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Intracellular Protein Degradation: From a Vague Idea through the Lysosome and the Ubiquitin-Proteasome System and onto Human Diseases and Drug Targeting - PubMed

  • ️Sun Jan 01 2012

Intracellular Protein Degradation: From a Vague Idea through the Lysosome and the Ubiquitin-Proteasome System and onto Human Diseases and Drug Targeting

Aaron Ciechanover. Rambam Maimonides Med J. 2012.

Abstract

Between the 1950s and 1980s, scientists were focusing mostly on how the genetic code was transcribed to RNA and translated to proteins, but how proteins were degraded had remained a neglected research area. With the discovery of the lysosome by Christian de Duve it was assumed that cellular proteins are degraded within this organelle. Yet, several independent lines of experimental evidence strongly suggested that intracellular proteolysis was largely non-lysosomal, but the mechanisms involved have remained obscure. The discovery of the ubiquitin-proteasome system resolved the enigma. We now recognize that degradation of intracellular proteins is involved in regulation of a broad array of cellular processes, such as cell cycle and division, regulation of transcription factors, and assurance of the cellular quality control. Not surprisingly, aberrations in the system have been implicated in the pathogenesis of human disease, such as malignancies and neurodegenerative disorders, which led subsequently to an increasing effort to develop mechanism-based drugs.

Keywords: Ubiquitin; diseases; lysosome; proteasome; protein degradation.

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Figures

Figure 1
Figure 1

The lysosome. Ultrathin cryosection of a rat PC12 cell that had been loaded for 1 hour with bovine serum albumin (BSA)-gold (5-nm particles) and immunolabeled for the lysosomal enzyme cathepsin B (10-nm particles) and the lysosomal membrane protein LAMP1 (15-nm particles). Lysosomes are recognized also by their typical dense content and multiple internal membranes. Bar, 100 nm. Courtesy of Viola Oorschot and Judith Klumperman, Department of Cell Biology, University Medical Centre Utrecht, The Netherlands.

Figure 2
Figure 2

The four digestive processes mediated by the lysosome (from the upper left corner clockwise). (i) Specific receptor-mediated endocytosis, (ii) pinocytosis (non-specific engulfment of cytosolic droplets containing extracellular fluid), (iii) phagocytosis (of extracellular particles), and autophagy; (iv) microautophagy of intracellular proteins under basal conditions, and (v) macroautophagy of organelles under stress) (with permission from Nature Publishing Group; published originally in Ciechanover83).

Figure 3
Figure 3

APF-1/ubiquitin is shifted to high-molecular-mass compound(s) following incubation in ATP-containing crude cell extract. 125I-labeled APF-1/ubiquitin was incubated with reticulocyte crude fraction II in the absence (open circles) or presence (closed circles) of ATP, and the reaction mixtures were resolved via gel filtration chromatography. Shown is the radioactivity measured in each fraction. As can be seen, following addition of ATP, APF-1/ubiquitin becomes covalently attached to some component(s) in fraction II, which could be another enzyme of the system or its substrate(s) (with permission from Proceedings of the National Academy of the USA; published originally in Ciechanover et al.39).

Figure 4
Figure 4

Multiple molecules of APF-1/ubiquitin are conjugated to the proteolytic substrate, probably signaling it for degradation. To interpret the data described in the experiment depicted in Figure 2 and to test the hypothesis that APF-1 is conjugated to the target proteolytic substrate, 125I-APF-1/ubiquitin was incubated along with crude fraction II (Figure 3 and text) in the absence (lane 1) or presence (lanes 2–5) of ATP and in the absence (lanes 1 and 2) or presence (lanes 3–5) of increasing concentrations of unlabeled lysozyme. Reaction mixtures resolved in lanes 6 and 7 were incubated in the absence (lane 6) or presence (lane 7) of ATP, and included unlabeled APF-1/ubiquitin and 125I-labeled lysozyme. C1–C6 denote specific APF-1/ubiquitin-lysozyme adducts in which the number of APF-1/ubiquitin moieties bound to the lysozyme moiety of the adduct is increasing, probably from 1 to 6. Reaction mixtures were resolved via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and visualized following exposure to an X-ray film (autoradiography) (with permission from Proceedings of the National Academy of the USA; published originally in Hershko et al.40).

Figure 5
Figure 5

The ubiquitin-proteasome proteolytic system. Ubiquitin is activated by the ubiquitin-activating enzyme, E1 (1) followed by its transfer to a ubiquitin-carrier protein (ubiquitin-conjugating enzyme, UBC), E2 (2). E2 transfers the activated ubiquitin moieties to the protein substrate that is bound specifically to a unique ubiquitin ligase E3 (A and B). In the case of RING finger ligases, the transfer is direct (A3). Successive conjugation of ubiquitin moieties to one another generates a polyubiquitin chain (A4) that serves as the binding (A5) signal for the downstream 26S proteasome that degrades the target substrates to peptides (A6). In the case of HECT domain ligases, ubiquitin generates an additional thiol-ester intermediate on the ligase (B3) and only then is transferred to the substrate (B4). Successive conjugation of ubiquitin moieties to one another generates a polyubiquitin chain (B5) that binds to the 26S proteasome (B6) followed by degradation of the substrate to peptides (B7). Free and reusable ubiquitin is released by de-ubiquitinating enzymes (DUBs) (8).

Figure 6
Figure 6

The proteasome. The proteasome is a large, 26S, multicatalytic protease that degrades polyubiquitinated proteins to small peptides. It is composed of two subcomplexes: a 20S core particle (CP) that carries the catalytic activity and a regulatory 19S regulatory particle (RP). The 20S CP is a barrel-shaped structure composed of four stacked rings, two identical outer α rings and two identical inner β rings. The eukaryotic α and β rings are composed each of seven distinct subunits, giving the 20S complex the general structure of α1–7β1–7β1–7α1–7. The catalytic sites are localized to some of the β subunits. Each extremity of the 20S barrel can be capped by a 19S RP, each composed of 17 distinct subunits: 9 in a “base” subcomplex, and 8 in a “lid” subcomplex. One important function of the 19S RP is to recognize ubiquitinated proteins and other potential substrates of the proteasome. Several ubiquitin-binding subunits of the 19S RP have been identified; however, their biological roles or modes of action have not been discerned. A second function of the 19S RP is to open an orifice in the α ring that will allow entry of the substrate into the proteolytic chamber. Also, since a folded protein would not be able to fit through the narrow proteasomal channel, it is assumed that the 19S particle unfolds substrates and inserts them into the 20S CP. Both the channel opening function and the unfolding of the substrate require metabolic energy, and, indeed, the 19S RP “base” contains six different ATPase subunits. Following degradation of the substrate, short peptides derived from the substrate are released, as well as reusable ubiquitin. a: Electron microscopy image of the 26S proteasome from the yeast S. cerevisiae. b: Schematic representation of the structure and function of the 26SA proteasome (with permission from Nature Publishing Group; published originally in Ciechanover et al.83).

Figure 7
Figure 7

Some of the different functions of modification by ubiquitin and ubiquitin-like proteins. a: Proteasomal-dependent degradation of cellular proteins (see Figure 4). b: Mono- or oligoubiquitination targets membrane proteins to degradation in the lysosome/vacuole. c: Monoubiquitination, or d: a single modification by a ubiquitin-like (UBL) protein, SUMO for example, can target proteins to different subcellular destinations such as nuclear foci or the nuclear pore complex (NPC). Modification by UBLs can serve other, non-proteolytic, functions, such as protecting proteins from ubiquitination or activation of E3 complexes. e: Generation of a Lys–63-based polyubiquitin chain can activate transcriptional regulators, directly or indirectly (via recruitment of other proteins (protein Y; shown), or activation of upstream components such as kinases). Ub, ubiquitin; K, Lys; S, Cys (with permission from Nature Publishing Group; published originally in Ciechanover et al.83).

Figure 8
Figure 8

Aberrations in the ubiquitin-proteasome system and pathogenesis of human diseases. Normal degradation of cellular proteins maintains them in a steady-state level, though this level may change under various pathophysiological conditions (upper and lower right side). When degradation is accelerated due an increase in the level of an E3 (Skp2 in the case of p27, for example), or overexpression of an ancillary protein that generates a complex with the protein substrate and targets it for degradation (the human papillomavirus E6 oncoprotein that associates with p53 and targets it for degradation by the E6-AP ligase, or the cytomegalovirus-encoded ER proteins US2 and US11 that target MHC class I molecules for ERAD), the steady-state level of the protein decreases (upper left side). A mutation in a ubiquitin ligase (such as occurs in adenomatous polyposis coli (APC), or in E6-AP (Angelmans’ syndrome)) or in the substrate’s recognition motif (such as occurs in β-catenin or in ENaC) will result in decreased degradation and accumulation of the target substrate.

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