Helen C. Ardley

E3 ubiquitin ligases

The selectivity of the ubiquitin-26 S proteasome system (UPS) for a particular substrate protein relies on the interaction between a ubiquitin-conjugating enzyme (E2, of which a cell contains relatively few) and a ubiquitin-protein ligase (E3, of which there are possibly hundreds). Post-translational modifications of the protein substrate, such as phosphorylation or hydroxylation, are often required prior to its selection. In this way, the precise spatio-temporal targeting and degradation of a given substrate can be achieved. The E3s are a large, diverse group of proteins, characterized by one of several defining motifs. These include a HECT (homologous to E6-associated protein C-terminus), RING (really interesting new gene) or U-box (a modified RING motif without the full complement of Zn2+-binding ligands) domain. Whereas HECT E3s have a direct role in catalysis during ubiquitination, RING and U-box E3s facilitate protein ubiquitination. These latter two E3 types act as adaptor-like molecules. They bring an E2 and a substrate into sufficiently close proximity to promote the substrates ubiquitination. Although many RING-type E3s, such as MDM2 (murine double minute clone 2 oncoprotein) and c-Cbl, can apparently act alone, others are found as components of much larger multi-protein complexes, such as the anaphase-promoting complex. Taken together, these multifaceted properties and interactions enable E3s to provide a powerful, and specific, mechanism for protein clearance within all cells of eukaryotic organisms. The importance of E3s is highlighted by the number of normal cellular processes they regulate, and the number of diseases associated with their loss of function or inappropriate targeting.

The Ubiquitin-conjugating Enzymes UbcH7 and UbcH8 Interact with RING Finger/IBR Motif-containing Domains of HHARI and H7-AP1

Ubiquitinylation of proteins appears to be mediated by the specific interplay between ubiquitin-conjugating enzymes (E2s) and ubiquitin-protein ligases (E3s). However, cognate E3s and/or substrate proteins have been identified for only a few E2s. To identify proteins that can interact with the human E2 UbcH7, a yeast two-hybrid screen was performed. Two proteins were identified and termed human homologue of Drosophila ariadne (HHARI) and UbcH7-associated protein (H7-AP1). Both proteins, which are widely expressed, are characterized by the presence of RING finger and in between RING fingers (IBR) domains. No other overt structural similarity was observed between the two proteins. In vitrobinding studies revealed that an N-terminal RING finger motif (HHARI) and the IBR domain (HHARI and H7-AP1) are involved in the interaction of these proteins with UbcH7. Furthermore, binding of these two proteins to UbcH7 is specific insofar that both HHARI and H7-AP1 can bind to the closely related E2, UbcH8, but not to the unrelated E2s UbcH5 and UbcH1. Although it is not clear at present whether HHARI and H7-AP1 serve, for instance, as substrates for UbcH7 or represent proteins with E3 activity, our data suggests that a subset of RING finger/IBR proteins are functionally linked to the ubiquitin/proteasome pathway.

UCH‐L1 aggresome formation in response to proteasome impairment indicates a role in inclusion formation in Parkinson's disease

Aggresomes are associated with many neurodegenerative disorders, including Parkinsons disease, and polyglutamine disorders such as Huntingtons disease. These inclusions commonly contain ubiquitylated proteins. The stage at which these proteins are ubiquitylated remains unclear. A malfunction of the ubiquitin/proteasome system (UPS) may be associated with their formation. Conversely, it may reflect an unsuccessful attempt by the cell to remove them. Previously, we demonstrated that overexpression of Parkin, a ubiquitin‐protein ligase associated with autosomal recessive juvenile Parkinsonism, generates aggresome‐like inclusions in UPS compromised cells. Mutations in the de‐ubiquitylating enzyme, UCH‐L1, cause a rare form of Parkinsonism. We now demonstrate that overexpression of UCH‐L1 also forms ribbon‐like aggresomes in response to proteasomal inhibition. Disease‐associated mutations, which affect enzymatic activities, significantly increased the number of inclusions. UCH‐L1 aggresomes co‐localized with ubiquitylated proteins, HSP70, γ‐tubulin and, to a lesser extent, the 20S proteasome and the chaperone BiP. Similar to Parkin inclusions, we found UCH‐L1 aggresomes to be surrounded by a tubulin rather than a vimentin cage‐like structure. Furthermore, UCH‐L1 aggregates with Parkin and α‐synuclein in some, but not all inclusions, suggesting the heterogeneous nature of these inclusion bodies. This study provides additional evidence that aggregation‐prone proteins are likely to recruit UPS components in an attempt to clear proteins from failing proteasomes. Furthermore, UCH‐L1 accumulation is likely to play a pathological role in inclusion formation in Parkinsons disease.

Proteomic analysis of increased Parkin expression and its interactants provides evidence for a role in modulation of mitochondrial function.

Parkin is an ubiquitin‐protein ligase (E3), mutations of which cause juvenile onset – autosomal recessive Parkinsons disease, and result in reduced enzymic activity. In contrast, increased levels are protective against mitochondrial dysfunction and neurodegeneration, the mechanism of which is largely unknown. In this study, 2‐DE and MS proteomic techniques were utilised to investigate the effects of increased Parkin levels on protein expression in whole cell lysates using in an inducible Parkin expression system in HEK293 cells, and also to isolate potential interactants of Parkin using tandem affinity purification and MS. Nine proteins were significantly differentially expressed (±2‐fold change; p<0.05) using 2‐DE analysis. MS revealed the identity of these proteins to be ACAT2, HNRNPK, HSPD1, PGK1, PRDX6, VCL, VIM, TPI1, and IMPDH2. The first seven of these were reduced in expression. Western blot analysis confirmed the reduction in one of these proteins (HNRNPK), and that its levels were dependent on 26S proteasomal activity. Tandem affinity purification/MS revealed 14 potential interactants of Parkin; CKB, DBT, HSPD1, HSPA9, LRPPRC, NDUFS2, PRDX6, SLC25A5, TPI1, UCHL1, UQCRC1, VCL, YWHAZ, YWHAE. Nine of these are directly involved in mitochondrial energy metabolism and glycolysis; four were also identified in the 2‐DE study (HSP60, PRDX6, TPI1, and VCL). This study provides further evidence for a role for Parkin in regulating mitochondrial activity within cells.

The aggravating role of the ubiquitin-proteasome system in neurodegeneration.

Association of protein inclusions or aggregates within brain tissues of patients with neurodegenerative disorders has been widely reported. These inclusions are commonly characterised both by the presence of ubiquitylated proteins and the sequestration of components of the ubiquitin–proteasome system (UPS). Such observations have led to the proposition that the UPS has a direct role in their formation. Indeed, the presence of ubiquitylated proteins and UPS components in inclusions may reflect unsuccessful attempts by the UPS to remove aggregating proteins. Whether the physical presence of inclusions causes cell death or, conversely, whether they are non‐toxic and their presence reflects a cellular protective mechanism remains highly controversial.

The Role of Ubiquitin-Protein Ligases in Neurodegenerative Disease

Alzheimer’s disease and Parkinson’s disease are the most common neurodegenerative conditions associated with the ageing process. The pathology of these and other neurodegenerative disorders, including polyglutamine diseases, is characterised by the presence of inclusion bodies in brain tissue of affected patients. In general, these inclusion bodies consist of insoluble, unfolded proteins that are commonly tagged with the small protein, ubiquitin. Covalent tagging of proteins with chains of ubiquitin generally targets them for degradation. Indeed, the ubiquitin/proteasome system (UPS) is the major route through which intracellular proteolysis is regulated. This strongly implicates the UPS in these disease-associated inclusions, either due to malfunction (of specific UPS components) or overload of the system (due to aggregation of unfolded/mutant proteins), resulting in subsequent cellular toxicity. Protein targeting for degradation is a highly regulated process. It relies on transfer of ubiquitin molecules to the target protein via an enzyme cascade and specific recognition of a substrate protein by ubiquitin-protein ligases (E3s). Recent advances in our knowledge gained from the Human Genome Mapping Project have revealed the presence of potentially hundreds of E3s within the human genome. The discovery that parkin, mutations in which are found in at least 50% of patients with autosomal recessive juvenile parkinsonism, is an E3 further highlights the importance of the UPS in neurological disease. To date, parkin is the only E3 confirmed to have a direct causal role in neurodegenerative disorders. However, a number of other (putative) E3s have now been identified that may cause disease directly or interact with neurological disease-associated proteins. Many of these are either lost or mutated in a given disease or fail to process disease-associated mutant proteins correctly. In this review, we will discuss the role(s) of E3s in neurodegenerative disorders.

Human homologue of ariadne promotes the ubiquitylation of translation initiation factor 4E homologous protein, 4EHP.

Human homologue of Drosophila ariadne (HHARI) is a RING‐IBR‐RING domain protein identified through its ability to bind the human ubiquitin‐conjugating enzyme, UbcH7. We now demonstrate that HHARI also interacts with the eukaryotic mRNA cap binding protein, translation initiation factor 4E homologous protein (4EHP), via the N‐terminal RING1 finger of HHARI. HHARI, 4EHP and UbcH7 do not form a stable heterotrimeric complex as 4EHP cannot immunoprecipitate UbcH7 even in the presence of HHARI. Overexpression of 4EHP and HHARI in mammalian cells leads to polyubiquitylation of 4EHP. By contrast, HHARI does not promote its own autoubiquitylation. Thus, by promoting the ubiquitin‐mediated degradation of 4EHP, HHARI may have a role in both protein degradation and protein translation.

Molecular analysis of the presenilin 1 (S182) gene in "sporadic" cases of Alzheimer's disease: identification and characterisation of unusual splice variants.

Abstract: Mutations of the presenilin 1 (PS‐1) gene at the Alzheimers disease (AD) FAD3 locus on chromosome 14q24.3 are responsible for the majority of familial early‐onset AD. As genes responsible for familial forms of AD are obvious candidates for further investigation in “sporadic” disease, we performed a molecular analysis of PS‐1 transcripts extracted from brain tissues of a series of histologically confirmed cases of “sporadic” AD (n = 10) and also from histologically “normal” (non‐Alzheimer) age‐matched brain controls (n = 5). No sequence changes in the PS‐1 coding sequence were detected after analysis by reverse transcription‐PCR. This suggests that the frequency of mutations in the PS‐1 (S182) coding region in “sporadic” Alzheimers disease is very low. However, we demonstrated that the PS‐1 gene is highly variably spliced. One splice variant involves the 5′ untranslated region of the PS‐1 gene only and hence encodes for normal PS‐1. Six further splice variants involve coding regions of the PS‐1 gene and result in truncated proteins lacking specific transmembrane domains. Most of these variants do not coincide with recognized sites of introns in the PS‐1 gene. One of these variants, resulting in the loss of transmembrane domain TM‐VII, was found only in an AD patient.

Ubiquitin-Protein Ligases - Novel Therapeutic Targets?

Intracellular protein degradation is a tightly regulated process that in many cases is controlled by protein ubiquitylation. The ubiquitin pathway is a major route by which cells not only remove normal proteins at the appropriate time but also abnormally folded normal or mutant, cytoplasmic and membrane, proteins. This has led to a major impetus to identify constituents of the pathway. The key components that regulate substrate ubiquitylation are the ubiquitin-protein ligases. Ligases come in many forms, from single proteins to very large multiprotein complexes. Specificity of targeting can be modulated by the requirement for post-translational modifications such as phosphorylation, hydroxylation or oxidation of the substrate and, in some cases, the ligase itself. The requirement for substrate modification prior to ubiquitylation allows the same ligase to target different substrates within the same cell at different times. Abnormal intracellular protein processing is a common feature of many human diseases including neurodegenerative diseases and cancer. It may not represent the causative factor that initiates the disease process but may be a downstream regulator of the toxic effect. These abnormalities often arise from the loss of a key protein-protein interaction. As a consequence, mutated proteins can have very different half-lives from their normal counterparts. This can affect the levels of their activity and/or lead to the formation of protein aggregates (inclusion bodies/aggresomes). In this review, we aim to highlight examples of diseases where abnormal protein ubiquitylation is proposed to be a key regulator of the disease process. The recent success of the proteasome inhibitor Bortezomib (PS-341) for treatment of relapsed, refractory myeloma suggests that the modulation of individual ubiquitin-protein ligase activities with synthetic agents may represent a novel approach that has enormous potential for the treatment of a wide range of diseases.

The aggravating role of the ubiquitin-proteasome system in neurodegenerative disease.

Intraneuronal inclusion bodies are key pathological features of most age-related neurodegenerative disorders including Parkinsons disease and Alzheimers disease. These inclusions are commonly characterized both by the presence of ubiquitinated proteins and the sequestration of components of the UPS (ubiquitin-proteasome system). Unfortunately, as we age, the efficiency of the UPS declines, suggesting that the presence of ubiquitinated proteins and UPS components in inclusions may reflect unsuccessful attempts by the (failing) UPS to remove the aggregating proteins. Whether the physical presence of inclusions causes cell death or, conversely, whether they are non-toxic and their presence reflects a cellular protective mechanism remains highly controversial. Animal and in vitro model systems that allow detailed characterization of the inclusions and their effects on the cell have been developed by us and others. Identification of the mechanisms involved in inclusion formation is already aiding the development of novel therapeutic strategies to prevent or alleviate aggregate-associated neurodegenerative diseases.