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Cartoon depiction of cotranslational folding of a polypeptide. The nascent polypeptide assumes secondary structure as it emerges from the ribosome during biosynthesis. Three intermediates show different stages of folding, with chaperones and folding catalysts playing key roles. The process involves posttranslational oligomeric assembly and degradation pathways.
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Cartoon depiction of cotranslational folding of a polypeptide.The nascent polypeptide is shown assuming secondary structure as itemerges from the ribosome during the process of biosynthesis. The earliestintermediate, I1, is not well-stabilized by extensive tertiary interactions andis in equilibrium with multiple conformations. The second intermediateshown, I2, is the N-terminal domain; more extensive tertiary interactionswill allow this intermediate to be more stable. The final intermediate, I3,depicts the structure of the full-length polypeptide immediately prior torelease from the ribosome with the C-terminal domain not yet fully packed.Chaperones and/or folding catalysts (CH and FC) may interact with eitherthe nascent intermediate structures or with the full-length product (M*)following release from the ribosome. The final stages of folding from M* tonative monomer, Mn, occur following release. Association of monomeric unitsinto oligomeric structures, O, may occur posttranslationally as depicted or,as discussed in the text, may involve nascent polypeptides. The structurethat was used to develop this cartoon was of one subunit of the bacterialluciferase b2 homodimer (66).
A scenario for GHRendocytosis and downregulation:(1) GH binds and dimerizes twoGHRs, which causes recruitmentand activation of Jak2; Jak2phosphorylates the GHR, itself, andSTATs. Signal transduction isrelayed to the nucleus and genes areactivated; (2) specific ubiquitinconjugases (E2) and ligases (E3)dock onto the UbE motif andubiquitinate the GHR and possiblyother attached proteins; (3) theE2/E3s activate the clathrin-coatedendocytosis machinery, servingeither as adaptors for clathrin or asconnectors to AP2 adaptors; (4)transport to early and lateendosomes; (5) degradation of GHand GHR by proteasomes andlysosomes. Journal of Cell Science 112, 1417-1423 (1999)
Ubiquitination motifs Journal of Cell Science 112, 1417-1423 (1999)
Primjer: Degradacija ciklinatijekom staničnog ciklusa
The ubiquitin system of S. cerevisiae The yeast ubiquitin genes, two of which (UBI1 and UBI2) contain introns, encode fusion proteins of ubiquitin (yellow rectangles) to itself (UBI4) or to one of the two specific ribosomal proteins (UBI1–UBI3) (red and blue rectangles). These fusion proteins are cleaved by deubiquitinating enzymes, yielding mature ubiquitin. Thioester bonds between ubiquitin and the active-site Cys residues of ubiquitin-specific enzymes. The conjugation of ubiquitin to other proteins involves a preliminary ATP-dependent step, in which the last residue of ubiquitin (Gly76) is joined, through a thioester bond, to a Cys residue in the ubiquitin-activating (E1) enzyme encoded by UBA1. The activated ubiquitin is transferred to a Cys residue in one of at least 13 distinct ubiquitin-conjugating (E2) enzymes encoded by the UBC family genes, and from there to a Lys residue of an ultimate acceptor protein (yellow oval). This last step and the formation of a multi-ubiquitin chain (black ovals) require participation of another component, called E3 (the names of some of the yeast E3 proteins are included). A targeted, ubiquitinated protein substrate is processively degraded to short peptides by the ATP-dependent 26S proteasome.
The N-end rule pathway N-degron Notations in the yeast (a) and mouse (b) pathways show type 1 (purple) and type 2 (red) primary, secondary (light blue) and tertiary (green) destabilizing N-terminal residues; yellow ovals indicate the rest of a protein substrate. a, The in vivo half-lives of X-gals, -galactosidase-based test proteins in S. cerevisiae (right). X-gal proteins bearing stabilizing N-terminal residues (black) are metabolically stable (t1/2, more than 20 h). The tertiary destabilizing residues N (Asn) and Q (Gln) are converted into secondary destabilizing residues D (Asp) and E (Glu) by N-terminal amidohydrolase (Nt-amidase), encoded by NTA1. D and E are conjugated to R (Arg), one of the primary destabilizing residues, by Arg–RNA protein transferase (R-transferase), encoded by ATE1. b, In the mammalian N-end rule pathway, the deamidation step is mediated by two distinct enzymes, NtN-amidase and NtQ-amidase, specific for N-terminal Asn and Gln residues, respectively54. In vertebrates, the set of secondary destabilizing residues contains not only Asp and Glu but also Cys (C), which is a stabilizing residue in yeast55. In mammals but not in yeast, Ala (A), Ser (S) and Thr (T) are primary (type 3) destabilizing residues48. c, S. cerevisiae UBR1 has two binding sites for the primary destabilizing N-terminal residues of either proteins or short peptides. The type 1 site is specific for basic N-terminal residues Arg, Lys and His. The type 2 site is specific for bulky hydrophobic N-terminal residues Phe, Leu, Trp, Tyr and Ile. UBR1 contains yet another substrate-binding site (i), which targets proteins bearing internal (non-N-terminal) degrons. In yeast, these proteins include the CUP9 repressor57. A complex of UBR1 and the ubiquitin-conjugating (E2) enzyme RAD6 produces a substrate-linked multi-ubiquitin chain
20S Proteasome The catalytic core of the proteasome(20S proteasome) is built up of seven differenta and seven different b-subunits arranged as a cylindrical777 7 complex in four stacked rings. Vertebrates possess three additional -subunits that are induced by interferon- (IFN-). Degradation of folded proteins requirescooperation of the 20S-core protease with the 19Scap, a structure that canbind to both ends of the barrel, resulting in the 26Sproteasome. The 19S cap contains at least 20 subunits,some of which are involved in the recognitionand removal of ubiquitin. It also possesses anATPase activity that helps to unfold and feed thesubstrate into a chamber in the 20S core that harboursthe active sites. Further complexity arisesfrom an additional 11S regulatory complex,also upregulated upon stimulation with IFN-.Two forms of the 11S regulator exist: a heterooligomericring made up of REGa and REGb subunitsand a homo-oligomeric REGg particle. The 11S regulator can replace the 19S complex and results in activation and altered peptidase activity upon binding to the 20S proteasome in vitro.
Intracellular targeting of proteasome Trends in Cell Biology,10:268, 2000. Multiubiquitinated membrane proteins target the proteasome to the endoplasmic reticulum (ER). Ubiquitin-activating enzyme E1 hydrolyses ATP and forms a high-energy thioester linkage between acysteine of its active site and the C-terminus of ubiquitin. Activated ubiquitin is then transferred to aubiquitin-conjugating enzyme E2. These enzymes function in concert with ubiquitin protein ligases,E3, and attach ubiquitin to the substrate protein. Isopeptidases associated with the 19S cap or in thecytosol disassemble the multiubiquitin chain and ubiquitin can be recycled.
Perfect use of imperfection Nature 404, 709 - 710 (2000) Figure 1 A use for protein waste. A significant amount (30% or more) of protein production is faulty and results in the formation of defective ribosomal products (DRiPs). Cellular quality-control mechanisms ensure that DRiPs are immediately removed by becoming tagged with a small molecule called ubiquitin (Ub) and targeted to the proteasome, which chops the DRiPs up into small peptides. Samples of the peptides are transported by the transporter associated with antigen presentation (TAP) into the endoplasmic reticulum (ER), loaded on MHC class I molecules and transported to the cell surface for recognition by T cells. Peptides from fully functional proteins that have aged are presented to T cells in the same way. But the DRiP short cut ensures that the immune system can get a head start on fighting infection, because it does not have to wait for proteins (such as those produced in virus-infected cells) to age.
