Protein Folding Diseases PDF

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Protein mis-folding diseases: Cystic Fibrosis: Cystic Fibrosis is one of the most discussed and researched genetic disorders of modern times. It’s a disorder that affects the lungs mainly but also the pancreas, liver, kidneys and intestine. Long term issues include difficulty breathing and coughing ...

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Protein mis-folding diseases: Cystic Fibrosis: Cystic Fibrosis is one of the most discussed and researched genetic disorders of modern times. It’s a disorder that affects the lungs mainly but also the pancreas, liver, kidneys and intestine. Long term issues include difficulty breathing and coughing up mucus due to frequent lung infections. CF is inherited in an autosomal recessive manner meaning that it is caused by the presence of mutations in both copies of the gene for cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is involved in production of sweat, digestive fluids and mucus. When it’s not functional, secretions which are usually thin and not viscous become thick. Cause: CF is caused by a mutation in CFTR. There are many different disease causing mutations, but the most common one by far is the DeltaF508, which is the deletin of three nucleotides encoding for a phenylalanine at the 508 position. His mutation accounts for 66-70% of CF cases worldwide and 90% in the US, however, there are over 1500 mutations known to cause CF. Only one functioning copy of CFTR is needed to negate the effects of one defective allele. CFTR is found at the q31.2 locus on chromosome 7, is 230,000 bp long and creates a protein that is 1,480 amino acids long. Structurally CFTR is known as an ABC gene (ATP-Binding Casette). The product of this gene is the CFTR protein which is a chloride ion channel important in synthesising sweat, digestive juices and mucus. It contains two ATP-hydrolysing domains, which allows the protein to use energy in the form of ATP. It also contains two domains comprising of six alpha helices apiece, which allow the protein to cross the cell membrane. A regulatory binding site on the protein allows it to be activated by phosphorylation. The C-terminus of the protein is anchored to the cytoskeleton by a PDZ domain interaction. Pathophysiology: Different mutations cause different defects in CFTR protein, sometimes giving a milder or more severe phenotype. These proteins are hence the targets for drugs that can sometimes restore their function. DeltaF508 creates a protein that mis-folds and as such is not appropriately transported to the cell membrane, resulting in its untimely degradation via the Unfolded Protein Response. Other mutations result in truncations due to premature termination of protein synthesis. Other mutations produce proteins that don’t use energy in the form of ATP normally, d not allow chloride, iodide and thiocyanate to cross the membrane appropriately, and are degraded at a faster rate than normal. Mutations may also lead to fewer copies of the CFTR protein being produced. CFTR is a protein anchored in the outer membrane of many exocrine cells all around the body as well as in the lungs. It spans the membrane and acts as a channel connecting the cytoplasm to the surrounding fluid. The channel is primarily responsible for controlling the movement of halogens from inside to the outside of the cell; however in sweat glands it facilitates the movement of chloride from the sweat duct into the cytoplasm. Most of the damage in CF is due to blockage of the narrow passages of affected organs with

thickened secretions. These blockages lead to remodelling and infection in the lung, damage by accumulated digestive enzymes in the pancreas, blockage of the intestines by thick faeces etc. The widely accepted theory suggests that defective ion transport leads to dehydration in the airway epithelia, thickening mucus. In airway epithelial cells, the cilia exist in between the cell’s apical surface and mucus in a layer known as the airway surface layer. The flow of ions from the cell and into this layer is determined by channels such as CFTR. CFTR not only allows chloride ions to be drawn from the cell into the ASL, but it also regulates another channel called ENac, which allows sodium ions to leave the ASL ad enter the respirator epithelium. CFTR normally inhibits ENac, but if the CFTR is defective then sodium flows freely from the ASL and into the cell. As water follows the sodium by osmosis, the depth of ASL will be depleted and the cilia will be left in the mucus layer. As cilia cannot effectively move in this kind of thick, viscous environment, mucociliary clearance is deficient and a build-up of mucus occurs, clogging small airways. The accumulation of more viscous nutrient-rich mucus in the lungs allows bacteria to hide from the body’s immune system, causing repeat respiratory infections. Management: There are currently no known cures for CF, but there are several treatment methods currently in practice. The cornerstones of management are proactive treatment of airway infections and encouragement of good nutrition and active lifestyle. Many CF sufferers are on one or more antibiotics at all times, even when healthy, to prophylactically suppress infection. Prolonged therapy often requires hospitalisation and insertion of a more permanent IV. Inhaled therapy with antibiotics such as tobramycin is often given for months at a time to improve lung function by impeding the growth of colonised bacteria. Antibiotics can also be given orally. Research: Gene therapy has been explored as a potential cure for CF. This is where there is an attempt to restore genetic normality in the afflicted cells of the patients’ body, hence restoring the normal production of CFTR. So far, little benefit has been seen from this kind of therapy. Most CF gene research is based around trying t place a normal copy of the CFTR gene into the affected cells. To prevent symptoms found in the lungs, only 5-10% of normal CFTR expression is needed. Multiple approaches for delivering this gene have been tested, such as liposomes and viral vectors in animal models and clinical trials. However, both were found to be inefficient treatment methods, mainly because of poor vector uptake. Small molecules are another potential treatment for CF. These molecules aim at compensating various mutations in the CFTR gene. One approach is to develop drugs that get the ribosome to overcome the stop codon of truncation mutations and synthesise a full-length CFTR protein (around 10% f CF is down to a protein truncation). These drugs target mutations such as G542X, which consists of the amino acid glycine in position 542 being replaced with a stop codon. Aminoglycoside antibiotics interfere with protein synthesis and error-correction. In some cases, they can cause the cell to overcome a premature stop codon by inserting a random amino acid, thereby allowing expression of the full-length protein. Precision Medicine Approaches:

Subclasses of CF are defined according to the functional effects of specific genetic variants of CFTR. 6 subclasses are defined: - I = No functional protein (G542x) - II = Trafficking defect (DeltaF508) - III = Defective regulation (G551D) - IV = Decreased conductance (R117H) - V = Reduced synthesis (3120+1G>A) - VI = Reduced stability (Q1412x) The first drug approved for a subclass of CF was Ivacaftor, which increases the opening probability of channels on the cell surface. It was initially approved for class III CF, for which the trafficking of CFTR is fine, but the regulation is defective. The most comment variant, DeltaF508, results in the destruction of a misfolded CFTR protein (Class II). For this a combination of Lumacaftor (to enhance intracellular processing and trafficking) and Ivacaftor may be optimal. In this, Lumacaftor aids in the correct trafficking of mutant CFTR by providing a proteasomal escape. However, when the defective CFTR reaches the cell surface, it has a gating abnormality similar to G551D and hence Ivacaftor, as it does in G551D patients, aids by increasing channel opening times.

Creuzfeldt-Jakob Disease: CJD is an incurable and fatal neurodegenerative disease in humans. It is sometimes compared to ‘mad cow disease’ or bovine spongiform encephalopathy (BSE) but only Variant CJD (vCJD) is acquired from BSE. CJD is caused by a transmissible protein called a prion; these are misfolded proteins that replicate by converting their correctly folded counterparts to their misfolded structure. CJD causes rapid degradation of neuronal tissue and causes small pores to develop in the brain, giving it the appearance of a kitchen sponge under the microscope, hence ‘spong’iform. Prognosis is poor with 15% of patients surviving for 2 or more years. Direct causes of death include heart failure, respiratory failure, pneumonia and other infections. There are four classifications of CJD: - Variant CJD acquired from BSE by consuming food with the prions present. - Sporadic CJD caused by spontaneous misfolding of prion-protein in an individual. This accounts for 85% of cases of CJD. - Familial CJD caused by an inherited mutation in the prion-protein gene. This accounts for the majority of the other 15% of CJD cases. - Iatrogenic CJD caused by contamination with tissue from an infected person, such as from a blood transfusion. Cause: It’s caused by prions. Prions are proteins that occur naturally in neurons of the CNS. The protein they’re made of is called PrP and is normally found on the membranes of cells. It has 209 amino acids, one disulphide bond, a molecular mass of ~36kDa and a mainly alpha-helical structure. They’re thought to affect signalling processes, damaging neurons and resulting in degeneration that causes spongiform appearance in the brain. Prions are dangerous as they promote the refolding of native prion protein in the disease

state. The number of misfolded prion proteins increases exponentially and the process leads to a large quantity of insoluble protein in affected cells. This mass of misfolded proteins disrupt neuronal cell function and causes cell death. Mutations in the gene for PrPC can cause misfolding of the dominantly alpha-helical regions to Beta pleated sheets. This disables the ability of the protein to undergo digestion. Once the prion I stransmitting it invades the brain and induces other PrP molecules to misfold in a self-sustaining feedback loop.

Sickle Cell Disease: SCD is a group of blood disorders, the most common is Sickle Cell Anaemia SCA. This results in abnormal haemoglobin in the red blood cells, leading to a rigid sickle-cell like shape. Problems in SCA usually begin around 5-6 months of age. Health problems include pain (sickle cell crisis), anaemia, swelling in the hands and feet, bacterial infections and stroke. Average life expectancy is 40-60 years. SCD occurs when a person inherits two abnormal copies of the haemoglobin gene on chromosome 11. A person with one copy does not have the disease but is said to possess sickle-cell trait, they are carriers. Diagnosis is by a blood test. As of 2015 4.4 million people have SCD and around 80 million are carriers. 80% of SCD cases are believed to be in Sub-Saharan Africa. Genetics: Normal people have normal haemoglobin consisting of 4 monomers, 2 alpha and 2 beta chains. In SCA both beta-globin subunits are replaced with haemoglobin S. SCDs have an autosomal recessive pattern of inheritance (as in cystic fibrosis). In individuals homozygous for HgbS, long chain polymers of HbS distort the shape of the RBC from the normal smooth biconcave disc shape to ragged and full of spikes, making it fragile and susceptible to breakages in the capillaries. The sickle cell disease occurs when the sixth amino acid is changed from Glutamate to Valine, known as E6V. Valine is hydrophobic which causes the haemoglobin molecule to collapse in on itself occasionally. When enough collapses the cell becomes sickle shaped. The gene defect is known to be a single nucleotide polymorphism (A to T) of the Beta-globin gene. This is normally a benign mutation in normal oxygen concentration, but under conitions of low oxygen concentration, HbS polymerises. The deoxy form of HB exposes a hydrophobic patch on the protein between the E and F helices, the hydrophobic Val at position 6 of the beta chain associates with the hydrophobic patch, causing HBs molecules to aggregate and form fibrous precipitates. Pathophysiology: Loss of RBC elasticity is central to the pathophysiology of SCD. Normally RBCs are quite elastic, which allows the to deform and squeeze through tight cappilaries. In SCD, low-oxygen tension promotes RBC sickling and repeated sickling can damage the cell membrane and decerease overall elasticity of the cell. These cells fail to return to normal shape when oxygen levels are restored, as a consequence, these rigid cells cannot deform when they pass through capillaries and lead to vessel occlusion and ischaemia. The actual anaemia of the illness is caused by haemolysis. Although the marrow attempts to compensate by creating new RBCs, it does not match the rate at which sickle cells are destroyed. Healthy RBCs function for 90-120 days, but sickled cells last only 10-20 days.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3882043/...