Post Translational Modification- Definition, Processing

Proteins are the workhorses of the cell, performing a variety of functions such as catalysis, transport, signaling, and regulation. However, proteins are not always ready to function right after they are synthesized by ribosomes. Many proteins undergo further changes after translation, which are collectively called post-translational modifications (PTMs).

PTMs are covalent and usually enzymatic alterations of amino acid side chains or protein termini that occur after the protein has been translated from mRNA. PTMs can modify the chemical properties, conformation, stability, interactions, and localization of proteins, thereby affecting their activity and function. PTMs can also create new functional groups or binding sites on proteins that are not encoded by the genetic code.

There are more than 400 different types of PTMs that have been identified in various organisms, ranging from bacteria to humans. Some of the most common and well-studied PTMs include proteolysis, phosphorylation, glycosylation, sulfation, methylation, hydroxylation, acetylation, ubiquitination, and SUMOylation. These modifications can occur on specific amino acids or protein termini, depending on the enzymes and cofactors involved.

PTMs are essential for many cellular processes and biological functions. They can regulate protein folding, stability, degradation, trafficking, localization, and interactions. They can also modulate enzyme activity, signal transduction pathways, gene expression, chromatin structure, DNA repair, cell cycle progression, and cell death. Dysregulation or malfunction of PTMs can lead to various diseases such as cancer, diabetes, neurodegeneration, and immune disorders.

Therefore, understanding the mechanisms and functions of PTMs is crucial for advancing our knowledge of protein biology and human health. In this article, we will review some of the major types of PTMs and their roles in protein processing and function. We will also discuss some of the tools and methods for identifying and predicting PTMs in proteins.

Post-translational modifications (PTMs) can occur in different cellular compartments, depending on the type and function of the modification. Some PTMs are co-translational, meaning they occur during or immediately after the synthesis of the polypeptide chain by the ribosome. Other PTMs are post-translational, meaning they occur after the polypeptide chain has been released from the ribosome.

The most common location for co-translational modifications is the endoplasmic reticulum (ER), a membrane-bound organelle that serves as a site for protein synthesis and folding. The ER contains various enzymes and chaperones that catalyze and assist in the modification and maturation of newly synthesized proteins. Some of the co-translational modifications that occur in the ER are:

• Signal peptide cleavage: The removal of a short amino acid sequence at the N-terminus of the polypeptide chain that directs the protein to the ER membrane or lumen.
• N-linked glycosylation: The addition of a carbohydrate chain to the amide nitrogen of asparagine residues in a specific consensus sequence. This modification helps in protein folding, stability, sorting and recognition.
• Disulfide bond formation: The formation of covalent bonds between cysteine residues in the polypeptide chain. This modification stabilizes the tertiary and quaternary structure of proteins and confers resistance to reducing agents.

The most common location for post-translational modifications is the Golgi apparatus, a membrane-bound organelle that serves as a site for protein sorting and packaging. The Golgi apparatus consists of a series of flattened sacs called cisternae, where proteins undergo further modifications before being transported to their final destinations. Some of the post-translational modifications that occur in the Golgi apparatus are:

• O-linked glycosylation: The addition of a carbohydrate chain to the hydroxyl group of serine or threonine residues in the polypeptide chain. This modification can affect protein stability, activity, localization and interactions.
• Sulfation: The addition of a sulfate group to tyrosine residues in the polypeptide chain. This modification enhances protein-protein interactions, especially with extracellular matrix components and growth factors.
• Prenylation: The addition of a lipid group (such as farnesyl or geranylgeranyl) to cysteine residues near the C-terminus of the polypeptide chain. This modification targets proteins to the plasma membrane or other membrane-bound organelles.

Other locations for post-translational modifications include the cytoplasm, the nucleus, the mitochondria and other organelles. Some examples of these modifications are:

• Phosphorylation: The addition of a phosphate group to serine, threonine or tyrosine residues in the polypeptide chain. This modification regulates protein activity, localization and interactions by creating or disrupting binding sites for other molecules.
• Methylation: The transfer of a methyl group to lysine or arginine residues in the polypeptide chain. This modification affects protein-DNA interactions, chromatin structure and gene expression.
• Acetylation: The addition of an acetyl group to lysine residues in the polypeptide chain. This modification alters protein-DNA interactions, chromatin structure and gene expression.
• SUMOylation: The attachment of a small ubiquitin-like modifier (SUMO) protein to lysine residues in the polypeptide chain. This modification modulates protein activity, stability, localization and interactions.
• Ubiquitination: The attachment of one or more ubiquitin proteins to lysine residues in the polypeptide chain. This modification marks proteins for degradation by the proteasome or alters their activity, localization and interactions.

As you can see, post-translational modifications can occur in various locations within the cell and have diverse effects on protein structure and function. These modifications increase the complexity and diversity of the proteome and play important roles in cellular processes such as signaling, metabolism, differentiation and disease.

Proteolysis is the process of breaking down proteins into smaller fragments called peptides or amino acids. Proteolysis can occur during or after protein synthesis, and it can have various effects on the structure, function and regulation of proteins.

One type of proteolysis that occurs during protein synthesis is the cleavage of signal peptides. Signal peptides are short sequences of amino acids that direct the newly synthesized protein to its proper location within the cell, such as the endoplasmic reticulum (ER), the Golgi apparatus, the plasma membrane or the extracellular space. Signal peptides are recognized and removed by signal peptidases, which are enzymes located in the membrane of the ER or other organelles. The removal of signal peptides allows the protein to fold properly and interact with other proteins or molecules in its destination.

Another type of proteolysis that occurs after protein synthesis is the activation or inactivation of proteins by removing specific parts of their polypeptide chains. This can alter the shape, stability, activity or interactions of the protein, and it can also generate new functional peptides from a single precursor protein. For example, insulin is synthesized as a single polypeptide chain called preproinsulin, which contains a signal peptide, a B chain, a C peptide and an A chain. The signal peptide is cleaved in the ER, producing proinsulin. Proinsulin is then transported to the Golgi apparatus, where it is further cleaved by proteases called prohormone convertases, which remove the C peptide and generate two active peptides: insulin A chain and insulin B chain. These two peptides form disulfide bonds and constitute the mature insulin hormone, which is stored in secretory vesicles and released into the bloodstream when needed.

Proteolysis can also regulate the degradation of proteins by marking them for destruction by proteasomes or lysosomes. Proteasomes are large complexes of proteases that degrade proteins that are tagged with ubiquitin, a small protein that acts as a molecular signal for protein quality control. Ubiquitin can be attached to proteins by enzymes called ubiquitin ligases, which recognize specific amino acid sequences or modifications on the target protein. Ubiquitination can target proteins for degradation when they are damaged, misfolded, mutated or no longer needed by the cell. Lysosomes are membrane-bound organelles that contain various hydrolytic enzymes that degrade proteins and other macromolecules that are delivered to them by endocytosis, autophagy or phagocytosis. Lysosomal proteolysis can degrade extracellular proteins that are taken up by the cell, as well as intracellular proteins that are sequestered in vesicles called autophagosomes.

Proteolysis is an essential post-translational modification that modulates the diversity, complexity and functionality of the proteome. Proteolysis can affect various aspects of protein biology, such as folding, sorting, activation, inactivation, degradation and signaling.

Insulin is a hormone that regulates blood glucose levels by stimulating the uptake of glucose by cells. Insulin is synthesized in the beta cells of the pancreas as a precursor protein called preproinsulin. Preproinsulin consists of a signal peptide, an A chain, a B chain, and a C peptide. The signal peptide directs the protein to the endoplasmic reticulum (ER), where it is cleaved off by a signal peptidase. The remaining protein is called proinsulin.

Proinsulin undergoes further processing in the ER and the Golgi complex, where it is folded and disulfide bonds are formed between the A and B chains. The C peptide acts as a linker between the two chains and also facilitates the correct folding of proinsulin. In the Golgi complex, proinsulin is packaged into secretory vesicles, where it is cleaved by proteases called prohormone convertases (PC1 and PC2). These enzymes remove the C peptide and generate mature insulin, which consists of two polypeptide chains (A and B) connected by disulfide bonds.

The C peptide and insulin are stored together in the secretory vesicles until they are released into the bloodstream in response to high glucose levels. The C peptide has no known biological activity, but it can be used as a marker of insulin secretion. Insulin binds to its receptor on the target cells and triggers a cascade of events that leads to glucose uptake and metabolism.

Insulin synthesis and activation is an example of how post-translational modifications can affect the structure, function, and regulation of a protein. Proteolysis is essential for generating the active form of insulin from its precursor protein. Without proteolysis, insulin would not be able to bind to its receptor and exert its effects on glucose homeostasis. Proteolysis also ensures that insulin is released in a controlled manner, preventing excessive or insufficient secretion of the hormone.

Phosphorylation: addition of phosphate groups

Phosphorylation is the process of adding one or more phosphate groups to a protein, usually at the hydroxyl groups of serine, threonine or tyrosine residues. Phosphate groups are negatively charged and can alter the shape and charge of the protein, affecting its interactions with other molecules. Phosphorylation is one of the most common and important post-translational modifications in animal cells, as it regulates many aspects of protein function, such as activity, localization, stability and interactions.

Phosphorylation is mediated by enzymes called kinases, which transfer a phosphate group from a donor molecule, usually adenosine triphosphate (ATP), to a specific amino acid residue on the target protein. The reverse reaction, the removal of a phosphate group from a protein, is catalyzed by enzymes called phosphatases. The balance between kinases and phosphatases determines the phosphorylation state of a protein and its functional consequences.

Phosphorylation can have different effects on different proteins, depending on the context and the site of modification. For example, phosphorylation can activate or inhibit enzymes by changing their catalytic activity or their substrate affinity. Phosphorylation can also alter the subcellular localization of proteins by creating or disrupting binding sites for other molecules, such as transporters, anchors or receptors. Phosphorylation can also modulate the interactions between proteins by changing their conformation or their affinity for other proteins or ligands. Phosphorylation can also affect the stability of proteins by influencing their degradation by proteases or ubiquitin-mediated pathways.

Phosphorylation plays a key role in many cellular processes, such as signal transduction, cell cycle regulation, metabolism, gene expression, apoptosis and differentiation. Many diseases are associated with abnormal phosphorylation of proteins, such as cancer, diabetes, neurodegeneration and inflammation. Therefore, understanding the mechanisms and functions of phosphorylation is essential for biomedical research and drug development.

Glycosylation is the process of attaching carbohydrate molecules, also known as glycans, to proteins or lipids. Glycosylation can occur in two ways: N-linked or O-linked. N-linked glycosylation involves the attachment of glycans to the nitrogen atom of asparagine residues in the polypeptide chain. O-linked glycosylation involves the attachment of glycans to the oxygen atom of serine or threonine residues in the polypeptide chain.

Glycosylation is one of the most common and complex post-translational modifications of proteins. It occurs in the endoplasmic reticulum (ER) and the Golgi apparatus, where various enzymes catalyze the addition, removal and modification of glycans. Glycosylation can affect the folding, stability, trafficking, localization, interactions and functions of proteins.

Glycosylation plays a crucial role in many biological processes, such as:

• Cell-cell recognition and communication: Glycans on the cell surface can act as ligands for receptors on other cells or molecules, mediating cell adhesion, signaling and immune responses. For example, blood group antigens are determined by the type of glycans on red blood cells.
• Protein quality control: Glycans can serve as markers for protein folding and degradation. For example, misfolded proteins in the ER are recognized by a lectin called calnexin, which binds to specific N-linked glycans and facilitates their proper folding or disposal.
• Protection from proteases: Glycans can shield proteins from proteolytic cleavage by blocking the access of proteases to their substrates. For example, mucins are heavily glycosylated proteins that form a protective layer on epithelial surfaces.
• Modulation of protein activity and function: Glycans can alter the conformation, stability, affinity and specificity of proteins, thereby influencing their biological activity and function. For example, insulin receptor is a glycoprotein that undergoes N-linked glycosylation in the ER and O-linked glycosylation in the Golgi. The glycans on the insulin receptor affect its binding to insulin and its signaling cascade.

Glycosylation is a dynamic and diverse process that generates a large variety of glycoforms (different glycan structures on the same protein). The diversity and complexity of glycosylation is influenced by several factors, such as:

• Genetic factors: The expression and activity of glycosyltransferases (enzymes that transfer glycans to proteins) and glycosidases (enzymes that remove glycans from proteins) are regulated by genes. Mutations or polymorphisms in these genes can affect the type and amount of glycans on proteins.
• Environmental factors: The availability and composition of sugar nucleotides (the precursors of glycans) can vary depending on the nutritional status, metabolic state and stress level of the cell. This can affect the synthesis and modification of glycans on proteins.
• Cellular factors: The localization and trafficking of proteins within the ER and Golgi can affect their exposure to different enzymes and substrates involved in glycosylation. This can affect the maturation and processing of glycans on proteins.

Glycosylation is a highly regulated and coordinated process that adds another layer of complexity and functionality to proteins. Glycosylation is essential for normal cellular physiology and homeostasis, as well as for various pathological conditions such as cancer, inflammation, infection and congenital disorders. Therefore, understanding the mechanisms and consequences of glycosylation is important for advancing biomedical research and developing novel diagnostic and therapeutic strategies.

Sulfation is a post-translational modification that involves the transfer of a sulfate group from a donor molecule to a specific amino acid residue in a protein. The most common target for sulfation is tyrosine, which can accept a sulfate group on its phenolic hydroxyl group. Sulfation can also occur on other amino acids, such as serine, threonine, cysteine and histidine.

Sulfation is catalyzed by a family of enzymes called tyrosylprotein sulfotransferases (TPSTs), which are located in the trans-Golgi network (TGN) of the cell. TPSTs use 3`-phosphoadenosine-5`-phosphosulfate (PAPS) as the sulfate donor, and transfer the sulfate group to the tyrosine residue in a specific peptide sequence. There are two isoforms of TPSTs in humans, TPST-1 and TPST-2, which have different substrate specificities and tissue distributions.

Sulfation plays an important role in modulating the function and interaction of various proteins, especially those involved in cell signaling, adhesion, and extracellular matrix formation. For example, sulfation of tyrosine residues in chemokine receptors enhances their binding affinity and signaling response to chemokines, which are molecules that regulate the migration and activation of immune cells. Sulfation of tyrosine residues in proteoglycans, such as heparan sulfate and chondroitin sulfate, affects their interaction with growth factors, cytokines, and other molecules that regulate cell growth and differentiation. Sulfation of tyrosine residues in coagulation factors, such as factor V and factor VIII, influences their activity and stability in blood clotting.

Sulfation is a reversible modification that can be removed by sulfatases, which are enzymes that hydrolyze the sulfate ester bond. The balance between sulfation and desulfation is critical for maintaining the proper function and regulation of sulfated proteins. Abnormalities in sulfation or sulfatase activity have been implicated in various diseases, such as cancer, inflammation, infection, neurological disorders, and metabolic disorders. Therefore, sulfation is an important post-translational modification that modulates the structure and function of many proteins in the cell.

Methylation is a post-translational modification that involves the transfer of one-carbon methyl groups to nitrogen or oxygen atoms of amino acid side chains. Methylation can increase the hydrophobicity of the protein and can neutralize a negative charge when bound to carboxylic acids. Methylation is mediated by enzymes called methyltransferases and S-adenosyl methionine (SAM) is the primary methyl group donor.

Methylation can occur on different amino acids, such as lysine, arginine, histidine, glutamate, aspartate and proline. The most common targets are lysine and arginine residues, which can be mono-, di- or tri-methylated. Methylation of lysine and arginine can affect the interaction of proteins with DNA, RNA and other proteins, and can regulate gene expression, chromatin structure, transcription, splicing and DNA repair. For example, histone methylation is a key epigenetic mark that influences gene expression by altering the accessibility of chromatin to transcription factors.

Methylation can also occur on non-histone proteins, such as p53, NF-kB, STAT3 and ESR1. Methylation of these proteins can modulate their activity, stability, localization and interaction with other molecules. For example, methylation of p53 at lysine 372 by SETD8 enhances its stability and transcriptional activity. Methylation of NF-kB at arginine 30 by PRMT1 enhances its DNA binding and transcriptional activation. Methylation of STAT3 at arginine 31 by CARM1 inhibits its dimerization and nuclear translocation. Methylation of ESR1 at lysine 302 by SET7/9 enhances its interaction with coactivators and estrogen response element.

Methylation is a reversible modification that can be removed by enzymes called demethylases. Demethylases can be classified into two families: lysine-specific demethylases (LSDs) and Jumonji C (JmjC) domain-containing demethylases. LSDs use flavin adenine dinucleotide (FAD) as a cofactor and can remove mono- and di-methyl groups from lysine residues. JmjC demethylases use iron and alpha-ketoglutarate as cofactors and can remove all three methyl groups from lysine and arginine residues.

Methylation is a dynamic and regulated process that plays an important role in various biological functions, such as gene expression, protein-protein interaction, protein stability and signal transduction. Abnormal methylation patterns can lead to diseases such as cancer, neurological disorders and metabolic disorders.

Hydroxylation is the biological process of adding a hydroxy group (-OH) to a protein amino acid. Protein hydroxylation is one type of post-translational modification that involves the conversion of a -CH group into a -COH group. These hydroxylated amino acids are involved in the regulation of some important factors called transcription factors. Among the 20 amino acids, the two amino acids regulated by this method are proline and lysine.

Proline hydroxylation occurs mainly in collagen, the most abundant protein in mammals. Collagen is composed of three polypeptide chains that form a triple helix. The hydroxylation of proline residues enhances the stability and strength of the collagen fibers by forming hydrogen bonds within and between the chains. Proline hydroxylases are enzymes that catalyze this reaction using oxygen, iron and vitamin C as cofactors. Deficiency of vitamin C can lead to scurvy, a disease characterized by impaired collagen synthesis and bleeding gums.

Lysine hydroxylation occurs in various proteins, such as histones, tubulin and elastin. Histones are proteins that package and organize DNA in the nucleus. The hydroxylation of lysine residues in histones can affect their interaction with DNA and other proteins, thereby influencing gene expression and chromatin structure. Tubulin is a protein that forms microtubules, which are essential for cell division and movement. The hydroxylation of lysine residues in tubulin can modulate its polymerization and stability. Elastin is a protein that provides elasticity and resilience to tissues such as skin, lungs and blood vessels. The hydroxylation of lysine residues in elastin can facilitate its cross-linking and assembly into elastic fibers.

Hydroxylation is also involved in the biosynthesis of some hormones and neurotransmitters, such as dopamine, epinephrine, norepinephrine and serotonin. These molecules are derived from the amino acid tyrosine, which undergoes hydroxylation at different positions by different enzymes. For example, tyrosine hydroxylase converts tyrosine to L-DOPA, which is then converted to dopamine by aromatic L-amino acid decarboxylase. Dopamine can be further hydroxylated to norepinephrine by dopamine beta-hydroxylase, and norepinephrine can be methylated to epinephrine by phenylethanolamine N-methyltransferase. Serotonin is synthesized from tryptophan, which is first hydroxylated to 5-hydroxytryptophan by tryptophan hydroxylase, and then decarboxylated by aromatic L-amino acid decarboxylase.

Hydroxylation is a key post-translational modification that affects the structure, function and activity of many proteins and molecules in the cell. It is regulated by various factors, such as oxygen availability, cofactor levels, enzyme expression and activity, and feedback mechanisms. Dysregulation of hydroxylation can lead to various diseases, such as cancer, cardiovascular disorders, neurodegeneration and fibrosis.

Apart from the modifications mentioned above, there are many other types of post-translational modifications that can affect the structure and function of proteins. Some of them are:

• SUMOylation: This is the covalent attachment of a small protein called SUMO (small ubiquitin-like modifier) to the lysine residues of the target protein. SUMOylation can regulate various aspects of protein activity, such as transcription, DNA repair, nuclear transport and cell cycle progression. SUMOylation is reversible and can be removed by specific proteases called SENPs (sentrin-specific proteases).
• Disulfide bond formation: This is the formation of a covalent bond between two cysteine residues in a protein. Disulfide bonds can stabilize the tertiary and quaternary structure of proteins and also play a role in redox signaling and regulation. Disulfide bonds can be formed in the endoplasmic reticulum (ER) or in the extracellular space by enzymes called protein disulfide isomerases (PDIs).
• Lipidylation: This is the addition of lipid groups to the amino acid side chains of proteins. Lipidylation can increase the hydrophobicity and membrane association of proteins and also affect their localization, trafficking and interactions. There are different types of lipids that can be attached to proteins, such as fatty acids, prenyl groups, glycosylphosphatidylinositol (GPI) anchors and cholesterol.
• Acetylation: This is the transfer of an acetyl group from acetyl-CoA to the amino group of lysine residues in proteins. Acetylation can modulate various functions of proteins, such as DNA recognition, protein-protein interaction, protein stability and enzyme activity. Acetylation is reversible and can be removed by enzymes called deacetylases.
• Prenylation: This is the addition of a prenyl group (a type of lipid) to the cysteine residues near the C-terminus of proteins. Prenylation can enhance the membrane association and subcellular localization of proteins and also influence their interactions with other proteins and signaling molecules. There are two types of prenyl groups that can be attached to proteins: farnesyl and geranylgeranyl.

These are some examples of other post-translational modifications that can occur in proteins. There are many more types of modifications that have been discovered and are still being studied. Post-translational modifications add another layer of complexity and diversity to the proteome and have important implications for cellular functions and diseases.

Post-translational modifications (PTMs) are covalent changes of proteins that occur after their synthesis by ribosomes. PTMs can alter the chemical properties, conformation, interactions, localization and lifespan of proteins, thereby regulating their functions in various cellular processes . PTMs are essential for the proper functioning of many proteins, especially those involved in signal transduction, metabolism, cell cycle, apoptosis and immune response .

Some of the significance of PTMs are:

• Protein folding: PTMs can aid in the correct folding of proteins by facilitating the formation of disulfide bonds, glycosylation or hydroxylation . For example, insulin is synthesized as a precursor protein that undergoes proteolytic cleavage and disulfide bond formation to generate the mature and active hormone.
• Protein stability: PTMs can modulate the stability and degradation of proteins by affecting their susceptibility to proteases or ubiquitination . For example, glycosylation can protect proteins from proteolytic cleavage by blocking the access of enzymes to their substrates. Phosphorylation can also regulate the stability of proteins by altering their interactions with other molecules or targeting them for degradation.
• Protein activity: PTMs can activate or inhibit the enzymatic or binding activity of proteins by changing their conformation or affinity for substrates or ligands . For example, phosphorylation is a common mechanism to regulate the activity of kinases, transcription factors and receptors by inducing conformational changes or creating docking sites for other molecules.
• Protein function: PTMs can modulate the function of proteins by influencing their localization, trafficking, sorting or secretion . For example, lipidation can anchor proteins to the cell membrane or vesicles, while prenylation or acetylation can affect their subcellular distribution. Glycosylation can also determine the sorting or secretion of proteins by interacting with specific lectins in the endoplasmic reticulum or Golgi apparatus.

PTMs are important components in cell signaling, as they can mediate the transmission and integration of signals from various stimuli. PTMs can also generate diversity and complexity in the proteome, as they can create different forms of proteins with distinct properties and functions. Therefore, PTMs are essential for the regulation of protein function and cellular homeostasis.

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