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Post-translational Modification
Every protein forms a unique three-dimensional structure that allows the protein to be active. Some proteins need further modification after translation before they reach their final active state.
Post-translational modification involves the enzymatic and covalent modification of proteins and is an important process in cell signalling. Protein modifications occur at the C- or N-terminus (the two ends) of the polypeptide chain. They can also occur on the side chains (the part of each amino acid that differs from the others). A covalent bond is a strong chemical bond in which atoms share electrons.
There are hundreds of ways proteins can be modified, but in this article we are only going to discuss the most common mechanisms of post-translational modifications of proteins.
Phosphorylation is one of the most common ways of modifying a protein after translation. It regulates enzymes, receptors and many other types of protein. The process of phosphorylation is reversible and involves adding a phosphate group to specific amino acids such as serine, threonine and tyrosine; the phosphate group is donated from ATP.
Phosphorylation is catalysed by enzymes called protein kinases. These transfer the terminal phosphate group of ATP to the hydroxyl group (an -OH group) on serine, threonine and tyrosine amino acids.
Phosphorylation can be reversed by protein phosphatases. These enzymes remove the phosphate group from the protein through hydrolysis (splitting a bond using water).
Diagram - The process of phosphorylation and dephosphorylation
Creative commons source by Petaholmes [CC BY-SA 4.0 (https://creativecommons.org/licenses/by-sa/4.0)]
Why phosphorylation of proteins is highly effective:
- Adding a phosphate group gives the enzyme or protein two negative charges. This changes the electrostatic interactions (the attraction and repulsion between charged parts), so new interactions can form. These can change the activity of the enzyme and how well it binds its substrate.
- The phosphate group can form hydrogen bonds.
- The rate of phosphorylation and dephosphorylation can change, either taking less than a second or many hours. This allows the number of phosphorylated proteins to change depending on the needs of the physiological processes taking place.
- As ATP is used as an energy currency and in phosphorylation, this links the regulation of metabolism and the energy status of the cell together.
- Phosphorylation can lead to highly amplified effects within the cell. One activated kinase can phosphorylate many target proteins in a short space of time. The activated proteins may also be enzymes, which can go on to catalyse further reactions. Each step in a cascade of kinases (kinases that activate other kinases) multiplies the signal, so a small initial signal can have a large effect.
Proteins can be phosphorylated abnormally and this can lead to diseased states. Diseases such as Alzheimer’s disease, Parkinson’s disease and cancer have been linked to abnormal protein phosphorylation.
Another mechanism of post-translational modification is glycosylation, and this process involves the covalent attachment of a carbohydrate to a protein. It is one of the most complicated processes of post-translational modification due to the large number of enzyme-dependent steps involved. The donor molecule is a glycosyl group. This is a mono- or oligosaccharide (a single sugar or a short chain of sugars). It forms a glycosidic bond (the bond that joins a sugar to another molecule) with the acceptor molecule, which in this case is a protein.
There are a number of subtypes of glycosylation which we will discuss further:
N-linked glycosylation is a type of glycosylation that starts in the endoplasmic reticulum and is remodelled in the Golgi apparatus. A single sugar or a short chain of sugars (a mono- or oligosaccharide) is attached to a nitrogen atom in the side chain of the asparagine amino acid. N-linked glycosylation requires energy and usually occurs to proteins bound for the membrane or proteins that are going to be secreted.
Diagram - N-linked glycosylation with an asparagine residue in blue
Creative commons source by Lizziechka [CC BY-SA 4.0 (https://creativecommons.org/licenses/by-sa/4.0)]
O-linked glycosylation involves glycans (sugar chains) being attached to the hydroxyl groups (-OH groups) of serine or threonine amino acids. Mucin-type O-linked glycosylation, which builds the sugar chains on mucins (the main proteins in mucus secretions), starts in the Golgi apparatus. O-linked glycosylation is also important in the production of proteoglycans (proteins heavily coated with sugar chains), which are a component of the extracellular matrix. Not all O-linked glycosylation starts in the Golgi apparatus. Collagen is glycosylated in the endoplasmic reticulum, and many proteins in the nucleus and cytoplasm carry a single sugar, N-acetylglucosamine (O-GlcNAc).
Diagram - O-linked glycosylation with a serine residue in blue
Public Domain source by Tpirojsi
There are other types of glycosylation such as S-linked glycosylation and C-linked glycosylation, but we are not going to discuss these types in this article.
Why glycosylation is an important type of post-translational modification:
- Glycosylation helps proteins to fold correctly. It also provides protein stability, which helps the protein carry out its function.
- Glycosylation has been shown to be important in how proteins interact with other molecules.
- For example, glycosylation can affect how a ligand (a molecule that binds to a receptor) interacts with its receptor. It can also affect how the receptor interacts with the cell signalling mechanisms inside the cell.
- In both cases, glycosylation can change the biological response of the cell.
- Glycosylation plays an important role in determining the blood group of a person. The blood group is determined by the presence or absence of glycosyltransferases (enzymes that add sugars to other molecules). These enzymes catalyse the final stage of transferring a sugar to the H antigen (the basic sugar marker on the outer surface of the red blood cell). The sugar that is added, or its absence, determines the person’s blood group.
It is easy to get confused about the difference between glycosylation and glycation, so we shall go through the differences now:
- Glycation is when a sugar molecule is attached to a protein not under enzymatic control. Glycosylation is the attachment of a sugar to a protein under enzyme control, using activated sugar donors (sugars joined to a carrier molecule that makes them reactive).
- Glycation often occurs in the blood, where simple sugars such as glucose are found and can attach to haemoglobin without an enzyme. This forms glycated haemoglobin. It can increase the number of free radicals (highly reactive molecules that can damage cells) within the red blood cell.
- Glycated haemoglobin or HbA1c reflects the average blood glucose level over the previous 8 to 12 weeks. It is used to monitor how well a patient with diabetes mellitus is controlling their glucose levels. In adults, an HbA1c of 48 mmol/mol (6.5%) or more is one of the criteria for persistent hyperglycaemia (a high blood glucose level that does not settle). Persistent hyperglycaemia is used to diagnose diabetes mellitus. HbA1c should not be used for diagnosis in some groups, including children and young people under 18, pregnant women, women within 2 months postpartum (after giving birth) and people who have had symptoms of diabetes for less than 2 months. It should be interpreted with caution in people with abnormal haemoglobin, severe anaemia or a recent blood transfusion. (Check out our article on Diabetes Mellitus).
Acetylation is another example of post-translational modification and it is an important process that occurs in cell biology. In proteins, acetylation usually involves the addition of an acetyl group (CH3CO-) to an amino group (an -NH2 group). This can be the free amino group at the N-terminal or the amino group on the side chain of a lysine amino acid. Enzymes catalyse the reaction.
There are two forms of protein acetylation: N-terminal acetylation and lysine acetylation.
N-terminal acetylation is regarded as irreversible, because no enzyme that removes it has been found. It is important for the function and regulation of a variety of proteins. The process involves the addition of an acetyl group to the free amino group present at the N-terminal of the polypeptide chain.
Diagram - The process of N-terminal acetylation
Creative commons source by Ybs.Umich [CC BY-SA 4.0 (https://creativecommons.org/licenses/by-sa/4.0)]
Acetylation of lysine residues involves acetyl-CoA (a coenzyme that carries acetyl groups), which donates its acetyl group to lysine. The acetyl becomes attached to an amino group present on the side chain of a lysine residue.
An example of lysine acetylation is the acetylation of histones (proteins that DNA wraps around) and p53 (a tumour suppressor protein that responds to DNA damage). Lysine amino acids in the tail of the histone protein, which extends from its core, are acetylated. This makes the chromatin less condensed, so more transcription can take place. The acetylation of p53 is important for its activation. Once activated, p53 can halt the cell cycle, allow damaged DNA to be repaired, or start apoptosis.
Disulphide bonds are strong bonds formed by the oxidation of the thiol groups (R-SH, sulphur-containing groups) of two cysteine amino acids. In proteins that are made for secretion, disulphide bonds form in the lumen of the endoplasmic reticulum.
Disulphide bonds are important in stabilising the tertiary structure (the overall three-dimensional shape) of single polypeptide chains. They also stabilise multi-subunit proteins (proteins made of more than one chain), such as antibodies and insulin. Many proteins that are secreted into the extracellular space contain disulphide bonds. Most cytosolic proteins (proteins in the fluid part of the cytoplasm) do not, because the cytosol is a reducing environment (one that tends to break disulphide bonds rather than form them). Proteins with many disulphide bonds tend to be more resistant to external forces, such as heat and detergents. They are also less likely to denature (lose their shape).
Image - Structure of cysteine
Creative commons source by bigblue0092 [CC BY-SA 4.0 (https://creativecommons.org/licenses/by-sa/4.0)]
Image - Polypeptide chain with disulphide bonds
Creative commons source by Jü [CC BY-SA 4.0 (https://creativecommons.org/licenses/by-sa/4.0)]
Many proteins can be modified post-translation by specific proteolysis (cutting the polypeptide chain with a protease, an enzyme that cuts proteins) to produce the final active form of the protein. This can include removing the signal peptide (a short stretch of amino acids that directs the protein to where it is needed). It can also include removing the N-terminal methionine (the first amino acid in the chain), or converting an inactive protein to its active form.
For some proteins, the first version that is made is called the preproprotein (a precursor that carries a signal peptide and a further section that keeps it inactive). It is cleaved to produce the proprotein (an inactive precursor). Further modification or cleavage then produces the final functional form.
For example, albumin, a protein found in blood, is synthesised as preproalbumin and then the signal peptide is removed to give proalbumin. To form the final functional protein (albumin), the first 6 amino acids (the N-terminal propeptide) are removed from proalbumin.
Proteins may contain a signal peptide. This is usually at the N-terminal (the start of the chain). The sequence allows the protein to be directed to an organelle or to be secreted out of the cell. Once the protein passes through a membrane such as that of the endoplasmic reticulum, an enzyme called signal peptidase removes the signal peptide.
Removal of the N-Terminal Methionine
Methionine aminopeptidase (an enzyme that cuts off the first amino acid) removes the initiator methionine (the first amino acid in a newly made chain) from many newly synthesised proteins. Cleavage depends strongly on the side-chain size of the second amino acid. A small neighbouring amino acid, such as glycine or alanine, gives the enzyme access to the first peptide bond (the bond joining two amino acids). A bulky one tends to prevent cleavage. The initiator methionine is therefore retained in some proteins.
Cleavage of Precursor Proteins
Some proteins are synthesised in the form of their precursors, such as zymogens (inactive enzyme precursors) and prohormones (inactive hormone precursors). These are then cleaved to give rise to the final active form of the protein.
One example of this process is the synthesis of proteases (enzymes that cut other proteins). They are made in an inactive form so that they can be stored in cells safely, without damaging the cell that makes them. The inactive proteases are stored in the cell. When they are needed, they are released and then activated in the correct place. An example of a zymogen is trypsinogen. It is made in the pancreas and released into the duodenum (the first part of the small intestine). There, an enzyme called enteropeptidase cuts off a short piece of trypsinogen. This changes the shape (conformation) of the protein and completes the active site, so the enzyme trypsin is fully active.
Another example of this process is the synthesis of insulin. The hormone is synthesised as preproinsulin, which then has the signal peptide removed to produce proinsulin. Disulphide bonds form between the A and B chains in the endoplasmic reticulum, while the C chain (the connecting chain) still joins them. These give stability to the protein molecule. Proinsulin is then packaged into immature secretory granules (vesicles that store the hormone) at the trans-Golgi network (the exit side of the Golgi apparatus). Inside the granules, enzymes called prohormone convertases (PC1/3 and PC2) and carboxypeptidase E cut out the C chain. This produces mature insulin and the free C chain (C-peptide). The granules are stored in the cytoplasm of pancreatic beta cells until the cell is stimulated to release insulin. These cells sit in the islets of Langerhans (clusters of hormone-making cells in the pancreas).
Diagram - The structure of mature insulin
Creative commons source by Zappys Technology Solution, edited by Dr. Peter Parkinson [CC BY-SA 4.0 (https://creativecommons.org/licenses/by-sa/4.0)]
Diagram - Process of insulin production
Creative commons source by Fred the Oyster [CC BY-SA 4.0 (https://creativecommons.org/licenses/by-sa/4.0)]
Most polypeptide hormones are first made as larger precursor polypeptides, which are then cleaved to produce the smaller active hormone. A precursor that contains several different smaller polypeptides is called a polyprotein. An example is pro-opiomelanocortin (POMC), a large precursor that contains many different smaller polypeptides. Depending on the tissue in the human body, the cleavage of this polyprotein can give rise to a variety of hormones. Examples are adrenocorticotropic hormone (ACTH, which acts on the adrenal glands) and beta-melanocyte-stimulating hormone (beta-MSH, which acts on pigment-producing cells).
Diagram - The derivatives of pro-opiomelanocortin
Public Domain source by Klaus Hoffmeier
As mentioned at the beginning of the article, there are many ways in which a protein can undergo post-translational modification. Here are some other forms of post-translational modification:
- Lipidation: attachment of lipids to a protein. The lipid often anchors the protein to a membrane, such as the cell membrane.
- Alkylation: attachment of an alkyl group (a small chain of carbon and hydrogen atoms, such as a methyl or ethyl group) to an amino acid.
- Hydroxylation: attachment of a hydroxyl group (an -OH group) to the side chain of an amino acid such as proline, lysine or asparagine.
Constitutive and Regulatory Secretory Pathways
A protein that is destined for secretion into the extracellular environment can be delivered to the plasma membrane by one of two pathways. These are the constitutive secretory pathway (always on, with no trigger needed) and the regulatory secretory pathway (also called the regulated pathway, which needs a trigger).
Proteins such as immunoglobulins, collagen, albumin and plasma membrane receptors are synthesised in the cell and packaged into vesicles. The vesicles then fuse with the plasma membrane in an unregulated manner and are released into the extracellular space. These are examples of the constitutive secretory pathway, because these proteins are constantly produced.
Proteins released by the regulatory secretory pathway include insulin, ACTH (adrenocorticotropic hormone) and trypsinogen, the inactive precursor of trypsin. This pathway involves the protein being synthesised in the cell then packaged into a storage vesicle. A signal, such as a hormone or neurotransmitter binding to a receptor on the plasma membrane, starts a signalling pathway inside the cell. This makes the vesicles that contain the protein fuse with the plasma membrane and release the protein into the extracellular space. For insulin released in response to glucose, the trigger is a rise in calcium inside the beta cell.
Collagen is a fibrous protein found in connective tissue such as tendons, ligaments, bone and cartilage. The protein is also a part of the loose connective tissue which helps to provide structure to the internal organs.
The fibrous protein is produced mainly by cells called fibroblasts, which are found in connective tissue. Other cells, such as osteoblasts, also make collagen. As well as synthesising collagen, fibroblasts also produce ground substance, a shapeless gel-like matrix that is found between cells and fibres in connective tissue.
The basic unit of the fibrous protein collagen is tropocollagen (a rod-shaped molecule made of three chains). Collagen is formed of three polypeptide chains that form a right-handed triple helix structure. The triple helical structure gives the protein high tensile strength (resistance to being pulled apart). It also makes the protein non-compressible and non-extensible (it does not stretch), which gives strength to the connective tissue where the protein is found. The fibrous protein is stabilised by hydrogen bonds between the polypeptide chains.
Image - The triple helical structure of collagen
SimpleMed original by Dr. Peter Parkinson
Every third amino acid in the polypeptide chain of tropocollagen is glycine. Proline and hydroxyproline are also common in the other positions, making up about 22% of all the amino acids in the chain. Glycine's side chain is a single hydrogen atom. This is small enough to fit into the middle of the helix.
Image - The structure of glycine
Public Domain Source by Benjah-bmm27
Collagen is a great example of post-translational modification as there are a number of ways in which the protein is modified. We are now going to discuss the steps involved in the synthesis and modification of collagen.
- The polypeptide chains are synthesised at ribosomes on the endoplasmic reticulum (ER) and then the chains enter the lumen of the ER.
- Once the polypeptide chains have entered the lumen of the ER, the signal sequence is cleaved by signal peptidase (an enzyme that removes the signal sequence).
- Proline and lysine amino acids are hydroxylated (an -OH group is added) by the enzymes prolyl hydroxylase and lysyl hydroxylase. Hydroxyproline helps to stabilise the triple helix. Stereoelectronic effects play a key role here (the -OH group shifts the electrons in the proline ring, which favours the shape needed for the helix). Hydroxylysine provides sites for glycosylation and cross-linking (covalent links between collagen molecules).
- Whilst in the lumen of the ER, there is the addition of N-linked oligosaccharides.
- In the ER lumen, galactose, or glucose linked to galactose, is added to hydroxylysine residues. This collagen O-linked glycosylation occurs before triple-helix formation.
- When three polypeptide chains align, there is the formation of disulphide bonds.
- In the lumen of the ER, the triple helical shape of procollagen (the collagen precursor molecule) forms from the C- to N-terminus.
- After triple-helix formation, procollagen passes through the Golgi apparatus and is packaged into secretory vesicles for transport to the plasma membrane.
- At the plasma membrane, procollagen is secreted out of the cell via the process of exocytosis.
- Sections of amino acids at the N- and C-terminus (the propeptides) do not form the triple helical shape. Once the procollagen molecule is secreted out of the cell, the propeptides are removed by procollagen N-proteinase (ADAMTS2) and procollagen C-proteinase (BMP1), which are enzymes that cut proteins. This forms the molecule tropocollagen.
- After the collagen molecules associate with each other laterally, covalent cross-linking between the molecules occurs to form fibrils (thin threads of collagen). This process is catalysed by lysyl oxidase, an enzyme that needs copper (Cu2+ ions) and a built-in cofactor (a helper molecule) called lysine tyrosylquinone for its activity.
- Finally, the fibrils aggregate together to form collagen fibres.
Diagram - The process of collagen synthesis and the post-translational modification it goes under
Collagen synthesis diagram by Mfigueiredo. Source. Licensed under CC BY-SA 3.0 Unported.
Scurvy is caused by a lack of vitamin C. It is rare today but still occurs, mainly in people whose diets are restricted or who have risk factors such as an eating disorder or alcohol or drug dependence. Historically, scurvy is best known as a disease of sailors.
Collagen prolyl hydroxylases hydroxylate proline residues, while separate lysyl hydroxylases hydroxylate lysine residues. Both enzyme groups require vitamin C and Fe2+ for their activity. Hydroxyproline helps to stabilise the collagen triple helix. Hydroxylysine provides sites for glycosylation and contributes to collagen cross-linking. Vitamin C deficiency impairs these hydroxylation reactions, producing fragile connective tissue. Symptoms of scurvy include: feeling very tired and weak; feeling irritable and sad; joint, muscle or leg pain; swollen or bleeding gums; and easy bruising. Red or blue spots can appear on the skin, usually on the legs and feet. They may be less noticeable on brown or black skin.
Scurvy is treated with vitamin C. Mild cases are often treated by adding vitamin C to the diet. A GP may also recommend vitamin C supplements (ascorbic acid) by mouth until the patient feels better. The British National Formulary (BNF) dose for an adult is not less than 250 mg daily in divided doses. Most people feel better within days and go on to make a full recovery. The GP may refer the patient to a specialist for treatment, support or advice, depending on what is causing the scurvy.
Image - A patient suffering from swollen, bleeding gums due to scurvy
Historical image from Modern surgery, general and operative (1919), via Internet Archive Book Images. Source. The source marks the image CC0 (Public Domain Dedication).
EDS is a group of genetic connective tissue disorders. There are 13 types. A genetic mutation has been identified for most types, but not for hypermobile EDS (hEDS), which is the most common type. Hypermobile means that the joints move more than normal. There is currently no test to confirm whether someone has hEDS. The form of EDS that the patient will have depends on which of the genes involved in the structure, processing or production of collagen is affected. Symptoms vary by type. Patients may have stretchy skin (skin hyperextensibility), joint dislocations, scoliosis (a curved spine) and osteoarthritis (damage to the joints). Aortic dissection (a tear in the wall of the aorta) is a feature of vascular EDS, in which the blood vessels can split open. Scoliosis is a feature of kyphoscoliotic EDS.
There is no cure for EDS and no specific treatment, but it is possible to manage many of the symptoms with support and advice. Care depends on the type. Patients may be helped by physiotherapy, occupational therapy and counselling. For certain types of EDS, regular scans carried out in hospital can detect problems with internal organs.
Image - Skin hyperextensibility (stretchy skin) in a patient with Ehlers-Danlos Syndrome
Creative commons source by Ellen C. Kelleher [CC BY-SA 4.0 (https://creativecommons.org/licenses/by-sa/4.0)]
Edited by: Dr. Thomas Burnell
Reviewed by: Dr. Marcus Judge
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