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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.

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Presentation on theme: "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."— Presentation transcript:

1 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.

2 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.

3 The role of N -linked glycosylation in ER protein folding. 3

4 The unfolded protein response in yeast

5 The export and degradation of misfolded ER proteins

6 Protein translocation

7 ENDOSITOSis

8 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

9 Folding in vitro vs. in vivo folding by dilution in buffer protein denatured in a chaotrope folded protein in vitroin vivo folding folded protein

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 intramolecular misfolding X X X X intermolecular aggregation X X X X X X incorrect molecular interactions & loss of activity incorrect molecular interactions & loss of activity exposed hydrophobic residues 3-10

11 EukaryotesArchaeaBacteria --Trigger Factor NAC - Hsp70 system[Hsp70 system]Hsp70 system prefoldin - chaperonins (group II) chaperonins (Group I) small Hsps [small Hsps] Hsp90-[Hsp90] AAA ATPases --SecB --[PapD/FimC] Hip, Hop, Bag, clusterin, cofactors A-E, calnexin, calreticulin, etc. etc. -- Overview of chaperone families: Distribution

12 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

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14 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

15 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

16 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.

17 Ubiquitinylation – Proteosome Degradation E3 determines protein substrate

18 8.42 The ubiquitin-proteasome pathway

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21 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

22 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.

23 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)

24 Ubiquitin AA Sequence MQIFV K TLTG KTITLEVEPS DTIENVKA K I QDKEGIPPDQ QRLIFAG K QL EDGRTLSDYN IQ K ESTLHLV LRLR GG 48 63 6

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27 Proteasome-1 Proteasome-3 Proteasome-4

28 Roles of Ubiquitination

29 Different Types of Ubiquitin Tags

30 Transmembrane Proteins Regulated by Ub-dependent Sorting In metazoans: Neurotransmission:Ion channels: AMPA glutamate receptorsENaC Glycine receptorsClC-5 Cell-cell contacts:Immune molecules E-cadherindownregulated by viruses: OccludinMHC class I B7-2 Developmental patterning:ICAM-1 DeltaCD4 Notch Roundabout

31 Poly-Ub Chains Ub K K 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 K63

32 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

33 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

34 ENaC Subunits

35 Regulation of ENaC Surface Stability

36 Ub-like Proteins  SUMO-1 (sentrin, smt-3)  1996 – covalent modification – RanGAP1  RanGAP1 nearly quantitative modified  Cytosolic RanGAP1 to nuclear pore  Activate shuttling factor

37 Ubiquitin-like Proteins:

38 Ubiquitin Superfold and Ubiquitons Ub – blue SUMO-1 – green NEDD8 - red UB αβ roll suprfold

39 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

40 Competition/Regulation SUMO Reactive Oxygen Species: Oxidizes reactive thiols on SUMO enzymes Uba1/Aos1- S – S – Ubc9 Thus: SUMO can not attach and proteins not Sumoylated

41 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

42 Peptide generation in the class I pathway

43 Proteasome specificity NetChop is the best available cleavage method  www.cbs.dtu.dk/services/NetChop-3.0


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