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

misfoldingXX

XX

intermolecular

aggregation

XX

XX

XX

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 DegradationE3 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 LRLRGG48

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 enzymesUba1/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