9.3 Post-translational Modifications

Post-translational Modifications and Protein Function

Proteins don't reach their final functional form the moment translation ends. After the ribosome releases a polypeptide, chemical modifications reshape its structure, stability, location, and activity. These post-translational modifications (PTMs) massively expand the functional diversity of the proteome beyond what the genome alone encodes, and they allow cells to respond rapidly to signals without synthesizing new proteins from scratch.

PTMs regulate processes like cell signaling, cell cycle progression, apoptosis, and differentiation. The most common types are phosphorylation, glycosylation, ubiquitination, acetylation, and methylation.

Post-translational modifications in protein function

PTMs are chemical changes made to proteins after the mRNA-to-amino-acid translation step is complete. A few key properties to understand:

• Where they occur: On specific amino acid side chains (e.g., phosphorylation on serine, threonine, or tyrosine), at the N-terminus (e.g., acetylation), or at the C-terminus (e.g., prenylation).
• Reversibility varies: Some PTMs are reversible, like phosphorylation and acetylation, which makes them useful as molecular switches. Others are irreversible, like proteolytic cleavage and disulfide bond formation.

PTMs expand what the proteome can do. A single gene can give rise to many functionally distinct protein forms depending on which modifications are added. This means cells can alter protein behavior (structure, degradation resistance, organelle targeting, enzymatic activity) in response to hormones or stress without waiting for new transcription and translation.

PTMs also drive specific regulatory mechanisms:

• Cell signaling: Phosphorylation cascades relay signals from receptors to downstream targets
• Cell cycle control: Cyclin phosphorylation governs progression through cell cycle checkpoints
• Apoptosis: Caspase cleavage activates the programmed cell death pathway
• Protein-protein interactions: Specialized domains like SH2 domains recognize phosphotyrosine residues, linking modified proteins to new binding partners

Types of post-translational modifications

Phosphorylation is the addition of a phosphate group ($PO_4^{3-}$) to serine, threonine, or tyrosine residues. Protein kinases (such as PKA, PKC, and MAPKs) catalyze this reaction, and protein phosphatases (PP1, PP2A) reverse it. Because it's reversible, phosphorylation acts as a rapid on/off switch. It regulates enzyme activity (e.g., glycogen synthase), protein-protein interactions (e.g., 14-3-3 protein binding), and signal transduction (e.g., receptor tyrosine kinases).

Glycosylation attaches carbohydrate groups to proteins. There are two main types:

• N-linked glycosylation: Sugars attach to asparagine residues, beginning in the endoplasmic reticulum (ER)
• O-linked glycosylation: Sugars attach to serine or threonine residues, primarily in the Golgi apparatus

Glycosylation affects protein folding, stability, and how proteins are recognized by other molecules. Antibodies and many hormones are glycoproteins, and proteoglycans in the extracellular matrix depend on glycosylation for their function.

Ubiquitination involves attaching ubiquitin, a small 76-amino-acid protein, to lysine residues on a target protein. This requires a three-enzyme cascade:

1. E1 (activating enzyme): Activates ubiquitin in an ATP-dependent step
2. E2 (conjugating enzyme): Carries the activated ubiquitin
3. E3 (ligating enzyme): Transfers ubiquitin to the target protein, providing substrate specificity

The outcome depends on the type of ubiquitin chain. Polyubiquitination with K48-linked chains marks proteins for degradation by the 26S proteasome. Monoubiquitination plays roles in signaling, and K63-linked chains are involved in protein trafficking.

Acetylation adds an acetyl group ($CH_3CO$) to lysine residues. Histone acetyltransferases (HATs) add the group, and histone deacetylases (HDACs) remove it. On histones, acetylation neutralizes the positive charge on lysine, weakening histone-DNA interactions and opening chromatin for transcription. Acetylation also modifies non-histone proteins: acetylation of p53 can prevent its ubiquitination, stabilizing the protein. Bromodomain-containing proteins specifically recognize and bind acetylated lysine residues.

Methylation adds one or more methyl groups ($CH_3$) to lysine or arginine residues. Methyltransferases (often containing SET domains) add the groups, while demethylases (LSD1, JmjC-domain proteins) remove them. The functional effect depends on context: trimethylation of histone H3 at lysine 4 (H3K4me3) activates transcription, while trimethylation at lysine 9 (H3K9me3) represses it. This position-dependent effect is a common exam topic.

Effects of modifications on proteins

Protein stability. PTMs can either protect or doom a protein. Ubiquitination (K48-linked chains) targets proteins for proteasomal degradation. Glycosylation, on the other hand, can shield proteins from proteolytic cleavage and improve their solubility, extending their functional lifespan.

Protein localization. PTMs serve as targeting signals that direct proteins to specific compartments. Phosphorylation can expose a nuclear localization signal (NLS), sending a protein into the nucleus, or expose a nuclear export signal (NES), pushing it out. Glycosylation routes proteins into the secretory pathway through the ER and Golgi, ultimately directing them to the cell surface or for secretion.

Protein activity. PTMs modulate enzyme function by inducing conformational changes. Phosphorylation can activate kinases like PKA and Src, or inactivate others like GSK3β. Acetylation can alter the DNA-binding affinity of transcription factors such as p53 and FOXO, tuning their regulatory output.

Role of chaperones in folding

Chaperones are proteins that help other proteins fold correctly and assemble into their functional conformations. They're especially important during translation (when the polypeptide is still emerging) and under stress conditions like heat shock, when existing proteins are at risk of unfolding.

Chaperones work by binding to exposed hydrophobic regions on unfolded or partially folded proteins, preventing those regions from clumping together into nonfunctional aggregates. They then release the protein through ATP-dependent cycles, giving it repeated opportunities to reach its native conformation.

Two major categories of chaperones:

• HSP70 family (DnaK in bacteria): ATP-dependent chaperones that bind hydrophobic stretches of unfolded proteins and cycle between binding and release
• Chaperonins (GroEL/GroES in bacteria; TRiC/CCT in eukaryotes): Large barrel-shaped complexes that provide an enclosed chamber where a protein can fold in isolation, away from the crowded cytoplasm

Chaperones are also central to the cell's quality control system. If a protein repeatedly fails to fold correctly, chaperones can redirect it for degradation. For example, the E3 ubiquitin ligase CHIP cooperates with HSP70 to ubiquitinate terminally misfolded proteins, sending them to the proteasome.

This system maintains proteostasis (protein homeostasis). Without it, misfolded proteins accumulate and form toxic aggregates. Failures in proteostasis are linked to neurodegenerative diseases like Alzheimer's and Parkinson's, where misfolded protein aggregates are a hallmark of pathology.