Post-translational modification (PTMs) refers to a host of chemical modifications that occur on proteins after they have been synthesized from the ribosome. These modifications play critical roles in protein structure, function, localization, and stability, and are essential for the regulation of various cellular processes. It is a biochemical process which needs to be understood when designing mammalian cell expression systems. In this article, we will explore the significance and types of post-translational modifications.
Post-translational modifications are diverse and can occur at various sites on a protein, including amino acids such as serine, threonine, tyrosine, lysine, arginine, and histidine. They can involve enzymatic addition or removal of functional groups, or alterations in the protein’s structure through covalent modifications. Some of the major types of PTMs include phosphorylation, glycosylation, acetylation, methylation, ubiquitination, and proteolytic cleavage.
Phosphorylation of Proteins
Phosphorylation is one of the most common and extensively studied post-translational modifications. It involves the addition of a phosphate group to the side chains of serine, threonine, or tyrosine residues in proteins, catalyzed by protein kinases. Phosphorylation is a reversible modification and is crucial for the regulation of protein activity, cellular signaling pathways, and protein-protein interactions. It can influence protein conformation, enzyme activity, protein-protein interactions, and subcellular localization, thus modulating various cellular processes such as cell growth, differentiation, and apoptosis.
Glycosylation
Glycosylation is the addition of sugar moieties to proteins, forming glycoproteins. This modification occurs in the endoplasmic reticulum and Golgi apparatus and plays a crucial role in protein folding, stability, trafficking, and recognition. Glycosylation can affect protein solubility, immune recognition, and interaction with other proteins. It is involved in diverse biological processes, including cell-cell adhesion, cell signaling, and immune response.
Acetylation
Acetylation is the addition of an acetyl group to the amino terminus or lysine residues of proteins. It is a reversible modification catalyzed by acetyltransferases and deacetylases. Acetylation can modulate protein-protein interactions, DNA binding, protein stability, and enzymatic activity. It has been extensively studied in the context of histone proteins, where acetylation plays a crucial role in chromatin structure and gene expression regulation. Moreover, acetylation is involved in the regulation of diverse cellular processes, including metabolism, cell cycle progression, and cellular stress responses.
Methylation
Methylation involves the addition of a methyl group to amino acids, primarily lysine and arginine residues. It is a reversible modification catalyzed by protein methyltransferases and demethylases. Methylation can affect protein-protein interactions, gene expression, and signal transduction pathways. For example, histone methylation can regulate gene transcription by altering chromatin structure and recruiting specific protein complexes to the DNA.
The Addition of Ubiquitin
Ubiquitination is the attachment of ubiquitin, a small protein, to target proteins, marking them for degradation by the proteasome or altering their localization and function. Ubiquitination is a highly regulated process that involves the sequential action of three enzymes: E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), and E3 (ubiquitin ligase). Ubiquitination plays a crucial role in protein turnover, quality control, cell cycle progression, DNA repair, and signal transduction.
Proteolytic Cleavage of Proteins
Proteolytic cleavage is the specific cleavage of proteins by proteases, resulting in the generation of smaller protein fragments. This modification can activate or inactivate proteins, regulate protein-protein interactions, and produce bioactive peptides. Examples of proteolytic cleavage include the activation of zymogens into active enzymes and the processing of precursor proteins into mature forms, such as the conversion of proinsulin to insulin.
The Role of Disulphide Linkages In Protein Conformation
In post-translational modification (PTM), disulphide bond formation is a critical process that occurs in the maturation of many secretory and membrane proteins. Disulphide bonds are covalent bonds formed between two cysteine residues within a protein, and they play a crucial role in stabilizing the protein’s structure and function.
Oxidative Environment
Disulphide bond formation primarily occurs in the endoplasmic reticulum (ER), which provides an oxidative environment necessary for the process. The ER contains enzymes and cofactors that promote the formation of disulfide bonds, such as protein disulphide isomerase (PDI) and oxidized glutathione (GSSG).
Signal Peptide Cleavage
Many secretory and membrane proteins are synthesized with a signal peptide, which directs their transport into the ER. Upon entry into the ER, the signal peptide is cleaved off by a signal peptidase, exposing the newly synthesized protein to the ER lumen.
Formation of disulphide bonds
Inside the ER lumen, the protein folds, and disulfide bond formation takes place. This process is facilitated by the action of PDI and other ER-resident oxidoreductases. PDI catalyzes the oxidation of cysteine residues, transferring disulphide bonds between cysteine pairs or between different protein molecules. The precise mechanism by which PDI achieves this is still under investigation but most likely involves a series of thiol-disulfide exchange reactions.
Isomerization and Proofreading
During disulfide bond formation, incorrect or non-native disulfide bonds can form due to the presence of multiple cysteine residues within a protein. PDI can act as an isomerase, rearranging these incorrect disulfide bonds to form the correct ones. This proofreading process ensures that the protein attains its native conformation.
Quality Control
The ER has a stringent quality control system that monitors protein folding and ensures that only properly folded proteins proceed through the secretory pathway. Proteins that fail to form correct disulphide bonds or attain the appropriate structure may be retained in the ER, refolded, or targeted for degradation to maintain cellular homeostasis.
Disulphide Bond Reduction
In some cases, disulphide bonds may need to be reduced to allow further modifications or to enable protein unfolding and degradation. This reduction is facilitated by ER-resident enzymes like PDI, which can break disulphide bonds, converting them into free cysteine residues.
Post-Translational Modification of Proteins
Post-translational modification is essential for the proper functioning and regulation of proteins in cells. They expand the functional diversity of the proteome by adding chemical modifications that alter protein structure, stability, localization, and interaction properties. Phosphorylation, glycosylation, acetylation, methylation, ubiquitination, and proteolytic cleavage are just a few examples of the wide range of post-translational modifications that occur in cells. Understanding the roles and dynamics of these modifications is crucial for unraveling complex cellular processes and developing therapeutic strategies targeting specific PTMs.
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