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Exosomes in Cancer Disease, Progression, and Drug Resistance
Published in Vladimir Torchilin, Handbook of Materials for Nanomedicine, 2020
Taraka Sai Pavan Grandhi, Rajeshwar Nitiyanandan, Kaushal Rege
Tetraspanins (CD63, CD9, CD81, and CD82) are integral membrane protein components that are highly enriched in exosomes. They organize into tetraspanin-enriched domains by interacting with lipids, other cytosolic and transmembrane proteins [33, 38]. Cytoplasmic and transmembrane protein interactome of tetraspanins is considered another major source of protein loading into the exosomes [33]. Sphingosine and fatty acid containing ceramides have been shown to be essential in exosome biogenesis [39]. It is well known that ceramides can induce stable and spontaneous curvature in lipid bilayer membranes (liposomes), and play an essential role in inward budding of late endosomal limiting membrane for ILV generation [39]. Kajimoto et al. [34] showed that continuous metabolization of ceramide into sphingosine 1-phosphate (S1P) activates inhibitory G protein-coupled S1P receptors, essential for successful cargo sorting into ILVs in HeLa cells; S1P signaling was shown to play a role in the sorting of the tetraspanin CD63 protein into exosomes [34].
The Emerging Role of Exosome Nanoparticles in Regenerative Medicine
Published in Harishkumar Madhyastha, Durgesh Nandini Chauhan, Nanopharmaceuticals in Regenerative Medicine, 2022
Zahra Sadat Hashemi, Mahlegha Ghavami, Saeed Khalili, Seyed Morteza Naghib
It should be noted that the biogenesis pathway of exosomes and their distinction fromother cell-derived vesicles were identified due to the existence of lysosomal surface protein (LAMP), tetraspanins (CD81, CD9, and CD63), heat shock proteins (Hsc70), and also some fusion proteins such as Annexin, CD9, and flotillin in the exosomal membrane (Caby et al. 2005; Andaloussi et al. 2013; Conde-Vancells et al. 2008; Mohammadpour and Majidzadeh-A 2020). The transport (ESCRT) process requires the endosomal sorting complex, which is a collection of proteins necessary for formation and sorting of cargo into exosomes.
Extracellular Vesicles (EVs)
Published in Peixuan Guo, Kirill A. Afonin, RNA Nanotechnology and Therapeutics, 2022
Alice Braga, Giulia Manferrari, Jayden A. Smith, Stefano Pluchino
Nevertheless, a compelling re-examination of EV heterogeneity recently reported by Jeppesen et al. (2019) has cast doubt on the specificity of some well-accepted exosomal markers. The authors have employed a combination of high-resolution density gradient fractionation and Direct Immunoaffinity Capture (DIC) to purify bona fide exosomes, thus proposing a surprising reassessment of exosome composition and establishing a differential distribution of protein, RNA, and DNA between small extracellular vesicles (sEVs), a broad category inclusive of exosomes, and higher-density non-vesicular (NV) extracellular matter. EVs isolated by DIC of canonical tetraspanin exosome markers (CD63, CD81, and/or CD9) were found to be absent in many of the previously widely accepted exosomal markers and cargo constituents (van Niel, D’Angelo, and Raposo 2018; Kowal et al. 2016). While ALIX was abundant in DIC-isolated exosomes, TSG101 was absent and instead attributed to another class of sEVs called ARMMs (see the next section on MVs). Furthermore, the RAB GTPases, while implicated in the trafficking of MVBs to the plasma membrane for exosome release, were found to be a poor marker of exosomes. High-abundance cytosolic proteins such as glyceraldehyde 3-phosphate dehydrogenase (GAPDH), pyruvate kinase M1/2 (PKM), enolase 1 (ENO1), and 14-3-3 are widely acknowledged as typical components of the exosome lumen. However, Jeppesen et al. instead found these proteins enriched in non-exosomal sEV or even NV fractions. This suggests that the uptake of cytosolic constituents during classical exosome biogenesis is not the result of a random sampling of the cytosol but rather a highly controlled process. Moreover, this study distinguished sEVs (including exosomes) by their lack of nuclear or cytosolic microRNA (miRNA) protein machinery (such as Drosha, DiGeorge syndrome critical region 8 (DGCR8), Dicer, TAR RNA binding protein (TRBP), and glycine-tryptophan protein of 182 kDa (GW182)). These new findings refute the idea that exosomes hold the ability to carry out cell-independent miRNA biogenesis as previously suggested (Melo et al. 2014). Indeed, Jeppesen et al. found that many of the most abundant miRNAs showed substantial 3ˊ-trimming and were associated with the NV fraction, rather than being present in exosomes (or any other vesicles). Additionally, neither exosomes nor any other type of sEVs were found associated with double-stranded DNA in the extracellular environment, suggesting that sEVs might not be directly involved in chromatin release (Jeppesen et al. 2019). In summary, classical tetraspanin-enriched exosomes might contain only a limited repertoire of the diversity of biomolecules present in the extracellular milieu and thus a better elucidation of the heterogeneity of sEVs, and indeed NV fractions, is essential (Pluchino and Smith 2019).
Latest advances in extracellular vesicles: from bench to bedside
Published in Science and Technology of Advanced Materials, 2019
Tomofumi Yamamoto, Nobuyoshi Kosaka, Takahiro Ochiya
EVs have been observed in all body fluids, such as blood, urine, saliva, sputum, breast milk, semen, and cerebrospinal fluid [13–19]. EVs have been shown to contain miRNAs, mRNA, DNAs, and proteins within the lipid bilayer [11]. This bilayer construction, which is very stable, enables EVs to circulate intact through body fluids, and thus, EVs can transfer their components to distant recipient cells. The accumulating data have indicated that the contents, size, and membrane composition of EVs are highly heterogeneous, dynamic and depend on the cellular source, state, and environmental conditions. EVs have some exosomal markers, including members of the tetraspanin family (CD9, CD63, CD81), members of the endosomal sorting complex required for transport (ESCRT) (TSG101, Alix), heat shock proteins (Hsp60, Hsp70, Hsp90), and RAB proteins (RAB27 A/B) [20,21]. Cells release heterogeneous vesicles of different sizes and intracellular origins, including small EVs formed inside endosomal compartments and EVs of various sizes budding from the plasma membrane [22]. Differential separation by immuno-isolation using either CD63, CD81, or CD9 was proposed. Several classically used exosome markers, like major histocompatibility complex (MHC), flotillin, and Hsp70 proteins, are similarly present in all EVs, and they also identified proteins specifically enriched in small EVs, and defined a set of five protein categories displaying different relative abundance in distinct EV populations. In this way, understanding the heterogeneity of EVs is important for EV-based therapy, since each EV has difference of their biological function on their membrane surface.
No-stain protein labeling as a potential normalization marker for small extracellular vesicle proteins
Published in Preparative Biochemistry & Biotechnology, 2023
Anjugam Paramanantham, Rahmat Asfiya, Siddharth Das, Grace McCully, Akhil Srivastava
We further investigated the NSPL to demonstrate the viability of the method in quantifying the change in expression of a protein of interest (tetraspanins protein CD81) resulting from silencing the gene using RNA interference technology (siRNA). Further, to corroborate the results obtained through this method, samples from the same CD81 silencing experiment were analyzed by an established and robust quantitative proteomic method, MASS-Spectrometry (Bruker timsTOF pro2 mass spectrometer). CD81 protein was evaluated in si-CD81-H1299 cells and in sEVs derived from them (siCD81-sEVs). Western blot containing the samples was subjected to NSPL method (Figure 5A). After total protein labeling, the blot was probed for the protein of interest (CD81). The results from the western blot showed a reduction in CD81 expression in si-CD81-H1299 cells compared with the control H1299 cells. The same trend was also observed in sEVs isolated from corresponding silenced and non-silenced control cells (Figure 5B). The band quantification for each sample obtained through the NSPL method and analyzed through the ImageJ tool supports the blot data and showed an 0.2 and 0.0189-fold reduction in CD81 proteins in si-CD81-H1299 cells and si-CD81-sEVs, respectively (Figure 5C). Further, the results were corroborated by spectral count data for CD81 in sEVs obtained from mass spectrometry that shows a difference of 80 spectral counts between two samples (untreated control sEVs and si-CD81 treated sEVs) (Figure 5D). Thus, these results confirm that the NSPL method could be effectively used for measuring the band intensity in a western blot, and hence, can aid in quantifying change in protein expression without any housekeeping protein.
Advances of engineered extracellular vesicles-based therapeutics strategy
Published in Science and Technology of Advanced Materials, 2022
Hiroaki Komuro, Shakhlo Aminova, Katherine Lauro, Masako Harada
Since all types of cells secrete EVs, their recovery has been reported in all bodily fluids [20], including blood [21], urine [22], saliva [23], breast milk [24], semen [25], bronchoalveolar lavage fluid [26], synovial fluid [27], cerebrospinal fluid [28], and amniotic fluid [29]. EVs are broadly classified into three types: exosomes, microvesicles, and apoptotic bodies (Figure 1). The classification of EV can be associated with their biogenesis. Exosomes (50–150 nm) derive from the inward budding of the endosomal compartment, multivesicular bodies (MVBs). Upon fusion with the cell membrane, exosomes are released to the extracellular space and mediate intercellular communication by transferring bioactive molecules such as RNAs, proteins, and lipids between cells in living organisms [30]. Tetraspanins belong to a cell surface protein family with four transmembrane domains required for intraluminal vesicle formation [31]. CD9, CD63, and CD81 are tetraspanin proteins found enriched in exosomes and often used as EV surface markers [12]. Microvesicles (100–1000 nm), on the other hand, are secreted directly from the plasma membrane. Microvesicles are not as well studied or defined as the other EV types, resulting in more broad characteristics. Apoptotic bodies (100–5000 nm) are produced from apoptotic cells when they undergo programmed cell death. Numerous studies illustrate the heterogeneity of these EVs and missing pieces of information left uncovered, implicating the change in descriptions in the future [12]. For example, there is no defined method or unique maker to separate one EV type from another. A report suggested that 100,000 g ultracentrifugation (UC) co-isolates two EV subtypes defined by specific proteins [32], proposing the importance of considering heterogeneity and diversity among EV types in the study design. Furthermore, some EV subtypes are named after the cell from which they are secreted, such as cardiosome (cardiovascular-origin) and oncosome (oncology-origin) [33–36], though ISEV strongly suggests that the generic term EV be widely adopted [37]. Moreover, the guidelines published by ISEV in 2018 recommend referring to small EVs (sEVs) (<100 nm or <200 nm) or >200 nm as medium/large EVs (m/lEVs) EVs based on their physical characteristic, size and density [12]. This article will adhere to the term ‘EV’ in place of exosome or the names used in the original literature to avoid terminology ambiguity.