Alan L. Schwartz

Ubiquitin‐mediated proteolysis: biological regulation via destruction

The ubiquitin proteolytic system plays an important role in a broad array of basic cellular processes. Among these are regulation of cell cycle, modulation of the immune and inflammatory responses, control of signal transduction pathways, development and differentiation. These complex processes are controlled via specific degradation of a single or a subset of proteins. Degradation of a protein by the ubiquitin system involves two successive steps, conjugation of multiple moieties of ubiquitin and degradation of the tagged protein by the 26S proteasome. An important question concerns the identity of the mechanisms that underlie the high degree of specificity of the system. Substrate recognition is governed by a large family ubiquitin ligases that recognize the substrates, bind them and catalyze/facilitate their interaction with ubiquitin. BioEssays 22:442—451, 2000.

Targeting Proteins for Destruction by the Ubiquitin System: Implications for Human Pathobiology

Cellular proteins are in a dynamic state maintained by synthesis and degradation. The ubiquitin proteolytic pathway is responsible for the degradation of the bulk of cellular proteins including short-lived, regulatory, and misfolded/denatured proteins. Ubiquitin-mediated proteolysis involves covalent attachment of multiple ubiquitin molecules to the protein substrate and degradation of the targeted protein by the 26S proteasome. Recent understanding of the molecular mechanisms involved provides a framework to understand a wide variety of human pathophysiological states as well as therapeutic interventions. This review focuses on the response to hypoxia, inflammatory diseases, neurodegenerative diseases, and muscle-wasting disorders, as well as human papillomaviruses, cervical cancer and other malignancies.

The ubiquitin conjugation system is required for ligand-induced endocytosis and degradation of the growth hormone receptor.

The ubiquitin‐dependent protein degradation system has recently been implicated in downregulation of signal transducing receptors. Growth hormone receptor (GHR) cDNA was transfected into Chinese hamster ovary cells, which exhibit a temperature‐sensitive defect in ubiquitin conjugation (CHO‐ts20), as well as into wild‐type cells (CHO‐E36). Upon binding of growth hormone (GH), two GHR polypeptides dimerize and initiate signal transduction. In CHO‐E36 and in CHO‐ts20 at the permissive temperature the GHR was ubiquitinated and degraded in a GH‐dependent fashion. However, at the non‐permissive temperature in CHO‐ts20 cells, neither GH‐dependent uptake nor degradation of the GHR was observed, while in CHO‐E36 cells both GHR uptake and degradation were accelerated. Incubation of CHO‐E36 cells with inhibitors of endosomal/lysosomal function (NH4Cl, bafilomycin A1) markedly reduced ligand‐induced GHR degradation. Our results indicate that a functional ubiquitin conjugating system is required for GH‐induced endocytosis and that degradation of both the exoplasmic and cytoplasmic portions of the GHR occurs within the endosomal/lysosomal compartment.

39 kDa receptor-associated protein is an ER resident protein and molecular chaperone for LDL receptor-related protein.

The low density lipoprotein receptor‐related protein (LRP) is a multifunctional endocytic receptor with the ability to bind and endocytose several structurally and functionally distinct ligands. A 39 kDa receptor‐associated protein (RAP) inhibits all ligand interactions with LRP in vitro. In the present study, we demonstrate that RAP is an endoplasmic reticulum (ER) resident protein. The tetrapepetide sequence HNEL at the C‐terminus of RAP is both necessary and sufficient for RAP retention within the ER. Metabolic labeling combined with cross‐linking studies show that RAP interacts with LRP in vivo. Pulse‐chase analysis reveals that this association is transient early in the secretory pathway and coincides with LRP aggregation and reduced ligand binding activity. Both internal triplicated LRP binding domains on RAP and multiple RAP binding domains on LRP appear to contribute to the aggregation of LRP and RAP. Dissociation of RAP from LRP results from the lower pH encountered later in the secretory pathway and correlates with an increase in LRP ligand binding activity. Taken together, our results thus suggest that RAP functions intracellularly as a molecular chaperone for LRP and regulates its ligand binding activity along the secretory pathway.

The ubiquitin-mediated proteolytic pathway: mode of action and clinical implications

Proteolysis via the ubiquitin system plays important roles in a variety of basic cellular processes. Among these are regulation of cell cycle and division, modulation of the immune and inflammatory responses, and development and differentiation. In all cases studied, these complex processes are mediated via degradation or processing of a single or a subset of specific proteins. Ubiquitin‐mediated degradation of a protein involves two discrete and successive steps: (1) conjugation of multiple moieties of ubiquitin to the protein, and (2) degradation of the conjugated protein by the 26S proteasome complex with the release of free and reutilizable ubiquitin. In a few cases, it has been reported that ubiquitination targets membrane‐anchored proteins to degradation in the lysosome/vacuole. An important yet largely unresolved problem involves the mechanisms that endow the system with the high degree specificity and selectivity toward its many substrates. These are determined by a large family of ubiquitin‐protein ligases that recognize different primary and/or secondary/post‐translational motifs in the different substrates and by a wide array of modifying enzymes, such as protein kinases, and ancillary proteins, such as molecular chaperones, that render them susceptible for recognition by the ligases via modification or association with protein substrates. With the broad spectrum of protein substrates and the complex enzymatic machinery involved in targeting them, it is not surprising that the system was recently implicated in the pathogenesis of several important diseases. In addition, genetic studies in animals underscore the role of the system in normal development. We briefly review the enzymatic cascade involved in ubiquitin‐mediated degradation, describe some of the structural motifs identified by the conjugating machinery, and summarize recent developments in the involvement of the system in the pathogenesis of selected disease states. J. Cell. Biochem. Suppl. 34:40–51, 2000.

α2-Macroglobulin Complexes with and Mediates the Endocytosis of β-Amyloid Peptide via Cell Surface Low-Density Lipoprotein Receptor-Related Protein

Abstract: A primary histopathological feature of Alzheimers disease is the accumulation of β‐amyloid (Aβ) in the brain of afflicted individuals. However, Aβ is produced continuously as a soluble protein in healthy individuals where it is detected in serum and CSF, suggesting the existence of cellular clearance mechanisms that normally prevent its accumulation and aggregation. Here, we demonstrate that Aβ forms stable complexes with activated α2‐macroglobulin (α2M⋆), a physiological ligand for the low‐density lipoprotein receptor‐related protein (LRP) that is abundantly expressed in the CNS. These α2M⋆/125I‐Aβ complexes are immunoreactive with both anti‐Aβ and anti‐α2M IgG and are stable under various pH conditions, sodium dodecyl sulfate, reducing agents, and boiling. We demonstrate that α2M⋆/125I‐Aβ complexes can be degraded by glioblastoma cells and fibroblasts via LRP, because degradation is partially inhibited by receptor‐associated protein (RAP), an antagonist of ligand interactions with LRP. In contrast, the degradation of free 125I‐Aβ is not inhibited by RAP and thus must be mediated via an LRP‐independent pathway. These results suggest that LRP can function as a clearance receptor for Aβ via a physiological ligand.

Identification of a novel ubiquitin conjugation motif, required for ligand-induced internalization of the growth hormone receptor.

In addition to its role in selective protein degradation, the conjugation of ubiquitin to proteins has also been implicated in the internalization of plasma membrane proteins, including the α‐factor receptor Ste2p, uracil permease Fur4p, epithelial sodium channel ENaC and the growth hormone receptor (GHR). Binding of GH to its receptor induces receptor dimerization, resulting in the activation of signal transduction pathways and an increase of GHR ubiquitination. Previously, we have shown that the ubiquitin conjugation system mediates GH‐induced GHR internalization. Here, we present evidence that a specific domain of the GHR regulates receptor endocytosis via the ubiquitin conjugation system. This ubiquitin‐dependent endocytosis (UbE) motif consists of the amino acid sequence DSWVEFIELD and is homologous to sequences in other proteins, several of which are known to be ubiquitinated. In addition, we show that GH internalization by a truncated GHR is independent of the presence of lysine residues in the cytosolic domain of this receptor, while internalization still depends on an intact ubiquitin conjugation system. Thus, GHR internalization requires the recruitment of the ubiquitin conjugation system to the GHR UbE motif rather than the conjugation of ubiquitin to the GHR itself.

Alterations in the protein composition of maturing phagosomes.

We investigated the protein composition of J774-E clone macrophage phagosomes isolated at different stages of phagolysosome biogenesis. Phagosomes formed by internalizing antibody-coated Staphylococcus aureus for 3 min followed by chase for 0, 4, 9, or 15 min were isolated by density gradient centrifugation. Enrichment and purity of the phagosome preparations were quantitated by radiolabeled ligand recovery, enzyme markers, and electron microscopy. One-dimensional SDS-PAGE analyses of the isolated phagosomes revealed virtually identical protein compositions. However, Western blot analyses with antibodies directed against selected proteins of known itineraries along the endocytic pathway demonstrated distinct differences in phagosome protein compositions. Accumulating within the maturing phagosome were the 31-kD subunit of the vacuolar proton pump, cathepsin D,beta-glucuronidase, the cation dependent mannose 6-phosphate receptor, and LAMP-1. Decreasing within the maturing phagosome were the FcII receptor, the mannose receptor, and alpha-adaptin. These results indicate that although the macrophage phagosomes total protein composition changes little during phagolysosome formation, the maturing phagosome both receives and eliminates, possibly by protein recycling, specific membrane and sequestered proteins.

The ubiquitin-mediated proteolytic pathway: mechanisms of recognition of the proteolytic substrate and involvement in the degradation of native cellular proteins.

Ubiquitin modification of a variety of protein targets within the cell plays important roles in many cellular processes. Among these are regulation of gene expression, regulation of cell cycle and division, involvement in the cellular stress response, modification of cell surface receptors, DNA repair, import of proteins into mitochondria, uptake of precursors into neurons, and biogenesis of mitochondria, ribosomes, and peroxisomes. The best studied modification occurs in the ubiquitin‐mediated proteolytic pathway. Degradation of a protein via the ubiquitin system involves two discrete steps. Initially, multiple ubiquitin molecules are covalently linked in an ATP‐dependent mode to the protein substrate. The targeted protein is then degraded by a specific and energy‐dependent high molecular mass protease into free amino acids, and free and reutilizable ubiquitin is released. In addition, stable mono‐ubiquitin adducts are also found in the cell, for example, those involving nucleosomal histones. Despite the considerable progress that has been made in elucidating the mode of action and roles of the ubiquitin system, many problems remain unsolved. For example, little is known on the signals that target proteins for degradation. Although mechanistic aspects of recognition via the N‐terminal residue have been studied thoroughly, it is clear that the vast majority of cellular proteins are targeted by other signals. The identity of the native cellular substrates of the system is another important, yet unresolved, problem: only few proteins have been recognized so far as substrates of the system in vivo. The scope of this review is to discuss the mechanisms involved in ubiquitin activation, selection of substrates for conjugation, and degradation of ubiquitin‐conjugated proteins in the cell‐free system. In addition, we shall summarize what is currently known of the physiological roles of ubiquitin‐mediated proteolysis in vivo.—Ciechanover, A.; Schwartz, A. L. The ubiquitin‐mediated proteolytice pathway: mechanisms of recognition of the proteolytic substrate and involvement in the degradation of native cellular proteins. FASEB J. 8: 182‐191; 1994.

Degradation of the E7 human papillomavirus oncoprotein by the ubiquitin-proteasome system: targeting via ubiquitination of the N-terminal residue

The E7 oncoprotein of the high risk human papillomavirus type 16 (HPV-16), which is etiologically associated with uterine cervical cancer, is a potent immortalizing and transforming agent. It probably exerts its oncogenic functions by interacting and altering the normal activity of cell cycle control proteins such as p21WAF1, p27KIP1 and pRb, transcriptional activators such as TBP and AP-1, and metabolic regulators such as M2-pyruvate kinase (M2-PK). Here we show that E7 is a short-lived protein and its degradation both in vitro and in vivo is mediated by the ubiquitin-proteasome pathway. Interestingly, ubiquitin does not attach to any of the two internal Lysine residues of E7. Substitution of these residues with Arg does not affect the ability of the protein to be conjugated and degraded; in contrast, addition of a Myc tag to the N-terminal but not to the C-terminal residue, stabilizes the protein. Also, deletion of the first 11 amino acid residues stabilizes the protein in cells. Taken together, these findings strongly suggest that, like MyoD and the Epstein Barr Virus (EBV) transforming Latent Membrane Protein 1 (LMP1), the first ubiquitin moiety is attached linearly to the free N-terminal residue of E7. Additional ubiquitin moieties are then attached to an internal Lys residue of the previously conjugated molecule. The involvement of E7 in many diverse and apparently unrelated processes requires tight regulation of its function and cellular level, which is controlled in this case by ubiquitin-mediated proteolysis.