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
in vitro
in vivo
protein denatured
in a chaotrope
folding by dilution
in buffer
folded
protein
folding
folded
protein
3-10
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
exposed X
hydrophobic
X
residues
intramolecular
X
X
misfolding
incorrect
molecular
interactions
&
loss of activity
X
X
intermolecular
XX X
X
aggregation
Overview of chaperone families:
Distribution
Eukaryotes
Archaea
NAC
NAC
Hsp70 system
[Hsp70 system]
prefoldin
prefoldin
chaperonins (group II) chaperonins (group II)
small Hsps
small Hsps
Hsp90
AAA ATPases
AAA ATPases
Hip, Hop, Bag, clusterin,
cofactors A-E, calnexin,
calreticulin, etc. etc.
Bacteria
Trigger Factor
Hsp70 system
chaperonins (Group I)
[small Hsps]
[Hsp90]
AAA ATPases
SecB
[PapD/FimC]
-
The Unfolded Protein Response (UPR)
Hsp4 (grp78)
grp170
XBP-1
IRE-1
• 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.
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 resynthesized) 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 Nterminal 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
Ubiquitination
1) 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

19S Subunit


Base and Lid
Contains subunits with
known and unknown
functions



Tetra-Ub (K48)
recognition
Deubiquitination activity
Protein unfolding activity
(Chaperone function)

20S Subunit


Barrel
Contains 6 proteolytic
sites




2x Tryptic
2x Chymotryptic
2x Peptidylglutamylpeptidase
Linearized protein
required
Ubiquitin AA Sequence
MQIFVKTLTG KTITLEVEPS DTIENVKAKI
6
QDKEGIPPDQ QRLIFAG
KQL EDGRTLSDYN
48
IQ
KESTLHLV LRLRGG
63
Proteasome-1
Proteasome-3
Proteasome-4
Roles of Ubiquitination
Different Types of Ubiquitin Tags
Transmembrane Proteins Regulated by Ub-dependent Sorting
In metazoans:
Neurotransmission:
AMPA glutamate receptors
Glycine receptors
Cell-cell contacts:
E-cadherin
viruses:
Occludin
Developmental patterning:
Delta
Notch
Roundabout
Ion channels:
ENaC
ClC-5
Immune molecules
downregulated by
MHC class I
B7-2
ICAM-1
CD4
Poly-Ub Chains
K48 Linkage
K
K63
Signal to
proteosome
K48, Ub4
K63 Linkage
K48
K
Ub
Ub
Ub
Ub
Ub
Cell Signaling
K63
Peters, J.M. 1998
Ubiquitin and the Biology of the Cell
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 αβ roll suprfold
Ub – blue
SUMO-1 – green
NEDD8 - red
SUMO

SUMO


Shared characteristics



SUMO-1 & SUMO-2/3
C-terminal -GG essential for conjugation
Affix to lysine residues in target
NOT directly associated with proteasomal
degradation
Competition/Regulation
SUMO
Reactive Oxygen Species: Oxidizes reactive thiols on SUMO
Uba1/Aos1- S – S – Ubc9
Thus: SUMO can not attach and proteins not Sumoylated
Examples of SUMO function
SUMO Effect
PROTEIN

RanGAP

Causes nuclear translocation

IkB

Blocks Ub-conjugation site, prevents
degradation

c-Jun

Inhibits transcriptional activity

p53 and mdm2

Blocks mdm2 self-ubiquitination, prevents degradation

SUMO-p53 in DNA binding domain   apoptotic activity
Peptide generation in the class I pathway
•
NetChop is the best available cleavage method
 www.cbs.dtu.dk/services/NetChop-3.0
Proteasome
specificity