Biochemistry Lecture 13 PDF

OTHER PROTEIN MODIFICATION REACTIONS Activation of zymogens by Proteolytic cleavage Addition of isoprenyl groups (Farnesylation of a Cys residue in an oncogene, the Ras protein) Oligosaccharide linkages in glycoproteins Formation of chymotrypsin and trypsin from their zymogens, chymotrypsinogen and...

OTHER PROTEIN MODIFICATION REACTIONS Activation of zymogens by Proteolytic cleavage Addition of isoprenyl groups (Farnesylation of a Cys residue in an oncogene, the Ras protein) Oligosaccharide linkages in glycoproteins Formation of chymotrypsin and trypsin from their zymogens, chymotrypsinogen and trypsinogen. The bars represent the amino acid sequences of the polypeptide chains, with numbers indicating the relative positions of the residues (the amino-terminal residue is number 1). Residues at the termini of the polypeptide fragments generated by cleavage are indicated below the bars. Note that in the final active forms, some numbered residues are missing. Recall that the three polypeptide chains (A, B, and C) of chymotrypsin are linked by disulfide bonds. (a) O-linked oligosaccharides have a glycosidic bond to the hydroxyl group of Ser or Thr residues (pink), illustrated here with GalNAc as the sugar at the reducing end of the oligosaccharide. One simple chain and one complex chain are shown. (b) N-linked oligosaccharides have an N-glycosyl bond to the amide nitrogen of an Asn residue (green), illustrated here with GlcNAc as the terminal sugar. Three common types of oligosaccharide chains that are N-linked in glycoproteins are shown. A complete description of oligosaccharide structure requires specification of the position and stereochemistry (α or β) of each glycosidic linkage. PROTEIN TARGETING AND DEGRADATION Proteins move from site of synthesis (cytosol in general, but also mitochondrial matrix) to: – Enter a subcellular compartment – Become part of the membrane – Exit a cell Most have a signal sequence at or near N-terminus Some proteins are targeted for degradation DIRECTING PROTEINS TO THE EDOPLASMIC RETICULUM (ER) AND BEYOND As peptide emerges from ribosome, signal sequence is bound by signal recognition particle (SRP) SRP/ribosome/RNA complex delivered to the ER lumen – Some protein modification takes place here (glycosylation, etc.) Some proteins stay in the ER. Other proteins are transported in vesicles to Golgi apparatus – Proteins are sorted in the Golgi for membrane insertion, for secretion or for other organelles (lysosomes etc.), in ways poorly understood Proteins targeted for mitochondria (and chloroplast) bind chaperone proteins in the cytosol and are delivered to receptors on the exterior of the organelle DIRECTING PROTEINS TO THE ER Postranslational modification in many eukaryotic proteins begins in the Endoplasmic Reticulum: The signal sequence Amino-terminal signal sequences of some eukaryotic proteins that direct their translocation into the ER. The hydrophobic core (yellow) is preceded by one or more basic residues (blue). The short-side-chain residues immediately precede (to the left of, as shown here) the cleavage sites (indicated by red arrows). The signal sequence is ultimately cleaved from the rest of the polypeptide DIRECTING PROTEINS TO THE ER The signal sequence directs the ribosome to the SRP and the ER Directing eukaryotic proteins with the appropriate signals to the endoplasmic reticulum. This process involves the SRP cycle and the translocation and cleavage of the nascent polypeptide. SRP is a rod-shaped complex containing a 300 nucleotide RNA (7SL-RNA) and six different proteins (combined Mr 325,000). One protein subunit of SRP binds directly to the signal sequence, obstructing elongation by sterically blocking the entry of aminoacyl-tRNAs and inhibiting peptidyl transferase (elongation step). Another protein subunit binds and hydrolyzes GTP. These steps are not shown here. The SRP receptor is a heterodimer of α (Mr 69,000) and β (Mr 30,000) subunits, both of which bind and hydrolyze multiple GTP molecules during this process. IN THE ER, PROTEINS ARE FURTHER MODIFIED IN SEVERAL WAYS Removal of signal sequence, folding, disulfide bonds formation, glycosylation… Example: Glycosylation Tunicamycin: Antibiotic that mimics the structure of UDP-GlcNac and blocks the first step of glycosylation Synthesis of the core oligosaccharide of glycoprotein: The core oligosaccharide is built up by the successive addition of monosaccharide units. 1,2 The first steps occur on the cytosolic face of the ER. 3 Translocation moves the incomplete oligosaccharide across the membrane (mechanism not shown), and 4 completion of the core oligosaccharide occurs within the lumen of the ER. The precursors that contribute additional mannose and glucose residues to the growing oligosaccharide in the lumen are dolichol phosphate derivatives. In the first step in the construction of the N-linked oligosaccharide moiety of a glycoprotein, 5,6 the core oligosaccharide is transferred from dolichol phosphate to an Asn residue of the protein within the ER lumen. The core oligosaccharide is then further modified in the ER and the Golgi complex in pathways that differ for different proteins. The five sugar residues shown surrounded by a beige screen (after step 7) are retained in the final structure of all N-linked oligosaccharides. 8 The released dolichol pyrophosphate is again translocated so that the pyrophosphate is on the cytosolic face of the ER, then 9 a phosphate is hydrolytically removed to regenerate dolichol phosphate. TARGETING PROTEINS FOR THE LYSOSOMES, PLASMA MEMBRANE OR SECRETION Proteins are moved from the ER to the cis side of the Golgi complex in transport vesicles. Sorting occurs primarily in the trans side of the Golgi complex. Sorting for the lysosome: Phosphorylation of mannose residues on lysosome-targeted enzymes: - N-Acetylglucosamine phosphotransferase recognizes some as yet unidentified structural feature of hydrolases destined for lysosomes. TARGETING AND IMPORT OF PROTEINS INTO THE NUCLEUS Proteins for nucleus have a nuclear localization sequence (NLS) – A NLS (4 to 8 residues) is not cleaved after the protein is targeted The nuclear envelope can break down (mitosis) and proteins will need to re-enter nucleus – A NLS may be located anywhere along the protein sequence – A NLS sequence is variable but includes several consecutive basic residues (Lys and Arg) Binds importin a and b and a GTPase called Ran Complex docks at a nuclear pore and is imported TARGETING AND IMPORT OF PROTEINS INTO THE NUCLEUS Signal sequences for nuclear transport are not cleaved NPC 1 A protein with an appropriate nuclear localization signal (NLS) is bound by a complex of importin α and β. 2 The resulting complex binds to a nuclear pore (NPC) and translocates. 3 Inside the nucleus, dissociation of importin β is promoted by the binding of Ran-GTP. 4 Importin α binds to Ran-GTP and CAS (Cellular Apoptosis Susceptibility Protein), releasing the nuclear protein. 5 Importin α and β and CAS are transported out of the nucleus and recycled in the cytosol. They are released in the cytosol when Ran hydrolyzes its bound GTP and becomes Ran-GDP. 6 Ran-GDP is bound by NTF2, and transported back into the nucleus. 7 RanGEF promotes the exchange of GDP for GTP in the nucleus, and Ran-GTP is ready to process another NLS-bearing protein-importin complex. Electron micrograph of the surface of the nuclear envelope, showing numerous nuclear pores (NPCs). PROTEIN DEGRADATION IS INEVITABLE Half-lives of proteins range from seconds to days to months to even a lifetime, – Defective proteins are short-lived, as are many metabolism regulatory proteins that respond to rapidly changing needs – Hemoglobin is long-lived (about 3 months).This feature is used for measuring long term glycemia in diabetes (HbA1c) – Cristallins (a lifetime), But almost all are eventually degraded MECHANISMS OF DEGRADATION In E. coli, proteases such as Lon (for “long form”, an ATP-dependent serine protease), ClpX, ClpAP etc. and FtsH hydrolyze defective (misfolded, truncated etc…) or short-lived proteins/peptides. In eukaryotes, the ubiquitin-proteasome system: – Proteins are linked to the protein ubiquitin via activating enzyme E1, conjugating enzyme E2, and ligating enzyme E3. – Ubiquinated proteins are cleaved by the 26 S proteasome complex Ubiquitin is very highly conserved protein PROTEIN TARGETING FOR DEGRADATION VIA ATTACHMENT OF UBIQUITIN Three-step pathway by which ubiquitin is attached to a protein. Two different enzyme-ubiquitin intermediates are involved (E1-U and E2-U). 1. The free carboxyl group of ubiquitin’s carboxyl-terminal Gly residue first becomes linked to an E1-activating enzyme via a thioester. 2. The ubiquitin is then transferred to an E2 conjugating enzyme. 3. An E3 ligase ultimately catalyzes the transfer of the ubiquitin from E2 to the target protein, linking ubiquitin through an amide (isopeptide) bond to an ε-amino group of a Lys residue of the target protein. Additional cycles produce polyubiquitin, a covalent polymer of ubiquitin subunits that targets the attached protein for destruction in eukaryotes. Multiple pathways of this sort, with different protein targets, are present in most eukaryotic cells. Defects in ubiquitination pathways lead to diseases states UBIQUITINATED PROTEINS ARE DEGRADED BY THE 26S PROTEASOME The 26S proteasome, highly conserved in all eukaryotes, is made of the 20S core particle and the 19S regulatory particle. (a) The core 20S particle consists of four rings arranged to form a barrel-like structure that has the protease activity. - Each of the inner rings has seven different β subunits (dark brown), three of which have protease activities. - The outer rings each have seven different α subunits (light brown). (b) The regulatory 19S particle( gray) binds ubiquitinated protein (blue), unfolds them and translocates them into the core particle, where they are degraded to peptides of 3 to 25 amino acids.