Prion disease, Alzheimer's disease, PTM, ion exchange chromatography and SDS-PAGE electrophoresis Flashcards
Prion disease definition
Prion protein is a structurally abnormal form of naturally occurring cellular protein
- Creutzfeldt-Jakob disease (CJD) – ingestion of meat from BSE cattle
- Bovine spongiform encephalopathy (BSE a.k.a. mad cow disease)
Alzheimer disease defintion
Accumulation of β-amyloid protein in brain of patients
- Slow progressing
- Characterized by the misfolding, aggregation and gain of toxicity of β-amyloid and tau protein
Similarities between Alzheimer’s disease and Prion disease
Cause neurodegeneration to the body, cause central nervous system to degenerate. Neural function will be affected.
Misfolded proteins aggregates into insoluble plaques.
Creutzfeldt-Jakob disease (CJD)
ingestion of meat from BSE cattle
Bovine spongiform encephalopathy
Mad Cow Disease
Amyloid
fibrous protein aggregation
Human prion disease
- The normal, cellular PrP, denoted PrPC, is converted into PrPSc
- A portion of its α-helical and coil structure is refolded into ß-sheet.
- Autocatalytic conversion (not requiring nucleic acids for replication).
- Due to mutation in the gene responsible for prion production.
- Accumulation of misfolded prion (PrPsc)
- Human Prion diseases may present as genetic or infectious disorders via consuming food containing prions
- Invariably fatal.
- Characterized by Dementia, motor disturbances, visual disturbances, loss of neurons
PrPsc characteristics
Talk about protease sensitivity, predominant secondary structure, solubility in water, infectious PrPsc
Protease resistant unlike PrPc protease sensitive
Beta sheet rich 30% alpha helix and 45% beta sheet
unlike PrPc 40% alpha helix and little to no beta sheet.
By a change in the protein structure from a predominantly α–helix to a ß-sheet structure
PrPsc is infectious allow refolding of PrPc to PrPsc due to polymerization.
PrPc is water soluble due to higher levels of alpha helix
but PrPsc is non-water insoluble due to higher levels of beta sheets.
What happens to the brain in human prion disease?
brain shrinks due to more PrPsc being increasingly deposited, the functional areas of the brain will decrease and degradation can occur very rapdily
Alzheimer Disease
Alzheimer’s disease is an irreversible, progressive brain disease that slowly destroys memory and thinking skills.
Although the risk of developing AD increases with age – in most people with AD, symptoms first appear after age 60
– AD is not a part of normal aging. It is caused by a fatal disease that affects the brain.
The brains of people with AD have an abundance of two abnormal structures:
- β-amyloid plaques, which are dense deposits of protein and cellular material that accumulate outside and around nerve cells
- Neurofibrillary tangles, which are twisted fibers that build up inside the nerve cell
Beta-amyloid plaques
Amyloid precursor protein (APP) is the precursor to amyloid plaque. APP is a normal protein but folded wrongly – trigger abnormal proteolytic cleavage.
- Wrongly folded APP sticks through the neuron membrane.
- Enzymes (β-secretase) cut the APP into fragments of protein, including beta-amyloid (long fiber protein, consists of β-sheets)
- Beta-amyloid fragments clumps to form insoluble plaques, disrupting the work of neurons. This affects the areas of the brain.
Amyloid plaque formation
Because APP is folded wrongly, it will be cut by β-secretase abnormally – plaque
- The accumulation of plague is abnormal
- Insoluble and can’t be eliminated
Deposition around the neurons, thus disrupting the neuron transmission
Neurofibrillary Tangles
Neurofibrillary tangles are abnormal accumulations of a protein called tau that collect inside neurons.
Healthy neurons are supported internally by structures called microtubules, which help guide nutrients and molecules from the cell body to the axon and dendrites.
In healthy neurons, tau normally binds to and stabilizes microtubules.
In Alzheimer’s disease, tau proteins detach from microtubules and stick to other tau molecules, forming threads that eventually join to form tangles inside neurons and blocking the neuron’s transport system
Summary of Alzheimer’s disease
Plaques– deposits of the protein beta-amyloid that accumulate in the spaces between nerve cells
Tangles – deposits of the protein tau that accumulate inside of nerve cells
In a nut shell, neurons have an internal support structure partly made up of microtubules. A protein called tau helps stabilize microtubules. In AD, tau changes, causing microtubules to collapse, and tau proteins clump together to form neurofibrillary tangles.
APP is folded wrongly, cut by beta-secretase abnormally, resulting in a accumulation of insoluble plaque that is deposited around the neuron that affects neural transmission.
Post translational modifications purpose
A key mechanism to increase proteomic diversity. After protein is synthesized, proteins can undergo several PTMs to create many different types of proteins, which exponentially increase the diversity of the proteins.
It can occur during synthesis or after synthesis.
Modify side chains or break the bonds between amino acids
Critical to the localization and functional capability of final protein product.
Protein separation methods (2 ways)
SDS-PAGE, Ion-exchange chromatography
SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis)
Proteins have varying charges and complex shapes, therefore may not migrate into the gel at similar rates.
Proteins are usually denatured in the presence of a detergent such as sodium dodecyl sulfate (SDS) which coats the proteins with a negative charge.
The electrophoretic migration rate through a gel is therefore determined only by the size of the complexes and not by the charge differences.
Molecular weights are determined by simultaneously running marker proteins of known molecular weight to compare and estimate the size of the proteins.
How to conduct SDS-PAGE
Cast the gel vertically Load the samples Known standard is loaded simultaneously Samples move from top to bottom A standard graph can be plotted to estimate the size of the protein
Ion exchange chromatography
Proteins - made up of twenty common amino acids. Some of these amino acids possess side groups (“R” groups) which are either positively or negatively charged.
Protein has more positive charges than negative charges - basic protein
More negative charges than the positive charges - acidic protein
Protein contains a predominance of ionic charges - bound to a support that carries the opposite charge.
Basic protein (positively charged) - bind to a negatively-charged support
Acidic protein (negatively charged) - bind to a positively-charged support
Ion exchange: Purification of proteins according to change of working pH
Principle of ion exchange chromatography
- Retention by attraction of opposite charge
- Allows molecules to be separated based upon their charge
- Mobile phase (at different pH/strengths) - Carry sample together
- Stationary phase
- Anion exchanger – positively charged
- Cation exchanger – negatively charged
- Anion exchanger – will bind with negatively-charged protein
- Elute positively-charged protein
- Cation exchanger – will bind with positively-charged protein
- Elute negatively-charged protein
- Elute by change of pH to neutralize charged group on either solute or stationary phase.
- Elute by pH or salt gradient to enhance separation.
- Isoelectric point (pI)
- Washing of unbound protein
- Retention time/flow rate
- Proteins move through the column at rates determined by their net charge at their pH being used . With cation exchangers, proteins with a more net negative charge move faster and elute earlier.
