Detailed Notes on Cytosolic Protein Modification and Chaperones

Cytosolic Events: Protein Modification and Chaperones

Introduction

  • Many eukaryotic proteins must be sorted to different intracellular destinations.
  • Key questions include:
    • How does a protein reach its correct location?
    • How does it achieve a folded conformation upon arrival?
    • How is the protein activated?
    • How is a damaged or function-completed protein removed?
  • These questions are addressed through protein modification.

Protein Synthesis in the Cytosol

  • Most protein synthesis in eukaryotic cells begins on free cytosolic ribosomes, with exceptions for mitochondrial and plastid translation.
  • There's a common pool of ribosomal subunits in the cytosol.
  • These subunits assemble ribosomes on mRNAs encoding cytosolic proteins, which remain free in the cytosol.
  • Multiple ribosomes assemble, forming free cytosolic polyribosomes (polysomes).
  • In eukaryotic cells, most protein synthesis occurs in the cytosol.

Macromolecular Crowding

  • The cytoplasm in a eukaryotic cell is a crowded environment.
  • The sizes, shapes, and concentrations of macromolecules are significant, while small molecules are not shown.
  • Crowding effects are substantial; reaction rates and equilibria in test tubes can differ significantly from those within cells.
  • Ellis RJ (2001). "Macromolecular crowding: obvious but underappreciated". Trends Biochem. Sci. 26 (10): 597–604.

Nascent Proteins and Aggregation

  • Newly synthesized proteins are in a non-native, aggregation-prone conformation.
  • Multiple ribosomes translate a single mRNA simultaneously, leading to nascent proteins being in close proximity.
  • This proximity increases the risk of aggregation.

Molecular Chaperones

  • Molecular chaperones are cellular proteins that ensure proper polypeptide folding.
  • Unfolded and misfolded proteins are protease-sensitive, non-functional, and prone to aggregation.
  • Folded proteins are stable, resistant to proteases, and functional.
  • Ellis RJ. (1987) Proteins as molecular chaperones. Nature;328:378-9.

Hsc70 and Hsp40 Chaperone System

  1. Hsp40 Recognition: Hydrophobic patches on nascent/unfolded proteins are recognized by Heat shock protein 40 family members (Hsp40 co-chaperone).
  2. Substrate Delivery: Hsp40 delivers the substrate to ATP-bound (OPEN conformation) Heat shock cognate protein 70 (Hsc70 chaperone) and stimulate its ATPase activity.
  3. Hsc70 Shielding: ATP hydrolysis results in ADP-bound (CLOSED conformation) Hsc70 shielding the hydrophobic patches of the substrate, preventing aggregation, and allowing time for the hydrophilic parts of the substrate to fold.
  4. Substrate Release: Upon nucleotide exchange, Hsc70 adopts its open conformation, releasing the substrate, which may then snap into its final conformation.
  • For many proteins, chaperones are necessary for correct folding.

Studying Chaperone Interactions

  • In vitro recapitulation is used to study chaperone interactions.
  • Heating a target protein (PrT) at, for example, 45^\circ C for 15 minutes leads to aggregation.
  • Heating in the presence of Hsp40 increases the soluble fraction: heating in the presence of Hsc70 has a larger effect and Hsp40 and Hsc70 together have even more effect.
  • Maximal solubilization requires Hsp40, Hsc70, and ATP.

Hsc70 and Client Protein Fate

  • Hsc70 shields the hydrophobic regions of its clients, reducing the aggregation of nascent or unfolded proteins.
  • A partially-folded Hsc70 client protein may:
    • Be released and find its stable conformation.
    • Be passed on to other chaperones for further folding and/or assembly into multimeric complexes.
    • Be transported to a lysosome (e.g., via autophagy).
    • Be passed to proteasomes for degradation.
  • Both productive (FOLDING, ACTIVATION) and non-productive (DESTRUCTION, INACTIVATION) fates can occur from an Hsc70-bound state.

Regulation of Hsc70 Client Release

  • A nucleotide exchange factor (NEF) binds the Hsc70:client complex and removes ADP from the nucleotide-binding site of Hsc70.
  • This promotes nucleotide exchange, allowing ATP to enter the nucleotide-binding site of Hsc70.
  • Hsc70:ATP adopts an OPEN conformation, releasing the client protein.
  • Examples of NEFs for Hsc70 include BAG-1, BAG-2, and HSPBP1.
  • Other Hsc70 co-chaperones include Hsp40 family members and CHIP (E3 ubiquitin ligase).

Further Chaperone Interactions

  • After Hsc70, client proteins may interact with:
    • Hsp90 dimer, providing a platform for further protein folding and assembly of multimeric complexes.
    • Chaperonins (14, 16, or 18 x Hsc60, depending on species), providing a cage that isolates small (<70 kDa) folding proteins (e.g., tubulin, actin) from the cytosol, with a residence time of ~10 seconds.

Determination of Protein Fate

  • There is an interacting, competing network of co-chaperones that determines the fate of a chaperone client.
  • The prevailing concentrations of Hsc70 and Hsp90 co-chaperones determine the proportion of an unfolded/misfolded protein that can gain a stable conformation versus the proportion that is destroyed.
  • Co-chaperones make decisions by competing to release chaperone clients.
  • The fate of chaperone client proteins is not pre-determined.
  • Spooner et al., (2008) Cytosolic chaperones influence the fate of a toxin dislocated from the endoplasmic reticulum. Proc Natl Acad Sci USA;105:17408-13.
  • Examples:
    • HOP transfers clients from Hsc70 to Hsp90.
    • BAG-1 releases Hsc70 clients at the proteasome, favoring destruction.
    • BAG-2 releases clients away from the proteasome, favoring folding.
    • HIP competes with NEFs, maintaining the Hsc70: client interaction.
    • Hsp40 co-chaperones show substrate specificity.

Hsc70 as Disaggregase

  • Some Hsc70 co-chaperones convert it into a disaggregase.
  • Hsc70 in the presence of the Hsp40 DNAJB1 and a NEF Apg2 can disaggregate Tau, αSyn and huntingtin exon 1 amyloid fibres
  • Beton et al (2022). Cooperative amyloid fibre binding and disassembly by the Hsp70 disaggregase. EMBO J;41(16):e110410.

Summary of Cytosolic Molecular Chaperones

  • Cytosolic molecular chaperones can:
    • Prevent aggregation of unfolded proteins: e.g., Hsc70 binds hydrophobic regions of a client, delaying folding of these regions until the hydrophilic parts of the target protein have gained structure.
    • Provide a controlled environment for folding: e.g., chaperonins form a cage that encloses the target protein, allowing folding in a protected environment away from the cytosol; they may even aid folding directly.
    • Permit assembly and disassembly of multimeric complexes: e.g., histone complexes, clathrin cages, α-synuclein fibres, etc.
    • Direct proteins with folding problems for destruction: e.g., the Hsc70 co-chaperone BAG-1 can engage a Hsc70:client complex with the proteasome and the lysosome.

Engaging the Proteasome

Architecture of the Proteasome

  • Proteasomes are abundant in the cytosol and nucleoplasm (approximately 30,000; constitute 1-2% of total cellular protein).
  • The 20S core particles are cylinders with three proteolytic activities: chymotrypsin-like, trypsin-like, and peptidylglutamyl-peptide hydrolyzing (caspase-like).
  • The active sites are inside the barrel, encoded by the β subunits.
  • Each 20S core has 19S caps, regulatory particles (RP), at one or both ends.

Targeting Proteins to the Proteasome

  • Ubiquitin (Ub) is a conserved 76 amino acid protein found in all eukaryotic cells.
  • Cytosolic proteins destined for proteasomal degradation are usually marked for destruction by covalent addition of a chain of Ub molecules (polyubiquitylation), allowing them to be bound by the 19S RP.
  • A chain of four Ub proteins signifies a doomed protein: tetra-Ub is a degradation signal.
  • Monoubiquitinated receptors are targeted to lysosomes instead.

Ubiquitination Process Step-by-Step

  1. Activation: Ub is activated by an E1 ubiquitin-activating enzyme.
  2. Conjugation: Activated Ub is transferred to an E2 ubiquitin-conjugating enzyme.
  3. Ligation: The E2-Ub conjugate associates with an E3 ubiquitin ligase.
  4. Binding: The E3-E2-Ub conjugate binds the target protein.
  5. Transfer: Ub is transferred to the target protein.

Specificity of E1s, E2s, and E3s

  • There are approximately 9 E1 enzymes in mammalian cells, which are vital enzymes.
  • There are over 30 E2 enzymes. Each can select their own E3s, providing some substrate specificity.
  • There are hundreds of different E3 ligases. Each type selects its target proteins by recognizing some specific feature, such as:
    • Extended residence in a chaperone system
    • Their N-terminus
    • Misfolded regions
    • Exposure of a degradation signal
  • E3s effectively control the stability of proteins involved in key cellular processes, including timing key transitions in the cell cycle, circadian rhythms, development, signaling, and immunity.

Ubiquitin Attachment and Chain Formation

  • Ubiquitin is normally added covalently to the side chain of an available lysine residue on the target molecule.
  • The process is repeated using side chains of lysine residues in ubiquitin until a chain of at least 4 Ub is completed.
  • This multi-ubiquitin (4 or more) chain is a degradation signal.
  • Polyubiquitylated proteins can be bound by the proteasomal 19S RP.

Targeting the Proteasome and Destruction: Step-by-Step

  1. Polyubiquitylated proteins bind to the 19S regulatory particle of the proteasome.
  2. The RP uses ATP to generate energy to unfold the target protein and feed it into the 20S core. Deubiquitylases (DUBs) remove Ub molecules and return them to a common pool for recycling.
  3. Three proteolytic activities are encoded by the β subunits of the 20S core.
  4. The target protein is degraded into small peptides (typically 7–9 amino-acid residues long, though they can range from 4 to 25 residues), which are ejected from the proteasome.

UPS Summary

  1. Ubiquitin (Ub) is activated by an E1 ubiquitin-activating enzyme.
  2. Ub is transferred to an E2 ubiquitin-conjugating enzyme.
  3. Ub-conjugated E2 selects an E3 ubiquitin ligase.
  4. The E3-E2-Ub complex selects its target and transfers Ub to a lysine residue on the target protein (T).
  5. The process is repeated until T is tetra-ubiquitylated.
  6. The Ub-Target protein binds the proteasome RP and is degraded in the core; deubiquitylases (DUBs) recycle Ub.

The Proteasome's Unexpected Role

  • Pietroni et al., (2013) The proteasome cap RPT5/Rpt5p subunit prevents aggregation of unfolded ricin A chain. Biochem J, 453:435-45.
  • The proteasome is not just a destructive machine; it has a fail-safe mechanism.
  • One of the RP subunits acts as a chaperone that directs some clients for destruction and allows re-folding of others back to their native conformation.

Consequences of UPS Failure

  • The UPS (ubiquitin-proteasome system) is relevant for proteins that fail to fold correctly, normal turnover of cytosolic proteins, proteins whose concentrations must change rapidly (e.g., cyclins), viral proteins, and misfolded proteins ejected from the ER.
  • When proteasomes or E3s fail, proteins that would normally be destroyed accumulate instead.
  • This can lead to the formation of aggregates, e.g., in neurons of people with Parkinson’s and Alzheimer’s diseases.
  • If cell cycle proteins are not degraded properly, it can lead to cell proliferation (as in cancer).
  • Overactive proteasomes have been implicated in autoimmune diseases including systemic lupus erythematosus and rheumatoid arthritis.

Other Cytosolic Post-Translational Modifications

1. Proteolytic Cleavage

  • Example: Activation of effector proteases of apoptosis by proteolytic cleavage and subunit rearrangement.

2. Addition of Lipids

  • Permits membrane targeting.
  • Many regulatory proteins (e.g., Rabs that regulate membrane traffic) are modified by the addition of lipids.
  • Rabs are doubly prenylated (prenylated lipids are either a 15C farnesyl or a 20C geranylgeranyl).
  • When the prenyl groups are masked by GDI (GDP dissociation inhibitor), Rab-GDP is cytosolic.
  • Following nucleotide exchange, GDI dissociates, and the prenyl groups of Rab-GTP enter the target membrane.

3. Phosphorylation

  • Addition/removal of phosphates can alter the activity of a protein; phosphates can activate or inactivate.
  • Example: Control of CDK activation.

Studying Phosphorylation

  • In vitro recapitulation.
  • SCH772984 is an inhibitor of ERK. Nuclear entry of cyclin B1 requires phosphorylation by ERK.
  • U0126 is an inhibitor of MEK, a MAP kinase kinase. The Ras-Raf-Mek-Erk pathway.

Complex Phosphorylation: Example of p53

  • Phosphorylated p53 protein tetramerizes and binds DNA, where it acts as a trans-activator of a huge number of genes.
  • Its regulation by kinases that respond to stress (u.v. light, heat, osmotic shock, DNA damage, hypoxia) is highly complex, with 24 phosphorylation sites known.

4. ADP Ribosylation

  • Addition of one or more ADP-ribose residues to a protein (can occur in prokaryotes as well as eukaryotes).
  • ADP-ribosylated proteins have roles in cell signalling, DNA repair, and apoptosis.
  • Some bacterial toxins interfere with these processes.
    • Cholera toxin is an ADP-ribosylase, targeting G proteins and interfering with signalling.
    • Diphtheria toxin is an ADP-ribosylase that targets EF-2 (elongation factor 2) and interferes with protein synthesis.

5. Methylation

  • Protein methylation typically takes place on arginine (R) or lysine (K) residues in the protein sequence.
  • Histone methylation is catalyzed by histone methyltransferases.
  • Methylated histones can act epigenetically to repress or activate gene expression.