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Lecture 3

BIOC 212 Lecture Notes - Lecture 3: Ribonucleoprotein, Glycophorin, Asparagine


Department
Biochemistry
Course Code
BIOC 212
Professor
Jason Young
Lecture
3

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BIOC 212/Winter 2011/Lecture 3
- glucocorticoid receptors belong to a family of steroid hormone receptors; respond to hormones by
activating/repressing genes in an inflammatory reaction
- ligand binding domain recognizes hormones; DNA-binding domain binds to GR promoter elements GRE -->
N-terminal activation domain regulated transcription of genes
TPR Domain Co-Chaperones
- usually bind only Hsp90; are adaptors that connect chaperones to different protein complexes/locations
FKBP52 --> Pplase, steroid receptor chaperones
Tom40 --> mitochondrial import
CHIP --> ubiquitin ligase
GR LBD – hydrophobic steroid is bound in the interior of the LBD (ligand-binding domain); necessary to
maintain native state; without corticosteroids, LBD can't fold stably
- chaperones keep LBD partially folded so it can bind hormones; other domains of GR remain folded
Sequential Action of Chaperones – LBD is folded by a defined sequence of chaperones:
Hsp40/Hsc70 --> Hsc70/Hsp90 --> Hsp90/FKBP52/p23
- without hormone, GR continues to cycle through the system; with hormone GR becomes a stable dimer
HSFI – is a transcription factor, regulator of the heat shock response
- has DNA-binding, transcription activation and trimerization domains
- inactive HSFI is a monomer; active it is a trimer and recognizes HSE (heat shock element) promoters
Heat Shock Response – heat shock followed by a recovery period triggers a typical response:
1. Translation is inhibited immediately and recovers approximately 1hr after HS
2. Transcription of Hsp is upregulated for 6-12 hours; other proteins are down-regulated
3. Approximately 24 hours later, everything returns to normal
Regulation of HSF – inactive HSF1 monomer mimics unfolded protein --> bound by chaperone Hsp90
- after HS, Hsp90 binds unfolded proteins and HSFI is free to trimerize and activate transcription
- phosphorylation of HSF1 further controls activation
- HSF1 is down-regulated by binding of excess chaperones to the monomer form

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Protein Degradation – ubiquitin-mediated protein degradation by proteasomes in the cytosol
- ubiqutin is a small 8kDa protein, covalently bonded to lysine sidechains of itself and proteins
- polyubiquitin chains target a protein for proteasome degradation but not ubiquitin itself
Ubiquitation Enzymes: E1 – ubiquitinal enzyme attaches Ub in a chemically reactive state on U2
E2 – ubiquitin conjugating enzyme transfers Ub to substrate
E3 – ubiquitin ligase provides substrate specificity of ubiquitination
- there are many E3, fewer E1 and E2 --> they control degradation
- Ub C-terminus carboxyl group is covalently linked to lysine sidechains via an isopeptide bond
- substrate can have multiple ubiquitination sites (many, but not ALL lysine, depends on accessibility)
Ubiquitin Linkages – can be linked to either itself or lysine 48 or 63
- polyubiquitin chains (K48 linkage) target proten for proteasome degradation
- K63 linkages activate different signals; poly/monoubiquitin
Monoubiquitination – histone regulation
Multiubiquitination - endocytosis
Polyubiquitination --> K48 linkage: proteasomal degradation
K63 linkage: DNA repair
Proteasome Subunits Core: 2 outer rings of 2 similar alpha subunits, 2 inner rings of similar beta units
- beta subunits have protease activity on their inside surface
- a 19s cap attaches to the outer rings:
- base with 6 AAA-family ATPase subunits --> protein “unfoldase”
- has a lid with non-ATPase subunits, poly-Ub receptors, Ub hydrolases
AAA family – ATP-dependent proteins with many functions
- the proteasome is large, has a central 20s cyliner and 19s caps
- cylinder and caps = 26s proteasome, ~2.5MDa, performs general degradation in cytosol, nucleus, ER
Proteasome Function – lid recognizes poly-Ub chain; Ub hydrolases remove poly-Ub and pass it to base
- base units use ATPase activity to unfold substrate and feed it into the 20s core
- 3 proteolytic subunits inside the core cleave substrate into fragments at basic/acidic/hydrophobic sites

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BIOC 212/Winter 2011/Lecture 4
Lecture 4: Protein Folding 4.
Jan 12, 2011 by Dr. Jason Young
Protein Modifications
Amino acids, building blocks of proteins, are different by their side chains, which can be covalently
modified for various cellular functions. Those modifications include
Phosphorylation (on tyrosine, for example)
Acetylation (on lysine or arganine)
Ubiquitination (usually on lysine 48, be discussed later)
Methylation (arganine, for example)
Lipidation
Modifications can change surface or conformation of protein:
Modifications can be used as regulation of proteins
Enzymes that put those modification on are turn on/off by different signals
In some cases, a modification creates or blocks a binding site for other proteins
ex: ubiquitination creates binding site for more ubiquitin moieties
many modifications are regulated and reversible.
Modifications on Amino acids
Amino acids modifications
Lys (reversible modifications; on the primary amine)
Acetylation
Methylation (mono, di, tri)
ubiquitination
Arg (reversible)
methyation (mono or di)
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