Protein Folding and Processing The classic principle of protein folding is that all the information...
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Transcript of Protein Folding and Processing The classic principle of protein folding is that all the information...
Protein Folding and Processing
The classic principle of protein folding is that all the information required for a protein to adopt the correct three-dimensional conformation is provided by its amino acid sequence.
Molecular chaperones are proteins that facilitate the folding of other proteins.
Two specific families of chaperone proteins act in a general pathway of protein folding in both prokaryotic and eukaryotic cells – Heat shock proteins and Chaperonins.
Unfolded polypeptide chains are shielded from the cytosol within the chamber of the chaperonin.
Action of chaperones during translation and Transport
chains that are still being translated on ribosomes, thereby preventing incorrect folding or aggregation of the amino-terminal portion of the polypeptide before synthesis of the chain is finished.
• Chaperones also stabilize unfolded polypeptide chains during their transport into subcellular organelles.
The role of N-linked glycosylation in ER protein folding.
3
The unfolded protein response in yeast
The export and degradation of misfolded ER proteins
Protein translocation
ENDOSITOSis
Protein folding in the cell
Basics- cell compartments, molecular crowding: cytosol, ER, etc.
Folding on the ribosome- co-translational protein folding
Molecular chaperones- concepts, introduction- intramolecular chaperones- chemical chaperones- protein chaperones
Folding in vitro vs. in vivo
folding by dilutionin buffer
protein denaturedin a chaotrope
foldedprotein
in vitro in vivo
folding
foldedprotein
Problem: non-native proteins
• non-native proteins expose hydrophobic residues that are normally buried within the ‘core’ of the protein
• these hydrophobic amino acids have a strong tendency to interact with other hydrophobic (apolar) residues
- especially under crowding conditions
intramolecular
misfolding
XX
XX
intermolecular
aggregation
XX
XX
XX
incorrectmolecular
interactions&
loss of activity
incorrectmolecular
interactions&
loss of activity
exposedhydrophobic
residues
3-10
Eukaryotes Archaea Bacteria- - Trigger Factor
NAC NAC -Hsp70 system [Hsp70 system] Hsp70 system
prefoldin prefoldin -chaperonins (group II) chaperonins (group II) chaperonins (Group I)
small Hsps small Hsps [small Hsps]Hsp90 - [Hsp90]
AAA ATPases AAA ATPases AAA ATPases- - SecB- - [PapD/FimC]
Hip, Hop, Bag, clusterin, cofactors A-E, calnexin,
calreticulin, etc. etc.- -
Overview of chaperone families:Distribution
IRE-1
XBP-1
The Unfolded Protein Response (UPR)
• The UPR occurs when proteins are misfolded in the endoplasmic reticulum (ER).
• Reducing agents, such as DTT, interfere with disulfide bond formation while drugs can interfere with glycosylation; both agents cause proteins to misfold in the ER thus triggering the UPR.
• The product of the ire-1 gene is the sensor of misfolded proteins and when activated removes an intron from the pre mRNA from the xbp-1 gene.
• Active xbp-1 protein (from spliced mRNA) activates the genes that code for ER chaperones, such as hsp-4.
Hsp4 (grp78)grp170
PROTEIN TURNOVER AND AMINO ACID CATABOLISM
Degradation of proteins
1) dietary proteins- amino acids- pepsin in stomach- pancreatic proteases- aminopeptidase N- other peptidases
2) endogenous proteins- protein turnover: synthesis, degradation, resynthesis- damaged proteins- half-lives of proteins: depend on amino-terminal residues
Cellular Protein Degradation
• Lysosomal• Nonspecific• Endocytosis• Foreign proteins• Energy favorable to degrade proteins
• Non-lysosomal• Specificity, requires ATP• Conditions of stress• Ubiquitin-proteosomal pathway• 26S proteosome• Role in cellular processes/signaling
Protein turnover; selective degradation/cleavage Individual cellular proteins turn over (are degraded and re-synthesized) at different rates. E.g., half-lives of selected enzymes of rat liver cells range from 0.2 to 150 hours. N-end rule: On average, a protein's half-life correlates with its N-terminal residue. Proteins with N-terminal Met, Ser, Ala, Thr, Val, or
Gly have half lives greater than 20 hours. Proteins with N-terminal Phe, Leu, Asp, Lys, or Arg
have half lives of 3 min or less.PEST proteins having domains rich in Pro (P), Glu (E), Ser (S), Thr (T), are more rapidly degraded than other proteins.
Ubiquitinylation – Proteosome Degradation
E3 determines protein substrate
8.42 The ubiquitin-proteasome pathway
Ubiquitination1) ubiquitin- a 8.5 kd protein (76 residues)- formation of an isopeptide bond with ε-amino group of lysine of the proteins - a tag for destruction - polyubiquitin: a strong signal for degradation 2) enzymes for ubiquitination- E1 (ubiquitin-activating enzyme) - E2 (ubiquitin-conjugating enzyme)- E3 (ubiquitin-protein ligase)- variation: E3 > E2 > E1: more finely tuned substrate discrimination- HPV (human papilloma virus) activates a specific E3 enzyme: tumor suppressor protein p53
Regulation of ubiquitination: Some proteins regulate or facilitate ubiquitin conjugation. Regulation by phosphorylation of some target proteins has been observed. E.g., phosphorylation of PEST domains activates ubiquitination of proteins rich in the PEST amino acids. Glycosylation of some PEST proteins with GlcNAc has the opposite effect, prolonging half-life of these proteins.
19S and 20S Proteasome Subunits Characteristics
20S Subunit Barrel Contains 6
proteolytic sites 2x Tryptic 2x Chymotryptic 2x Peptidylglutamyl-
peptidase
Linearized protein required
19S Subunit Base and Lid Contains subunits with
known and unknown functions Tetra-Ub (K48)
recognition Deubiquitination activity Protein unfolding activity
(Chaperone function)
Ubiquitin AA Sequence
MQIFVKTLTG KTITLEVEPS DTIENVKAKI
QDKEGIPPDQ QRLIFAGKQL EDGRTLSDYN
IQKESTLHLV LRLRGG
48
63
6
Proteasome-1
Proteasome-3
Proteasome-4
Roles of Ubiquitination
Different Types of Ubiquitin Tags
Transmembrane Proteins Regulated by Ub-dependent Sorting
In metazoans:Neurotransmission: Ion channels:AMPA glutamate receptors ENaCGlycine receptors ClC-5
Cell-cell contacts: Immune molecules E-cadherin downregulated by
viruses:Occludin MHC class I
B7-2Developmental patterning: ICAM-1Delta CD4NotchRoundabout
Poly-Ub Chains
Ub
K
K
Ub
UbUb
Ub
UbUb
Ub
Ub
K48 Linkage
K63 Linkage
K63
K48
Peters, J.M. 1998Ubiquitin and the Biology of the Cell
Signal to proteosome
K48, Ub4
Cell Signaling
K63
ENaC function
• Major ion channel that controls salt and fluid resorption in the kidney
• Mutations in the PPXY motif cause accumulations of channels at the cell surface and result in Liddle’s syndrome, and inherited form of hypertension
ENac surface Stability
• Nedd 4 (HECT ligase)-negatively regulates ENaC surface stability
• Nedd4 WW domains bind PPXY motif of ENaC subunits
• Nedd4 also interacts with serum and glucocorticoid-regulated kinase
(SGK)• SGK contains two PPXY motifs that bind to Nedd4 WW domains
• SGK-dependent Nedd4 P inhibits the Nedd4-ENaC interaction
• therefore, Nedd4 P increases ENaC at the cell surface
ENaC Subunits
Regulation of ENaC Surface Stability
Ub-like Proteins
SUMO-1 (sentrin, smt-3) 1996 – covalent modification – RanGAP1 RanGAP1 nearly quantitative modified Cytosolic RanGAP1 to nuclear pore Activate shuttling factor
Ubiquitin-like Proteins:
Ubiquitin Superfold and Ubiquitons
Ub – blueSUMO-1 – greenNEDD8 - red
UB αβ roll suprfold
SUMO SUMO
SUMO-1 & SUMO-2/3
Shared characteristics C-terminal -GG essential for conjugation Affix to lysine residues in target
NOT directly associated with proteasomal degradation
Competition/RegulationSUMO
Reactive Oxygen Species: Oxidizes reactive thiols on SUMO enzymes
Uba1/Aos1- S – S – Ubc9
Thus: SUMO can not attach and proteins not Sumoylated
Examples of SUMO function
RanGAP
IkB
c-Jun
p53 and mdm2
Causes nuclear translocation
Blocks Ub-conjugation site, prevents degradation
Inhibits transcriptional activity
Blocks mdm2 self-ubiquitination, prevents degradation
SUMO-p53 in DNA binding domain apoptotic activity
PROTEIN SUMO Effect
Peptide generation in the class I pathway
Proteasome specificity• NetChop is the best available cleavage
method www.cbs.dtu.dk/services/NetChop-3.0