CHAPERONE PROTEINS
Chaperones involved in protein folding are defined as- proteins that aid other proteins in acquiring the native active formation, but are not part of the final protein structure. The two main chaperones are classical chaperones and carbohydrate binding chaperones. Classical chaperones are found throughout the cell but carbohydrate binding chaperones are specific to the ER.
ER-associated Degradation
Newly made membrane and secretory proteins enter the ER unfolded via channels called translocons. When in the lumen, chaperones and modifying enzymes aid in post-translational modification to their native conformation where they'll exit the ER. Many factors affect the degree of misfolding, such as mutations, sub stochiometric levels of binding partners and chaperone shortages. Misfolded proteins are often retained in the ER, becoming clients of ER-associated degradation (ERAD). ERAD doesn't have one universal pathway, instead many branches for different misfolded protein classes sharing a universally similar sequence - substrate recognition, retrotranslocation, ubiquitination and degradation1.
Post-Translational Modification of Proteins and their sequences
Post translational modification of proteins and their constituent amino acid sequences can trigger major alterations to the function of a protein and how it communicates with the wider cell. It can be triggered both a positive and a negative change, for example triggering a signalling cascade to synthesise fibrin in blood clots, or inactivation of an enzyme.
Modification can take place through a plethora of modifying enzymes. One of the most common is the Protein Kinase, which phosphorylates specific amino acids in protein residues. Protein kinases transfer the gamma phosphate from ATP (high energy) onto tyrosine, serine, threonine or histidine residues.
Phosphorylation
Addition reactions
Methyl groups (CH3) may be added to lysine and arginine residues, (usually, due to the nitrogen containing element of these residues) but may also be added to the C- and N- termini of proteins. An important example includes the m6A methyltransferases (N-6 adenine-specific DNA methylase) which methylate specific DNA sequences in order to prevent a host degrading its own genome by restriction enzymes [4].
Acetyl groups (CH3CO) are added to alpha amino groups of the first amino acid residue on a protein from acetyl CoA (N-Acetylation). This is catalysed by N-Acetyl Transferases, and has a key role in phase II metabolism (involving metabolite conjugation for byproduct secretion in the liver [5].
Lipid groups may be added to proteins in a process called lipidation (or prenylation). Mediation of protein-protein and protein-membrane interactions is triggered by a type of lipidation: Farnesylation. This involves the addition of an isoprenyl group to a cysteine residue. Another process, palmitoylation, increases the affinity of a protein to the inner plasma membrane of a cell by the addition of a palmitic acid to a cysteine residue [6].
Glycosylation, the addition of a carbohydrate (glycan) to a hydroxyl or other functional group on a protein residue, has many applications in the cell. There are many different types, including:
N-linked - attachment of glycans to nitrogens of asparagine or arginine. Critical for glycoprotein folding.
O-linked - attachment of glycans to hydroxyl of serine, threonine, tyrosine, hydroxylysine or hydroxyproline.
C-linked - attachment of glycans to a carbon on a tryptophan
Phosphoglycans - attachment to the phosphate of a phosphoserine
All of these types of glycosylation play a key role in cell-cell adhesion and communication, and works tightly with the immune system - lectin presentation on the cell surface allows the immune system to differentiate between blood types, and allows for agglutination of blood cells (haemagglutination) [7].
Proteins may also have hydroxyl groups (-OH) added by hydroxylation. This reaction is catalysed by hydroxylases, such as prolyl 3-hydroxylase [8]. This enzyme is key to hydroxylation of proline residues in collagen; without the addition of hydroxyl groups, the gamma-c atom that forms hydroxyproline would not stabilise the alpha helices found in collagens' secondary structure. The most common residues to be hydroxylated are lysine and proline, forming hydroxylysine and hydroxyproline, which are free to make strong electronegative attractions with other elements of the protein when folding [9].
Proteolytic Modification
Proteins are often broken down into constituent amino acids by proteolysis due to mis-folding, or mis-incorporation of an incorrect amino acid into the sequence. Proteins may also be broken down in recycling of unneeded proteins in order to synthesise required proteins. Proteolysis may also be required to cleave a protein to turn it into an active state, for example with Insulin. A precursor is synthesised and cleaved at two c-terminal domains to produce an active insulin molecule.Another example includes the blood clot cascade. Proteases catalyse the reactions, but proteolysis can also occur through cellular conditions, such as high temperatures or low pH [10].
Carboxylation, Amidation and Ubiquitination
Carboxylation primarily occurs in proteins involved in the blood clotting pathway. Proteins such as Factor II and VII, along with Protein C have CO2 added to glutamate residues (forming gamma-carboxyglutamate) by the action of gamma-glutamyl carboxylase. This addition of carbon dioxide is essential in activating protein in the cascade. Another example includes RuBisCo, a major constituent of the Calvin Cycle [11].
Amidation can be a major post-translational modification event - the addition of amide groups (-NH2) to C-termini of proteins reduces the proteins' sensitivity to proteolysis, thus extending the protein half life. In addition, amidation has the ability of targeting proteins towards their receptors as it alters affinity. Associated enzymes include Peptidylglycine alpha-hydroxylating monooxygenase and peptidyl alpha-hydroxyglycine alpha-amidating lyase [12].
Ubiquitination involves addition of ubiquitin, a small 8.6kDa protein, to protein sequences. Ubiquitin has associations with essential cellular processes, such as DNA repair, gene expression, endocytosis and proteasomal degradation. When a protein undergoes ubiquitination,, the ubiquitin tags the protein for specific targets, either marking it for proteasomal degradation or preventing/promoting protein-protein interactions. Ubiquitin-conjugating enzyme (E2) is a key enzyme in marking proteins with ubiquitin for degradation [13].
