Presentation on theme: "Protein Folding and Processing"— 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.To be useful, polypeptides must fold into distinct three-dimensional conformations, and in many cases multiple polypeptide chains must assembled into a functional complex.-3dimensional conformations of proteins result from interactions between the side chains of their constituent amino acids.-The classic principle of protein folding suggests that all the info. Required for a protein to fold into the correct 3D conformation is encoded or provided by its amino acid sequence. It is now known that this is not quite the case. The proper folding is actually mediated by other proteins….these proteins are called chaprerone protiens.-Chaperones function to assist the assembly process, they do not convey additional information necessary for folding into the correct conformation. The folded conformation is determined by the amino acid sequence.-They seem to function by stabilizing unfolded or partially folded polypeptides that are intermediates along the pathway leading to the final correctly folded state. Chaperones stabilize unfolded proteins which prevents the accumulation of incorrectly folded proteins. As you know protein misfolding could have very bad consequences. For example, misfolded proteins can aggregate to form insoluble fibers called amyloid fibers. These fibers accumulate in the extracellular spaces and within cells and are characteristic of several neurodegenerative disease such as Parkinson’s and Alzheimer’s disease.-There are 2 specific types of chaperone proteins……heat shock protiens and chaperonins. They are found in the cytosol and subcellular organelles of eukaryotic cells.-HSP stabilize and facilitate the refolding of protiens that have bee partially denatured as a reslt of expsoure to elevated temperatures. They bind short hydrophobic regions of unfolded polypeptides. This maintains the polypeptide chain in an unfolded conformation and prevents aggregation.-Chaperonins function to protect unfolded polypeptides from proteolysis in the cytoplasm. Chaperonins consist of multiple protein subunits arranged in two stacked rings to form a double chambered structure. This allows protein folding to occur without aggregation of unfolded segments.
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.cell4e-fig jpg Here are two examples of chaperone usage.-In the top figure, chaperones are bound to nascent polypeptide peptide chains that are still being translated on ribonsomes. They function to prevent incorrect folding and aggregation of the amino-terminal portion of the polypeptide before synthesis of the entire chain is complete. Generally protiens fold into domains of amino acids, so it is necessary to prevent aberrant folding or aggregation with other proteins.-Chaperones also stabilize unfolde polypeptide chains during their transport into subcellular organelles like during the transfer of protiens into the mitochondria from the cytosol. Proteins are transported across the mitochondrial membrane in partially unfolded conformations that are stabilized by chaperones in the cytosol. Chaperones in the mitochondria then facillitate the transfer of the polypeptide chain across the membrane and its subsequent folding within the organelle.Chaperones also stabilize unfolded polypeptide chains during their transport into subcellular organelles.
3 The role of N-linked glycosylation in ER protein folding.
8 Protein folding in the cell Basics- cell compartments, molecular crowding: cytosol, ER, etc.Folding on the ribosome- co-translational protein foldingMolecular chaperones- concepts, introduction- intramolecular chaperones- chemical chaperones- protein chaperones
9 Folding in vitro vs. in vivo protein denaturedin a chaotropefolding by dilutionin bufferfoldingfoldedproteinfoldedprotein
10 Problem: non-native proteins 3-10Problem: 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 conditionsintramolecularmisfoldingXintermolecularaggregationincorrectmolecularinteractions&loss of activityexposedhydrophobicresidues
11 Overview of chaperone families: Distribution EukaryotesArchaeaBacteria-Trigger FactorNACHsp70 system[Hsp70 system]prefoldinchaperonins (group II)chaperonins (Group I)small Hsps[small Hsps]Hsp90[Hsp90]AAA ATPasesSecB[PapD/FimC]Hip, Hop, Bag, clusterin, cofactors A-E, calnexin, calreticulin, etc. etc.
12 The Unfolded Protein Response (UPR) Hsp4 (grp78)grp170XBP-1IRE-1The 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.
14 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 residues
15 Cellular Protein Degradation LysosomalNonspecificEndocytosisForeign proteinsEnergy favorable to degrade proteinsNon-lysosomalSpecificity, requires ATPConditions of stressUbiquitin-proteosomal pathway26S proteosomeRole 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 cell4e-fig jpgUbiquitination is a multi-step proces used in eukaryotic cells to mark cytosolic and nuclear proteins for rapid proteolysis. Ubiquitin is a 76 amino acid polypeptide that is highly conserved in all eukaryotes. It marks proteins for degradation by attaching to the amino group of the side chain of a lysine residue. Then several more ubiquitins are added to form a multiubiquitin chain. The resulting polyubiquinated proteins are recognized and degraded by a large multisubunit protease complex called the proteasome. During the process of proteolysis in the proteasome, ubiquitin is released so that it can be recycled and used again.-Here are the specifics of ubiquination:-Proteins are marked for rapid degradation by the covalent attachment of several molecules of ubiquitin. Ubiquitin is first activated by the enzyme E1. Actiavated ubiquitin is thane transferred to one of several different ubiquitin-conjugating enzyme E2. Then a ubiquitin ligase (E3) associates with E2 and directs the transfer of ubiquitin oto a specific target protein. This results in the addition of multiple ubiquitins and polyubiquinataed proteins are degraded by the proteasome.-Although ubiquitination typically targets proteins for degradation, in some cases the addition of ubiquitin serves other functions such as serving as a marker for endocytosis or the addition of ubiquitin related proteins.
21 Ubiquitination 1) ubiquitin - a 8.5 kd protein (76 residues) formation of an isopeptide bond with ε-amino group of lysineof the proteins- a tag for destruction- polyubiquitin: a strong signal for degradation2) enzymes for ubiquitination- E1 (ubiquitin-activating enzyme)- E2 (ubiquitin-conjugating enzyme)- E3 (ubiquitin-protein ligase)- variation: E3 > E2 > E1: more finely tuned substrate discriminationHPV (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 19S SubunitBase and LidContains subunits with known and unknown functionsTetra-Ub (K48) recognitionDeubiquitination activityProtein unfolding activity (Chaperone function)20S SubunitBarrelContains 6 proteolytic sites2x Tryptic2x Chymotryptic2x Peptidylglutamyl peptidaseLinearized protein required
30 Transmembrane Proteins Regulated by Ub-dependent Sorting In metazoans:Neurotransmission: Ion channels:AMPA glutamate receptors ENaCGlycine receptors ClC-5Cell-cell contacts: Immune moleculesE-cadherin downregulated by viruses:Occludin MHC class IB7-2Developmental patterning: ICAM-1Delta CD4NotchRoundabout
31 Poly-Ub Chains K48 Linkage K63 K K63 Linkage K48 K Signal to proteosomeK48, Ub4K63 LinkageK48KUbUbCell SignalingK63UbUbPeters, J.M. 1998Ubiquitin and the Biology of the CellUb
32 ENaC functionMajor ion channel that controls salt and fluid resorption in the kidneyMutations 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 stabilityNedd4 WW domains bind PPXY motif of ENaC subunitsNedd4 also interacts with serum and glucocorticoid-regulated kinase (SGK)SGK contains two PPXY motifs that bind to Nedd4 WW domainsSGK-dependent Nedd4 P inhibits the Nedd4-ENaC interactiontherefore, Nedd4 P increases ENaC at the cell surface