Extracellular vesicles in liver pathobiology: Small particles with big impact.

Extracellular vesicles (EVs) are nanometer-sized, membrane-bound vesicles released by cells into the extracellular milieu. EVs are now recognized to play a critical role in cell-to-cell communication. EVs contain important cargo in the form of proteins, lipids, and nucleic acids and serve as vectors for delivering this cargo from donor to acceptor or target cell. EVs are released under both physiologic and pathologic conditions, including liver diseases, and exert a wide range of effects on target cells. This review provides an overview on EV biogenesis, secretion, cargo, and target cell interactions in the context of select liver diseases. Specifically, the diverse roles of EVs in nonalcoholic steatohepatitis, alcoholic liver disease, viral hepatitis, cholangiopathies, and hepatobiliary malignancies are emphasized. Liver diseases often result in an increased release of EVs and/or in different cargo sorting into these EVs. Either of these alterations can drive disease pathogenesis. Given this fact, EVs represent a potential target for therapeutic intervention in liver disorders. Because altered EV composition may reflect the underlying disease condition, circulating EVs can be exploited for diagnostic and prognostic purposes as a liquid biopsy. Furthermore, ex vivo modified or synthesized EVs can be engineered as therapeutic nano-shuttles. Finally, we highlight areas that merit further investigation relevant to understanding how EVs regulate liver disease pathogenesis. (Hepatology 2016;64:2219-2233).

Biophysical investigations with MARCKS-ED: dissecting the molecular mechanism of its curvature sensing behaviors.

Curved membranes are a common and important attribute in cells. Protein and peptide curvature sensors are known to activate signaling pathways, initiate vesicle budding, trigger membrane fusion, and facilitate molecular transport across cell membranes. Nonetheless, there is little understanding how these proteins and peptides achieve preferential binding of different membrane curvatures. The current study is to elucidate specific factors required for curvature sensing. As a model system, we employed a recently identified peptide curvature sensor, MARCKS-ED, derived from the effector domain of the myristoylated alanine-rich C kinase substrate protein, for these biophysical investigations. An atomistic molecular dynamics (MD) simulation suggested an important role played by the insertion of the Phe residues within MARCKS-ED. To test these observations from our computational simulations, we performed electron paramagnetic resonance (EPR) studies to determine the insertion depth of MARCKS-ED into differently curved membrane bilayers. Next, studies with varied lipid compositions revealed their influence on curvature sensing by MARCKS-ED, suggesting contributions from membrane fluidity, rigidity, as well as various lipid structures. Finally, we demonstrated that the curvature sensing by MARCKS-ED is configuration independent. In summary, our studies have shed further light to the understanding of how MARCKS-ED differentiates between membrane curvatures, which may be generally applicable to protein curvature sensing behavior.

Multivalency amplifies the selection and affinity of bradykinin-derived peptides for lipid nanovesicles.

The trimer of a bradykinin derivative displayed a more than five-fold increase in binding affinity for phosphatidylserine-enriched nanovesicles as compared to its monomeric precursor. The nanovesicle selection is directly correlated with multivalency, which amplifies the electrostatic attraction. This strategy may lead to the development of novel molecular probes for detecting highly curved membrane bilayers.

MARCKS-ED peptide as a curvature and lipid sensor.

Membrane curvature and lipid composition regulates important biological processes within a cell. Currently, several proteins have been reported to sense and/or induce membrane curvatures, e.g., Synaptotagmin-1 and Amphiphysin. However, the large protein scaffold of these curvature sensors limits their applications in complex biological systems. Our interest focuses on identifying and designing peptides that can sense membrane curvature based on established elements observed in natural curvature-sensing proteins. Membrane curvature remodeling also depends on their lipid composition, suggesting strategies to specifically target membrane shape and lipid components simultaneously. We have successfully identified a 25-mer peptide, MARCKS-ED, based on the effector domain sequence of the intracellular membrane protein myristoylated alanine-rich C-kinase substrate that can recognize PS with preferences for highly curved vesicles in a sequence-specific manner. These studies further contribute to the understanding of how proteins and peptides sense membrane curvature, as well as provide potential probes for membrane shape and lipid composition.

Detection of highly curved membrane surfaces using a cyclic peptide derived from synaptotagmin-I.

The generation of highly curved membranes is essential to cell growth, division, and movement. Recent research in the field is focused to answer questions related to the consequences of changes in the topology of the membrane once it is created, broadly termed as membrane curvature sensing. Most probes that are used to study curvature sensing are intact membrane active proteins such as DP1/Yop1p, ArfGAP1, BAR domains, and Synaptotagmin-I (Syt1). Taking a cue from nature, we created the cyclic peptide C2BL3C based on the membrane penetration C2B loop 3 of Syt1 via "Click" chemistry. Using a combination of spectroscopic techniques, we investigated the peptide-lipid interactions of this peptide with synthetic phospholipid vesicles and exosomes from rat blood plasma. We found that the macrocycle peptide probe was selective for lipid vesicles with highly curved surfaces (d < 100 nm). These results suggested that C2BL3C functions as a selective detector of highly curved phospholipid bilayers.

Constant pressure-controlled extrusion method for the preparation of Nano-sized lipid vesicles.

Liposomes are artificially prepared vesicles consisting of natural and synthetic phospholipids that are widely used as a cell membrane mimicking platform to study protein-protein and protein-lipid interactions, monitor drug delivery, and encapsulation. Phospholipids naturally create curved lipid bilayers, distinguishing itself from a micelle. Liposomes are traditionally classified by size and number of bilayers, i.e. large unilamellar vesicles (LUVs), small unilamellar vesicles (SUVs) and multilamellar vesicles (MLVs). In particular, the preparation of homogeneous liposomes of various sizes is important for studying membrane curvature that plays a vital role in cell signaling, endo- and exocytosis, membrane fusion, and protein trafficking. Several groups analyze how proteins are used to modulate processes that involve membrane curvature and thus prepare liposomes of diameters <100 - 400 nm to study their behavior on cell functions. Others focus on liposome-drug encapsulation, studying liposomes as vehicles to carry and deliver a drug of interest. Drug encapsulation can be achieved as reported during liposome formation. Our extrusion step should not affect the encapsulated drug for two reasons, i.e. (1) drug encapsulation should be achieved prior to this step and liposomes should retain their natural biophysical stability, securely carrying the drug in the aqueous core. These research goals further suggest the need for an optimized method to design stable sub-micron lipid vesicles. Nonetheless, the current liposome preparation technologies (sonication, freeze-and-thaw, sedimentation) do not allow preparation of liposomes with highly curved surface (i.e. diameter <100 nm) with high consistency and efficiency, which limits the biophysical studies of an emerging field of membrane curvature sensing. Herein, we present a robust preparation method for a variety of biologically relevant liposomes. Manual extrusion using gas-tight syringes and polycarbonate membranes, is a common practice but heterogeneity is often observed when using pore sizes <100 nm due to due to variability of manual pressure applied. We employed a constant pressure-controlled extrusion apparatus to prepare synthetic liposomes whose diameters range between 30 and 400 nm. Dynamic light scattering (DLS), electron microscopy and nanoparticle tracking analysis (NTA) were used to quantify the liposome sizes as described in our protocol, with commercial polystyrene (PS) beads used as a calibration standard. A near linear correlation was observed between the employed pore sizes and the experimentally determined liposomes, indicating high fidelity of our pressure-controlled liposome preparation method. Further, we have shown that this lipid vesicle preparation method is generally applicable, independent of various liposome sizes. Lastly, we have also demonstrated in a time course study that these prepared liposomes were stable for up to 16 hours. A representative nano-sized liposome preparation protocol is demonstrated below.