Christopher G. Adda

Comparative proteomics evaluation of plasma exosome isolation techniques and assessment of the stability of exosomes in normal human blood plasma

Exosomes are nanovesicles released by a variety of cells and are detected in body fluids including blood. Recent studies have highlighted the critical application of exosomes as personalized targeted drug delivery vehicles and as reservoirs of disease biomarkers. While these research applications have created significant interest and can be translated into practice, the stability of exosomes needs to be assessed and exosome isolation protocols from blood plasma need to be optimized. To optimize methods to isolate exosomes from blood plasma, we performed a comparative evaluation of three exosome isolation techniques (differential centrifugation coupled with ultracentrifugation, epithelial cell adhesion molecule immunoaffinity pull‐down, and OptiPrepTM density gradient separation) using normal human plasma. Based on MS, Western blotting and microscopy results, we found that the OptiPrepTM density gradient method was superior in isolating pure exosomal populations, devoid of highly abundant plasma proteins. In addition, we assessed the stability of exosomes in plasma over 90 days under various storage conditions. Western blotting analysis using the exosomal marker, TSG101, revealed that exosomes are stable for 90 days. Interestingly, in the context of cellular uptake, the isolated exosomes were able to fuse with target cells revealing that they were indeed biologically active.

Proteome profiling of exosomes derived from human primary and metastatic colorectal cancer cells reveal differential expression of key metastatic factors and signal transduction components

Exosomes are small extracellular 40–100 nm diameter membrane vesicles of late endosomal origin that can mediate intercellular transfer of RNAs and proteins to assist premetastatic niche formation. Using primary (SW480) and metastatic (SW620) human isogenic colorectal cancer cell lines we compared exosome protein profiles to yield valuable insights into metastatic factors and signaling molecules fundamental to tumor progression. Exosomes purified using OptiPrep™ density gradient fractionation were 40–100 nm in diameter, were of a buoyant density ∼1.09 g/mL, and displayed stereotypic exosomal markers TSG101, Alix, and CD63. A major finding was the selective enrichment of metastatic factors (MET, S100A8, S100A9, TNC), signal transduction molecules (EFNB2, JAG1, SRC, TNIK), and lipid raft and lipid raft‐associated components (CAV1, FLOT1, FLOT2, PROM1) in exosomes derived from metastatic SW620 cells. Additionally, using cryo‐electron microscopy, ultrastructural components in exosomes were identified. A key finding of this study was the detection and colocalization of protein complexes EPCAM‐CLDN7 and TNIK‐RAP2A in colorectal cancer cell exosomes. The selective enrichment of metastatic factors and signaling pathway components in metastatic colon cancer cell‐derived exosomes contributes to our understanding of the cross‐talk between tumor and stromal cells in the tumor microenvironment.

A Phase 1 Trial of MSP2-C1, a Blood-Stage Malaria Vaccine Containing 2 Isoforms of MSP2 Formulated with Montanide® ISA 720

Background In a previous Phase 1/2b malaria vaccine trial testing the 3D7 isoform of the malaria vaccine candidate Merozoite surface protein 2 (MSP2), parasite densities in children were reduced by 62%. However, breakthrough parasitemias were disproportionately of the alternate dimorphic form of MSP2, the FC27 genotype. We therefore undertook a dose-escalating, double-blinded, placebo-controlled Phase 1 trial in healthy, malaria-naïve adults of MSP2-C1, a vaccine containing recombinant forms of the two families of msp2 alleles, 3D7 and FC27 (EcMSP2-3D7 and EcMSP2-FC27), formulated in equal amounts with Montanide® ISA 720 as a water-in-oil emulsion. Methodology/Principal Findings The trial was designed to include three dose cohorts (10, 40, and 80 µg), each with twelve subjects receiving the vaccine and three control subjects receiving Montanide® ISA 720 adjuvant emulsion alone, in a schedule of three doses at 12-week intervals. Due to unexpected local reactogenicity and concern regarding vaccine stability, the trial was terminated after the second immunisation of the cohort receiving the 40 µg dose; no subjects received the 80 µg dose. Immunization induced significant IgG responses to both isoforms of MSP2 in the 10 µg and 40 µg dose cohorts, with antibody levels by ELISA higher in the 40 µg cohort. Vaccine-induced antibodies recognised native protein by Western blots of parasite protein extracts and by immunofluorescence microscopy. Although the induced anti-MSP2 antibodies did not directly inhibit parasite growth in vitro, IgG from the majority of individuals tested caused significant antibody-dependent cellular inhibition (ADCI) of parasite growth. Conclusions/Significance As the majority of subjects vaccinated with MSP2-C1 developed an antibody responses to both forms of MSP2, and that these antibodies mediated ADCI provide further support for MSP2 as a malaria vaccine candidate. However, in view of the reactogenicity of this formulation, further clinical development of MSP2-C1 will require formulation of MSP2 in an alternative adjuvant. Trial Registration Australian New Zealand Clinical Trials Registry 12607000552482

Phosphoinositide-mediated oligomerization of a defensin induces cell lysis

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

Plasmodium falciparum merozoite surface protein 2 is unstructured and forms amyloid-like fibrils

Several merozoite surface proteins are being assessed as potential components of a vaccine against Plasmodium falciparum, the cause of the most serious form of human malaria. One of these proteins, merozoite surface protein 2 (MSP2), is unusually hydrophilic and contains tandem sequence repeats, characteristics of intrinsically unstructured proteins. A range of physicochemical studies has confirmed that recombinant forms of MSP2 are largely unstructured. Both dimorphic types of MSP2 (3D7 and FC27) are equivalently extended in solution and form amyloid-like fibrils although with different kinetics and structural characteristics. These fibrils have a regular underlying beta-sheet structure and both fibril types stain with Congo Red, but only the FC27 fibrils stain with Thioflavin T. 3D7 MSP2 fibrils seeded the growth of fibrils from 3D7 or FC27 MSP2 monomer indicating the involvement of a conserved region of MSP2 in fibril formation. Consistent with this, digestion of fibrils with proteinase K generated resistant peptides, which included the N-terminal conserved region of MSP2. A monoclonal antibody that reacted preferentially with monomeric recombinant MSP2 did not react with the antigen in situ on the merozoite surface. Glutaraldehyde cross-linking of infected erythrocytes generated MSP2 oligomers similar to those formed by polymeric recombinant MSP2. We conclude that MSP2 oligomers containing intermolecular beta-strand interactions similar to those in amyloid fibrils may be a component of the fibrillar surface coat on P. falciparum merozoites.

Recombinant protein vaccines against the asexual blood stages of Plasmodium falciparum

It is now more than 25 years since asexual blood-stage antigens of Plasmodium falciparum were first expressed as recombinant proteins. Although many asexual blood-stage vaccine candidates have been identified none have yet been fully evaluated in clinical trials. The results of studies in animal models, and from in vitro studies with P. falciparum, indicate that antibody responses induced by many of these recombinant proteins can inhibit parasite development but so far the evidence that protection can be achieved in exposed human populations is limited. Recombinant forms of MSP2 and AMA1 expressed in E. coli have had significant effects in Phase II trials, although for both antigens the effect was against a subset of parasites expressing a form of these polymorphic antigens related to that in the vaccine. More knowledge of the antigenic structure of the native parasite antigens is required so that the recombinant protein constructs can be optimized to induce the correct antibody fine specificity. The very different structural characteristics of MSP2 and AMA1 are discussed, as are some approaches being taken to overcoming the problem of diversity in these antigens.

Solution Conformation, Backbone Dynamics and Lipid Interactions of the Intrinsically Unstructured Malaria Surface Protein MSP2

Merozoite surface protein 2 (MSP2), one of the most abundant proteins on the surface of the merozoite stage of Plasmodium falciparum, is a potential component of a malaria vaccine, having shown some efficacy in a clinical trial in Papua New Guinea. MSP2 is a GPI-anchored protein consisting of conserved N- and C-terminal domains and a variable central region. Previous studies have shown that it is an intrinsically unstructured protein with a high propensity for fibril formation, in which the conserved N-terminal domain has a key role. Secondary structure predictions suggest that MSP2 contains long stretches of random coil with very little alpha-helix or beta-strand. Circular dichroism spectroscopy confirms this prediction under physiological conditions (pH 7.4) and in more acidic solutions (pH 6.2 and 3.4). Pulsed field gradient NMR diffusion measurements showed that MSP2 under physiological conditions has a large effective hydrodynamic radius consistent with an intrinsic pre-molten globule state, as defined by Uversky. This was supported by sedimentation velocity studies in the analytical ultracentrifuge. NMR resonance assignments have been obtained for FC27 MSP2, allowing the residual secondary structure and backbone dynamics to be defined. There is some motional restriction in the conserved C-terminal region in the vicinity of an intramolecular disulfide bond. Two other regions show motional restrictions, both of which display helical structure propensities. One of these helical regions is within the conserved N-terminal domain, which adopts essentially the same conformation in full-length MSP2 as in corresponding peptide fragments. We see no evidence of long-range interactions in the full-length protein. MSP2 associates with lipid micelles, but predominantly through the N-terminal region rather than the C terminus, which is GPI-anchored to the membrane in the parasite.

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

ABSTRACT 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.

Random Sequence Libraries Displayed on Phage: Identification of Biologically Important Molecules

Phage display has become a widely used tool for the identification of proteins or peptides with affinity for a variety of biomolecules. The versatility, simplicity and cost effectiveness of this application has pervaded a wide variety of research areas. Although not without its limitations, phage display has provided a convenient methodology for obtaining ligands to study the function, structure and diagnostic or therapeutic potential of various macromolecules. This review highlights some recent research employing this technology that serves to illustrate its utility in various research and clinical applications.

EGCG disaggregates amyloid-like fibrils formed by Plasmodium falciparum merozoite surface protein 2

Merozoite surface protein 2 (MSP2), one of the most abundant proteins on the surface of Plasmodium falciparum merozoites, is a promising malaria vaccine candidate. MSP2 is intrinsically unstructured and forms amyloid-like fibrils in solution. As this propensity of MSP2 to form fibrils in solution has the potential to impede its development as a vaccine candidate, finding an inhibitor that inhibits fibrillogenesis may enhance vaccine development. We have shown previously that EGCG inhibits the formation of MSP2 fibrils. Here we show that EGCG can alter the β-sheet-like structure of the fibril and disaggregate pre-formed fibrils of MSP2 into soluble oligomers. The fibril remodelling effects of EGCG and other flavonoids were characterised using Thioflavin T fluorescence assays, electron microscopy and other biophysical methods.