Posttranslational Modifications

Why This Matters

Posttranslational modifications (PTMs) represent the cell's sophisticated toolkit for expanding the functional diversity of the proteome far beyond what the genome alone encodes. You're being tested on how cells use these chemical modifications to rapidly and reversibly control protein activity, localization, stability, and interactions—all without synthesizing new proteins. Understanding PTMs means grasping core concepts like signal transduction, protein turnover, epigenetic regulation, and protein folding/stability.

Think of PTMs as the cell's editing system: translation gives you a rough draft, but modifications create the final, functional product. The key insight for exams is that different PTMs achieve similar regulatory goals through distinct mechanisms—and knowing which modification does what (and why) will help you tackle FRQs that ask you to predict cellular outcomes when specific pathways are disrupted. Don't just memorize the modifications—know what biological problem each one solves.

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Reversible Covalent Switches: Rapid Signal Control

These modifications act as molecular on/off switches, allowing cells to respond to signals within seconds to minutes. The reversibility is key—enzymes that add the modification are paired with enzymes that remove it, creating dynamic regulatory circuits.

Phosphorylation

• Phosphate group addition to Ser, Thr, or Tyr residues—introduces negative charge and steric bulk that alter protein conformation and activity
• Kinases add, phosphatases remove—this enzyme pairing enables rapid, reversible signal switching in cascades like MAPK and PI3K/Akt
• Central to signal transduction—arguably the most common PTM, with ~30% of proteins phosphorylated at any given time

Acetylation

• Acetyl group addition to lysine residues—neutralizes the positive charge of lysine, weakening DNA-histone interactions
• HATs add, HDACs remove—histone acetyltransferases and deacetylases dynamically regulate chromatin accessibility
• Generally activates transcription—open chromatin structure allows transcription factor binding; also modifies non-histone proteins like p53

Methylation

• Methyl groups added to Lys or Arg residues—does not change charge but creates binding sites for reader proteins
• Context-dependent effects on gene expression—H3K4me3 activates transcription, while H3K9me3 represses it
• More stable than acetylation—methylation marks can persist through cell division, contributing to epigenetic memory

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Protein Tagging for Fate Determination

These modifications attach small proteins or protein-like molecules to targets, marking them for specific cellular fates. The "tag" itself carries information that other cellular machinery reads and acts upon.

Ubiquitination

• Ubiquitin attached to lysine residues via E1-E2-E3 enzyme cascade—polyubiquitin chains (especially K48-linked) target proteins for proteasomal degradation
• Regulates protein half-life and turnover—critical for cell cycle progression, removing cyclins at precise times
• Non-degradative roles exist—monoubiquitination and K63-linked chains regulate DNA repair, endocytosis, and NF-κB signaling

SUMOylation

• SUMO proteins attached to lysine residues—structurally similar to ubiquitin but functionally distinct
• Modulates localization and protein-protein interactions—often targets transcription factors and nuclear proteins
• Stress response and genome stability—SUMOylation increases during cellular stress and regulates DNA damage repair pathways

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Irreversible Activation: Proteolytic Processing

Unlike reversible modifications, proteolytic cleavage permanently alters protein structure. This "one-way switch" ensures commitment to activation and prevents inappropriate reversal.

Proteolytic Cleavage

• Peptide bond hydrolysis activates zymogens—inactive precursors like trypsinogen become active enzymes (trypsin) upon cleavage
• Cascade amplification in signaling—blood clotting and complement pathways use sequential proteolysis to amplify signals exponentially
• Prevents premature activity—digestive enzymes and caspases are synthesized inactive to protect the cell until appropriate triggers arrive

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Structural Modifications: Stability and Folding

These PTMs directly influence protein architecture, ensuring proper folding and long-term stability. They're particularly important for secreted and extracellular proteins that must survive harsh environments.

Disulfide Bond Formation

• Covalent $-S-S-$ bonds between cysteine residues—stabilizes tertiary and quaternary structure by cross-linking polypeptide regions
• Occurs in the ER for secreted proteins—the oxidizing environment of the ER lumen (vs. reducing cytoplasm) favors disulfide formation
• Critical for antibody function—immunoglobulins rely on interchain disulfide bonds to maintain their characteristic Y-shape

Hydroxylation

• Hydroxyl groups added to Pro and Lys residues—requires $\text{Fe}^{2+}$, $\alpha$-ketoglutarate, and vitamin C as cofactors
• Essential for collagen triple helix stability—hydroxyproline forms additional hydrogen bonds that stabilize the collagen structure
• Oxygen sensing via HIF regulation—prolyl hydroxylases hydroxylate HIF-1α under normoxia, targeting it for degradation; hypoxia inhibits this, stabilizing HIF

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Localization Signals: Membrane Targeting

These modifications anchor proteins to membranes or direct them to specific cellular compartments. The lipid or carbohydrate addition provides a physical "address tag" that determines where a protein functions.

Lipidation

• Lipid groups (myristoyl, palmitoyl, prenyl) attached to proteins—increases hydrophobicity for membrane association
• Myristoylation is typically irreversible; palmitoylation is reversible—this difference allows dynamic regulation of membrane localization
• Essential for Ras and other signaling proteins—farnesylation of Ras anchors it to the plasma membrane where it transduces growth signals

Glycosylation

• Carbohydrate chains attached to Asn (N-linked) or Ser/Thr (O-linked)—occurs in ER and Golgi during secretory pathway transit
• N-linked begins with 14-sugar precursor transferred en bloc—subsequent trimming and modification creates mature glycoproteins
• Critical for protein folding, stability, and recognition—glycans on cell surface proteins mediate cell-cell interactions and immune recognition (blood types, for example)

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Quick Reference Table

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Self-Check Questions

1. Which two PTMs both regulate transcription through histone modification but have opposite default effects on chromatin accessibility?

2. A protein needs to be rapidly activated in response to a growth factor, then quickly inactivated when the signal stops. Which PTM is most likely involved, and why would proteolytic cleavage be inappropriate here?

3. Compare ubiquitination and SUMOylation: both attach small proteins to lysine residues, so how would you determine which modification a target protein has received based on its cellular fate?

4. A patient presents with symptoms of scurvy. Which PTM is disrupted, what enzyme requires the missing cofactor, and how does this explain the connective tissue problems observed?

5. FRQ-style: Explain how the same amino acid residue (lysine) can be modified by acetylation, methylation, ubiquitination, or SUMOylation. What determines which modification occurs, and what are the functional consequences of each?