Unit 2.4: The Complex Proteome

Human Proteome:

• the complete collection of proteins expressed from the genetic information encoded in our DNA, also known as the genome.

•  a single gene can contribute to the production of multiple proteins or protein variants.

Diversity of the proteome due to 2 Reasons:

• Alternative splicing: allows a single gene to produce different mature mRNA transcripts.

• Post-translational modifications: alter proteins after they have been synthesized.

• These processes allow cells to produce proteins with different structures, activities, locations, and functions without requiring a separate gene for every protein form.

Compartmentalization:

• defining characteristics of eukaryotic cells is the compartmentalization of genetic information within the nucleus

• Compartmentalization allows cellular processes to occur in distinct environments and provides more precise control over cellular activities

Factors that can change the composition of the proteome:

These factors allow cells to adjust the protein they produce and their functions:

1. Developmental Stage of an Organism

2. The type of cell/tissue

3. internal cellular signals

4. external environmental signals

Body’s Response to Eating a Meal:

1. Stimulus = Increase in Blood Glucose Levels

2. Specialized pancreatic cells called beta cells, located in the islets of the pancreas, detect changes associated with elevated blood glucose.

3. When blood glucose is high, these cells increase the synthesis and secretion of insulin, a protein hormone that acts as an effectors signal by communicating with target cells

4. Insulin binds to receptors on target cells

5. Intracellular signalling pathways stimulate responses that help regulate blood glucose.

Glucose Absorption After a Meal:

• After a meal, carbohydrates are digested into simpler sugars, including glucose, which is absorbed into the bloodstream.

• Most glucose absorption occurs in the small intestine, where specialized epithelial cells have microvilli that increase the surface area available for absorption.

• Glucose crosses the intestinal epithelium and enters nearby blood vessels.

• The absorbed glucose then travels through the circulatory system to tissues throughout the body.

• This results in an increase in blood glucose, which provides a stimulus for the pancreatic beta cells to respond.

Insulin Biosynthesis:

• Increased glucose metabolism in pancreatic beta cells stimulates insulin secretion

• Pancreatic beta cells contain abundant rough endoplasmic reticulum (rough ER), which is visible in electron micrographs.

• Because insulin is a secreted protein, its synthesis is in the rough ER. 

• The insulin gene initially produces a precursor protein called preproinsulin that contains 110 AA and is longer than the mature, functional insulin molecule, which only contain 51 AA

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Mature Insulin Structure:

• Mature insulin consists of 2 chains of polypeptides called:

→ A Chain: 21 AA and B Chain: 30 AA → Add to 51

• the 2 chains are connected by disulfide bonds to form the functional insulin molecule

• The conversion from the precursor, preproinsulin to insulin involves severe post-translational processing in the rough ER, where the precursor is directed to by its N-terminal signal sequence.

Post-Translation Processing in the Rough ER of Preproinsulin:

1. The signal sequence emerges from the ribosome during translation.

2. A signal recognition particle (SRP) recognizes the signal sequence and directs the ribosome to the ER membrane.

3. The growing polypeptide enters the ER lumen through a protein-conducting channel.

4. The signal sequence is cleaved by a signal peptidase, after which the molecule is called proinsulin

5. Proinsulin then undergoes further folding and processing before it becomes mature insulin.

Proinsulin Folding and Maturation:

1. Proinsulin folds into its appropriate three-dimensional structure, which is assisted by molecular chaperones in the ER.

2. During folding, three disulfide bonds form to help stabilize the structure of the mature insulin

3. The folded proinsulin is then transported from the rough ER to the Golgi apparatus.

4. Further cleavage occurs here to form mature insulin dimer that contains both chain A and B, but also releases a new chain C.

Types of Post-Translational Modifications:

1. Cleavage

2. Protein folding

3. Disulfide Bond Formation

4. Covalent attachment of chemical groups (i.e. phosphorylation, methylation, acetylation)

5. Protein Degradation

Phosphorylation:

• a reversible post-translational modification that involves the covalent attachment of a phosphate group to serine, threonine or tyrosine amino acid residues in a protein by enzymes called kinases

Methylation:

• involves the addition of a methyl group to specific amino acid residues in a protein.

• can affect protein interactions, gene regulation, and other cellular processes.

Acetylation:

• the addition of an acetyl group to a protein

• It can influence protein stability, interactions, localization, and activity.

Receptors:

• proteins that recognize signaling molecules, often called ligands, and initiate cellular responses.

Insulin Receptor:

• The insulin receptor belongs to a family of receptors called receptor kinases.

• This receptor exists in a monomeric form.

Insulin Receptor Activity:

• When insulin binds to the extracellular surface of the insulin receptor kinases, it cause the two independent receptor monomers to pair up/dimerize.

• This activates the cytoplasmic domains of the receptor.

• The activated receptors phosphorylate each other at many regions of the receptor tails and leads to binding of other important cytoplasmic proteins.

• This initiates a cascade of events inside the cell and this signalling pathway ultimately helps regulate glucose uptake and metabolism.

Signalling and Glucose Uptake:

• Once insulin binds to its receptor, a signal transduction cascade takes place

• Downstream signaling proteins relay and amplify the signal (Signal Amplification)

• One important result = the movement of glucose transporter proteins, which allows these cells to take up more glucose from the bloodstream

Increasing glucose uptake from the cell allows lowering blood glucose levels.

Regulation of Intracellular Signalling:

To stop the process form carrying on unnecessarily, regulatory mechanisms are required called positive feedback loops and negative feedback loops.

Positive feedback:

• Positive-feedback loops reinforce or amplify a response, helping a signal increase or persist under appropriate conditions.

Negative feedback:

• Negative-feedback loops reduce signaling activity and help terminate a response once it is no longer needed.

Double-negative regulation:

• One inhibitor suppresses another inhibitor.

• Removing the inhibition on the second component can activate the pathway and provides another way for cells to regulate the intensity and duration of their responses.

Tissues that Contribute to Blood Glucose Regulation:

1. Adipose tissue: Takes up glucose and fatty acids and stores excess in the form of triglycerides.

2. Skeletal muscle: Takes up glucose for energy production and stores excess glucose as glycogen.

3. Liver: Take up glucose from the blood and store excess as glycogen. However, skeletal muscles absorb glucose more efficiently than liver cells.
   
   

Exons:

• Regions retained in the mature RNA after splicing (coding regions)
  
  

Intron:

• Regions removed during RNA processing (non-coding regions).
  
  

Alternative Splicing:

• During conventional RNA splicing, introns are removed and exons are joined together to produce mature mRNA.

• However, During alternative splicing, a single pre-mRNA can be processed in different ways to generate multiple mature mRNA transcripts.

Alternative Splicing at Various Junctions:

• During alternative splicing, some exons may be excluded during the splicing process (being removed much like the introns), leading to the production of many isoforms or different types of mature mRNA from the same pre-mRNA transcript

• This is because what it may recognize as an exon in this transcript may have been an intron in another transcript.

• Alternative splicing can lead to the production of different but related proteins.

Exon 11 spicing of the Insulin Receptor:

• occurs during the processing of the pre-mRNA that encodes the human insulin receptor, which contains 22 exons.

• in skeletal muscles, exon 11 is removed form the mature mRNA product along with transcript.

• translated into higher affinity version of the insulin receptor in muscle cells.

• muscles cells have the ideal insulin receptor isoform as this allows most glucose uptake.

• provides for the high-glucose requirement of muscle cells.

Exon 11 Retention of the Insulin Receptor:

• In contrast to skeletal cells, mature liver cells tend to retain the exon 11 during splicing of mRNA.

• this leads to a lower affinity version of the insulin receptor in liver cells.

Blood Glucose Regulation via Negative Feedback Loop;

1. After a meal, blood glucose rises. Pancreatic beta cells detect this change and release insulin, which stimulates glucose uptake and storage in responsive tissues.

2. As glucose is removed from the bloodstream, blood glucose levels begin to return toward their normal range.

3. The decline in blood glucose is detected by the pancreatic cells as they reduce the stimulus for insulin secretion.

This negative feedback loop prevent excessive or prolonged responses and maintains stable blood glucose levels by returning them to resting state by reducing the original stimulus.

Why Proteomic Complexity Matters?

• Changes in alternative splicing or protein processing can disrupt biological processes and contribute to disease.

• For example, insulin must be processed correctly to form a functional hormone capable of binding to its receptor.

• the insulin receptor is encoded by a single gene,

  in which alternate splicing results in one of two

  isoforms.

• If the insulin receptor isoform was incorrectly spliced during mRNA processing, there also would be no ability to activate glucose transport proteins allowing for the import of glucose from the blood stream at these target regions.

• A defect in either the insulin protein or the insulin receptor can lead to the inability to take up glucose, resulting in hyperglycemia and eventually diabetes