The Complex Proteome (Theme 2: Module 4) Flashcards
Human Proteome
the full number of proteins that are expressed by all the hereditary information in our DNA (genome)
our genome encodes for over
1,000,000 proteins - suggests that single genes can encode multiple proteins
eukaryotes are unique because segregation of genetic info occurs inside:
double membraned nuclear envelope
-Nucleus: transcription of DNA into RNA, along with RNA processing
-Double membrane of nucleus: continuous, evolved from membranous network of single-membrane ER
-Compartmentalization: allows for more intricate control in regulation of cellular processes
Following transcription in eukaryotes:
mRNA’s are prepared for translation
mature mRNA’s are exported out of the nucleus and into the cytosol where free or bound ER ribosomes facilitate translation into polypeptides
the complexity of the proteome is affected by:
the cascade of events that occur - alternative splicing and post-translational modifications
composition of the proteome can change in response to:
organisms developmental stage, in response to external/internal signals
Example of how cells detect changes (stimuli that result in cellular processes) in the environment
-following a meal, many cells would be sensitive and respond to the stimulus (eg.increase in blood glucose levels)
-these glucose levels are regulated in our bodies
-regulation occurs due to sensory responses in specialised beta islet cells of the pancreas that will lead to a cascade of events that can return blood glucose back to normal levels
-in response to high glucose levels, the pancreas will modulate the synthesis and secretion of an increased amount of its own signal
-insulin is the effector protein that is produced by the pancreatic beta cells - communicates with target cells, and therefore leads to a decrease in blood glucose levels
This is highly regulated process that depends on cell-to-cell communication
Following a meal, where is glucose absorbed? (2 places)
- mainly into the bloodstream
- some absorbs in the mouth across thin epithelial surfaces that are associated w/underlying blood vessels/capillaries - or a large amount of absorption occurs in microvilli cells of small intestine
Absorbed glucose is transported to:
microvilli cells of small intestine: immediately associates with very small blood vessels. They absorb the glucose that is found within the intestinal tract, from these absorbed glucose molecules are transported into the blood vessels. Glucose molecules will travel through the circulatory system
-after a meal, synthesized pancreatic beta cells are able to detect an increase in blood glucose levels and adjust the amount of synthesis and secretion of the insulin proteins
(insulin will act as an effector to help reduce blood glucose levels)
Glucose metabolism controls:
insulin biosynthesis (which is regulated at both transcriptional and translational levels)
glucose metabolism leads to an increase in the insulin gene transcription and mRNA translation
Dorothy Hodgin
used x-ray crystallography to determine the structure of the functional insulin protein
-it is made u of 2 amino acid chains: alpha chain (21 amino acids) , beta chain (30 amino acids)
Dimer
made up of 2 amino acid chains. these make up the functional insulin protein
post-translational modifications
processing of the insulin protein from a single polypeptide of 110 amino acids (preproinsulin) to a protein structure containing 2 polypeptides of 21 and 30 amino acids
Preproinsulin (110 amino acid precursor)
contains an N-terminal signal sequence which interacts w/signal recognition particles (SRP) to facilitate translocation of preproinsulin into the lumen of the rough ER
(originally it is processed through cleavage of the signal sequence, yielding a proinsulin molecule as a result)
further modification to obtain the mature insulin protein secreted from pancreatic beta cells
-why are post-translational modifications crucial
-proinsulin will undergo folding+formation of 3 disulphide bonds
-chaperone proteins: found within the rough E.R assist in protein folding
-folded proinsulin protein is transported from the rough E.R to golgi apparatus
-further cleavage occurs: forming mature insulin dimer (A+B chains) and in the process releases a small C-chain
Post-translational modifications are crucial because: N-terminal and C-terminal amino acid residues in the A & B chains are able to bind to the insulin receptors on the target cells
functional diversity of the proteome is increased by:
post-translational modifications (cleavage)
other modifications :covalent attachment of other molecules and/or degradation of entire proteins
3 types of covalent attachment:
- Phosphorylation: covalent attachment of phosphate group to serine, theonine, tyrosine amino acid residues in a protein (by kinases/enzymes)
- Methylation: involves the covalent addition of a methyl group
- Acetylation: involves the addition of an acetyl group to a specific amino acid residue in a protein
Receptors:
important proteins that receive and interpret information (such as signalling molecules like insulin), thousand of different types
Receptor kinases:
family of receptors that the insulin protein will bind to
-exist in monomeric forms
-enable cells in the body to transport glucose across the plasma membrane into the cytosol of the cell
-when a signal (ex:insulin) binds to a receptors monomer, a conformational change causes monomers to pair up (dimerize). leads to the activation of cytoplasmic domains of the receptor (which have the ability to act like kinase proteins)
Kinase proteins
engage in the phosphorylation of specific amino acids (phosphorylate each other at many regions on the receptor tails and can lead to blinding and activation of other important cytoplasmic proteins)
Result of intracellular signal amplification
(insulin induces the uptake of glucose)
extracellular insulin signal causes a series of cytoplasmic proteins to become sequentially activated and will lead to an intracellular response
leads to… activation of glucose transporter proteins at the cell surface, as result, absorption of glucose into the cell
How are intracellular signals amplified:
induction of cellular signals: occurs through activation of a series of diverse transducer and amplifier proteins that are downstream from the activated receptor, (however, many elements in the the signalling pathway can lead to.. )
activation of negative feedback loops: lead to intracellular signal termination
double negative feedback: inhibitor of the signal can also be inhibited. provides fine control in a cell in response to an extracellular signal
Tissues that have insulin receptor kinases (able to detect changes in blood glucose levels and can contribute to the absorption of glucose from the blood) :
- Fat cells in adipose tissue: take up glucose and fatty acids and store the excess as fats in the form of triglycerides
- Liver/muscle cells: able to take up glucose from the blood and store the excess as glycogen
Alternative Splicing
enables one pre-mRNA molecule to be spliced at different junctions to result in many different mature mRNA molecules that each contain different combinations of transcribed exons
some exons may be excluded leading to production of many isoforms or different types of mature mRNA from the same pre-mRNA transcript (occurs because sometimes what the spliceosome will sometimes recognize as an exon, can be identified as an intron in other primary transcripts )
elements of alternative splicing
exons: regions of pre-mRNA that are included in the mature mRNA
introns: parts of the pre-mRNA that are removed during the splicing process
splicing
non-protein coding introns are removed from the pre-mRNA, and exons are joined or spliced together to produce a mature mRNA that will then serve as a template for translation into a functional protein
alternative splicing helps:
in the regulation of gene expression.
(since the same primary transcript can be spliced in different ways to produce mature mRNA isoforms that can lead to the production of different, but related proteins)
example of alternative splicing
observed during the processing of the primary transcript of the gene that encodes for human insulin receptor:
-insulin receptor gene: 22 exons
-in skeletal muscle cells: exon II is removed from mature mRNA product and introns during splicing process
this mRNA isoform of insulin receptor will be translated into a higher affinity version of the insulin receptor in muscle cells
-skeletal muscles will be able to mount a higher response of glucose uptake in response to an insulin signal
what does the ideal insulin receptor inform to have at skeletal muscles do?
vs liver cells
contributes to lowering blood glucose levels, allows the muscle cells to absorb enough glucose to meet their high energy needs
Liver cells: produce an insulin receptor w/lower affinity to insulin (one key difference: during splicing, exon II is retained in the mature mRNA molecule. As a result, message in DNA blueprint is the same but alternative splicing leads to the processing of multiple different mRNA molecules and eventually, the translation of alternative protein isoforms)
Insulin acts as an effector signal
targets cells of the body to absorb glucose from the bloodstream
signal is regulated once glucose levels are returned to resting levels, there will be feedback to bring the entire system back to the starting/resing point
we will see a negative feedback loop: the drop in blood glucose will be detected by the pancreatic cells and there will be a decrease in the secretion of insulin (feedback of this sensor cell limits further response in the entire system)
example of how: Changes to splicing can lead to detrimental cellular effects
-if insulin was not processed correctly following translation, there may not be any ability for this protein to bind to the insulin receptors on target tissues
-if the insulin receptor protein was incorrectly spliced, there would be no ability to activate glucose transport proteins that allow for the import of glucose from the blood stream at these target regions
- defect in insulin protein/receptor can lead to the inability to take up glucose, resulting in hyperglycemia and diabetes
^^an example of how the complex proteome can very due to different patterns of gene expression and protein modification
