP2 binding and membrane permeabilization. This mechanism of innate defense may be conserved across defensins from different species. [ABSTRACT FROM AUTHOR]

Heparanase is a β-D-endoglucuronidase that cleaves heparan sulfate (HS), facilitating degradation of the extracellular matrix (ECM) and the release of HS-bound biomolecules including cytokines. The remodeling of the ECM by heparanase is important for various physiological and pathological processes, including inflammation, wound healing, tumour angiogenesis and metastasis. Although heparanase has been proposed to facilitate leukocyte migration through degradation of the ECM, its role in inflammation by regulating the expression and release of cytokines has not been fully defined. In this study, the role of heparanase in regulating the expression and release of cytokines from human and murine immune cells was examined. Human peripheral blood mononuclear cells treated ex vivo with heparanase resulted in the release of a range of pro-inflammatory cytokines including IL-1β, IL-6, IL-8, IL-10 and TNF. In addition, mouse splenocytes treated ex vivo with heparanase resulted in the release of IL-6, MCP-1 and TNF. A similar pattern of cytokine release was also observed when cells were treated with soluble HS. Furthermore, heparanase-induced cytokine release was abolished by enzymatic-inhibitors of heparanase, suggesting this process is mediated via the enzymatic release of cell surface HS fragments. As soluble HS can signal through the Toll-like receptor (TLR) pathway, heparanase may promote the upregulation of cytokines through the generation of heparanase-cleaved fragments of HS. In support of this hypothesis, mouse spleen cells lacking the key TLR adaptor molecule MyD88 demonstrated an abolition of cytokine release after heparanase stimulation. Furthermore, TLR4-deficient spleen cells showed reduced cytokine release in response to heparanase treatment, suggesting that TLR4 is involved in this response. Consistent with these observations, the pathway involved in cytokine upregulation was identified as being NF-κB-dependent. These data identify a new mechanism for heparanase in promoting the release of pro-inflammatory cytokines that is likely to be important in regulating cell migration and inflammation. [ABSTRACT FROM AUTHOR]

Heparanase is a β- d-endoglucuronidase that cleaves heparan sulphate, a key component of the ECM and basement membrane. The remodelling of the ECM by heparanase has been proposed to regulate both normal physiological and pathological processes, including wound healing, inflammation, tumour angiogenesis and cell migration. Heparanase is also known to exhibit non-enzymatic functions by regulating cell adhesion, cell signalling and differentiation. In this study, constitutive heparanase-deficient (Hpse−/−) mice were generated on a C57BL/6 background using the Cre/loxP recombination system, with a complete lack of heparanase mRNA, protein and activity. Although heparanase has been implicated in embryogenesis and development, Hpse−/− mice are anatomically normal and fertile. Interestingly, consistent with the suggested function of heparanase in cell migration, the trafficking of dendritic cells from the skin to the draining lymph nodes was markedly reduced in Hpse−/− mice. Furthermore, the ability of Hpse−/− mice to generate an allergic inflammatory response in the airways, a process that requires dendritic cell migration, was also impaired. These findings establish an important role for heparanase in immunity and identify the enzyme as a potential target for regulation of an immune response. [ABSTRACT FROM AUTHOR]

Histidine-rich glycoprotein (HRG) is a relatively abundant plasma protein that has been implicated in multiple biological processes including immunity, tumor progression, and vascular biology. However, current protocols for purifying HRG from plasma result in the copurification of contaminating proteins and raise questions over the validity of biological activities ascribed to HRG. In this study, we describe a two-step protocol for the large-scale purification of HRG from human plasma using a combination of metal affinity and ion exchange chromatography. The protocol employs a rapid and simple strategy to isolate highly purified HRG that minimizes proteolytic cleavage of the protein. The purification of HRG was assessed at each stage by measuring the amount of HRG immunoreactive protein using a specific monoclonal antibody against total protein, and demonstrated ∼1,000-fold purification with an overall yield of ∼32%. Mass spectrometry analysis demonstrated that plasma-derived HRG was free of contaminating proteins and gel electrophoresis showed it to have minimal proteolytic degradation. Characterization of protein by physical method showed that the protein exists as a single, monodisperse species. In contrast to the previous studies of HRG purified by different methods, HRG purified using the new procedure demonstrated a reduced profile of functions. Although the HRG retained binding to heparin and phosphatidic acid, it did not interact with necrotic cells or other cellular lipids. These data demonstrate that HRG does not exhibit the broad interactive properties that have been reported previously, suggesting that copurification of HRG-binding partners or other impurities are responsible for some of the reported functional properties. The findings in this study demonstrate that the new purification procedure can provide a ready source of pure HRG to assess ligand specificity and biological function of this important plasma protein. ©2013 IUBMB Life, 65(6):550-563, 2013 [ABSTRACT FROM AUTHOR]

The methylation of histones is a fundamental epigenetic process regulating gene expression programs in mammalian cells. Dysregulated patterns of histone methylation are directly implicated in malignant transformation. Here, we report the unexpected finding that the invasive extracellular matrix degrading endoglycosidase heparanase enters the nucleus of activated human T lymphocytes and regulates the transcription of a cohort of inducible immune response genes by controlling histone H3 methylation patterns. It was found that nuclear heparanase preferentially associates with euchromatin. Genome-wide ChIP-on-chip analyses showed that heparanase is recruited to both the promoter and transcribed regions of a distinct cohort of transcriptionally active genes. Knockdown and overexpression of the heparanase gene also showed that chromatin-bound heparanase is a prerequisite for the transcription of a subset of inducible immune response genes in activated T cells. Furthermore, the actions of heparanase seem to influence gene transcription by associating with the demethylase LSD1, preventing recruitment of the methylase MLL and thereby modifying histone H3 methylation patterns. These data indicate that heparanase belongs to an emerging class of proteins that play an important role in regulating transcription in addition to their well-recognized extra-nuclear functions. [ABSTRACT FROM AUTHOR]

Defensins form an integral part of the cationic host defence peptide (HDP) family, a key component of innate immunity. Apart from their antimicrobial and immunomodulatory activities, many HDPs exert multifaceted effects on tumour cells, notably direct oncolysis and/or inhibition of tumour cell migration. Therefore, HDPs have been explored as promising anticancer therapeutics. Human β-defensin 2 (HBD-2) represents a prominent member of human HDPs, being well-characterised for its potent pathogen-killing, wound-healing, cytokine-inducing and leukocyte-chemoattracting functions. However, its anticancer effects remain largely unknown. Recently, we demonstrated that HBD-2 binds strongly to phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2), a key mediator of defensin-induced cell death and an instructional messenger during cell migration. Hence, in this study, we sought to investigate the lytic and anti-migratory effects of HBD-2 on tumour cells. Using various cell biological assays and confocal microscopy, we showed that HBD-2 killed tumour cells via acute lytic cell death rather than apoptosis. In addition, our data suggested that, despite the reported PI(4,5)P2 interaction, HBD-2 does not affect cytoskeletal-dependent tumour cell migration. Together, our findings provide further insights into defensin biology and informs future defensin-based drug development. [ABSTRACT FROM AUTHOR]

The B cell antigen receptor (BCR) efficiently facilitates the capture and processing of a specific antigen for presentation on MHC class II molecules to antigen-specific CD4+ T cells (1). Despite this, the majority of B cells are thought to play only a limited role in CD4+ T cell activation because BCRs are clonotypically expressed. Here, we show, however, that activated B cells can, both in vitro and in vivo, rapidly donate their BCR to bystander B cells, a process that is mediated by direct membrane transfer between adjacent B cells and is amplified by the interaction of the BCR with a specific antigen. This results in a dramatic expansion in the number of antigen-binding B cells in vivo, with the transferred BCR endowing recipient B cells with the ability to present a specific antigen to antigen-specific CD4+ T cells. [ABSTRACT FROM AUTHOR]

Heparanase (HPSE) is an endoglycosidase that cleaves heparan sulfate (HS) and plays an important role in tumor metastasis, angiogenesis and inflammation. The regulation of HPSE expression and function is tightly controlled and the increasing use of the mouse as an animal model to define the role of HPSE in many physiological and pathological settings, makes understanding the regulatory mechanisms of HPSE in this species of fundamental importance. However, the expression distribution of the mouse Hpse gene and the mechanisms that regulate its transcription are poorly defined. In this study, the mouse Hpse gene was determined to encode for two mRNA transcripts of 1.9 and 3.2 kb in length with identical open reading frames that showed similar tissue expression distribution to the human HPSE. The mouse Hpse promoter was cloned and a 478-bp minimal promoter was identified that contained regulatory elements responsible for both basal promoter activity in mouse tumor cells as well as inducible activity in T cells. Mutagenesis and transactivation studies identified a functional site in the minimal promoter region for the transcription factor Early growth response gene 1 (Egr1). Interestingly, Egr1 acted differentially in mouse tumor cells, functioning in an activating or repressive manner in breast carcinoma or melanoma cells, respectively. Furthermore, the proximal region of the promoter, identified as important in the regulation of Hpse transcription, was shown to become accessible in T cells upon cell activation. Significantly, the maximal accessibility of the promoter occurred at 16 h post-stimulation, which correlated with the induction kinetics of Hpse mRNA expression. In summary, this study demonstrates that mouse Hpse is expressed and regulated in a similar manner to human HPSE and also provides some novel insights into mechanisms of Hpse gene regulation that are likely to be relevant to control of the human gene.Immunology and Cell Biology (2007) 85, 205–214. doi:10.1038/sj.icb.7100022; published online 9 January 2007 [ABSTRACT FROM AUTHOR]

Summary Haematopoietic cell-specific transmembrane-4 (HTm4) is a four-transmembrane protein most closely related to CD20 and the beta subunit of the high affinity receptor for IgE (FcℇRIβ). To date, it has only been described in humans, where it is expressed in haematopoietic cells of both myeloid and lymphoid lineages. The function of HTm4 is unknown; however, as for CD20 and FcℇRI-β, it is likely to play a role in signal transduction as part of a multi-subunit cell surface receptor complex. In this study, we report the cDNA cloning and expression distribution of mouse HTm4. The deduced mouse HTm4 protein is of 213 amino acids, and contains four putative transmembrane domains. Mouse HTm4 shows 62% overall amino acid identity with human HTm4; the transmembrane regions are highly conserved between both species (75% identity), whereas the N- and C-terminal and inter-transmembrane loop regions are more divergent (52%). Interestingly, the N-terminal domain of mouse HTm4 is predicted to be 23 amino acids shorter, and the C-terminal domain 23 amino acids longer, than that of human HTm4. Northern blot and reverse transcriptase (RT)-PCR analysis suggest that mouse HTm4 mRNA is expressed at low levels only in spleen, bone marrow and peripheral blood leucocytes. This is the first report of the cloning of HTm4 from a species other than human, and provides important sequence information towards the understanding of the function of this poorly characterized four-transmembrane molecule. [ABSTRACT FROM AUTHOR]

Histidine-rich glycoprotein (HRG) is a plasma protein of vertebrates that has been implicated in the regulation of several important biological functions, including the immune response and blood clotting. In the present study, we have isolated and determined the sequence of the cDNAs for both mouse and rat HRG. The deduced amino acid sequences of mouse and rat HRG are 525 and 510 amino acids, respectively, and they show the same three-domain structure that has been predicted for human HRG, with which they share high amino acid identity. Northern blot analysis indicates that the mouse HRG mRNA is 1.7 kb and is localized specifically to the liver. It has been suggested, somewhat controversially, that some immune cells, such as monocytes and megakaryocytes, also synthesize HRG. Reverse transcriptase-polymerase chain reaction analysis has failed to show any HRG mRNA in immune tissues of the mouse, including the spleen, thymus, lymph node, bone marrow and peripheral blood leucocytes. These data suggest that HRG expression by immune cells is due to the acquisition ofplasma HRG derived from the liver. Finally, genomic Southern blot analysis of the mouse HRG gene suggests that it is a single copy gene. Summary Histidine-rich glycoprotein (HRG) is a plasma protein of vertebrates that has been implicated in the regulation of several important biological functions, including the immune response and blood clotting. In the present study, we have isolated and determined the sequence of the cDNAs for both mouse and rat HRG. The deduced amino acid sequences of mouse and rat HRG are 525 and 510 amino acids, respectively, and they show the same three-domain structure that has been predicted for human HRG, with which they share high amino acid identity. Northern blot analysis indicates that the mouse HRG mRNA is 1.7 kb and is localized specifically to the liver. It has been suggested, somewhat controversially, that some immune cells, such as monocytes and megakaryocytes, als... [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]