Proteins

Question 1

Give examples of genes from which there is good correlation between mRNA and protein concentrations

Answer 1

Housekeeping genes

TCA enzyme genes

Question 2

Give examples of genes from which there is poor correlation between mRNA and protein concentrations

Answer 2

Genes for transcription factors, signalling and cell cycle

Question 3

What factors may give rise to the discrepancy between mRNA and protein concentrations?

Answer 3

Transcription can be regulated by the chromatin state of the DNA region containing the gene.
mRNA stability can be affected by both intrinsic factors of the sequence itself and extrinsic regulation e.g. miRNA
Translational efficiency denotes the amount of protein that is made from a transcript and is affected by ribosome occupancy and other phenomena such as codon usage.
Decay rates for proteins are very different from those for mRNA , both as a global average and for a specific gene
Protein turnover is the largest contributor to the discrepancy

Question 4

It is possible to predict protein abundance from mRNA. T/F?

Answer 4

False - owing to the complexity of regulation of protein production

Question 5

Give an example of a testing method for determining the total concentration of protein in a solution

Answer 5

Bicinchoninic acid assay (BCA assay)

Question 6

Describe the methodology for a BCA assay to determine total protein concentration in a solution

Answer 6

First, the peptide bonds in protein reduce copper 2+ ion to copper +. The amount of copper that is reduced in proportional to the amount of protein present in the solution.
Then, two molecules of Bbicinchoninic acid chelate with each copper+ ion forming a purple-coloured complex that strongly absorbs. light at wavelength 562 nm. Thus, by measuring the absorption spectra of the solution and comparing with a standard curve, the amount of protein present can be quantified.

Question 7

Give examples of investigative techniques which look at protein abundance, distribution and localisation that use monoclonal antibodies

Answer 7

ELISA
Western Blot
Immunoprecipitation
Immunohistochemistry
Epitope tagging

Question 8

Describe the methodology of ELISA

Answer 8

A specific antibody. is added to bind to the antigen and then a further antibody that is linked to an enzyme is added. Finally an enzyme substrate is added and the subsequent reaction produces a detectable signal, usually a colour change

Question 9

What is ELISA used for?

Answer 9

To detect and quantify a specific antigen in a liquid sample

Question 10

What are western blots used for?

Answer 10

Used to detect specific proteins in a sample of tissue, homogenate or extract
Used for qualitative detection of single proteins and protein modification
Semi-quantitative estimation of a protein

Question 11

What can be used to help quantify proteins in a western blot?

Answer 11

By applying a dilution series of a purified protein of known concentration - this will allow a more precise estimate of. target protein concentration

Question 12

Describe the methodology of a western blot

Answer 12

SDS-PAGE is commonly used for gel electrophoresis, here proteins are mixed with SDS detergent which binds to the protein and gives it size-dependent negative charge and consistent hydrodynamic properties. A tracking dye is also used to make the sample visible. SDS-page will determine protein size and subunit composition
Following electrophoresis, the sample is transferred to a membrane. Blocking agents are used to prevent non-specific antibody binding. The sample is then stained with antibodies specific to the target protein. The membrane is then stained with a secondary antibody which recognises the first antibody and allows detection through a. varies of methods (staining, immunofluorescence, radioactivity)

Question 13

What is immunoprecipitation?

Answer 13

A technique of precipitating a protein antigen out of solution using an antibody that specifically bonds to that particular protein

Question 14

What is immuoprecipitation used for?

Answer 14

Used to isolate and concentrate a particular protein from a sample containing many thousands of different proteins.

Question 15

What is the difference between immunohistochemistry and immunocytochemistry?

Answer 15

Immunohistochemistry uses tissue sections either embedded in paraffin or frozen to preserve morphology
Immunocytochemistry refers to the staining of isolated/cultured intact cells

Question 16

Describe the difference between chromogenic detection and immunofluorescence detection in immunohistochemistry

Answer 16

In chromogeneic detection an enzyme converts a soluble substrate into an insoluble coloured product at the antigen site
In immunofluorescence, fluorochrome conjugated reagent is bound, either directly or indirectly, to. the primary antibody and once excited, will emit light at a specific wavelength

Question 17

What is an epitope?

Answer 17

The part of an antigen molecule to which an antibody attaches itself

Question 18

Describe the methodology of epitope tagging

Answer 18

A known epitope is fused to a recombinant protein by means of genetic engineering. By choosing an epitope for which there is an antibody available, this technique makes it possible to detect proteins for which no antibody is available.

Question 19

What is epitope tagging particularly useful for?

Answer 19

Characterisation of newly discovered proteins and proteins of low immunogenicity

Question 20

Describe how a genome wide association study identified a gene involved in the pathogenesis of hypertension

Answer 20

An SNP (rs13333226) was identified in a genome wide association study with hypertension. This SNP exists upstream from the gene uromodulin. The minor allele of this gene (G) was shown to lower the risk of hypertension and lower urinary uromodulin excretion in hypertension patients in the BRIGHT study and the general population in the HERCULES study

Question 21

Explain the function of uromodulin in decreasing blood pressure

Answer 21

UMOD expression is restricted to the medullary region of the thick ascending limb - the region responsible for reabsorbing 25% of filtered salt - which is known to bind several cytokines. UMOD is trafficked to the sub apical space where it co-localises with NKCC2. It is thought that decreased. UMOD (due to the genetic variant), decreases sodium reabsorption in the thick ascending limb which decrease effective circulating volume to lower blood pressure.

Question 22

Describe how luciferase reporter assays can be used to investigate uromodulin and hypertension

Answer 22

Luciferase reporter assays are bio-luminescent reporter assays which consist of a luciferase reporter enzyme and a detection agent that provides the enzyme substrate. When the reporter enzyme and detection reagent are combined, the light emitted is proportional to reporter gene expression levels and is detected using luminometer.

Question 23

What is the benefit of doing proteome-wide analysis compared to single protein analysis?

Answer 23

Proteome-wide analysis allows us to examine the interrelationships between proteins to start to develop personalised treatments

Question 24

Briefly describe how SNPs can affect warfarin metabolism

Answer 24

The variable pharmacokinetics and efficacy of warfarin can be partially attributed to genetic variability of the liver enzyme responsible for the inactivation of warfarin - CYP 2CP.
The CYP2CP*2 polymorphism is associated with 30% less enzyme activation and 8-16% lower dosing, whilst the *3 allele is associated with 90% less enzyme activity and 20-36% lower dosing.
The net effect depends on whether an individual is hetero- or homo-zygous for the variant allele.
>200 SNPs of the CYP2CP gene have now been identified of which only ~30 are in the protein coding region. Many are associated with reduced enzyme activity

Question 25

Briefly describe the association between ethnicity and genetic variability in response to / metabolism of warfarin

Answer 25

Caucasians are more likely to. have CYP2CP alleles which are associated with higher dosage requirements.
A variant of the VKORC1 gene which results in a decreased ability to clot and therefore equates to lower warfarin dosing requirements is seen most commonly in African individuals

Question 26

Give an overview of the phenotypic variations in codeine metabolism and how these arise

Answer 26

Codeine is de-methylated to its active form (morphine) in the body by the enzyme CYP2D6. There are >80 allele variations of CYP2D6, which each have a different effect.
The phenotype is. based on functional impact and can. be classified as poor metaboliser, intermediate metaboliser, extensive metaboliser and ultra-rapid metaboliser.
The EM phenotype is most common. The frequency of these alleles again vary by ethnicity, with the ultra-metaboliser phenotype much more common in African individuals. These individuals have increased rapid formation of morphine which increases the risk of toxicity

Question 27

Genetic variations in CYP2D6 result in phenotypic variations in codeine metabolism. However, this enzyme also acts as 25% of all prescription drugs so the risk of adverse reactions is increased when individuals are taking multiple drugs. Drugs on which CYP2D6 act include…?

Answer 27

SSRIs
TCAs
Beta blockers
Opiates
Neuroleptics
Antiarrythmics

Question 28

Give an example of a specific cancer therapy which is designed based on the specific genetics of the cancer cell

Answer 28

HER2 is present on all human cells, but in certain types of cancer (1/5 of breast and stomach cancers), its levels are unusually high. Trastuzumab (Herceptin) is a monoclonal antibody based biological therapeutic and can be used to help control the growth of cancer cells that contain high amounts of HER2 by targeting these cells for destruction

Question 29

What are antibody microarrays?

Answer 29

These are a specific form of protein microarray - a high-throughput method used to track the interactions, activities and functions of proteins on a large scale

Question 30

Describe the methodology of antibody microarrays

Answer 30

A collection of capture antibodies are spotted and fixed on a solid surface and the interaction between the antibody and its target antigen is detected.

Question 31

Describe the application of antibody microarrays

Answer 31

They are often used for detecting protein expression from various bio fluids
One main focus in antibody array is biomarker discovery, specifically for cancer

Question 32

What is chromosomal immunoprecipitation?

Answer 32

A type of immunoprecipitation which investigates the interactions between proteins and DNA It aims to determine whether specific proteins are associated with specific genomic regions e.g. transcription factors on promoters.

Question 33

Describe the methodology of chromosomal immunoprecipitation

Answer 33

DNA and associated proteins on chromatin in living cells / tissue are crosslinked
DNA/protein complexes are sheared into fragments by sonification or nuclease digestion
Cross-linked DNA fragments associated with the protein(s) of interest are selectively immunoprecipitated fro the debris using a protein-specific antibody
Associated DNA fragments are purified and identified using PCR, DNA microarrays and molecular cloning and sequencing

Question 34

Describe the philosophy/methodology of ‘bottom up’ proteomics

Answer 34

The crude protein extract is first enzymatically digested then the peptides are separated using high performance liquid chromatography coupled to mass spectrometry, The identified peptides are then assembled to allow protein identification

Question 35

Describe the philosophy/methodology of ‘top-down’ proteomics

Answer 35

An intact protein is analysed by first separating proteins using 2D gel electrophoresis or mass spectrometry, then fragmentation occurs to allow protein identification

Question 36

Describe the basic methodology of high performance liquid chromatography

Answer 36

This is a method of separating proteins which relies on pumps to pass a pressurised liquid solvent containing the sample mixture through a column filled with a solid adsorbent material. Each component in the sample interacts differently with the adsorbent material causing different flow rates for different components leading to separation

Question 37

Briefly describe the different type of high performance liquid chromatography

Answer 37

Gel filtration - proteins are separated by size
Affinity - separation occurs due to the unique binding characteristics of a protein of interest for an affinity matrix in the column
Ion exchange - separation is base on charge: DEA E-cellulose is used for positive charge separation. and CM-cellulose is used for negative charge separation

Question 38

Describe the methodology of 2D gel electrophoresis.

Answer 38

The first round of gel electrophoresis separates proteins based on isoelectric point - the pH at which a molecule has net zero charge
The molecules are then perpendicularly separated in the second round, which separates based on molecular weight

Question 39

Describe the pros and cons of 2D gel electrophoresis

Answer 39

2DGE allows differential labelling and separation of different samples (e.g. disease and control) on the same gel
However, it is biased towards fairly abundant protein, is not suited for transmembrane proteins and does not always enable protein identification

Question 40

Describe how mass spectrometry can allow protein identification

Answer 40

Ionised biomolecules are accelerated in an electric field and enter the flight tube. During their flight in this tube, different molecules are separated according to their mass:charge ratio and therefore reach the detector at different times. In this way, each molecule yields a distinct signal.

Question 41

Describe the. accuracy of mass spectrometry in protein identification

Answer 41

Mass spectrometry is an analytical tool used for measuring the molecular mass of a sample to within an accuracy of 0.01%. This is sufficient to allow minor mass changes to be detected e.g. the substitution of one amino acid for another or a post-translational modification

Question 42

Describe the methodology of stable isotope labelling with amino acids in cell culture (SILAC)

Answer 42

Two populations of cells are cultured: one in a growth medium containing normal amino acids and the other containing amino acids labelled with stable. heavy isotopes. As the cells grow, they incorporate. the heavy amino acids into all of their proteins. All the proteins from both cell populations can then be combined and analysed together using mass spectrometry which can differentiate the normal and heavy amino acids.

Question 43

What are protein-protein interactions?

Answer 43

PPIs are physical contacts of high specificity established between two or more protein molecules. They allow proteins to work in complexes to carry out specific functions.

Question 44

Give an example of a method of identifying protein-protein interactions

Answer 44

Affinity purification coupled to mass spectrometry