Caplan 1 Flashcards
Describe Anfinsen’s experiment which demonstrated the principle of self assembly:
Took a purified protein, denatured it, and upon removal of denaturants observed the polypeptide to refold.
However, physical conditions in cells unsuitable for refolding due to molecular crowding and relatively high temperatures, resulting in aggregation rather than folding.
Molecular chaperone proteins evolved to:
promote folding and prevent aggregation
The folding of a polypeptide chain is a complex process that is thought to occur via various folding intermediates
- The burst phase (0-5 ms) involves:
The burst phase (0-5 ms) involves formation of secondary structure and collapse of the hydrophobic core.
The folding of a polypeptide chain is a complex process that is thought to occur via various folding intermediates.
- Intermediate phase (5-100 ms)
Intermediate phase (5-100 ms) involves formation of a molten globule intermediate, which has characteristics of both folded and unfolded proteins.
The folding of a polypeptide chain is a complex process that is thought to occur via various folding intermediates.
- The final rate-limiting step
The attainment of native structure. The final transition is marked by conversion of the molten globule via global repacking of hydrophobic side chains and the association of domains that were folded independently in the intermediate stages.
Molecular chaperones bind to and stabilize an otherwise unstable conformer of another protein, and facilitate its correct fate in vivo:
Be it folding, oligomeric assembly, transport to a particular subcellular compartment, or controlled switching between active and inactive conformations.
Describe the different families of molecular chaperones – notably those belonging to the Hsp70, Hsp60 (chaperonin) and Hsp90 families
All bind to and release polypeptides in a manner dependent on ATP binding, hydrolysis and nucleotide exchange. These molecular chaperones function in association with many co-chaperone proteins that regulate the reaction cycle of polypeptide binding and release by the chaperone. These regulated cycles promote polypeptide folding.
Describe Bacterial Chaperonin’s structure and function:
a homo-oligomer of 14 subunits (each of 60 kDa) arranged into 2 stacked rings each of 7 subunits. Each ring is structured like a donut with a 7-fold axis and a chamber.A smaller lid structure, also comprising 7 subunits, sits on top of one of the barrels. Unfolded proteins (orange) bind to the rim of the barrel and are displaced into the cavity by the lid structure. The protein can then fold in a sequestered and protected environment of the chamber. The lid dissociates due to changes in the conformation of the large subunit as ATP is hydrolyzed.
An Adaptive Response to Stress: Heat Shock Transcription Factor (Hsf)
Molecular chaperone gene transcription is controlled by Hsf which responds to the presence of unfolded protein or heat shock or
The Ubiquitin/Proteasome Pathway:
Protein degradation in the cytosol and nucleus is largely accomplished by the proteasome, a large gated protease.Proteasome substrates are targeted via covalent linkage to multiple copies of ubiquitin, a 7 kDa protein.
Two Additional proteasome activities
Two additional activities have been described in mammalian proteasomes (cleaving after branched chain amino acids and between small neutral amino acids). The products are peptides in the 7-9 amino acid range.
Proteasome Structure
The proteasome comprises a central catalytic core (20S proteasome) and a regulatory cap (19S) that together are called the 26S proteasome. The 20S core of eukaryotes is comprised of 2 copies each of 14 different subunits, although these fall into two categories of alpha-type and ß-type. Access to the channel is via the tunnel formed at the ends of the -subunit rings. It is thought that a single unfolded polypeptide transits into the proteasome at one end and is degraded processively in the central chamber.
The three activities of eukaryotic proteasomes
- chymotrypsin-like (cleaves after hydrophobic amino acids)
- trypsin-like (cleaves after basic amino acids),
- peptidyl-glutamyl peptide hydrolyzing activity (cleaves after acidic amino acids).
The 19S regulatory complex contains subunits that:
recognize ubiquitinylated substrates, deubiquitinylate the substrates; and prepare them for proteolysis via protein unfolding if necessary. The 19S complex is comprised of at least 15 subunits, six of which are AAA-ATPases that can perform unfolding.
Proteasomes are targets for chemotherapy.
Velcade is especially useful for multiple myeloma
- Ubiquitin and Ubiquitinylation
Ubiquitin is a 76-amino-acid protein that becomes covalently attached to polypeptides that are substrates for degradation. Ubiquitin is linked in linear chains where the carboxyl end of the terminal glycine becomes covalently attached to the epsilon amino group of lysine 48. The attachment of ubiquitin to proteins requires the action of three different types of enzymes called E1, E2 and E3.
- Ubiquitin and Ubiquitinylation*
- E1* is an enzyme that
carries out ATP-dependent activation of the C-terminal glycine in a two-step reaction. First a ubiquitin-adenylate is formed, followed by transfer of activated ubiquitin to a thiol site in E1. The high-energy thiol bond is important for Ub transfer to E2 enzymes. There are only a small number of E1 enzymes
- Ubiquitin and Ubiquitinylation*
- E2*
Ubiquitin and UbiquitinylationThe E2 enzymes, or ubiquitin conjugating enzymes, accept the ubiquitin from E1 and transfer it to the protein substrate in a reaction that requires E3, the ubiquitin protein ligase.
Ubiquitin and Ubiquitinylation
E3
E3 enzymes, or ubiquitin ligases, are a large and diverse protein family that specifies substrate selection
Ubiquitin and Ubiquitinylation
The process of quality control
integrates molecular chaperone actions with those of the ubiquitin/proteasome system. This is important for the clearance of misfolded proteins, which appear to be identified as such by molecular chaperones and then targeted to specific ubiquitin ligases. Chief among these is CHIP (C-terminal Hsp interacting protein), which binds directly to Hsp70 and catalyzes ubiquitinylation of misfolded proteins
Protein Aggregation
Aggregates occur when misfolded proteins overwhelm the ubiquitin/proteasome pathway. Aggregates represent amorphous assemblies of misfolded proteins bound together via hydrophobic interactions or ordered assemblies of amyloid fibres. Both types of aggregates are inaccessible to the proteasome and must be cleared by the autophagic system (see lecture AC4 for more information on autophagy). Some aggregates are toxic and pathogenic while others appear to be protective of cell viability
Prions
Prions are proteins that form toxic amyloids that are transmissible. Several late onset diseases are characterized by the presence of ordered aggregates, as follows:
Several late onset human diseases are characterized by the presence of protein aggregates. These include:
- Amyloid diseases and Alzheimer’s
- Parkinson’s disease (PD) and Lewy Bodies
- polyglutamine repeat diseases
- Prion diseases
- Amyloid diseases and Alzheimer’s
Amyloid is a conformation of proteins that involves a stacked ß-sheet. Amyloids form fibres that are very stable (see figure above, which shows an electron micrograph of amyloid fibres, and a schematic in the figure below). Many different proteins form amyloids and they are associated with many different diseases, the most common are late-onset neurodegenerative states including Alzheimer’s.
Alzheimer’s disease (AD) is characterized by extracellular amyloids of a peptide called Aß that is cleaved from the Alzheimers precursor protein (APP). AD is also characterized by intracellular deposits, or neurofibrillary tangles, of the microtubule binding protein, Tau. The form of Tau that aggregates is hyperphosphorylated.
- Parkinson’s disease (PD) and Lewy Bodies
PD involves loss of dopaminergic neurons in the substantia nigra. The disease is associated with presence of Lewy Bodies that are aggregates enriched in a-synuclein.
- polyglutamine repeat diseases
Some proteins contain stretches of polyglutamine (poly-Q), e.g., Androgen Receptor. In some cases, the triplet encoding Q (CAG) expands during replication. In proteins where this expands beyond ~ 36 Q residues (AR normally has 21-22 Q residues). Currently, nine different proteins are known where expansion of their polyQ results in neurotoxicity. The following list shows the name of the disease followed by the proteins involved in parentheses:
Huntington’s (Huntingtin)
SMBA (Spinal Muscular Atrophy, Androgen Receptor)
DRPLA (Atrophin 1)
SCA 1, 2, 3, 6, 7, 17 (spinocerebellar ataxias: Ataxin 1,2 3,7,17 and Tata box binding protein)
- Prion diseases
Prions are transmissible amyloids and cause disease in humans and other mammals. Transmission can occur by eating contaminated food. Human prion diseases include Creutzfeldt-Jakob disease and fatal familial insomnia. All known prion diseases attack the brain.
