Post-Translational Modifications (PTMs) and the Proteasome

Overview of PTMs

Post-translational modifications (PTMs) are chemical changes to proteins after translation, crucial for regulating protein function, localization, and stability.

• Common PTMs: Phosphorylation, ubiquitylation, acetylation, methylation, glycosylation.

• Amino Acid Targets:

  • Phosphorylation: Serine, threonine, tyrosine

  • Ubiquitylation: Lysine

  • Acetylation: Lysine

  • Methylation: Lysine, arginine

• Importance: PTMs regulate protein activity, interactions, localization, and degradation.

Phosphorylation

Phosphorylation is the addition of a phosphate group, typically to serine, threonine, or tyrosine residues, catalyzed by kinases.

• Chemical Reaction:

• Kinase Structure: Kinases have conserved catalytic domains; substrate specificity is determined by active site structure and regulatory domains.

• Functional Impact: Alters protein conformation, activity, and interactions.

Ubiquitylation and Proteasomal Degradation

Ubiquitylation is the covalent attachment of ubiquitin to lysine residues, marking proteins for degradation or altering their function.

• Process: Involves E1 (activating), E2 (conjugating), and E3 (ligase) enzymes.

• Linkage Diversity: Ubiquitin can be attached to different lysines (e.g., K48, K63), resulting in different cellular signals.

• Proteasome: A large, ATP-dependent protease complex that degrades polyubiquitylated proteins.

• Stages of Regulation:

  • Recognition: Ubiquitin tags and loosely folded regions are recognized.

  • Commitment: ATP hydrolysis drives substrate unfolding and translocation.

• Checks and Balances: Regulatory mechanisms ensure only appropriate proteins are degraded.

Cell Cycle Regulation

Stages and Checkpoints

The cell cycle consists of ordered phases (G1, S, G2, M) regulated by checkpoints to ensure proper cell division.

• Checkpoints: G1/S, G2/M, and metaphase-anaphase transitions.

• Purpose: Prevents progression if DNA is damaged or replication is incomplete.

Cyclin-Dependent Kinases (Cdks)

• Definition: Serine/threonine kinases that drive cell cycle transitions.

• Regulation by Cyclins: Cyclins bind Cdks, activating them at specific cell cycle stages.

• Regulation by PTMs: Phosphorylation and dephosphorylation modulate Cdk activity.

• Inactivation:

  • Inhibitory kinases (e.g., Wee1)

  • Cdk inhibitors (CKIs)

  • Ubiquitylation and proteasomal degradation

APC/C Complex

• Definition: Anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase that targets cell cycle proteins for degradation.

• Regulation: Activated by Cdh1 and Cdc20 at different cell cycle stages.

Experimental Techniques

• Flow Cytometry: Measures DNA content to assess cell cycle distribution.

• Western Blotting: Detects cell cycle proteins and their modifications.

• System Diagrams: Used to predict effects of perturbations (e.g., inhibition of Cdc25 phosphatase).

Translation and Ribosomes

Basic Concepts of Translation

Translation is the process by which ribosomes synthesize proteins using mRNA as a template.

• tRNA: Transfer RNAs carry amino acids and recognize codons via their anticodon loop.

• tRNA Synthesis: Aminoacyl-tRNA synthetases attach amino acids to tRNAs.

Ribosome Structure and Function

• Binding Sites: A (aminoacyl), P (peptidyl), and E (exit) sites.

• Peptide Elongation: Involves codon recognition, peptide bond formation, and translocation.

• Elongation Factors: Assist in tRNA selection and ribosome movement.

• Release Factors: Recognize stop codons and promote termination.

• rRNA: Catalyzes peptide bond formation and ensures translation fidelity.

Ribosome Profiling

• Definition: Technique to map ribosome positions on mRNAs genome-wide.

• Procedure: Treat cells with nucleases, isolate ribosome-protected fragments, and sequence them.

• Interpretation: Reveals translation rates and ribosome stalling sites.

Protein Targeting to the Endoplasmic Reticulum (ER) and Membrane Proteins

ER Protein Sorting

Proteins destined for secretion or membrane localization are targeted to the ER during translation.

• Major ER Roles: Protein folding, quality control, lipid synthesis, calcium storage.

• Folding: ER chaperones assist in proper protein folding after translocation.

ER Targeting Mechanism

• Signal Recognition Particle (SRP): Binds to ER signal sequence on nascent polypeptide.

• SRP Receptor: Anchors the complex to the ER membrane.

• Translocator: Channel through which the polypeptide enters the ER lumen.

• Signal Sequence: Hydrophobic stretch at N-terminus; also functions as start/stop-transfer sequence.

• Membrane Orientation: Determined by the position of signal and transfer sequences.

Cell Signaling

Principles of Cell Signaling

Cell signaling enables cells to sense and respond to their environment through receptor-mediated pathways.

• Receptor Classes:

  • Ligand-gated ion channels (e.g., NMDAR)

  • G protein-coupled receptors (GPCRs; e.g., Gs, Gi, Gq)

  • Enzyme-coupled receptors (e.g., receptor tyrosine kinases, EGFR)

• Second Messengers: Small molecules (e.g., cAMP, Ca2+) that relay signals inside the cell.

• Molecular Switches: Proteins that toggle between active/inactive states (e.g., GTPases).

• Specificity: Achieved by receptor-ligand binding, compartmentalization, and scaffolding proteins.

• Feedback:

  • Negative feedback dampens signaling (e.g., receptor desensitization).

  • Positive feedback amplifies responses (e.g., cell cycle transitions).

• Signaling Speed: Fast (e.g., exocytosis) vs. slow (e.g., cell cycle regulation).

GPCR and RTK Mechanisms

• GPCR Activation: Ligand binding induces conformational change, activating G proteins.

• RTK Activation: Ligand-induced dimerization and autophosphorylation initiate downstream signaling.

• Pathway Components: Include kinases, phosphatases, second messengers, and transcription factors.

Research Papers and Data Interpretation

• Collins et al., Yip et al., Fomicheva et al., Ingolia et al.: Review figures and notes from class for data interpretation and short answer questions.

• Application: Be prepared to analyze experimental data and relate findings to the above concepts.