Ubiquitinylation

The transcriptional activity of NRs is also modulated by various posttranslational modifications of the receptors themselves or of their coregulatory proteins. Phosphorylation, as well as several other types of modification, such as acetylation, SUMOylation, ubiquitinylation, and methylation, has been reported to modulate the functions of NRs, potentially constituting an important cellular integration mechanism. In addition to the modifications of the receptors themselves, such modifications have been reported for their coactivators and corepressors. Therefore, these different modes of regulation reveal an unexpected complexity of the dynamics of NR-mediated transcription. 

This cytoplasmic protein is phosphorylated by an IKK complex which is activated by cytokines, reactive oxygen species, and mitogens. Phosphorylated IkB can be ubiquitinylated and degraded, thus releasing its hold on NF-kB. Glucocorticoids affect many steps in this process, as described in the text. 

Iwai etal, 1995 Guo etal, 1995). It seems therefore highly probable that the proteosome (and also ubiquitinylation) is involved in IRP-2 degradation. 

Bloom, J., Amadoe, V., Baetolini, E., DeMaetino, G. and Pagano, M. Proteasome-mediated degradation of p21 via N-terminal ubiquitinylation. Cell, 2003, 3 35, 1-20. 

Ganoth, D., et ah. The cell-cycle regulatory protein Cksl is required For SCF( Skp2)-mediated ubiquitinylation of p27. Nat Cell Biol, 2001, 3(3),... 

Chromatin-modifying complexes are classified into two major groups (1) enzymes that conttol covalent modifications of the amino-terminal tails of histones (acetylation, methylation, phosphorylation, ubiquitinylation) (see Sections 1.3 and... 

Osoata G Adcock IM, Barnes PJ, Ito K. (2005) Oxidative stress causes HDAC2 reduction by nitration, ubiquitinylation and proteasomal degradation. Proc Am Thorac Soc 2 A755 (Abstr.)... 

The N-terminal tails of histone proteins are rich in arginine and lysine residues and undergo various types of posttranslational modifications. There are small modifications such as acetylation, methylation, phosphorylation but also the attachment of larger peptide groups such as ubiquitinylation and sumoylation [1]. This has an impact on chromatin structure and subsequently on gene transcription and the epigenetic maintenance of altered transcription after cell division [2],... 

In cells of the mammary gland, either in normal epithelial or in cancerous cells, the packaging of chromosomal DNA into chromatin restricts the access of the transcription machinery, thereby causing transcriptional repression. The basic N-termini of histones are subject to post-translational modifications, including lysine acetylation, lysine and arginine methylation, serine phosphorylation and ubiquitinylation [56]. It has been proposed in the histone code hypothesis that the intricate pattern of modifications of the N-terminal histone tail influences gene regulation [57]. 

Nandi D, Jiang H, Monaco JJ (1996) Identification of MECL-1 (LMP-10) as the third IFN-gamma-inducible proteasome subunit. J Immunol 156 2361-2364 Obin MS, Jahngen-Hodge J, Nowell T, Taylor A (1996) Ubiquitinylation and ubiq-uitin-dependent proteolysis in vertebrate photoreceptors (rod outer segments) Evidence for ubiquitinylation of Gt and rhodopsin.) Biol Chem 271 14473-14484 Otsu M, Urade R, Kito M, Omura F, Kikuchi M (1995) A possible role of ER-60 protease in the degradation of misfolded proteins in the endoplasmic reticulum. J Biol Chem 270 14958-14961... 

PCAs facilitate analysis of formerly unassayable processes like ubiquitinylation and histone (de-)acetylation in a live-cell context (4). It also significantly broadens the spectrum of assays around well-known targets such as GPCRs enabling analysis of GPCR homo-and heterodimerization and biased signaling (5). 

The ubiquitinylation of proteins is a complex process which involves several specific enzymatic reactions. Three sequential steps can be distinguished (fig. 2.15A) ... 

Fig. 2.15A. Pattern of ubiquitinylation of proteins and degradation in the proteosome. Ubiquitin (Ub)is initially activated by an enzyme El, whereby the C-terminal carboxyl group of ubiquitin becomes attached to a SH group of El via a thioester bond. Ubiquitin is then transferred from El-Ub to E2, from which it is transferred with the help of E3 to the target protein. Several ubiquitin molecules can attach to the target protein in a hnear or in a crosshnked fashion. The mono- or polyubiquitinylated protein is degraded to peptides in the 26S proteosome. In the above diagram the filled circles represent the ubiquitin residues attached to the target protein. K lysine residues of the target protein.

The third step of ubiquitinylation, the transfer of ubiquitin to the target protein, is catalyzed by a ubiquitin-protein-ligase, or E3 enzyme. In this reaction ubiquitin is linked by its C-terminal glycine in an amide isopeptide linkage to an e-NH2-group of the substrate proteins Lys residues. 

In other families of E3 enzymes, no intermediate E3-ubiquitin linkage can be demonstrated. In this case ubiquitin is transferred directly from E2 to the substrate protein. The E3 enzymes are nevertheless required for ubiquitinylation since the E3 enzymes are responsible for substrate selection and are foimd in tight complexes with the cognate E2 proteins. 

The binding of ubiquitinylated substrates requires the 19S complex of the proteasome, which possesses a ubiquitin binding site and several ATPase sites. It is assumed that the recognition and ATP-dependent unfolding of the substrate protein occurs within the 19S complex. 

This class of E3 enzymes recognizes the nature of the N-terminus of target proteins. As shown by Varshavsky (1992), yeast proteins may be selected for ubiquitinylation according to the N-end rule . Proteins containing basic or bulky-hydrophobic amino acids at the N-terminus are recognized by distinct N-end rule E3 enzymes of which the E3a enzyme (UBRl protein in yeast) is best characterized. 

The discovery of this family of E3 enzymes started from the studies on the targeted degradation of the p53 tumor suppressor protein. Ubiquitinylation and degradation of p53 can be mediated by the papillomavirus E6 oncoprotein (see below) in collaboration with a further protein, E6-AP (E6 associated protein). E6-AP was the first member of a large family of E3 enzymes, the Hect (homologous to E6-AP C-terminus ) domain family. These proteins contain an essential active site Cys residue near the C-terminus and one or several WW domains ( see Chapter 8.2.6). 

This example illustrates nicely how extracellular signals can induce the ubiquitinyla-tion and degradation of specific proteins. As shown by the processing of the pl05 precmsor, ubiquitinylation can be also used for partial proteolysis and for specific activation of a regulatory protein. 

Two types of E2/E3 complexes are of particular importance for cell cycle control. One complex, the SCF complex, is of outstanding importance for the Gl/S transition. The other, the anaphase-promoting complex or the cyclosome, is especially important for the course and control of mitosis. Common to both complexes is the variable collaboration with different proteins to mediate the ubiquitinylation of different substrates. 

In addition to phosphorylation, tau is also subjected to ubiquitinylation. Ubiquitin is a 76 amino acid protein that flags the proteins to be degraded in an ATP-dependent manner. Ubiquinated tau has been found in inclusion bodies found in Pick s disease or Parkinson s disease or in PHFs in AD. 

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