Unit 2 Study Guide: Post-Translational Modifications, Proteasome, Cell Cycle, Translation, Protein Targeting, and Cell Signaling

Post-Translational Modifications (PTMs) and the Proteasome

Overview of PTMs

Post-translational modifications (PTMs) are chemical changes to proteins after their synthesis, 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: $\text{Protein-OH} + \text{ATP} \rightarrow \text{Protein-O-PO}_3^{2-} + \text{ADP}$

• Kinase Structure: Kinases have conserved catalytic domains; their activity is regulated by activation loops, substrate binding sites, and regulatory subunits.

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

Ubiquitylation and Proteasomal Degradation

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

• Enzymatic Cascade:

  • E1: Ubiquitin-activating enzyme

  • E2: Ubiquitin-conjugating enzyme

  • E3: Ubiquitin ligase (confers substrate specificity)

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

• Proteasome Function: The 26S proteasome is a multi-subunit complex with multiple protease active sites, capable of degrading a wide range of polyubiquitylated proteins.

• Regulation:

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

  • Commitment: ATP hydrolysis drives substrate unfolding and translocation.

  • Checks and Balances: Regulatory subunits ensure specificity and prevent unwanted degradation.

Cell Cycle Regulation

Stages and Checkpoints

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

• Checkpoints: G1/S, G2/M, and metaphase/anaphase checkpoints monitor DNA integrity and spindle attachment.

Cyclin-Dependent Kinases (Cdks)

• Definition: Cdks are serine/threonine kinases that drive cell cycle progression.

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

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

• Inactivation Mechanisms:

  • Inhibitory kinases (e.g., Wee1)

  • Cdk inhibitors (CKIs)

  • Ubiquitylation and proteasomal degradation

APC/C Complex

• APC/C (Anaphase Promoting Complex/Cyclosome): E3 ubiquitin ligase that targets cell cycle proteins for degradation.

• Regulators: Cdh1 and Cdc20 confer substrate specificity 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 Structure: Cloverleaf structure with anticodon loop and amino acid attachment site.

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

Ribosome Structure and Function

• Binding Sites: A (aminoacyl), P (peptidyl), and E (exit) sites coordinate tRNA binding and peptide elongation.

• Peptide Elongation Steps:

  1. Aminoacyl-tRNA enters A site.

  2. Peptide bond formation catalyzed by rRNA (peptidyl transferase activity).

  3. Translocation moves tRNAs and mRNA through the ribosome.

• Elongation Factors: Assist in tRNA selection and translocation (e.g., EF-Tu, EF-G in prokaryotes).

• Release Factors: Recognize stop codons and promote polypeptide release.

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

Ribosome Profiling

• Technique: Sequencing of ribosome-protected mRNA fragments to map translation in vivo.

• Interpretation: Reveals translation rates and ribosome occupancy on mRNAs.

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-resident chaperones assist in protein folding and prevent aggregation.

ER Targeting Mechanism

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

• SRP Receptor: Anchors SRP-ribosome complex to ER membrane.

• Translocator: Channel through which polypeptide enters ER lumen.

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

• Membrane Orientation: Determined by position and number of signal and stop-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 pathways)

  • 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 Mechanisms:

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

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

• Signaling Speed: Fast (e.g., ion channel opening) vs. slow (e.g., gene expression changes in cell cycle).

GPCR Activation Mechanism

• Ligand binding induces conformational change in GPCR.

• Activated GPCR promotes GDP-GTP exchange on G protein α subunit.

• G protein subunits activate downstream effectors (e.g., adenylyl cyclase).

RTK Signaling

• Ligand binding induces receptor dimerization and autophosphorylation.

• Phosphorylated RTKs recruit signaling proteins to propagate the signal.

• Perturbations (e.g., mutations, inhibitors) can alter pathway output.

Summary Table: Key PTMs and Their Features

PTM	Target Amino Acid(s)	Enzyme	Function
Phosphorylation	Ser, Thr, Tyr	Kinase	Regulates activity, signaling
Ubiquitylation	Lys	E1, E2, E3 ligases	Degradation, signaling
Acetylation	Lys	Acetyltransferase	Regulates chromatin, protein stability
Methylation	Lys, Arg	Methyltransferase	Gene regulation, signaling

Additional info:

• Some details (e.g., specific examples of signaling pathways, or figures from referenced papers) are not included due to lack of content in the original file.

• For paper-specific questions (Collins et al., Yip et al., Fomicheva et al., Ingolia et al.), review class notes and figures as directed.