Assignment #2 - The Structure of Insulin

Insulin Structure

Insulin is a small protein with a molecular weight of about 5800 Daltons. As mentioned in the previous blog posting, insulin consists of two peptide chains (A and B) that contain 51 amino acids in total. However, The insulin mRNA, which is coded for by the INS gene, is translated as a single chain precursor called preproinsulin which has 110 amino acids and removal of its signal peptide during insertion into the endoplasmic reticulum generates proinsulin (Bowan,1999). The proinsulin molecule contains 86 amino acids. Proinsulin differs from insulin in that it contains a connecting peptide, or C-peptide, of 35 amino acids (number may very between species) that connects the carboxy terminus of the B-chain to the amino terminus of the A-chain. The C-peptide has basic residues at each end, Arg-Arg at the amino terminus and Lys-Arg at the carboxy terminus. The Arg-Arg and Lys-Arg residues at either ends are the sites of enzymatic cleavage in the conversion of proinsulin to insulin. This occurs in the Golgi complex (Talwar et al., 2006).

To show how insulin can vary slightly from species to species, I prepared an alignment of preproinsulin mRNA sequences from four different species; 2 large mammals, a small mammal and a non-mammal.

Figure 1: The protein sequence alignment of preproinsulin in four different species. (Alignment produced using ClustalW2 software).

In figure 1, the preproinsulin sequence alignments of four species are compared; a *  means that the amino acids in all sequences are exact matches, : means that the sequences are very similar, .   means the sequences are slightly similar, while no symbol means the sequences are completely different. The table below summarizes the similarities between the alignments.

Table 1:  The similarity scores from comparing the alignments of the preproinsulin sequences produced in figure 1. (Table obtained from ClustalW software)

As shown in table 1, the most similar sequences are the cow and boar. This is plausible because cows and boars are both large mammals with similar lifestyles so most of the sequence is conserved. The largest differences come when the mammals are compared to the non-mammalian jungle foul. However, 65% is still a relatively high conservation. The amino acid sequence of insulin is highly conserved among vertebrates, and insulin from one mammal almost certainly is biologically active in another. Even today, many diabetic patients are treated with insulin extracted from pig pancreas (Bowan, 1999). Both the B and A-chain sequences are highly conserved in mammals and birds, with the exception of those found in the hystricomorph rodents (guinea pig, chinchilla, etc.). Even the fish insulins maintain greater than 50% homology with mammalian insulins (Permutt et al.,1984).

Relationship Between Structure and Activity

 Insulin's two chains are connected by two disulfide bridges. One bridge is found at position 7 on both chains and the other bridge is located at position 20 of chain A and position 19 of chain B. The disulfide bridges are essential for biological activity. In the three-dimensional structure of insulin the A-chain terminal residues are on the surface of the molecule. These residues are invariant and play a large role in the stability, conformation, and activity of the insulin molecule and are therefore always conserved. The C-peptide covers this region in proinsulin which explains the inactivity of proinsulin. The A₂₀ Cys and the A₂₁ Asn are always conserved as well. Any modification to these residues will result in the loss of receptor binding ability and of biological activity (Espinal, 1981). There are also several residues in the B-chain that are equally as important such as B₁ and B₂₉. The removal of eight of the C-terminal residues of the B-chain will also reduce the biological activity of insulin (Talwar et al., 2006). The biological activity of insulin is known to be closely related to these eight C-terminal residues (Shanghai Insulin Research Group, 1973).

Figure 2: The double chain structure of insulin. (Blue = A-chain, Yellow= B-chain, Grey= disulphide bonds)

The above dual chain structure and disulphide bridges are conserved in all species that have been studied which indicates that this structure is a functional requirement for the biological activity of insulin (Espinal, 1981).





*References can be accessed through links on pictures and citations.*

Assignment #1 - Insulin - Hormone Origin, Structure, and Function

Origin

Insulin is a small protein (peptide) hormone that was discovered through the efforts of several Canadian scientists; Banting, Macleod, Collip, and Best in 1921. Insulin is produced by beta cells which are located in the pancreas in clusters called islets of Langerhans. In the formation of insulin, the messenger RNA transcript is translated into an inactive protein called preproinsulin. Preproinsulin contains an amino-terminal signal sequence. This amino-terminal sequence is required for the precursor hormone to be able to pass through the endoplasmic reticulum membrane where it can undergo post-translational processing. The parts of the hormone that are not required for the biologically active hormone are clipped away during the post-translational processing. Once in the endoplasmic reticulum, the preproinsulin signal sequence is proteolytically removed to produce proinsulin. "Once the post-translational formation of three vital disulfide bonds occurs, specific peptidases cleave proinsulin. The final product of the biosynthesis is mature and active insulin. Finally, insulin is packaged and stored in secretory granules, which accumulate in the cytoplasm, until release is triggered" (Cartailler, J.P., 2012). This process is shown in figure 1.

Figure 1: The Synthesis of Insulin from Preproinsulin

 

Structure

Insulin is a 5.8 kDa peptide hormone that consists of two peptide chains (A and B) that contain 51 amino acids in total. The A chain contains 31 amino acids while the B chain contains 20 amino acids (Derewenda et al., 1989). The two peptide chains are linked by three disulphide bonds (Brange et al., 1993). The amino acid sequences as well as the placement of the disulphide bonds can be seen in Figure 2. Although insulin is active as a monomer, during its synthesis and storage it assembles into dimers and to hexamers when zinc is present (Derewenda et al, 1989). The hexamer formation is inactive but is much more stable than the active monomer form (Allison, K., 2010). Being stored as a hexamer allows large amounts of insuling to be readily available. The insulin molecule is synthesized as a single chain proinsulin molecule as mentioned previously. In the proinsulin molecule, an additional C peptide connects the carboxylic end of the B chain with the amino terminus of the A chain. The A chain forms a helical segment followed by a turn and then makes a second helical segment anti-parallel to the first one. The B chain forms a much defined alpha-helix at the N-terminal part, followed by a turn and a beta-sheet (Derewenda et al., 1989). The three dimensional structure of insulin can be seen in figures 3 and 4.

 

Figure 2: The amino acid sequence of insulin chains A and B and the placement of disulphide bonds

 

Figure 3: A structural model of an insulin monomer

 

  

 

Figure 4: A structural model of an insulin hexamer

 

Function 



 The main function of insulin is to maintain glucose homeostasis. Insulin causes muscle and fat to increase their uptake of glucose and inhibits glucose production by the liver making it the main regulator of blood glucose concentration. This is especially important after a meal. When blood glucose levels are elevated, insulin is produced and secreted by the pancreas and causes cells to take in more glucose and store it as glycogen until plasma glucose levels begin to descend. Insulin acts in a negative feed back with another hormone, glucagon. Glucagon has the opposite effects of insulin. When blood glucose levels are too low, glucagon is released from the alpha cells of the pancreas which stimulates gluconeogenesis, the break down of glycogen to glucose, and the release of glucose back into circulation resulting in a rise in blood glucose levels. Figure 5 shows how insulin and glucagon work together to maintain blood glucose levels.
 
 

 Figure 5: The negative feedback loop of  insulin and glucagon

                                                                                                                                        

 

"Insulin increases glucose uptake in cells by stimulating the translocation of the glucose transporter GLUT4 from intracellular sites to the cell surface" (Saltiel et al., 2001). Insulin action is initiated when it binds to and activates its cell-surface receptor. This cell-surface receptor consists of two α subunits and two β subunits that are linked by disulphide bonds to form an α2β2 heterotetrameric complex. Insulin binds to the extracellular α subunits, which then transmits a signal across the plasma membrane to activate the intracellular tyrosine kinase domain of the β subunit. The receptor then undergoes a series of intramolecular transphosphorylation reactions in which one β subunit phosphorylates its adjacent partner on specific tyrosine residues _{(Pessin et. al, 2000).}

"Insulin also stimulates cell growth and differentiation, and promotes the storage of substrates in fat, liver and muscle by stimulating lipogenesis, glycogen and protein synthesis, and inhibiting lipolysis, glycogenolysis and protein breakdown" (Saltiel et al., 2001).The actions of insulin are not completely understood yet and are still being studied.

 

 

 

 

Insulin is used to treat diabetes, an illness that is caused by too little insulin and/or a resistance to insulin. There are two types of diabetes. Type 1 occurs when the pancreas does not produce enough insulin or does not produce any insulin. Daily injections of insulin are required to treat type 1 diabetes. Type 1 is usually diagnosed in children and teens. The insulin can come in the form of a needle or via a pump that attaches to the patients side. Type 2 diabetes is usually caused by a poor diet resulting in high blood glucose levels. The muscle, liver, and fat cells do not respond to insulin the way they should meaning they are stimulated by insulin to uptake glucose. This is known as insulin resistance. Type 2 diabetes is usually diagnosed in adults who are overweight and is treated mostly through a change in diet and exercising to lower blood sugar levels (NCBI, 2012).

 

 

 

 

Figure 6: Healthy insulin producing beta-cells (left)  vs. damaged insulin producing beta-cells (left)

 

 

 

*References can be accessed through links on pictures and citations.*