Protein Trafficking (BY450 SC 2024) PDF

Protein processing & trafficking DR ANGEL A SHEERIN Learning outcomes  Be able to understand post translation modifications and what they mean  Be able to understand protein trafficking  Be able to understand protein degradation  Be able to relate all this information to a known patholo...

Protein processing & trafficking DR ANGEL A SHEERIN Learning outcomes  Be able to understand post translation modifications and what they mean  Be able to understand protein trafficking  Be able to understand protein degradation  Be able to relate all this information to a known pathology The basics From peptide chain to functioning protein To be useful polypeptides must fold into distinct three- dimensional conformations, often involving multiple polypeptide chains This is critical to a protein's functionality This is mediated by chemical bonds between individual amino acids on the chain(s) Chemical Bonds/interactions Stabilize Protein Structure Non-covalent and covalent bonds Added during protein modification and trafficking processes Figure 03.14: Five classes of bonds that stabilize protein structure. Representati ve Diseases Associated with Protein Aggregation  Representati ve Diseases Associated with trafficking issues Ø Autosomal dominant polycystic kidney disease (ADPKD) Ø Dent’s disease Ø Cystic fibrosis Core processes in folding  The role of chaperon es -To be able to fold correctly, assistance is needed. Picture credit:http://cdn.c.photoshelter.com/img-get/I0000gV9qvmcQr2Q/s/900/720/Social-Cartoons- Punch-Magazine-Ken-Pyne-1980-11-26-954.jpg Overview of their function  Molecular chaperone therefore have several functions: 1. Behaviour 2. Guidance 3. Prevention 4. Protection Example of chaperone action during translation Chaperones stabilise the growing N terminus of a polypeptide chain, stabilizing, so it can fold correctly upon completion of synthesis. FIGURE: COOPER (2000), FIGURE 7.17 Example of chaperone action during protein transport Mitochondrial chaperones facilitate transport and subsequent folding of the polypeptide chain within the organelle. FIGURE: COOPER (2000), FIGURE 2.17 Example of chaperone action during ER protein folding FIGURE: COOPER (2000), FIGURE 11.6  Molecular chaperones Protein Prokaryotes Eukaryotes family Hsp70 DnaK Hsc73 (cytosol) BiP (endoplasmic reticulum) SSC1 (mitochondria) ctHsp70 (chloroplasts) Hsp60 GroEL TriC (cytosol) Hsp60 (mitochondria) Cpn60 (chloroplasts) Hsp90 HtpG Hsp90 (cytosol) Grp94 (endoplasmic reticulum) Cooper (2000), Table 7.2 Enzymes Protein disulfide isomerase (PDI) Enzymes P E P T I DY L P R O LY L I S O M E RA S E (PPIASE) Direction- trafficking of proteins Where do I go? P R O T E I N T R A F F I C K I N G ( TA R G E T I N G ) A N D P R O T E I N S O RT I N G  What does my tag tell me? Unmodified Different types of protein Signal peptide Destination For; possibilities Secretion For; Golgi For; Nucleus Post- Co-translational translational Protein Trafficking – different pathways – depends on how protein is formed and its destination  Proteins are translated and deposited into the RER A very broad overview of Once processed, proteins secretory translocated from the RER to the Golgi apparatus pathway Some are modified in the Golgi via specific enzymes cis-maturation model- process of movement from cis to trans in the golgi apparatus Image reference: http://www.nature.com/horizon/proteinfolding/highli ghts/images/s2_nonspec1_f1.jpg After reaching the trans Golgi network (TGN), then the bud off and are transported to the final destination Signal sequence in translocation- co-translational targeting Protein Trafficking – Post translational targeting Maintained in unfolded state by cytosolic chaperones. Signal sequence recognised by distinct receptor proteins Sec62/63 Cytosolic chaperones drive protein into the translocon. Internal chaperone BiP acts in a ratchet fashion to draw the protein into the lumen.  Protein Trafficking – ER Membrane Proteins Transmembrane protein with cleavable signal sequence Subunits of the transmembrane translocon separate and protein anchored into membrane http://youtu.be/PUy_Em5dXmc Modifications in the ER  Protein Trafficking – The Endoplasmic Reticulum The endoplasmic reticulum, or ER, is a network of membrane enclosed tubules and sacs that extends from the nuclear membrane throughout the cytoplasm. The rough ER is covered by ribosomes on its outer surface and is involved in protein Brian Cambron, Kaskaskia College, Illinois metabolism. The transitional ER is involved in protein processing and is where vesicles exit to the Golgi apparatus. The smooth ER is involved in lipid, rather than protein,  Glycosylati on Post- translation al protein Attachmen t of lipids modificatio ns Acylation, phosporylation, Other sulfation, methylation… Glycosylatio n vPlays a role in protein folding vAddition of carbohydrate chains vTargets proteins for delivery to subcellular locations vForms recognition sites in cell- cell interactions Most glycoproteins destined for secretion or incorporation into plasma membrane N-linked glycoprotein N-linked glycosylation is initially carried out in the rough ER O-linked glycoprotein O-linked modifications occur in the Golgi apparatus  Attachment of lipids GPI anchor Targeting and anchoring of proteins to plasma membrane Cytosolic face of plasma membrane;  N-myristoylation  Prenylation (e.g ras oncogene)  Palmitoylation Extracellular face of plasma membrane;  Addition of glycolipids e.g. GPI (Glycosylphosphatidylinositol) Cooper (2000), Figure 7.32 anchor  Additions/modifications Myrisoyl- SRC Prenyl grp- RAS Overview of modificatio ns Modifications in the Golgi Protein Trafficking – Golgi Organisation Modifications in the golgi Protein Trafficking – ER to Golgi Transport in vesicles ERGIC: E R - G O L G I I N T E R M E D I AT E C O M PA RT M E N T Transport in coated vesicles Materials are carried between compartments using coated vesicles. Protein coats have two distinct functions: ◦Cause the membrane to curve and form a vesicle. ◦Select the components to be carried by vesicle. Transport in coated vesicles Transport in coated vesicles Retrieval of ER proteins Proteins destined for the ER lumen are marked with the sequence Lys- Asp-Glu-Leu (KDEL). Sequences recognised by receptors in ERGIC or Golgi and after processing, they are selectively returned to the ER Protein destruction  Protein degradation Posttranslational control -protein degradation UPS Lysosomes (autophagy) Picture reference:http://www.asknature.org/images/uploads/strategy/6eb3fb3131f864f614cd88cf46e02f56/ lysosome_eharrington_internal.jpg The ubiquitin- proteasome pathway (UPS) Recognised and Ubiquitin: 76-aa degraded by Major pathway of polypeptide, highly multisubunit selective protein conserved in all protease complex, degradation eukaryotes called proteasome Proteasome structure  The ubiquitin- proteasome pathway Ubiquitin first activated by enzyme E1. Activated ubiquitin transferred to a ubiquitin-conjugating enzyme (E2). Ubiquitin transferred to a ubiquitin ligase (E3) and then to a target protein. Figure: Cooper (2000), Figure 7.39 Multiple ubiquitins added, and the polyubiquinated proteins degraded the proteasome The lysosome system Lysosomes are membrane enclosed organelles that contain an array of digestive enzymes, including several proteases Containment prevents uncontrolled degradation of cell contents Allow cells to recycle components of nonessential proteins and organelles, also extensive tissue remodelling during development The Lysosome system Figure 7.41The lysosome system Lysosomes contain various digestive enzymes, including proteases. Lysosomes take up cellular proteins by fusion with autophagosomes, which are formed by the enclosure of areas of cytoplasm or organelles (e.g., a mitochondrion) in fragments of the endoplasmic reticulum. This fusion yields a Ref: The Cell: A Molecular Approach. 2nd edition. phagolysosome, which digests the contents of the autophagosome.  Lysosomal system in full flow….. Autophagy Autophagy = uptake of cytoplasmic areas or organelles into vesicles which fuse with lysosomes Autophagy regulated in response to availability of nutrients & during development of multicellular organisms Protein Trafficking – How does this relate to disorders  Normal protein trafficking in cells NORMAL TRAFFICKING WHEN TRAFFICKING GOES WRONG Disorder examples: X  Autosomal dominant polycystic kidney disease (ADPKD)  Nephrogenic diabetes insipidus (NDI).  Dent’s disease  Cystic fibrosis Schaeffer et al. 2014. Protein trafficking defects in inherited kidney diseases. NDT; 29: PP.iv33-iv44 ER retention /Gogi retention Retained in the ER Called back to the ER  Mistargeting Effecting protein targeting - dysregulation can lead to disease Figure: Defective endocytosis/Defe ctive degradation Endocytosis is a fundamental cellular process How might these mutations cause disease/conditions related to protein folding and/or trafficking –using cystic fibrosis as an example  Importance of Cystic fibrosis folding  Mucus secretion-cystic fibrosis Ø Can cause respiratory problems Ø Effects FEV Ø Can contribute to infections Ø CFTR plays a role Ø Hydration plays a role Secretory pathway in action for mucin secretion Schematic representation of the biosynthesis and secretion of mucin glycoproteins in a goblet or mucus cell.  UPS system in Cystic fibrosis Nery, FC et al (2011), Nat Commun.  Classes of CFTR Mutation I II III IV V Defective Defective Defective Defective Reduced Production Processing Regulation Conductance Amounts  CFTR Modulator Therapies rrectors- trafficking enhancers Figure from: https://www.cftrscience.com/sites/all/themes/cftrscience/images/pages/section-1/1.2_image.png  CFTR Modulator Therapies Potentiators- opening probabilities Figure taken directly from; Insights into the mechanisms underlying CFTR channel activity, the molecular basis for cystic fibrosis and strategies for therapy. Chiaw PK, Eckford PDW and Bear CE. Essays In Biochemistry (2011); 50 233-248; Ivacaftor in action  (2013) FEBS Journal 280:4395 “CTRF...Hub protein.........Significant number of post-translational modification.” “.....mutations in the CF transmembrane conductance regulator (CFTR) gene, which encodes a cAMP-activated anion channel that is expressed in the epithelia of the lung, pancreas, intestine, liver, sweat glands, and reproductive tract.”  References-Book chapters Karp, G. (2010), Cell biology (6th ed.). Wiley, Chapter 6 & 12 Cooper, G.M. & Hausman, R.E. (2009). The cell, a molecular approach (5th ed.). ASM Press, Washington, Chapters 8 (Folding & processing), 10 (Protein trafficking) Cooper, G.M. & Hausman, R.E. (2016). The cell, a molecular approach (5th ed.). ASM Press, Washington, Chapters 9 & 11. Lodish et al. (2007). Molecular Cell Biology, 6th Ed, Freeman; Companion website (with free animations): www.whfreeman.com/lodish Cooper, G.M. & Hausman, R.E. (2009). The cell, a molecular approach (5th ed.). ASM Press, Washington, Chapter 8 (protein degradation) Alberts et al. Essential cell biology 4th edition (2013). Plopper, G. Principles of cell biology (2015).