What are the Functions of Exosomal Proteins?

Exosomes are an individual class of biomolecules occupying a central place in the sweeping panorama of cell biology and medicine, with roles in intercellular signalling, physiological regulation and pathology. Such tiny but effective vesicles, with proteins of varying sizes both externally and internally, uncover complex ways in which cells interact, and provide new approaches to diagnosing and treating diseases. In this article, the categories, roles, and applications of exosome proteins for disease diagnosis and treatment are presented to give readers an understanding of exosome protein research.

Classification of Exosomal Proteins

Fig. 1. Exosomes are rich in tetraspanins, adhesion molecules, enzymes, scaffolds, RNA-binding proteins, RNAs, DNAs, and complex glycans (Pegtel DM, and Gould SJ, 2019).

• Integral exosomal membrane proteins: Exosomes are packed with tetraspanin family members, such as CD81, CD82, CD37, and CD63. These proteins are important for the trafficking of exosomes, their stability, and oligomerisation of other membrane proteins. Exosome markers include CD81 and CD63 and other markers, such as CD9, are also becoming increasingly important.
• Lipid-anchored outer membrane proteins: These proteins are anchored to the exosome membrane via glycosylphosphatidylinositol (GPI), such as CD39 and CD73. They are also involved in signal transduction and possibly receptor-mediated cellular regulation.
• Peripheral surface proteins: Exosomes contain a range of signalling surface proteins, such as Wnt proteins and their receptor/chaperone EVI (GPR177). They also transport bone morphogenetic proteins, transforming growth factor- Wnt proteins, tumor necrosis factor, TNF-dependent apoptotic ligand, FAS ligand, cytokines, and other signalling molecules. This diversity presents exosomes as versatile platforms for complex cell-to-cell communication. Exosome surfaces also contain extracellular matrix proteins such as fibronectin and tenascin C, which contribute to signalling and adhesion.
• Lipid-anchored inner membrane proteins: This category includes small GTPases (e.g., Rab, Ras, Rho) and other acylated proteins. Their anchoring and distribution influence exosomal transport pathways and are related to processes like viral infections.
• Scaffold proteins: Exosomal membranes are rich in scaffold molecules such as ERM proteins and Syntenin, which support the structure through interactions with membrane proteins. These scaffolds also bind to membrane lipids, regulating exosome formation and transport.
• Cofactors and heat shock proteins: Certain exosomal cofactors assist in protein folding and stabilization. Heat shock proteins like the HSP70 family regulate responses to protein unfolding and are involved in antigen presentation and immune modulation.
• Exosomal enzymes: Exosomes contain a variety of enzymes, such as lipases, metabolic enzymes, and glycosyltransferases, providing functions in metabolism and signal transduction. Their activity makes exosomes dynamic biochemical platforms, with notable enzyme activity in tumor cells.
• Soluble proteins and bulk inclusions: While most soluble proteins are not abundantly present, some exhibit functionality post exosome-cell fusion. Current therapeutic applications are being explored using photo-induced exosome release systems.

Functions of Exosomal Proteins

Fig. 2. A graphical representation of the protein composition of exosomes categorized as per the function performed (Mathivanan S, Ji H, et al., 2010).

• Signal transduction: Exosomal proteins, including receptors and ligands, can bind with target cell molecules to activate specific signaling pathways. For instance, epidermal growth factor receptors (EGFRs) in exosomes can initiate proliferation or differentiation signals via target cell receptors.
• Immune response regulation: Exosomes play a vital role in immune modulation. They can stimulate immune responses by presenting antigens like MHC molecules or suppress immune activity with molecules like PD-L1, influencing immune escape and surveillance. Heat shock proteins stabilize and fold proteins within exosomes, also involving antigen presentation and immune regulation.
• Substance transport: Exosomal membranes enable the protection and transport of unstable active substances, such as proteins and nucleic acids, which may be effectively delivered to target cells through membrane fusion or endocytosis, regulating metabolism or signaling pathways.
• Cell microenvironment shaping: Exosomes participate in extracellular matrix remodeling and microenvironmental regulation. By transporting protein hydrolases or adhesion molecules, they can affect cell migration, invasion, and tissue restructuring, especially within tumor microenvironments. Integrins, proteins facilitating cell adhesion, can influence tumor cell positioning and infiltration through exosomal mediation.
• Disease propagation and progression: Exosomal proteins, metabolites, and nucleic acids transferred to the target cell can disrupt biology and promote or prevent disease development (e.g, in cancer).
• Tissue repair: Exosomal proteins play a fundamental role in tissue repair, primarily by mediating intercellular communication, controlling cell activity, and providing important biomolecules to help accelerate repair and regeneration. Vascular endothelial growth factor (VEGF), for instance, has been demonstrated to induce angiogenesis in limb-ischemia models.

Applications of Exosomal Proteins in Disease Diagnosis and Treatment

As biomarkers

• Common exosomal markers: Exosomal markers such as CD9, CD63, CD81, HSP70, Alix and Tsg101 are commonly used for exosomal identity identification.
• Central nervous system disease markers: Some exosomal proteins can be used as biomarkers for neurological pathogens. EGFRvIII, for example, is heavily depleted of glioblastoma exosomes. Even misfolded proteins associated with Alzheimer's and Parkinson's diseases spread via exosomes, providing early diagnostic potential.
• Urogenital system cancer markers: Exosomes are also studied in urogenital cancers, with bladder cancer markers including TACSTD2 and EDIL-3, and prostate cancer markers like Annexin II and fatty acid synthase (FASN).
• Digestive system cancer markers: Exosomes are also used in the urogenital tract; bladder cancer markers include TACSTD2 and EDIL-3, and prostate cancer markers include Annexin II and fatty acid synthase (FASN).
• Respiratory system cancer markers: Proteins like EGFR and Src in lung cancer exosomes indicate roles in disease progression, with LMP1 being specific for nasopharyngeal carcinoma.
• Condition-dependent protein expression: Proteins that appear or disappear in exosomes under various physiological or pathological conditions differ from cell profiles, indicating potential as disease state indicators.

Disease treatment

• Cancer: Tumor antigen-encoded exosomes may activate antigen-presenting cells and incite an anti-tumor immune response. Engineered exosomes are able to release anti-tumor proteins directly into the cell, destroying the tumor.
• Cardiovascular disease: Exosomal proteins, including angiogenic factors such as VEGF, promote neovascularization and increase myocardial blood flow while inhibiting cardiomyocyte death and maintaining myocyte integrity. In addition, exosomes also contain anti-inflammatory agents and immunomodulatory molecules that suppress pro-inflammatory cytokine production, reducing local inflammation and preventing it from expanding.
• Neurodegenerative diseases: Exosomes with the ability to cross the blood-brain barrier can deliver neuroprotective proteins directly to damaged neurons, providing therapeutic benefits.

References

1. Mathivanan S, Ji H, et al. Exosomes: extracellular organelles important in intercellular communication. J Proteomics. 2010, 73(10):1907-20.
2. Pegtel DM, Gould SJ. Exosomes. Annu Rev Biochem. 2019, 88:487-514.

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