Detection and Isolation of Apoptotic Bodies to High Purity

Apoptotic bodies (ApoBDs), microvesicles and exosomes are the key members of the extracellular vesicle family, with ApoBDs being one of the largest type. ...

La Trobe University

Cationic antimicrobial peptides (CAPs) such as defensins are ubiquitously found innate immune molecules that often exhibit broad activity against microbial pathogens and mammalian tumor cells. Many CAPs act at the plasma membrane of cells leading to membrane destabilization and permeabilization. In this study, we describe a novel cell lysis mechanism for fungal and tumor cells by the plant defensin NaD1 that acts via direct binding to the plasma membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2). We determined the crystal structure of a NaD1:PIP2 complex, revealing a striking oligomeric arrangement comprising seven dimers of NaD1 that cooperatively bind the anionic headgroups of 14 PIP2 molecules through a unique 'cationic grip' configuration. Site-directed mutagenesis of NaD1 confirms that PIP2-mediated oligomerization is important for fungal and tumor cell permeabilization. These observations identify an innate recognition system by NaD1 for direct binding of PIP2 that permeabilizes cells via a novel membrane disrupting mechanism. DOI: http://dx.doi.org/10.7554/eLife.01808.001. 

Phosphoinositide-mediated oligomerization of a defensin induces cell lysis.

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. 

The Tomato Defensin TPP3 Binds Phosphatidylinositol (4,5)-Bisphosphate via a Conserved Dimeric Cationic Grip Conformation To Mediate Cell Lysis.

Cationic antimicrobial peptides (CAPs), including taxonomically diverse defensins, are innate defense molecules that display potent antimicrobial and immunomodulatory activities. Specific CAPs have also been shown to possess anticancer activities; however, their mechanisms of action are not well defined. Recently, the plant defensin NaD1 was shown to induce tumour cell lysis by directly binding to the plasma membrane phosphoinositide, phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). The NaD1-lipid interaction was structurally defined by X-ray crystallography, with the defensin forming a dimer that binds PI(4,5)P2 via its cationic β2-β3 loops in a 'cationic grip' conformation. In this study, we show that human β-defensin 3 (HBD-3) contains a homologous β2-β3 loop that binds phosphoinositides. The binding of HBD-3 to PI(4,5)P2 was shown to be critical for mediating cytolysis of tumour cells, suggesting a conserved mechanism of action for defensins across diverse species. These data not only identify an evolutionary conservation of CAP structure and function for lipid binding, but also suggest that PIP-binding CAPs could be exploited for novel multifunction therapeutics.

Human β-defensin 3 contains an oncolytic motif that binds PI(4,5)P2 to mediate tumour cell permeabilisation.

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.

Convergent evolution of defensin sequence, structure and function.

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.

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

Over 200 billion cells undergo apoptosis every day in the human body in order to maintain tissue homeostasis. Increased apoptosis can also occur under pathological conditions including infection and autoimmune disease. During apoptosis, cells can fragment into subcellular membrane-bound vesicles known as apoptotic bodies (ApoBDs). We recently developed a flow cytometry-based method to accurately differentiate ApoBDs from other particles (e.g. cells and debris). In the present study, we aim to further characterize subsets of ApoBDs based on intracellular contents and cell type-specific surface markers. Utilizing a flow cytometry-based approach, we demonstrated that intracellular contents including nuclear materials and mitochondria are distributed to some, but not all ApoBDs. Interestingly, the mechanism of ApoBD formation could affect the distribution of intracellular contents into ApoBDs. Furthermore, we also showed that ApoBDs share the same surface markers as their cell of origin, which can be used to distinguish cell type-specific ApoBDs from a mixed culture. These studies demonstrate that ApoBDs are not homogeneous and can be divided into specific subclasses based on intracellular contents and cell surface markers. The described flow cytometry-based method to study ApoBDs could be used in future studies to better understand the function of ApoBDs.

Determining the contents and cell origins of apoptotic bodies by flow cytometry.

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)P], 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)P 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.

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

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.

X-ray structure of a carpet-like antimicrobial defensin-phospholipid membrane disruption complex.

Human defensins belong to a subfamily of the cationic antimicrobial peptides and act as a first line of defense against invading microbes. Their often broad-spectrum antimicrobial and antitumor activities make them attractive for therapeutic development; however, their precise molecular mechanism(s) of action remains to be defined. We show that human β-defensin 2 (HBD-2) permeabilizes  cell membranes via a mechanism targeting the plasma membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP). We determined the structure of HBD-2 bound to PIP, which revealed two distinct PIP-binding sites, and showed, using functional assays, that mutations in these sites ablate PIP-mediated fungal growth inhibition by HBD-2. Our study provides the first insight into lipid-mediated human defensin membrane permeabilization at an atomic level and reveals a unique mode of lipid engagement to permeabilize cell membranes.

Thanh Kha Phan

Department of Biochemistry and Genetics,
La Trobe Institute for Molecular Science,
Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science

La Trobe University

Detection and Isolation of Apoptotic Bodies to High Purity

Thanh Kha Phan

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biochemistry and Genetics,La Trobe Institute for Molecular Science,Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science

La Trobe University

Detection and Isolation of Apoptotic Bodies to High Purity

Department of Biochemistry and Genetics,
La Trobe Institute for Molecular Science,
Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science