G551D Mutations and CFTR Protein

G551D Mutations

• G551D mutations are classified as Class III mutations and are also known as gating mutations.
• Occur in an estimated 6% of Cystic Fibrosis cases.
• The mutations result in CFTR protein being expressed at the cell surface membrane, but it is unable to transport enough chloride ions due to impaired nucleotide binding.
• The mutation disrupts ATP activation and regulation of the CFTR protein.

CFTR Protein Function and Implications

• The pancreatic duct cells secrete bicarbonate into the duct lumen via an apical membrane Cl2/HCO3 2 exchanger.
• The CFTR protein is responsible for the recycling of chloride ions into the lumen through a paracellular route.
• The electrochemical gradient established by chloride movement through the CFTR drives the movement of sodium and water into the ducts.
• Dysfunctional CFTR protein leads to altered electrolyte and fluid transport within the pancreas, lungs, and other organs.

Ivacaftor

• Ivacaftor directly binds to CFTR, likely on membrane spanning domains.
• It improves chloride transport by promoting ATP-independent gating and stabilizing the protein while the channel is open, delaying channel closure.

Cholesterol

• Cholesterol is an alicyclic compound containing 27 carbon atoms, a hydroxyl group at C3, a double bond between C5 and C6, an eight-membered hydrocarbon chain attached to carbon 17 in the D-ring, a methyl group (carbon 19) attached to carbon 10, and a second methyl group (carbon 18) attached to carbon 13.
• Cholesterol is a normal component of most body tissues, particularly the brain, nervous system, liver, and blood.
• Cholesterol is incorporated into the phospholipid bilayer of cell membranes, making them stronger, more flexible, but less permeable to water-soluble substances such as ions and monosaccharides.
• The polar hydroxyl group of cholesterol is oriented towards the surface of the membrane, while the hydrocarbon tail and steroid nucleus lie in the hydrophobic core.
• Cholesterol participates in forming sex hormones, adrenal hormones, vitamin D, and bile salts.
• Some cholesterol is transformed into vitamin D and some are coupled with proteins to become lipoproteins.

Cholesterol Metabolism

• Around one-third of plasma cholesterol exists in the free (or unesterified) form.
• The remaining two-thirds exist as cholesterol esters.
• The liver synthesizes cholesterol and secretes it as cholesterol esters, biliary cholesterol, or bile acids.
• Cholesterol esters are more hydrophobic than free cholesterol, and their production in the liver is catalyzed by acylCoA–cholesterol acyl transferase (ACAT).
• Animals are the primary source of dietary cholesterol, particularly meats, poultry, seafood, and dairy products.
• Plants do not contain cholesterol.

Lipoproteins

• After consumption and absorption, triglycerides and cholesterol are re-esterified in the intestinal mucosal cells and then coupled with various apoproteins, phospholipids, and unesterified cholesterol into lipoprotein particles called chylomicrons.
• Chylomicrons are composed of 90% TAG, 5% cholesterol, 3% phospholipids, and 2% protein.
• The major apolipoproteins of chylomicrons are apolipoprotein B48 (apoB48), apoCII, and apoE.
• Apoproteins contribute to the hydrophilicity and structural stability of the particle and have other functions: They activate certain enzymes required for normal lipoprotein metabolism, and they act as ligands on the surface of the lipoprotein that target specific receptors on peripheral tissues that require lipoprotein delivery for their innate cellular functions.
• Chylomicrons are secreted into intestinal lymph, entering the bloodstream through the thoracic duct, and bind to the wall of capillaries in adipose and skeletal muscle tissue.
• ApoCII activates lipoprotein lipase (LPL), an enzyme that hydrolyzes chylomicrons, leading to the release of free fatty acids derived from core triacylglycerides for use by target cells.

Remnant Chylomicrons

• Remnant chylomicrons are high in cholesterol esters and characterized by the presence of apoproteins B, CIII, and E.
• They are cleared from the circulation by binding of their E apoprotein to a receptor present only on the surface of hepatic cells.
• Subsequently, the bound remnants are taken to the inside of hepatic cells by endocytosis.

Low-Density Lipoprotein (LDL)

• LDL is taken up into cells via apo B-100 receptor-mediated endocytosis.
• Intracellular cholesterol intake is regulated via three feedback mechanisms: LDL-receptor expression, ACAT-mediated storage of cholesterol, and HMG CoA reductase (regulates endogenous production of cholesterol).
• Defects in the LDL receptor are associated with familial hypercholesterolaemia.
• This is exacerbated by upregulated intracellular production of cholesterol due to reduced inhibition of HMG CoA reductase.

Oxidized LDL (ox-LDL)

• When present in excess, LDL can infiltrate and accumulate within the vascular endothelium, particularly at sites of endothelial damage.
• LDL becomes oxidised (ox-LDL) at the apoprotein B-100 by reactive oxygen species in the subendothelial space.
• Accumulation of ox-LDL promotes migration of circulating monocytes into the vascular wall, where they become active macrophages and phagocytose ox-LDL.
• Upon phagocytosis of ox-LDLs, macrophages form atherogenic foam cells with reduced immune function.
• Macrophages recognise LDLs via a scavenger receptor, which is not down-regulated by incoming cholesterol.
• Foam cells continue to ingest ox-LDL until they undergo lysis, releasing their contents (including cholesterol and pro-inflammatory mediators).

Atherosclerosis

• Cholesterol is precipitated and forms diffuse atheromatous plaques within the arterial tunica intima.
• This contributes to the development of an atheroma, which expands into the luminal space leading to vessel narrowing (stenosis).
• Atheroma formation is exacerbated by deposition and crystallization of circulating calcium in atheromatous regions, as well as accumulation of fibrogenic substances, further contributing to sclerosis and narrowing of the vessel wall.

Epithelial-Mesenchymal Transition (EMT)

• EMT is characterized by changes in cell morphology, motility, and invasion.
• During EMT, cells acquire motility and invasive abilities.
• Cells express matrix metalloproteinases (MMPs), which can degrade the extracellular matrix (ECM) proteins, enabling further invasive behavior.
• Cells that have undergone EMT acquire resistance to senescence and apoptosis.

Metastasis

• Metastasis involves cancer cells leaving the primary tumour site and migrating to secondary sites via the bloodstream and/or lymphatic system.
• Cancer cells break away from the primary tumour, attach to and degrade proteins in the surrounding ECM, enabling them to escape.
• Different types of cancer tend to spread to particular organs and tissues, driven by soluble signal molecules such as chemokines and transforming growth factor beta (TGF-β).
• Metastasis requires invasion, which is enabled by EMT.

Cancer Cell Invasion and Metastasis

• Carcinoma cells in a primary tumour lose cell-cell adhesion mediated by E-cadherin repression, break through the basement membrane with increased invasive properties, and enter the bloodstream.
• When circulating tumour cells (CTCs) exit the bloodstream to form micrometastases, they undergo MET for clonal outgrowth.

Platelets and EMT

• Platelets in the blood can initiate the induction of EMT in cancer cells.
• When recruited to a site in the blood vessel, platelets release pro-EMT factors like TGF-β.
• Released TGF-β enhances invasiveness and promotes metastasis of cancer cells in the tumour.

Explore the implications of G551D mutations classified as Class III mutations, which are significant in cystic fibrosis cases. This quiz delves into the CFTR protein's function, its role in chloride ion transport, and the effects of Ivacaftor treatment in managing this mutation.