Label-Free Measurement of CD63 Positive Extracellular Vesicles Using Terahertz Chemical Microscopy.

Extracellular vesicles (EVs) are small cellular organelles involved in intracellular signaling and cell-to-cell interactions. Recent studies suggested that exosomes may have potential applications in the diagnosis and treatment of cancer and neurodegenerative diseases. In this study, extracellular vesicles of the human nonsmall cell lung cancer cell line H1299 and the unlabeled antiCD63 antibody were imaged using a new label-free terahertz chemical microscopy (TCM) technique to detect changes in the terahertz wave amplitude. To verify the high specificity of the protein biomarkers and the sensitivity of the biosensor surface, we also confirmed the selective binding of the antibody to the antigen, bovine serum albumin, and cancer cells. We also performed real-time measurements of the interaction between EVs from the H1299 cell and the antiCD63 antibody, which showed that the amount of change in the terahertz intensity increased with increasing concentration and the time to saturation decreased. Finally, to reuse the used biosensors (sensing plates), plasma-oxygen cleaning was used, and the activity of the biosensor surface was confirmed by terahertz microscopy and atomic force microscopy and was found to be reusable after less than 3 min of cleaning. Consequently, terahertz chemical microscopy was able to detect the presence or absence of antigen-antibody binding and its reaction rate and binding strength.

Liquid Biopsy for Pancreatic Cancer by Serum Extracellular Vesicle-Encapsulated Long Noncoding RNA HEVEPA.

OBJECTIVES: The role of long noncoding RNAs (lncRNAs) in pancreatic ductal adenocarcinoma (PDAC) remain unclear. Extracellular vesicle (EV)-encapsulated RNAs could be effective targets for liquid biopsy. We aimed to identify previously unknown EV-encapsulated lncRNAs in PDAC and establish highly accurate methods for isolating EVs. MATERIALS AND METHODS: Extracellular vesicles were isolated using existing and newly developed methods, namely, PEViA-UC and PEViA-IP, from serum samples of 20 patients with PDAC, 22 patients with intraductal papillary mucinous neoplasms, and 21 healthy individuals. Extracellular vesicle lncRNA expression was analyzed using digital PCR. RESULTS: Gene expression analysis using cDNA microarray revealed a highly expressed lncRNA, HEVEPA , in serum EVs from patients with PDAC. We established PEViA-UC and PEViA-IP using PEViA reagent, ultracentrifugation, and immunoprecipitation. Although detection of EV-encapsulated HEVEPA using existing methods is challenging, PEViA-UC and PEViA-IP detected EV HEVEPA , which was highly expressed in patients with PDAC compared with non-PDAC patients. The detection sensitivity for discriminating PDAC from non-PDAC using the combination of HEVEPA and HULC , which are highly expressed lncRNAs in PDAC, and carbohydrate antigen 19-9 (CA19-9), was higher than that of HEVEPA , HULC , or CA19-9 alone. CONCLUSIONS: Extracellular vesicle lncRNAs isolated using PEViA-IP and CA19-9 together could be effective targets in liquid biopsy for PDAC diagnosis.

Sialyl-Tn antigen facilitates extracellular vesicle-mediated transfer of FAK and enhances motility of recipient cells.

Protein glycosylation plays a pivotal role in tumour development by modulating molecular interactions and cellular signals. Sialyl-Tn (sTn) antigen is a tumour-associating carbohydrate epitope whose expression correlates with metastasis and poor prognosis of various cancers; however, its pathophysiological function is poorly understood. Extracellular vesicles (EVs) derived from cancer cells act as a signal mediator amongst tumour microenvironments by transferring cargo molecules. sTn antigen has been found in the glycans of EVs, thereby the functional relevance of sTn antigen to the regulation of tumour microenvironments could be expected. In the present study, we showed that sTn antigen induced TP53 and tumour suppressor-activated pathway 6 (TSAP6) and consequently enhanced EV production. Besides, the genetic attenuation of TSAP6 resulted in the reduction of the EV production in the sTn antigen expressing cells. The enhanced EV production in the sTn antigen-expressing cells consequently augmented the delivery of EVs to recipient cells. The produced EVs selectively and abundantly encased focal adhesion kinase and transferred it to EV-recipient cells, and thus, their cellular motility was enhanced. These findings would contribute to facilitate the elucidation of the pathophysiological significance of the sTn antigen in the tumour microenvironments and tumour development.

Utility of Claudin-3 in extracellular vesicles from human bile as biomarkers of cholangiocarcinoma.

Extracellular vesicles (EVs) are released from all cells. Bile directly contacts bile duct tumor; bile-derived EVs may contain high concentrations of cancer biomarkers. We performed a proteomic analysis of human bile-derived EVs and identified a novel biomarker of cholangiocarcinoma (CCA). EVs were isolated using ultracentrifugation, and chelating agents, ethylenediaminetetraacetic acid and ethylene glycol tetraacetic acid (EDEG) and phosphate buffered saline (PBS) were used as dissolution solutions. Bile was collected from 10 CCA and 10 choledocholithiasis (stones) cases. Proteomic analysis was performed; subsequently, ELISA was performed using the candidate biomarkers in a verification cohort. The vesicles isolated from bile had a typical size and morphology. The expression of exosome markers was observed. RNA was more abundant in the EDEG group. The proportion of microRNA was higher in the EDEG group. EDEG use resulted in the removal of more contaminants. Proteomic analysis identified 166 proteins as CCA-specific. ELISA for Claudin-3 revealed statistically significant difference. The diagnostic accuracy was AUC 0.945 and sensitivity and specificity were 87.5%. We report the first use of EDEG in the isolation of EVs from human bile and the proteomic analysis of human bile-derived EV-proteins in CCA. Claudin-3 in bile-derived EVs is a useful biomarker for CCA.

Tubby-like protein superfamily member PLSCR3 functions as a negative regulator of adipogenesis in mouse 3T3-L1 preadipocytes by suppressing induction of late differentiation stage transcription factors.

PLSCR3 (phospholipid scramblase 3, Scr3) belongs to the superfamily of membrane-associated transcription regulators named Tubby-like proteins (TULPs). Physiological phospholipid scrambling activities of PLSCRs in vivo have been skeptically argued, and knowledge of the biological functions of Scr3 is limited. We investigated the expression of Scr3 during differentiation of mouse 3T3-L1 preadipocytes by Western blotting (WB) and by reverse-transcription and real-time quantitative PCR (RT-qPCR). The Scr3 protein decreased during 3T3-L1 differentiation accompanied by a reduction in the mRNA level, and there was a significant increase in the amount of Scr3 protein secreted into the culture medium in the form of extracellular microvesicles (exosomes). On the other hand, Scr3 expression did not significantly decrease, and the secretion of Scr3 in 3T3 Swiss-albino fibroblasts (a parental cell-line of 3T3-L1) was not increased by differentiation treatment. Overexpression of human Scr3 during 3T3-L1 differentiation suppressed triacylglycerol accumulation and inhibited induction of the mRNAs of late stage pro-adipogenic transcription factors [CCAAT/enhancer-binding protein α (C/EBPα) and peroxisome proliferator-activated receptor γ (PPARγ)] and X-box-binding protein 1 (XBP1). Expression of early stage pro-adipogenic transcription factors (C/EBPß and C/EBPδ) was not significantly affected. These results suggest that Scr3 functions as a negative regulator of adipogenesis in 3T3-L1 cells at a specific differentiation stage and that decrease in the intracellular amount of Scr3 protein caused by reduction in Scr3 mRNA expression and enhanced secretion of Scr3 protein appears to be important for appropriate adipocyte differentiation.

ALG-2-interacting Tubby-like protein superfamily member PLSCR3 is secreted by an exosomal pathway and taken up by recipient cultured cells.

PLSCRs (phospholipid scramblases) are palmitoylated membrane-associating proteins. Regardless of the given names, their physiological functions are not clear and thought to be unrelated to phospholipid scrambling activities observed in vitro. Using a previously established cell line of HEK-293 (human embryonic kidney-293) cells constitutively expressing human Scr3 (PLSCR3) that interacts with ALG-2 (apoptosis-linked gene 2) Ca²âº-dependently, we found that Scr3 was secreted into the culture medium. Secretion of Scr3 was suppressed by 2-BP (2-bromopalmitate, a palmitoylation inhibitor) and by GW4869 (an inhibitor of ceramide synthesis). Secreted Scr3 was recovered in exosomal fractions by sucrose density gradient centrifugation. Palmitoylation sites and the N-terminal Pro-rich region were necessary for efficient secretion, but ABSs (ALG-2-binding sites) were dispensable. Overexpression of GFP (green fluorescent protein)-fused VPS4B(E235Q), a dominant negative mutant of an AAA (ATPase associated with various cellular activities) ATPase with a defect in disassembling ESCRT (endosomal sorting complex required for transport)-III subunits, significantly reduced secretion of Scr3. Immunofluorescence microscopic analyses showed that Scr3 was largely localized to enlarged endosomes induced by overexpression of a GFP-fused constitutive active mutant of Rab5A (GFP-Rab5A(Q79L)). Secreted Scr3 was taken up by HeLa cells, suggesting that Scr3 functions as a cell-to-cell transferable modulator carried by exosomes in a paracrine manner.

Prediction of a new ligand-binding site for type 2 motif based on the crystal structure of ALG-2 by dry and wet approaches.

ALG-2 is a penta-EF-hand Ca(2+)-binding protein and interacts with a variety of intracellular proteins. Two types of ALG-2-binding motifs have been determined: type 1, PXYPXnYP (X, variable; n = 4), in ALIX and PLSCR3; type 2, PXPGF, in Sec31A and PLSCR3. The previously solved X-ray crystal structure of the complex between ALG-2 and an ALIX peptide containing type 1 motif showed that the peptide binds to Pocket 1 and Pocket 2. Co-crystallization of ALG-2 and type 2 motif-containing peptides has not been successful. To gain insights into the molecular basis of type 2 motif recognition, we searched for a new hydrophobic cavity by computational algorithms using MetaPocket 2.0 based on 3D structures of ALG-2. The predicted hydrophobic pocket designated Pocket 3 fits with N-acetyl-ProAlaProGlyPhe-amide, a virtual penta-peptide derived from one of the two types of ALG-2-binding sites in PLSCR3 (type 2 motif), using the molecular docking software AutoDock Vina. We investigated effects of amino acid substitutions of the predicted binding sites on binding abilities by pulldown assays using glutathione-S-transferase -fused ALG-2 of wild-type and mutant proteins and lysates of cells expressing green fluorescent protein -fused PLSCR3 of wild-type and mutants. Substitution of either L52 with Ala or F148 with Ser of ALG-2 caused loss of binding abilities to PLSCR3 lacking type 1 motif but retained those to PLSCR3 lacking type 2 motif, strongly supporting the hypothesis that Pocket 3 is the binding site for type 2 motif.

The ALG-2 binding site in Sec31A influences the retention kinetics of Sec31A at the endoplasmic reticulum exit sites as revealed by live-cell time-lapse imaging.

ALG-2, a member of the penta-EF-hand protein family, interacts Ca²+-dependently with a COPII component, Sec31A. In this study, we first established HeLa cells stably expressing green fluorescent protein-fused ALG-2 (GFP-ALG-2) and red fluorescent protein-fused Sec31A (Sec31A-RFP). After inducing Ca²+-mobilization, the cytoplasmic distribution of GFP-ALG-2 changed from a diffuse to a punctate pattern, which extensively overlapped with the Sec31A-RFP-positive structures, indicating that ALG-2 is recruited to the endoplasmic reticulum exit sites (ERES) in living cells. Next, overlay experiments with biotin-labeled ALG-2 were done to dissect the ALG-2 binding site (ABS). They revealed that a sequence comprising amino acid residues 839-851 in the Pro-rich region was necessary and sufficient for direct binding to ALG-2. Finally, fluorescence recovery after photobleaching analysis indicated that the ABS deletion reduced the high-affinity population of Sec31A to the ERES, suggesting that the ABS is one of the key determinants of the retention kinetics of Sec31A at ERES.

Molecular basis for defect in Alix-binding by alternatively spliced isoform of ALG-2 (ALG-2DeltaGF122) and structural roles of F122 in target recognition.

BACKGROUND: ALG-2 (a gene product of PDCD6) belongs to the penta-EF-hand (PEF) protein family and Ca2+-dependently interacts with various intracellular proteins including mammalian Alix, an adaptor protein in the ESCRT system. Our previous X-ray crystal structural analyses revealed that binding of Ca2+ to EF3 enables the side chain of R125 to move enough to make a primary hydrophobic pocket (Pocket 1) accessible to a short fragment of Alix. The side chain of F122, facing a secondary hydrophobic pocket (Pocket 2), interacts with the Alix peptide. An alternatively spliced shorter isoform, designated ALG-2DeltaGF122, lacks Gly121Phe122 and does not bind Alix, but the structural basis of the incompetence has remained to be elucidated. RESULTS: We solved the X-ray crystal structure of the PEF domain of ALG-2DeltaGF122 in the Ca2+-bound form and compared it with that of ALG-2. Deletion of the two residues shortened alpha-helix 5 (alpha5) and changed the configuration of the R125 side chain so that it partially blocked Pocket 1. A wall created by the main chain of 121-GFG-123 and facing the two pockets was destroyed. Surprisingly, however, substitution of F122 with Ala or Gly, but not with Trp, increased the Alix-binding capacity in binding assays. The F122 substitutions exhibited different effects on binding of ALG-2 to other known interacting proteins, including TSG101 (Tumor susceptibility gene 101) and annexin A11. The X-ray crystal structure of the F122A mutant revealed that removal of the bulky F122 side chain not only created an additional open space in Pocket 2 but also abolished inter-helix interactions with W95 and V98 (present in alpha4) and that alpha5 inclined away from alpha4 to expand Pocket 2, suggesting acquirement of more appropriate positioning of the interacting residues to accept Alix. CONCLUSIONS: We found that the inability of the two-residue shorter ALG-2 isoform to bind Alix is not due to the absence of bulky side chain of F122 but due to deformation of a main-chain wall facing pockets 1 and 2. Moreover, a residue at the position of F122 contributes to target specificity and a smaller side chain is preferable for Alix binding but not favored to bind annexin A11.

The mechanism of Ca2+-dependent recognition of Alix by ALG-2: insights from X-ray crystal structures.

Alix [ALG-2 (apoptosis-linked gene 2)-interacting protein X] was originally identified as a protein that interacts with ALG-2, a member of the penta-EF-hand Ca(2+)-binding protein family. ALG-2 binds to its C-terminal proline-rich region that contains four tandem repeats of PXY (where X represents an uncharged amino acid). Recent X-ray crystal structural analyses of the Ca(2+)-free and Ca(2+)-bound forms of ALG-2, as well as the complex with an Alix oligopeptide, have revealed a mechanism of Ca(2+)-dependent binding of ALG-2 to its target protein. Binding of Ca(2+) to EF3 (third EF-hand) enables the side chain of Arg(125), present in the loop connecting EF3 and EF4 (fourth EF-hand), to move sufficiently to make a primary hydrophobic pocket accessible to the critical PPYP (Pro-Pro-Tyr-Pro) motif in Alix, which partially overlaps with the GPP (Gly-Pro-Pro) motif for binding to Cep55 (centrosome protein of 55 kDa). The fact that ALG-2 forms a homodimer and each monomer has one peptide-binding site indicates the possibility that ALG-2 bridges two interacting proteins, including Alix and Tsg101 (tumour susceptibility gene 101), and functions as a Ca(2+)-dependent adaptor protein.

Structural basis for Ca2+ -dependent formation of ALG-2/Alix peptide complex: Ca2+/EF3-driven arginine switch mechanism.

ALG-2 belongs to the penta-EF-hand (PEF) protein family and interacts with various intracellular proteins, such as Alix and TSG101, that are involved in endosomal sorting and HIV budding. Through X-ray crystallography, we solved the structures of Ca(2+)-free and -bound forms of N-terminally truncated human ALG-2 (des3-20ALG-2), Zn(2+)-bound form of full-length ALG-2, and the structure of the complex between des3-23ALG-2 and the peptide corresponding to Alix799-814 in Zn(2+)-bound form. Binding of Ca(2+) to EF3 enables the side chain of Arg125, present in the loop connecting EF3 and EF4, to move enough to make a primary hydrophobic pocket accessible to the critical PPYP motif, which partially overlaps with the GPP motif for the binding of Cep55 (centrosome protein 55 kDa). Based on these results, together with the results of in vitro binding assay with mutant ALG-2 and Alix proteins, we propose a Ca(2+)/EF3-driven arginine switch mechanism for ALG-2 binding to Alix.

Identification of Alix-type and Non-Alix-type ALG-2-binding sites in human phospholipid scramblase 3: differential binding to an alternatively spliced isoform and amino acid-substituted mutants.

ALG-2, a prototypic member of the penta-EF-hand protein family, interacts with Alix at its C-terminal Pro-rich region containing four tandem PXY repeats. Human phospholipid scramblase 3 (PLSCR3) has a similar sequence (ABS-1) in its N-terminal region. In the present study, we found that ALG-2 interacts with PLSCR3 expressed in HEK293 cells in a Ca(2+)-dependent manner by co-immunoprecipitation, pulldown with glutathione S-transferase (GST) fused ALG-2 and an overlay assay using biotin-labeled ALG-2. The GST fusion protein of an alternatively spliced isoform of ALG-2, GST-ALG-2(DeltaGF122), pulled down green fluorescent protein (GFP)-fused PLSCR3 but not GFP Alix. Deletion of a region containing ABS-1 was not sufficient to abrogate the binding. A second ALG-2-binding site (ABS-2) was essential for interaction with ALG-2(DeltaGF122). Real-time interaction analyses with a surface plasmon resonance biosensor using synthetic oligopeptides and recombinant proteins corroborated direct Ca(2+)-dependent binding of ABS-1 to ALG-2 and that of ABS-2 to ALG-2 as well as to ALG-2(DeltaGF122). The sequence of ABS-2 contains multiple prolines and two phenylalanines, among which Phe(49) was found to be critical, because its substitution with Ala or Tyr caused a loss of binding ability by pulldown assays using oligopeptide-immobilized beads. ALG-2-interacting proteins were classified into two groups based on binding ability to ALG-2(DeltaGF122): (i) isoform-non-interactive (ABS-1) types, including Alix, annexin A7, annexin A11, and TSG101 and (ii) isoform-interactive (ABS-2) types including PLSCR3, PLSCR4 and Sec31A. GST-pulldown assays using single amino acid-substituted ALG-2 mutants revealed differences in binding specificities between the two groups, suggesting structural flexibility in ALG-2-ligand complex formation.