Exosomes and Wnt Proteins

Exosomes

• Exosomes are extracellular vesicles with a diameter of 40-100 nm.
• Specific exosomal proteins like Alix, flotillin, TSG101, and CD63 can be used as biomarkers.
• Exosomes have a unique lipid bilayer composed of cholesterol, sphingolipids, ceramide, and glycerophospholipids with long and saturated fatty acid chains.
• The endosomal sorting complexes required for transport (ESCRT) facilitates membrane remodeling, resulting in MVB formation and budding.
• Proteins entering MVBs are tagged with ubiquitin.
• Exosomes protect secretion molecules from degradation in the ECM, making them useful for carrying miRNA.

Wnt Proteins and Exosomes

• Exosomes can carry Wnt proteins on their surface, inducing Wnt signaling activity in targeted cells.
• Ultracentrifugation separates cellular components based on spin timing and speeds.
• Sucrose gradients can further separate molecules of similar sizes based on their densities.
• The Wnt cargo receptor Evi is required for Wnt protein release on exosomes.
• Secreted and exosome forms of Wnt are considered active.
• Bafilomycin and GW4869 are inhibitors of exosome biogenesis, but won’t completely deplete Wnt levels experimentally.
• A retromer/SNX3 complex sorts Evi and Wnt, determining if they are packaged onto exosomes, recycled to the lysosomal pathway, or sent to the GA.

Hedgehog Carriers

• Hh and its co-receptor Ihog are transported via cytonemes (filopodia) in exovesicles characteristic of exosomes.

Exosomes in Cancer Metastasis

• Exosome-delivered EGFR regulates liver microenvironments, promoting gastric cancer liver metastasis.
• EGFR, located in the cytoplasmic membrane, plays a role in tumorigenesis and development.
• Gastric cancer can metastasize in the liver via exosomes delivering EGFR.
• HGF is involved with early liver morphogenesis and linked with tumor development, with its upregulation as a biomarker for cancer prognosis.
• c-MET is the receptor of HGF and is widely expressed in various cancers.
• c-MET, not HGF, is highly expressed in liver metastases of GC and binds with liver HGF.
• Exosomes from GC cells activate liver HGF by suppressing miRNA 26a/b.
• miRNA 26a/b normally suppresses HGF mRNA, but its suppression causes HGF levels to increase.
• There’s a correlation between specific exosome markers and different stages of GC/liver cancer.
• EGFR is more present in serum gastric cancer cells and their exosomes, with exosomes from SGC delivering EGFR into the membrane of mixed primary liver cells.
• EGFR in exosomes of gastric cancer cells increases the relative abundance of EGFR.
• EGFR activates liver HGF by suppressing miRNA 26a/b, with an anti-correlation between miRNA 26 a/b and EGFR.

Luciferase Assay

• A luciferase assay assesses if a specific mRNA is a direct target of a miRNA.
• A bioluminescent molecule is added downstream of the mRNA gene.
• If the miRNA binds to the 3’UTR of the mRNA, it is degraded, releasing less photons of light (less luciferase activity).

miRNA Binding

• The 5’ region of the miRNA binds to the 3’UTR region of the mRNA seed site with complementarity.
• Liver cells increase HGF concentration, stimulating migration but not changes in gastric cancer cells.

Exosomes & Inflammatory Response

• Endogenous miR155 and miR146a regulate inflammation and are released from bone marrow-derived dendritic cells within exosomes, then taken up by recipient dendritic cells.
• Exogenous miRNA mediates target gene repression and reprograms the cellular response to endotoxin.
• miRNA 155 enhances inflammatory gene expression, while miRNA146a reduces it.
• miRNA is ready to silence genes immediately upon delivery because already loaded into the Ago proteins.
• Injection of exosomes containing these miRNAs into mice can elicit modifications in their immune response, even endocrine response.
• LPS (endotoxin) stimulates the transfer of miRNA 155.
• Even with LPS stimulation, the transfer of miRNA 155 is inhibited if exosomes are inhibited.
• Functional transfer of miRNA155 results in a decrease in gene expression.
• Seed site specificity gives rise to these effects.

Hedgehog Signaling

• Hh signaling is relevant to early embryogenesis/morphological development.
• In the absence of Hh ligand, Patched receptor inhibits Smoothened receptor.
• Hh molecule is activated by autocleavage into an active 20-kDA N-terminal fragment with lipid modifications.
• Active Hh can outcompete smoothened for its 12-pass membrane receptor Patched.
• In the inactive form, Gli transcription factor is held in place by a complex with proteins like Sufu, Kif7, PKA, GSK3 and CK1, this protein can tag Gli for patrial proteasomal degradation.
• Pathway activation allows Gli to dissociate, upregulating hedgehog-related genes in the nucleus.
• Early limb development involves a Shh gradient, with stem cells telling cells to proliferate.
• In cancer, cells mimic this gradient, making healthy cells divide.
• Shh ligand appears in 4 forms: heterodimer with a SCUBE2 chaperon protein, soluble multimer, lipoprotein, or exovesicle.
• Shh pathways involve scaffolding and cytoskeleton remolding during cellular signalling which is helpful in guiding these secondary messengers from the PM to the nucleus.
• Vertebrates have 3 Shh signaling mechanisms: free diffusion, directed transport via filopodia, and exo/endocytosis.

Filopodia Transport

• Filopodia contract and elongate until they find their target cells, then become rigid, allowing direct membrane contact.
• Filopodia have variable speeds and lengths.
• Actin-based molecular motors can move along these structures.
• Filopodia of Shh-producing cells interact with filopodia of Shh-receiving cells that have the necessary co-receptors.
• The ability of molecular motors to move back and forth along the filopodia helps with signal concentration and correction of signaling mistakes.

Wnt Signaling

• Involved in the formation of the body axis/bilateral symmetry during embryonic development and tissue regeneration.

• Wnt ligands are secreted lipid-modified glycoproteins activated via glycosylation and palmitoylation.

• In the "on" state:

  • Wnt binds to Frizzled (transmembrane receptor), recruiting LRP (transmembrane receptor).
  • Dishevelled protein inhibits GSK-3 and CK-1 kinases, preventing them from phosphorylating beta-catenin.
  • Beta-catenin dissociates, localizing to the nucleus and binding to TCF/LEF to turn on genes.

• In the "off" state:

  • GSK-3 and CK-1 tag beta-catenin for degradation, causing ubiquitination and proteasomal degradation.
  • Groucho binds to TCF/LEF, blocking any accidental Wnt.

• Wnt ligand undergoes lipid modification in the ER (PORC enzyme) and is secreted via the Golgi apparatus with a molecular chaperon in an endosome.

YAP/TAZ Incorporation

• YAP/TAZ (Hippo signaling pathway) act as secondary messengers and there is cross talk between these two pathways.
• YAP/TAZ are associated with beta TcRP, beta catenin, GSK3 via axin 1
• Without Axin 1, YAP and TAZ localized to the nucleus.
• Wnt signaling should turn on YAP/TAZ gene expression.
• Wnt ligand increases both Wnt and YAP/TAZ signaling over time.
• LRP and YAP/TAZ bind to the same location on Axin 1, but binding of LRP to axin 1 causes YAP/TAZ to be pushed off the destruction complex.
• When they inhibited YAP/TAZ, there is less concentration of beta TcRP suggesting that YAP/TAZ are somehow involved in the default repressing signalling of Wnt.
• YAP/TAZ also help to recruit beta TcRP.

Angiogenesis/VEGF Signaling

• Physiological process in which blood vessels develop in adult tissues.
• The primary driver of angiogenesis is VEGF (the ligand), secreted by hypoxic cells, to create a concentration signalling gradient.
• VEGF binds to the VEGF receptor (receptor tyrosine kinase), initiating a phosphorylation cascade.
• VEGF receptor 2 is the main signalling receptor in endothelial blood cell development.
• NRP and HSPGs can also be co-receptors since VEGF can bind to them to form a multi protein complex.

VEGF Binding Effects

• VEGF + VEGFR can be encapsulated into an endosome to enter the cell cytoplasm to avoid over saturation of the signal.
• VEGF binding causes receptor phosphorylation, further phosphorylating PLC gamma.
• PLC gamma creates specific lipids via lipid modification that can act as secondary messengers:
  • DAG activates cPKC then RAF then MEK then ERK1/2 to turn of gene transcription related to cell migration, cell proliferation, homeostasis etc.
  • IP3 can activate Ca2+ which can activate calmodulin then calcineurin, then NFAT can dephosphorylate to also initiate gene transcription.
• Other pathways generated include nitric oxide for vasodilatation, loosening of tight junctions, and ECM remodeling.
• Tip cells guide blood vessel growth, following the VEGF signaling gradient.
• Tumors mimic VEGF secretion and generate pseudo tip cells to guide their own blood vessel growth.

VEGF Receptor Structure

• Explored the structure of the extracellular domain of the VEGF receptor: composed of seven immunoglobulin-like domains.
• The receptor without a ligand appeared as a monomer, adopting random configurations.
• One strand of the receptor binds to a ligand, increasing the affinity of the other strand, forming a tertiary complex.
• Domain 7 only binds once the ligand is bound to domain 2 and 3, showing that this conformation change in the structure is induced by the binding of the ligand to the receptor.
• Cross-over of the two VEGF receptor domains stabilizes the ligand.

TNFa

• TNFa signaling is involved in the systemic inflammation response, regulating cells of the immune system.
• TNFa is produced as a transmembrane protein and becomes a soluble version of active TNFa when cleaved by a protease.
• TNFa receptors are associated with the TRADD death domain, inducing caspases-mediated apoptosis (complex 2).
• TRADD complex via RIP kinase activates NFkb, MAPKs (complex 1) and MLKL (complex 3) transcription factors/pathways, also involved in inflammation response.
• TNFa activation can cause the upregulation/recruitment of more immune cells, and the upregulation of adhesion molecules in the respiratory epithelium.
• RIP kinase activation leads to worse survival outcomes in vivo, as it triggers the necrosis and apoptosis pathways.
• Different stimuli shape the IKK signal via positive and negative feedback loops (IKK is the main transcription factor for inflammatory genes).
• Strong TNF concentrations cause oscillations between on and off nuclear NFkB localization where as weaker concentrations have less oscillations. Different responses being triggered by different signaling concentrations.
• Cells activate in response to TNFa in a "digital manner".
• Persistent nuclear localization of NFkB (high TNFa concentration) is required for expression of later genes.

Interferons

• Interferons (IFN) are widely expressed cytokines with an antiviral and growth inhibitory effect.
• IFN are the first line of defense cytokines against viral infections.
• Three types of IFNs: Type 1 (mainly IFN alpha and beta), type 2 (mainly IFN gamma), and type 3 (mainly IFN lambda).
• IFNa receptor (IFNaRI + TYK2, IFNaR2 + JAK1).
• IFN gamma receptor (IFN gamma receptor 1 + JAK1, IFN gamma receptor 2 + JAK2).

IFN Signaling

• Pathogen recognition leads to IFN synthesis.
• IFNa binds to its receptor, phosphorylating STAT1 and STAT2, creating a dimer.
• This leads to the recruitment of IRF9 to form a tertiary complex, acting as a transcription factor for ISRE.
• IFN gamma signaling phosphorylates STAT1, creating a homodimer for GAS, triggering a more severe cellular response.
• There is cross talk between IFNa and IFN gamma pathways.
• IFNa alone is not enough to clear the hep c virus infection. Need a t-cell specific response to eliminate the virus or else there is a chronic infection.
• Gas transcripts via IFN gamma pathways are essential for clearing the virus.

miRNA's

• miRNAs are a type of non-coding RNA for proteins that performs functions as RNA molecules.
• miRNA does post translational modifications of mRNA.
• Biogenesis:
  • Drosha recognizes the primary microRNA structure and cuts haipins, the precursor microRNA then leaves the nucleus and is processes by Dicer and made into a complex with Argonaut via the RISC enzyme.
  • DGCR8 is an essential cofactor in the DROSHA microprocessor complex as it helps guide DROSHA to the proper cleavage site. The pre-miRNA leaves the nucleus via the XPO5 transporter which requires GTP. TRBP and PACT are essential cofactors in the Dicer cutting process.
  • Multiple miRNA can be produced from a larger primary microRNA structure as multiple hairpins can form.
• The microRNA is modified with a 5’ cap and a 3’ polyadeniline tail. Then can go on to cleave targets, accelerate deadenylating and translational inhibition.
• Dicer, drosha and argonaut are all endonucleases, cutting with the nucleic acid sequence.
• Argonaut is composed of 4 domains, called N, PAZ, Mid and PIWI, loaded with both the miRNA and mRNA with Mg2+ to catalyse the diester phosphate backbone.
• The miRNA binds to the 3’UTR of the mRNAs. If there is a lot of seed site complementarity then this will induce slicing and vice versa.
• The target mRNA are deadenylated and then are decaped and degraded by nucleases.

miRNA Regulations

• miRNAs can contribute to default repression by blocking the function of a transcription factor by maintaining a “zero” signal/default repressed signal blocking it’s function.
• Example of miRNA acting as a default repressor: miRNA 125 b can act as a default repressor in the response to DNA damage. In a normal cell, miRNA 125b targets residual p53 activity, avoiding apoptosis but when a signal occurs i.e. DNA damage miRNA125b is repressed allowing p53 to be activated and the cell can undergo apoptosis.
• Example of miRNA in default activation: miRNA 372 and 373 are activated by YAP/TAZ transcription factors. When the Hippo signal is off, YAP/TAZ are active but when a hippo signal occurs, LATS phosphorylates and inhibits YAP/TAZ but miRNA 372 and 373 can inhibit LATS to prevent signaling levels from getting too high.
• miRNA can also function as both tumor suppressor and oncogenes depending on what they are inhibiting the transcription of.
• For example, if the miRNA targets a mRNAs that code for tumor suppressors then they would be acting as an oncogene, activating cancer and vice versa.
• miRNA 122 interacts with sites in the 5’ and 3’ ends of HCV to act as a psuedo cap.

miRNA & Lipid Metabolism

• Lipid and cholesterol metabolism involve sterol binding proteins and sterol response element binding proteins (SREBPs) which can sense the changes in cellular sterol levels, and act as a transcription factor for SRE gene transcription related to lipid synthesis and uptake.
• SREBP1 turns on fatty acid synthesis to make other lipids.
• Cellular lipid levels can also be regulated via PPARS signalling. This PPAR signaling decreases plasma fatty acid and triglyceride levels by increasing cellular uptake.
• miRNA can act as positive and negative regulators of these pathways such as miRNA 27 a and b.
• HCV infections cause an increase in lipid synthesis (via SREBP) and a decrease in the breakdown in lipids (via PPARs) which results in a fatty liver disease.
• Both miRNA27a/b is upregulated by HCV but inhibit the HCV once the cell becomes to fatty.
• miRNA 27a/b can regulates JUN and HNF4a along with PPAR. miRNA 27 a/b upregulates JUN along with down regulating PPAR and HNFa:
  • JUN inhibits triglyceride degradation.

miRNA, HITS-CLIP and HCV

• One way of study miRNAs in the lab is by using HITS-CLIP which involves cross-linking and immunoprecipitation to identify mRNA targets of miRNAs. Can see these target sites by isolating the mRNA, miRNA and Ago risk complex by using an antibody against Argonaut.
• HITS CLIP (254 nm, more damaging).
• PARCLIP (more photoreactive, less damaging, in vivo). PAR-CLIP uses a 4SU uracil for the cross linking which is a more photoreactive molecule and can be feed in the diet of your in vivo animal model.
• miRNA 122 is interacting with the 5’UTR of the HCV genome. The HCV virus is removing/sequestering the cell’s normal miRNA122 pool along with it’s cellular function, using it it to protect itself from exonuclease degradation and has an effect in changing the host cells biology as it’s derepressing the miRNA targets (SPONGING EFFECT). Results in a shift in the equilibrium of regulation miRNAs play in the body, causing favoured mRNA translation.
• miRNA acts as a tumor suppressor in the liver, where chronic HCV infection is present.

miRNA & Ovarian Cancer

• Explored miRNA 450a as a tumor suppressor in ovarian cancer by regulating energy metabolism by repressing genes from mitochondrial oxidative phosphorylation complexes and the citric acid cycle.
• In ovarian cancer downregulation of miRNA 450 was found which is bad since it’s a tumor suppressor.
• Adding miRNA 450 a to a mouse in vivo reduces tumor volume and size.

ciRNA

• Circular RNAs are covalently closed and are considered non-coding RNAs but can have a role in cellular regulation.
• Circular RNA’s have lots of miRNA binding sites.
• CiRNA found predominantly expressed in excitatory neurons.
• ciRNAs have a role in upregulating miRNAs or in their stabilizations to move to axonal and synaptic cite for RNA processing (m7)
• For miRNA 671, CDR1as regulated it throughout sequestering/sponging.