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

Post-Translational Modifications (PTMs) and Proteasome

Overview of PTMs

Post-translational modifications (PTMs) are chemical changes to proteins after translation, crucial for regulating protein function, localization, and stability. PTMs target specific amino acids and include phosphorylation, ubiquitylation, acetylation, methylation, and others.

• Phosphorylation: Addition of a phosphate group, typically to serine, threonine, or tyrosine residues. Catalyzed by kinases.

• Ubiquitylation: Attachment of ubiquitin to lysine residues, marking proteins for degradation or altering their function.

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

Phosphorylation Mechanism

Phosphorylation is a reversible modification that alters protein conformation and activity.

• Chemical Reaction: Kinases transfer a phosphate group from ATP to the hydroxyl group of an amino acid.

$\text{Protein-OH} + \text{ATP} \rightarrow \text{Protein-O-PO}_3^{2-} + \text{ADP}$

• Kinase Structure: Kinases have conserved domains for ATP binding and substrate recognition, influencing specificity and activity.

Ubiquitylation and Proteasome Function

Ubiquitylation targets proteins for degradation by the proteasome, a multi-subunit protease complex.

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

• Diversity of Linkages: Ubiquitin can be attached to different lysines, creating distinct signals (e.g., K48 for degradation, K63 for signaling).

• Proteasome: Degrades polyubiquitylated proteins via multiple protease active sites.

• Regulation: Recognition and commitment stages involve ubiquitin, loosely folded regions, and ATP hydrolysis.

• Checks and Balances: Proteasome activity is tightly regulated to prevent unwanted protein degradation.

Cell Cycle Regulation

Stages and Checkpoints

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

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

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 ubiquitylation modulate Cdk activity.

• Inactivation: Inhibitory kinases, CKIs (Cdk inhibitors), and ubiquitylation can inactivate Cdks.

APC/C Complex

• APC/C: Anaphase-promoting complex/cyclosome, a ubiquitin ligase regulating mitosis.

• Regulation: Cdh1 and Cdc20 are co-activators controlling APC/C activity.

Experimental Techniques

• Cell Cycle Analysis: Methods include flow cytometry, immunoblotting, and live-cell imaging.

• Data Interpretation: Understanding perturbations (e.g., kinase inhibition) is key for analyzing cell cycle experiments.

Translation and Ribosomes

Basic Concepts of Translation

Translation is the process of synthesizing proteins from mRNA templates, occurring on ribosomes.

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

• Synthesis: tRNAs are transcribed and processed, then charged with amino acids by aminoacyl-tRNA synthetases.

• Decoding: Ribosomes read mRNA codons and facilitate peptide bond formation.

Ribosome Structure and Function

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

• Peptide Elongation: Involves tRNA entry, peptide bond formation, and translocation.

• Elongation Factors: Assist tRNA delivery and ribosome movement; release factors terminate translation.

• rRNA: Ribosomal RNA catalyzes peptide bond formation and ensures translation fidelity.

Ribosome Profiling

• Technique: Ribosome profiling uses deep sequencing to map ribosome positions on mRNAs.

• Data Interpretation: Reveals translation efficiency and identifies actively translated regions.

Protein Targeting to ER and Membrane Proteins

ER Transport and Protein Sorting

Proteins destined for secretion or membrane localization are targeted to the endoplasmic reticulum (ER).

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

• Chaperones: ER resident chaperones assist in protein folding and prevent aggregation.

Sequence of Events for ER-Bound Proteins

• Translation Initiation: Begins in cytosol; signal recognition particle (SRP) binds ER signal sequence.

• SRP Receptor: Directs ribosome to ER membrane.

• Translocator: Transfers nascent polypeptide into ER lumen or membrane.

ER Signal Sequence

• Definition: Short peptide sequence directing proteins to ER; similar properties to start- and stop-transfer sequences.

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

Cell Signaling

Principles of Cell Signaling

Cell signaling enables cells to respond to external and internal cues via 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., RTK, EGFR

• Second Messengers: Small molecules like cAMP, Ca2+ that propagate signals.

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

• Specificity: Achieved via receptor-ligand interactions, compartmentalization, and feedback mechanisms.

• Feedback: Negative feedback dampens signaling; positive feedback amplifies responses.

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

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

• RTK Signaling: Receptor dimerization and autophosphorylation initiate downstream pathways.

Research Papers and Data Interpretation

Collins et al., Yip et al., Fomicheva et al., Ingolia et al.

• Review Figures/Notes: Understand experimental design, data, and conclusions from class discussions.

• Short Answer Questions: Be prepared to explain key findings and interpret figures.

• Team Quiz: Collaborative analysis of Yip et al. paper.

• Guide Document: Use for Fomicheva et al. review.

Additional info: Some details about specific papers and experimental techniques were inferred based on typical genetics and cell biology curricula.