Post-Translational Processing & Sorting in ER PDF

Post-translational Processing & Sorting in ER Reading Material for the next 3 lectures: Molecular Biology of The Cell pp: 683-750  Objectives To describe protein glycosylation in the Endoplasmic reticulum (ER) To identify the importance of glycosyla...

Post-translational Processing & Sorting in ER Reading Material for the next 3 lectures: Molecular Biology of The Cell pp: 683-750  Objectives To describe protein glycosylation in the Endoplasmic reticulum (ER) To identify the importance of glycosylation of proteins To explain Quality control process and ER associated Protein degradation To describe carbohydrate modification in Golgi apparatus To describe targeting of lysosomal protein from Golgi to lysosome Protein synthesis/transport ER contains: a large number of ER resident proteins (chaperones) that facilitate protein folding and enzymes involved in protein/lipid synthesis. The soluble ER resident proteins contain a KDEL (retention signal) at the C terminal end of the protein. ER-resident protein receptors (KDEL receptor) KDEL receptor ensure recycling of ER-resident proteins back to ER Protein translocation into ER Which proteins are processed in ER? ER resident proteins (Calnexin, Calreticulin, BiP) Secreted proteins Membrane proteins Organelle proteins (Golgi, lysosomes, endosomes) Glycosylated proteins What processes take place in ER? Protein oligomerization Disulfide bond formation in polypeptides Addition of N-linked oligosaccharides to peptides Lipid synthesis Why proteins are glycosylated? Help in protein folding and maturation Important for protein stability Help protein sorting to its destination Change the biophysical properties of the proteins Important in protein-protein interaction Protein Glycosylation: - N-glycosylation on Aspargine: important for protein sorting and maturation - O-Glycosylation on Serine (Threonine): for changing biophysical properties of protein  N-glycosylation: - polypeptide chain is glycosylated on target asparagine amino acids. - The precursor oligosaccharide (shown in color) is attached only to asparagine in the sequences Asn-X-Ser and Asn-X-Thr (where X is any amino acid except proline). - These sequences occur much less frequently in glycoproteins than in non-glycosylated cytosolic proteins. - The five sugars (in the gray box) form the core region of this oligosaccharide. - The precursor oligosaccharide undergo extensive trimming in the Golgi apparatus. The precursor oligosaccharide is transferred from a dolichol lipid anchor to the asparagine as an intact unit in a reaction catalyzed by a transmembrane oligosaccharyl transferase enzyme. One copy of this enzyme is associated with each protein translocator in the ER membrane. Quality control in the ER - The ER chaperone proteins calnexin and calreticulin bind carbohydrate (lectin) of newly synthesized protein in ER. - They bind to incompletely folded proteins containing one terminal glucose on N-linked oligosaccharides, - They trapp these protein in the ER and recruit other ER enzymes to help fold the protein. - Removal of the terminal glucose by a glucosidase releases the protein from calnexin/calreticulin. - A glucosyl transferase is the crucial enzyme that determines whether the protein is folded properly or not: if the protein is still incompletely folded, the enzyme transfers a new glucose from UDP-glucose to the N-linked oligosaccharide, renewing the protein’s affinity for calnexin/calreticulin and retaining it in the ER. - The cycle repeats until the protein has folded completely. - Another ER chaperone, ERp57, collaborates with calnexin and calreticulin in retaining an incompletely folded protein in the ER. ERp57 recognizes free sulfhydryl groups, which are a sign of incomplete disulfide bond formation.  ER Associated Protein Degradation (ERAD) Misfolded proteins in the ER lumen are recognized and targeted to a translocator complex in the ER membrane. They first interact in the ER lumen with chaperones, disulfide isomerases, and lectins. They are then exported into the cytosol through the translocator. In the cytosol, they are ubiquitylated, deglycosylated, and degraded in proteasomes. Unfolded Protein Response (UPR) Signaling =ER Stress signaling Example of diseases developed due to misfolded proteins  Cystic Fibrosis mutations in CFTR (Chloride channel) leads to misfolded proteins and protein degradation. Some mutants (e.g. CFTR delta 508) results in a slowly folding protein which is not functional due to rapid protein degradation. Cells with this mutation grown at low temperature can produce functional channel by helping the mutated protein escape detection by UPR.  Gaucher Disease Gaucher disease results from a mutation in lysosomal acid β- glucosidase (catabolises glucosylceramide) Mutation in N370S results in lower catalytic activity and impaired exit from ER If the protein exits from ER it can function in lysosome. An inhibitor of this protein (iminosugar isofagomine) binds to active site and facilitate its folding and exit from ER (acts as a chemical chaperone). This will increase its availability in the lysosome. Washout of the drug results in restoring the activity of the mutated enzyme over time This study emphasizes an important role for ER in quality control and bypassing it using active-site specific inhibitors. Richard A. Steet, Stephen Chung, Brandon Wustman, Allan Powe†, Hung Do, and Stuart A. Kornfeld. PNAS 2006vol. 103no. 3713813–13818 Quality control in the ER Protein transport  Golgi The Golgi is comprised of distinct cisternae, each with a specific role in the modification of proteins. The function of the stack is determined by the enzymatic activities of each stack The Golgi is the site of final modifications of the carbohydrate side chains as well as other protein modifications. These modifications are dependent on sequential reactions. How is this achieved?  Processing of N-linked carbohydrate Removal and addition of various sugars on the branched chain N- linked carbohydrate leads to diversity of the side chain and variety in structure of mature protein. This diversity is used for selective recognition by receptors. The processing pathway is highly ordered: Step 1: Processing begins in the ER with the removal of the glucoses from the oligosaccharide initially transferred to the protein. Then a mannosidase in the ER membrane removes a specific mannose. The remaining steps occur in the Golgi stack. Step 2: Golgi mannosidase I removes three more mannoses. Step 3: N-acetylglucosamine transferase I then adds an N-acetylglucosamine. Step 4: Mannosidase II then removes two additional mannoses. This yields the final core of three mannoses that is present in a complex oligosaccharide. At this stage, the bond between the two N-acetylglucosamines in the core becomes resistant to attack by a highly specific endoglycosidase (Endo H). Since all later structures in the pathway are also Endo H-resistant, treatment with this enzyme is widely used to distinguish complex from high-mannose oligosaccharides. Step 5: Finally, as shown in Figure 13–30, additional N-acetylglucosamines, galactoses, and sialic acids are added. These final steps in the synthesis of a complex oligosaccharide occur in the cisternal compartments of the Golgi apparatus: three types of glycosyl transferase enzymes act sequentially, using sugar substrates that have been activated by linkage to the indicated nucleotide; the membranes of the Golgi cisternae contain specific carrier proteins that allow each sugar nucleotide to enter in exchange for the nucleoside phosphates that are released after the sugar is attached to the protein on the lumenal face. Note that, as a biosynthetic organelle, the Golgi apparatus differs from the ER: all sugars in the Golgi are assembled inside the lumen from sugar nucleotide, whereas in the ER, the N-linked precursor oligosaccharide is assembled partly in the cytosol and partly in the lumen, and most lumenal reactions use dolichol-linked sugars as their substrates.  Why proteins are glycosylated? Help in protein folding and maturation Important for protein stability Help protein sorting to its destination Change the biophysical properties of the proteins Important in protein-protein interaction O-linked glycosylation  O-linked glycosylation added to OH of Threonine. They are typically long, unbranched polymers (very large)  Proteoglycans - Proteoglycans have O-linked carbohydrates - Proteoglycans contain a large number of glucosaminoglycans. Part of the extracellular matrix. - They are long linear hydrophilic structures. - Play a role in cell-cell contact, as a reservoir of soluble growth factors, act as mechanical cushion (e.g. cartilage), and lining mucous membranes (component of muscins).  GPI anchor Immediately after the completion of protein synthesis, the precursor protein remains anchored in the ER membrane by a hydrophobic C-terminal sequence of 15–20 amino acids; the rest of the protein is in the ER lumen. Within less than a minute, an enzyme in the ER cuts the protein free from its membrane-bound C-terminus and simultaneously attaches the new C-terminus to an amino group on a preassembled GPI intermediate. The sugar chain contains an inositol attached to the lipid from which the GPI anchor derives its name. It is followed by a glucosamine and three mannoses. The terminal mannose links to a phosphoethanolamine that provides the amino group to attach the protein. The signal that specifies this modification is contained within the hydrophobic C-terminal sequence and a few amino acids adjacent to it on the lumenal side of the ER membrane. Because of the covalently linked lipid anchor, the protein remains membrane-bound, with all of its amino acids exposed initially on the lumenal side of the ER and eventually on the exterior of the plasma membrane. Protein transport Lysosomes The acid hydrolases are hydrolytic enzymes that are active under acidic conditions. An H+ ATPase in the membrane pumps H+ into the lysosome, maintaining its lumen at an acidic pH. Mannose 6-phosphate modification Lysosomal proteins (~100 enzymes) are modified by addition of N-acetyl glucosamine to the 6 position of the Mannose. The glucoseamine is then trimmed off to result in a protein with a mannose 6- phosphate residue. These proteins are soluble proteins and targeted to lysosomes. -mannose 6-phosphate acts as a recognition signal to send the protein to lysosome. The sequential action of two enzymes in the cis and trans Golgi network adds mannose 6-phosphate (M6P) groups to the precursors of lysosomal enzymes. The M6P-tagged hydrolases then segregate from all other types of proteins in the TGN because the M6P-modified lysosomal hydrolases bind M6P receptor in TGN. The clathrin-coated vesicles bud off from the TGN, shed their coat, and fuse with early endosomes. At the lower pH (acid) of the endosome, the hydrolases dissociate from the M6P receptors, and the empty receptors are retrieved in vesicles to the TGN for further rounds of transport. In the endosomes, the phosphate is removed from the M6P attached to the hydrolases, which may further ensure that the hydrolases do not return to the TGN with the receptor.  I-cell disease: lysosomal storage disease To late endosome M6P-R to the plasma P- transferase membrane Golgi TGN  I-cell disease: lysosomal storage disease To late endosome M6P-R soluble lysosomal enzymes are secreted Blood to the plasma P- transferase membrane Golgi TGN  Why proteins are glycosylated? Help in protein folding and maturation Important for protein stability Help protein sorting to its destination Change the biophysical properties of the proteins Important in protein-protein interaction Traffic signals for neutrophil localization in the vasculature Carbohydrate G protein coupled receptor Integrins ligand Selectin Chemoattractant Ig Family member Rolling Activation Adhesion  Diseases resulted from defects in protein processing and transport Cystic Fibrosis Atherosclerosis Diabetes (type II, adult onset) Viral Diseases (e.g., influenza) Alzheimer’s disease Prion diseases (e.g., Mad Cow) Lysosomal storage diseases (e.g. Niemann-Pick, I-Cell disease) And many others...  Objectives To describe protein glycosylation in the Endoplasmic reticulum (ER) To identify the importance of glycosylation of proteins To explain Quality control process and ER associated Protein degradation To describe carbohydrate modification in Golgi apparatus To describe targeting of lysosomal protein from Golgi to lysosome