Lecture 18 (Grimm)

Question 1

Protein Acetylation

Definition, Types, Mechanism & Function

Answer 1

Protein Acetylation
- Protein acetylation involves the addition of an acetyl group (CH₃CO) to the N-terminal α-amino group or lysine residues in proteins

Types
- N-terminal Acetylation: Catalyzed by N-terminal acetyltransferases (NATs). (irreversible as no known N-terminal deacetyltransferases exist)
- Lysine Acetylation: Catalyzed by lysine acetyltransferases (KATs (Reversible via lysine deacetylases (KDACs))

Mechanism
- Enzymes use acetyl-CoA as a donor to transfer the acetyl group.
- Acetylation neutralizes the positive charge on amino groups, altering local electrostatic properties and protein interactions.

Functions
N-terminal Acetylation
- Targets proteins for degradation or protects against it (polyubiquitination and proteasomal activity).
- Assists in protein folding.
- Facilitates protein-protein interactions and membrane targeting (helps anchor proteins to membranes)

Lysine Acetylation
- Regulates enzyme activity within signaling pathways.
- Influences protein degradation by affecting ubiquitination.
- Modulates protein synthesis by altering interaction with translation machinery.

Question 2

N-terminal Modifications during Translation in the Cytoplasm

Cotranslational Modifications

Answer 2

N-terminal Modifications during translation in the cytoplasm
- N-terminal modifications are crucial for determining protein stability, function, and readiness for additional post-translational modifications (PTMs)

In Bacteria
- Newly synthesized bacterial proteins start with an N-formylmethionine (fMet).
- Peptide deformylase (PDF) removes the formyl group, exposing the methionine for further processing.
- The exposed methionine is then cleaved by Methionine Aminopeptidase (MAP), creating a new N-terminal residue.

In Eukaryotes
- The initiator methionine is often cleaved by Methionine Aminopeptidase (MAP), creating a new N-terminal residue. (similar to bacteria but does not involve a formyl group)
- In some cases, the N-terminal methionine is directly acetylated by N-terminal acetyltransferases (NATs), skipping the removal step.

Question 3

N-terminal modifications after subcellular trafficking and during organelle-specific translation in mitochondria and plastids

Answer 3

N-terminal modifications after subcellular trafficking and during organelle-specific translation in mitochondria and plastids
Mitochondria
- Proteins synthesized in the cytosol are imported into mitochondria through the TOM (outer membrane) and TIM (inner membrane) complexes
- Once inside, the mitochondrial processing peptidase (MPP) cleaves targeting signals, exposing the N-terminus.
- Peptide Deformylase (PDF): Removes formyl groups from formylated methionine
- Methionine Aminopeptidase (MetAP): Cleaves the methionine, leaving a free N-terminal residue.
- N-terminal Acetylation (NTA): May occur if the protein’s N-terminus is acetylated post-import.

Plastids
- Proteins synthesized in the cytosol are transported into plastids through the TOC (outer chloroplast membrane) and TIC (inner chloroplast membrane) complexes.
- SPP (stromal processing peptidase) cleaves the targeting sequence, exposing the N-terminus.
- Peptide Deformylase (PDF): Removes formyl groups, similar to mitochondria.
- Methionine Aminopeptidase (MetAP): Removes methionine, exposing the second residue.
- Chloroplast N-terminal Acetyltransferase (cpNat): Acetylates the exposed N-terminal residue, stabilizing the protein.

Purpose of These Modifications
- Stabilization: Acetylation prevents degradation and enhances protein stability.
- Localization: Cleaving targeting signals ensures proper protein positioning within the organelle.
- Preparation for Further PTMs: Exposed N-termini can undergo additional modifications or interactions for specific functions

Question 4

Overview of N-terminal Modification
of Proteins

Answer 4

Overview of N-terminal Modification of Proteins
- N-terminal α-amino group is normally positively charged at neutral pH
- The chemical property ofthe N-terminus of proteins can be altered by modification (red labelled)

Question 5

Myristoylation

Answer 5

Myristoylation
- Myristoylation is a lipid modification where myristic acid (a 14-carbon saturated fatty acid) is covalently attached to the N-terminal glycine residue of a protein.

Cotranslational Myristoylation
- Methionine aminopeptidase (MetAP) removes the N-terminal methionine.
- Myristic acid is activated by linkage to coenzyme A (acyl-CoA) via acyl-CoA synthetase.
- N-myristoyltransferase (NMT) transfers myristic acid to the exposed glycine residue.

Posttranslational Myristoylation
- Proteins are cleaved by proteases to expose a glycine residue.
- NMT adds the myristic acid to this glycine residue.

Functions
- Myristoylation adds hydrophobicity, anchoring proteins to membranes
- Facilitates interactions between signaling proteins.
- Plays a role in cellular signaling and localization.