Lecture 17 (Grimm) Flashcards
Proteinfolding and posttranslational modification
Levels of Gene Regulation
Levels of Gene Regulation
Gene Regulation occurs at Multiple Levels
- Genomic, Transcription, RNA Processing/Translocation,
Translation and Posttranslation
- Eukaryotic gene expression is usually controlled at the Level of Initiation of Transcription
Posttranslational modifications (PTM)
Posttranslational modifications (PTM)
- Protein modifications that form a distinct structural form from the initial protein that arose by direct sequential construction of the polypeptide chain during translation.
Modifications can occur
- Simultaneous with Translation (cotranslational)
- Just prior to protein degradation
- Reversible or irreversible
- In prokaryotes and (more often) in Eukaryotes
Functions of Posttranslational Modifications
Functions of Posttranslational Modifications
- Correct folding and improved functional properties –> Modify activity and/or function
- Solubility (carbohydrates)
- Stabilization or Destabilization (carbohydrates, ubiquitination,
phosphorylation)
- Localization in different compartments
- Proteolytic processing (leader sequence removal, trimming
of precursors)
- Aging: Modification may identify the protein for degradation
- Regulation of molecular Interactions (phosphorylation, acetylation, acylation, glycosylation…)
Basic modes of posttranslational modification
Basic Modes of Posttranslational Modification
- Processing (Cleavage) of the backbone (processing from
large precursors to the biologically active product, e.g. peptide hormone, removal of N-terminal Met)
- Decay or processing of side chains (e.g. cis-trans Pro,
Hydroxy-Pro, methylation, acetylation)
- Attachments of “small” chemical modifying groups (phoshorylation 10% of all modifications, lipids, carbohydrates, …)
- Attachment of proteins (e.g. Ubiquitination, sumoylation)
Protein Methylation
PTM
Protein Methylation
- Methyl groups are added to specific amino acid residues, primarily arginine and lysine
Functions
- Increases Hydrophobicity
- May alter the charge of the protein ( e.g. if a carboxyl group of Glu is methylated)
Regulatory Impact:
- Direct action: Changes protein conformation and interactions (e.g., RNA binding).
- Indirect action: Methyl marks are recognized by effector proteins (‘Readers’) that mediate transcriptional changes.
- Cross-Talk with Other PTMs: Methylation competes with modifications like acetylation, e.g., at histone H3 sites (H3K9, H3K27).
Enzymatic Regulation
- Protein Arginine Methyltransferases (PRMTs): Enzymes that methylate arginine residues
- Protein Methyltransferases (PMTs): Regulate chromatin structure and nucleosome surfaces.
- Protein Demethylases (PDMs): Remove methyl groups and allow reversible modifications.
Oxidation of cysteine residues
Redox-based posttranslational modification (PTM)
Oxidation process of cysteine thiol groups (-SH) by reactive oxygen species (ROS)
- These modifications help regulate protein activity, protect against oxidative damage, and enable redox signaling in cells.
- Reversible modifications (disulfide bridges or glutathionylation) allow proteins to adapt to changing cellular conditions
Mechanism
- Disulfide Bridges: Bonds form either within the same protein (intramolecular) or between different proteins (intermolecular) for stability and regulation.
- Stepwise Oxidation: Sulfenic acid (-SOH) → Sulfinic acid (-SO₂H) → Sulfonic acid (-SO₃H) (irreversible damage).
- Glutathionylation: The thiol group can form a mixed disulfide bond with glutathione (GSH). This protects the protein from further damage and can later be reversed.
- Reversal: Enzymes like Thioredoxin (TRX), NAD(P)H-dependent thioredoxin reductase (NTRC) and Glutaredoxin (GRX) can reverse these oxidations restoring the original -SH state.
Direct Disulfide Reduction by Thioredoxin
Direct Disulfide Reduction by Thioredoxin
- Thioredoxin (TRX) is an enzyme that reduces disulfide bonds (S-S) back to free thiol groups (-SH) in cysteine residues.
Mechanism
1. TRX active site: Contains cysteine residues that attack the disulfide bond in the target protein, forming a mixed disulfide intermediate.
2. A second cysteine within TRX resolves the intermediate, releasing the reduced target protein.
3. Reduction is powered by electrons from NADPH, transferred via enzymes like thioredoxin reductase
Reduction of Thioredoxin by Thioredoxin
Reductase
Reduction of Thioredoxin by Thioredoxin Reductase
Ferredoxin-Linked Pathway (Plants and photosynthetic organisms)
1. Light excites electrons in photosynthetic systems, producing reduced ferredoxin.
2. Ferredoxin-Thioredoxin Reductase (FTR) transfers electrons from ferredoxin to oxidized TRX, reducing it.
3. Reduced TRX is now active, it can reduce disulfide bonds in target proteins by donating its electrons
NADPH-Linked Pathway (Animals and non-photosynthetic organisms)
1. NADPH acts as the electron donor.
2. NADPH-Thioredoxin Reductase (NTR) transfers electrons from NADPH to oxidized TRX.
3. Reduced TRX is ready to reduce disulfide bonds in target proteins.
Role of Fdx/Trx and NTRC in Photosynthesis and Starch Synthesis
Role of Fdx/Trx and NTRC in Photosynthesis and Starch Synthesis
Fdx/Trx System in Light Conditions
- Light reduces ferredoxin (Fdx) in the chloroplast via photosynthesis.
- Electrons are transferred to thioredoxin (Trx) via ferredoxin-thioredoxin reductase (FTR).
- Reduced Trx activates AGPase, enhancing starch synthesis.
NTRC in Dark or Low Light Conditions
- NADPH generated by the oxidative pentose-phosphate pathway (OPP) reduces NTRC.
- NTRC reduces Trx to activate AGPase, ensuring starch synthesis continues without light.
Leaf-to-Root Connection
- Photosynthesis in leaves produces sucrose, which is transported to roots.
- In roots, NTRC and Fdx/Trx regulate AGPase, converting sucrose to starch.
Functions
- Starch Biosynthesis
- ATP and Chlorophyll Synthesis
- Antioxidant Defense
- Plastid Gene Expression
- Cyclic Electron Transport
- Meristem Maintenance
Monothiol and dithiol Glutaredoxin (GRX)-dependent catalytic mechanisms
Monothiol and dithiol Glutaredoxin (GRX)-dependent catalytic mechanisms
Glutaredoxin (GRX) Functions
- Reduces disulfide bridges in proteins.
- Removes protein-glutathione adducts (deglutathionylation).
- Reduces hydrogen peroxide via redox mechanisms.
Mechanisms of GRX Action
Monothiol Mechanism
- GRX interacts with protein-glutathione (P-S-SG) to release reduced protein (P-SH).
- The glutathionylated GRX intermediate is regenerated by glutathione (GSH).
- Produces reduced protein and oxidized glutathione (GSSG).
Dithiol Mechanism
- GRX forms a disulfide intermediate with the protein disulfide bond (P-S-S-P).
- This intermediate is resolved using GSH, releasing the reduced protein (P-SH).
- Produces two reduced protein thiol groups.
Protein Phosphorylation
PTM
Protein Phosphorylation
- Involves adding a phosphate group to a protein by protein kinases, transferring it from ATP to an amino acid residue.
Function & Mechanism
- Signal Transduction: Acts as a key step in cellular response to environmental signals (light, pathogens, stress)
- Modifies protein activity, localization, conformation, binding affinity, and turnover.
- Cascade Amplification: Sequential phosphorylation through multiple proteins, including protein kinases that phosphorylate other proteins, can amplify signals.
- Protein Phosphatases remove phosphate groups, allowing dynamic regulation of protein function and reversibility of signals.
Molecular Remodeling of Photosystem I (PSI) and Photosystem II (PSII)
Molecular Remodeling of Photosystem I (PSI) and Photosystem II (PSII)
- Plants need to adjust the balance of energy distribution between PSI and PSII under different light conditions. This is managed by phosphorylation of light-harvesting complex II (LHCII), the antenna complex that collects light energy (Photosynthetic Adaptation)
- State 1: Energy is primarily directed to PSII.
- State 2: More energy is redistributed to PSI to balance the excitation energy between PSI and PSII.
Mechanism
- Unphosphorylated LHCIIs stabilize the PSII-LHCII megacomplex.
- Phosphorylation of LHCII trimer triggers dissociation of the megacomplex into discrete PSII-LHCII supercomplexes.
- Phosphorylation of other proteins in PSII (e.g., CP26, CP29, D2, CP43) displaces LHCIIs from PSII.
- Dissociated phosphorylated LHCII forms aggregates, releasing excess energy.
- Some LHCIIs re-associate with PSI, forming a PSI-LHCI/II supercomplex.
Function
- This redistribution of LHCII enhances PSI activity under conditions where PSII is saturated, preventing energy imbalance and protecting the photosynthetic machinery from damage.
Tetrapyrrole Pathway
Tetrapyrrole Pathway
- GUN4 regulates MgCh (Magnesium Chelatase) activity, a key enzyme in the pathway, enhancing the production of Mg-porphyrins, precursors for chlorophyll.
- In light, GUN4 activates MgCh and supports downstream conversion to chlorophyll via LPOR (Light-dependent Protochlorophyllide Oxidoreductase).
- In darkness, P-GUN4 adjusts MgCh activity to prevent overaccumulation of intermediates when light is unavailable.
- GUN4 phosphorylation ensures efficient chlorophyll production while preventing metabolic imbalances in varying light conditions.
Glycosylation of Proteins
PTM
Glycosylation of Proteins
- involves the addition of carbohydrate groups (glycans) to proteins.
Types of Glycosylation
- N-Glycosylation: Glycans linked to asparagine (Asn).
- O-Glycosylation: Glycans linked to serine (Ser) or threonine (Thr).
- C-Glycosylation: Glycans linked to tryptophan (Trp).
- Glypiation: Glycan core links a phospholipid and a protein.
- Phosphoglycosylation: Glycan binds to serine via a phosphodiester bond
Glycan assembly & Attachment
- Precursor Glycan Synthesis begins on the cytosolic face of the endoplasmic reticulum (ER).
- Structure is flipped into the ER lumen to complete assembly.
- Oligosaccharyltransferase (OSTase) transfers precursor glycan to the Asn residue of nascent proteins.
- Glycosylation efficiency depends on protein folding, as the accessibility of Asn residues determines whether they can be glycosylated.
- N-terminal residues are more glycosylated due to early folding and accessibility during ER transport, whereas folding reduces OSTase access to the C-terminal glycosylation consensus sequence.
Functions
- Assists protein folding and ensures quality control in the ER
- Plays a role in subcellular targeting, directing proteins to specific cell compartments
- Facilitates cell-cell recognition and signaling
