Protein Folding and Processing - PowerPoint Presentation

Slide1

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.Slide2

Action

of chaperones during translation and Transportchains 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.Slide3

The role of N-linked glycosylation in ER protein folding.

3Slide4

The unfolded protein response in yeast Slide5

The export and degradation of misfolded ER proteins Slide6

Protein translocationSlide7

ENDOSITOSisSlide8

Protein folding in the

cellBasics- cell compartments, molecular crowding: cytosol, ER, etc.Folding on the ribosome- co-translational protein foldingMolecular chaperones- concepts, introduction- intramolecular chaperones- chemical chaperones- protein chaperonesSlide9

Folding

in vitro vs. in vivo

folding by dilution

in buffer

protein denatured

in a chaotrope

folded

protein

in vitro

in vivo

folding

folded

proteinSlide10

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 conditionsintramolecular

misfolding

X

X

X

X

intermolecular

aggregation

X

X

X

X

X

X

incorrect

molecular

interactions

&

loss of activity

exposed

hydrophobic

residues

3-10Slide11

Eukaryotes

ArchaeaBacteria

-

-

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:

DistributionSlide12

IRE-1

XBP-1The 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)

grp170Slide13
Slide14

PROTEIN TURNOVER AND AMINO ACID CATABOLISM

Degradation of proteins1) dietary proteins- amino acids- pepsin in stomach- pancreatic proteases- aminopeptidase Nother peptidases2) endogenous proteins

- protein turnover: synthesis, degradation,

resynthesis

- damaged proteins

- half-lives of proteins: depend on amino-terminal residuesSlide15

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/signalingSlide16

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. Slide17

Ubiquitinylation – Proteosome Degradation

E3 determines protein substrateSlide18

8.42 The

ubiquitin-proteasome pathwaySlide19
Slide20
Slide21

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 p53Slide22

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. Slide23

19S and 20S

Proteasome Subunits Characteristics20S SubunitBarrelContains 6 proteolytic sites2x Tryptic2x Chymotryptic2x 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)Slide24

Ubiquitin AA Sequence

MQIFVKTLTG KTITLEVEPS DTIENVKAKI QDKEGIPPDQ QRLIFAG

K

QL EDGRTLSDYN

IQ

K

ESTLHLV LRLR

GG

48

63

6Slide25
Slide26
Slide27

Proteasome-1

Proteasome-3Proteasome-4Slide28

Roles of UbiquitinationSlide29

Different Types of Ubiquitin TagsSlide30

Transmembrane

Proteins Regulated by Ub-dependent SortingIn metazoans: Neurotransmission: Ion channels: AMPA glutamate receptors ENaC Glycine receptors ClC-5 Cell-cell contacts: Immune molecules

E-cadherin

downregulated

by viruses:

Occludin

MHC class I

B7-2

Developmental patterning:

ICAM-1

Delta CD4

Notch

RoundaboutSlide31

Poly-Ub Chains

Ub K K

Ub

Ub

Ub

Ub

Ub

Ub

Ub

Ub

K48 Linkage

K63 Linkage

K63

K48

Peters, J.M. 1998

Ubiquitin and the Biology of the Cell

Signal to proteosome

K48, Ub

4

Cell Signaling

K63Slide32

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 hypertensionSlide33

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 surfaceSlide34

ENaC

SubunitsSlide35

Regulation of ENaC Surface StabilitySlide36

Ub

-like ProteinsSUMO-1 (sentrin, smt-3)1996 – covalent modification – RanGAP1RanGAP1 nearly quantitative modifiedCytosolic RanGAP1 to nuclear poreActivate shuttling factorSlide37

Ubiquitin-like Proteins:Slide38

Ubiquitin Superfold and Ubiquitons

Ub – blueSUMO-1 – greenNEDD8 - redUB αβ roll suprfoldSlide39

SUMO

SUMOSUMO-1 & SUMO-2/3Shared characteristicsC-terminal -GG essential for conjugationAffix to lysine residues in targetNOT directly associated with proteasomal degradationSlide40

Competition/Regulation

SUMOReactive Oxygen Species: Oxidizes reactive thiols on SUMO enzymesUba1/Aos1- S – S – Ubc9Thus: SUMO can not attach and proteins not SumoylatedSlide41

Examples of SUMO function

RanGAPIkBc-Junp53 and mdm2Causes nuclear translocationBlocks Ub-conjugation site, prevents degradationInhibits transcriptional activityBlocks mdm2 self-ubiquitination, prevents degradationSUMO-p53 in DNA binding domain   apoptotic activity

PROTEIN

SUMO EffectSlide42

Peptide generation in the class I pathwaySlide43

Proteasome specificity

NetChop is the best available cleavage methodwww.cbs.dtu.dk/services/NetChop-3.0