Biochemistry Notes: Protein PTMs, Degradation, and Cell Cycle Regulation

Post-Translational Modifications (PTMs) and Protein Diversity

Definition: A post-translational modification is a covalent modification to a protein after it has been translated.
Common Types: Phosphorylation, ubiquitination, and acetylation.
Protein Processing: Any post-translational processing, such as the cleavage of peptide bonds to convert inactive proteins into active ones, is considered a PTM.
Purposes of PTMs: - Surface Changes: Alters the surface of a protein to change its binding partners. - Conformational Alterations: Used for regulation of protein activity. - Localization: Directs the protein to specific cellular compartments. - Stabilization: Maintains protein structural integrity.
Driving Complexity (The Proteome): - Transcriptional Level: Diversity starts with alternative promoters, alternative splicing, and mRNA editing, creating the transcriptome. - Translational Level: PTMs happen post-translation, resulting in the proteome, which drives the complexity and diversity of protein functions.
Specific Modifying Groups and Regulatory Functions: - Phosphate on Serine ( $Ser$ ), Threonine ( $Thr$ ), or Tyrosine ( $Tyr$ ): Drives the assembly of a protein into larger complexes. - Methyl on Lysine ( $Lys$ ): Helps create distinct regions in chromatin by forming mono-, di-, or trimethyl lysine in histones. - Acetyl on Lysine ( $Lys$ ): Helps to activate genes in chromatin by modifying histones; acetylation neutralizes the positive charge of lysine. - Palmityl Group on Cysteine ( $Cys$ ): The addition of this fatty acid drives protein association with the membrane. - Ubiquitin on Lysine ( $Lys$ ): - Monoubiquitin: Regulates the transport of membrane proteins in vesicles. - Polyubiquitin chain: Targets a protein for degradation by the proteasome.

Lipidation and Membrane Localization

General Function: Lipidation regulates cellular localization specifically at membranes.
Techniques to bring proteins to the lipid bilayer: - Myristoylation: Adding a myristoyl anchor to the N-terminal Glycine ( $Gly$ ) residue of a target protein via a lipid anchor to localize it near the membrane. - Palmitoylation (Palmdulation): Adding a fatty acid chain (palmitoyl anchor) via a thioester linkage between the Cysteine ( $Cys$ ) residue of a target protein and the lipid bilayer. - Prenylation: A farnesyl anchor is attached to the Cysteine ( $Cys$ ) residue of the target protein via a diester linkage.
Rationale for Membrane Proximity: Bringing proteins to the membrane ensures they are positioned to receive signals quickly. Receptors are activated by ligand binding at the outer surface; having the internal signaling protein close to the receptor saves time and increases efficiency.
Master Regulators: Signaling molecules that act as master regulators are heavily modified. - p53: Regulates life/death cell decisions; modified at the N-terminus via phosphorylation. - Tau: Another protein known to undergo significant modification.

Histone Modifications and Gene Regulation

Mechanism: Modifying histone proteins can "open up" the histone to modify the DNA sequence access, allowing transcription factors to attach.
Methylation vs. Acetylation: Acetylation of lysine neutralizes its charge. Methylation does not change the charge but changes protein-protein interactions.
The Histone Code: - Trimethyl on K9 ( $H3K9me3$ ): Associated with gene silencing and heterochromatin formation. - Trimethyl on K9 and K4: Associated with gene expression. - Trimethyl on K27 ( $H3K27me3$ ): Associated with gene silencing.
Specific Protein Example: The protein BPTF has a PHD domain that binds specifically to the H3K4me3 (histone 3 lysine 4 trimethyl) group. - BPTF acts as a transcriptional activator because the methylated lysine fits into a hydrophobic pocket of the PHD domain. - One target gene of BPTF is cMYC, an oncogene that controls cell growth and proliferation.

The Ubiquitin-Proteasome System

Ubiquitylation: Conjugation of ubiquitin (Ub) protein to a target protein.
Signaling Outcomes based on Ubiquitin Chains: - Monoubiquitylation: Signals histone regulation. - Multi-ubiquitylation: Signals endocytosis (bringing proteins into vesicles inside the cell). - Polyubiquitination via Lysine 48 ( $K48$ ): Signals the cell to degrade the protein via the 26S proteasome. - Lysine 63 ( $K63$ ) linked polyubiquitin chains: Signals DNA repair.
Bond Type: An isopeptide bond is formed between the target protein and Ub.
The Ubiquitin Conjugation Cycle: - E1 (Ub Activating Enzyme): Activates Ub using enthalpy from ATP hydrolysis (acts as a "supercharger"). - E2 (Ub Conjugating Enzyme): Receives the activated Ub from E1; looks for target proteins. - E3 (Ub Ligase): Required alongside E2 to ligate Ub to the target; determines high specificity and targets recognition. There are many more types of E3 enzymes than E1 or E2 to handle specific protein diversity.
Targeting for Degradation: - Exposed N-terminus: Specifically those with acetylated residues. - Degradation Signals (Degrons): Some proteins mask their degradation sites; signals can trigger unmasking. Proteases may cleave a peptide fragment to reveal a hidden degradation signal. E3 ligases may also recognize specific phosphorylated residues.
Reasons for Degradation: To remove misfolded proteins, regulate function tightly, or dispose of aged (senescent) or damaged proteins.
Subtypes of E3 Ligases: Ring, Hect, and RBR (Ring-between-Ring).
Deubiquitylating Enzymes (DUBs): Compete with E3 ligases. Functions include freeing Ub from precursors, limiting dwell time on proteasomes, and participation in kinetic competition at the proteasome.

The 26S Proteasome

Structure: - 19S Regulatory Subunits: Attached to the top and bottom of the core; involved in initial binding, Ub removal, and substrate unfolding via ATP-dependent steps. - 20S Proteolytic Core: The central cylinder where degradation is guaranteed.
Proteolytic Function: The 20S core contains various active sites with different protease activities to break a wide range of peptide bonds: - Sites that target acidic residues ( $Asp$ ). - Sites that target basic residues ( $Arg, Lys$ ). - Sites that target hydrophobic residues ( $Tyr, Trp, Phe$ ).
Regulatory Purpose: Active sites are sequestered in the center of the core so they do not accidentally degrade healthy cytosolic proteins.

The Cell Cycle and Checkpoints

Phases: - $G_0$ : Specialized resting state; post-mitotic; can be permanent. - $G_1$ : Growth phase; preparation for DNA synthesis. - $S$ (Synthesis): Chromosome duplication/DNA replication. - $G_2$ : Preparation for mitosis. - $M$ (Mitosis): Prophase, Metaphase, Anaphase, Telophase, and Cytokinesis. In human cells, the cycle lasts approximately $24\,\text{hours}$ .
Checkpoints (Binary Decisions): - $G_1/S$ Transition (Start/Restriction Point): Checks cell size, environment (nutrients/growth factors), density, and attachment before committing to DNA synthesis. - $G_2/M$ Transition: Checks if DNA is replicated properly and if the environment supports making two cells. - M-Phase Checkpoint (Spindle Assembly Checkpoint): Between metaphase and anaphase; ensures all chromosomes are attached to spindles and correctly aligned.

Cyclin-Dependent Kinases (Cdks) and Cyclins

Coordination: Cdks are activated by the binding of cyclin proteins. Cdks phosphorylate targets on Serine ( $Ser$ ) and Threonine ( $Thr$ ) motifs.
Cyclin Expression Patterns: - $G_1$ Cyclins: High in $G_1$ ; prompt expression of $G_1/S$ cyclins. - $G_1/S$ Cyclins: Activate Cdk late in $G_1$ ; levels fall in $S$ phase; commit cell to cycle entry. - S-Cyclins: Stimulate chromosome duplication. - M-Cyclins: Stimulate entry into mitosis at $G_2/M$ ; degraded during metaphase-anaphase transition.
Cdk Activation States: - Inactive: Cdk is alone; the T-loop (activation loop) physically blocks the active site. - Partially Active: Cyclin binds and pulls the T-loop away from the active site, allowing ATP entry. - Fully Active: CAK (Cdk-activating kinase) phosphorylates a Threonine residue on the T-loop, pinning it into a rigid open position with high binding affinity.
Inactivation Mechanisms: - Inhibitory Kinases: Wee1 adds inhibitory phosphates to the Cdk. - Inhibitory Proteins (CKIs): Proteins like p27 or p21 interfere with the active site. - Degradation: Ubiquitylation of cyclins.
M-Cdk Specific Functions: Induces spindle assembly, ensures sister chromatid attachment, triggers chromosome condensation, promotes nuclear envelope breakdown, and rearranges Golgi/actin cytoskeleton. - Positive Feedback: M-Cdk activates Cdc25 (a phosphatase that removes inhibitory phosphates from M-Cdk) and inhibits Wee1 (the kinase that adds them).

Ubiquitin Ligases in the Cell Cycle

SCF Complex: Acts at the $G_1/S$ transition. It destroys CKIs to allow Cdk activation and destroys $G_1/S$ cyclins in early $S$ phase. It recognizes phosphorylated targets via F-box subunits.
APC/C (Anaphase-Promoting Complex): Destroys $M$ and $S$ cyclins. - Cdc20 Association: Occurs in mid-mitosis. - Cdh1 Association: Occurs in late mitosis/early $G_1$ . - M-Cdks phosphorylate Cdh1 to prevent it from competing with Cdc20 during mid-mitosis. Once M-Cdks are inactivated, Cdh1 rebinds.

Protein Translation and the Ribosome

tRNA (Transfer RNA): - Length: Approximately $80\,\text{nucleotides}$ . - Structure: Cloverleaf structure folding into a 3D L-shape. - Ends: 3' end couples to an amino acid; anticodon pairs with mRNA codons. - Modifications: Includes Pseudouridine ( $\Psi$ ) and Inosine. Inosine allows one tRNA to recognize more than one codon (wobble position).
Codon Logic: There are $31$ tRNAs but $61$ codons. The 3rd position "wobble" allows for error tolerance.
tRNA Synthetase: Couples amino acids to tRNA via energy from ATP hydrolysis, forming an ester linkage at the 3'OH of the nucleotide sugar. Accuracy methods include: - High affinity for the correct amino acid. - Proofreading by flipping the amino acid into an editing site.
The Beetle and Jackbean Example: Jackbeans produce canavanine, an analog of arginine. Canavanine has a lower $pKa$ of its guanidino group, preventing salt bridge formation and leading to misfolding. Most bacteria can't tell them apart, but a specific beetle evolved a tRNA synthetase that discriminates against canavanine.
The Ribosome (A Ribozyme): - Prokaryotic Ribosome (70S): Consists of a 50S (large) and 30S (small) subunit. $2/3$ of the mass is RNA. - Sites: A (Aminoacyl), P (Peptidyl), and E (Exit).
Translation Steps: - Aminoacyl-tRNA binds to A site. - Peptide bond forms between P and A site amino acids. - Ribosome shifts; tRNA in P site moves to E and is ejected.
Elongation Factors (G-proteins): - EF-Tu (Prokaryotes): Binds charged tRNA. Correct base-pairing triggers GTP hydrolysis and release of the tRNA. Incorrect pairing leads to rejection (kinetic proofreading). - EF-G: Promotes translocation of the ribosome.
Energy Consumption: $1\,\text{ATP}$ for charging and $2\,\text{GTP}$ for elongation per amino acid.
Initiation and Termination: - Prokaryotes: Uses Shine-Dalgarno sequences; can be polycistronic. - Eukaryotes: Includes poly-A tail binding; scans for AUG. - Stop Codons: UAA, UAG, UGA. Release factors bind to the A-site; the ribosome uses $H_2O$ to catalyze the release of the peptide chain.
Selenocysteine: A recoding event where a segment preceding a UGA codon signals it to encode selenocysteine instead of a stop.

Ribosome Profiling and mRNA Footprinting

Technique: Uses RNA footprinting to map ribosomes.
Process: Treatment with Cyclohexamide (creates a $28-30\,\text{nucleotide}$ footprint), RNase digestion, and purification/sequencing of remaining mRNA fragments.
Applications: Identifies non-canonical start sites, measures translational efficiency, monitors speed, and observes how synthesis rates change under stress (e.g., heat shock causes arrest $30-60\,\text{codons}$ downstream of initiation).

The Endoplasmic Reticulum (ER) and Protein Translocation

Function: Site of lipid synthesis (Smooth ER) and protein synthesis (Rough ER). First step in the secretory pathway.
Modifications in ER: Primary location for disulfide bond formation and initial glycosylation (addition of sugar to Asparagine residues).
Signal Recognition Particle (SRP): A rodlike structure that binds the ER signal sequence as it emerges from the ribosome. It blocks the elongation factor site to pause translation, preventing the protein from being released into the cytosol until the ribosome binds to the ER membrane.
Sec61 Complex: The ER translocator channel. A water-filled pore that opens on the side to allow transmembrane domains to exit into the lipid bilayer.
Membrane Protein Orientation: - Positive-Inside Rule: The terminus with more positive charges is more likely to remain in the cytoplasm. - Tail-anchored proteins: Use the Get1, 2, 3 system. Get3 is an ATPase that captures hydrophobic C-terminal tails and brings them to the membrane markers Get1 and 2.
Chaperones: BiP (an $HSP70$ family member) assists in re-folding. Misfolded proteins are recognized by exposed non-polar residues. Chronically misfolded proteins are exported to the cytosol for proteasomal degradation.

Cell Signaling Mechanisms

Classes of Receptors: - Ion-Channel Coupled: Example: Glutamate binds to AMPARs (allows $Na^+$ entry to depolarize) which then allows NMDARs to open and allow $Ca^{2+}$ entry. - G-Protein Coupled Receptors (GPCRs). - Enzyme-Coupled Receptors.
Molecular Switches: - Phosphorylation: Regulated by $520$ kinases and $150$ phosphatases. - GTP-Binding: Regulated by GEF (exchanges GDP for GTP to turn on) and GAP (assists GTP hydrolysis to turn off).
GPCR Signaling: - $G_s$ : Activates adenylyl cyclase. - $G_i$ : Inhibits adenylyl cyclase. - Mechanism: Activated adenylyl cyclase produces cAMP, which activates PKA. PKA catalytic subunits enter the nucleus to activate CREB, leading to gene transcription. - Desensitization: GRK phosphorylates the receptor, allowing Arrestin to bind and lock the receptor in an inactive state.
Enzyme-Coupled Receptors (RTKs): - Dimerization: Signal molecules bring two kinase domains together. - Trans-phosphorylation: Kinase domains phosphorylate each other to create binding sites for downstream signaling proteins (e.g., PLC\gamma, PI3Kinase). - EGFR Exception: Not activated by trans-phosphorylation but by conformational change where one "activator" domain pushes a "receiver" domain into an active state.

Fomicheva Paper: CRISPR Screen and Cell Density

Goal: Identify genes regulating density-dependent proliferation.
Methods: - CRISPR KO Screen: Used 6 sgRNAs per gene for specificity/tilling. - BrdU: A thymidine analog that incorporates into replicating DNA; used to measure proliferation. - FUCCI System: Cdt1 (Red = $G_1$ ) and Geminin (Green = $S/G_2/M$ ) to visualize cell cycle phases.
Key Findings: - TRAF3 KO: Leads to over-proliferation at high densities. Organoids look tumor-like and less round. - Pathways: TRAF3 acts via the non-canonical NF-KB pathway (RelB becomes nuclear). It works independently of YAP/TAZ signaling. - Cell Cycle Impact: TRAF3 KO promotes $G_1$ entry by increasing Cyclin D1 levels. - DHB-mVenus: A biosensor for CDK2 activity (nuclear in $G_0$ , cytoplasmic when active). TRAF3 KO results in increased cytoplasmic distribution, indicating higher CDK2 activity and failure to enter $G_0$ arrest.