R. John Mayer

Ubiquitin and ubiquitin-like proteins as multifunctional signals.

Protein ubiquitylation is a recognized signal for protein degradation. However, it is increasingly realized that ubiquitin conjugation to proteins can be used for many other purposes. Furthermore, there are many ubiquitin-like proteins that control the activities of proteins. The central structural element of these post-translational modifications is the ubiquitin superfold. A common ancestor based on this superfold has evolved to give various proteins that are involved in diverse activities in the cell.

Ubiquitin-like protein conjugation and the ubiquitin–proteasome system as drug targets

The ubiquitin–proteasome system (UPS) and ubiquitin-like protein (UBL) conjugation pathways are integral to cellular protein homeostasis. The growing recognition of the fundamental importance of these pathways to normal cell function and in disease has prompted an in-depth search for small-molecule inhibitors that selectively block the function of these pathways. However, our limited understanding of the molecular mechanisms and biological consequences of UBL conjugation is a significant hurdle to identifying drug-like inhibitors of enzyme targets within these pathways. Here, we highlight recent advances in understanding the role of some of these enzymes and how these new insights may be the key to developing novel therapeutics for diseases including immuno-inflammatory disorders, cancer, infectious diseases, cardiovascular disease and neurodegenerative disorders.

Reduced stability of retinoblastoma protein by gankyrin, an oncogenic ankyrin-repeat protein overexpressed in hepatomas.

Hepatocellular carcinoma (HCC) is one of the most common cancers in Asia and Africa, where hepatitis virus infection and exposure to specific liver carcinogens are prevalent. Although inactivation of some tumor suppressor genes such as p53 and p16INK4Ahas been identified, no known oncogene is commonly activated in hepatocellular carcinomas. Here we have isolated genes overexpressed in hepatocellular carcinomas by cDNA subtractive hybridization, and identified an oncoprotein consisting of six ankyrin repeats (gankyrin). The expression of gankyrin was increased in all 34 hepatocellular carcinomas studied. Gankyrin induced anchorage-independent growth and tumorigenicity in NIH/3T3 cells. Gankyrin bound to the product of the retinoblastoma gene (RB1), increasing its phosphorylation and releasing the activity of the transcription factor E2F-1. Gankyrin accelerated the degradation of RB1 in vitro and in vivo, and was identical to or interacted with a subunit of the 26S proteasome. These results demonstrate the importance of ubiquitin–proteasome pathway in the regulation of cell growth and oncogenic transformation, and indicate that gankyrin overexpression contributes to hepatocarcinogenesis by destabilizing RB1.

Depletion of 26S Proteasomes in Mouse Brain Neurons Causes Neurodegeneration and Lewy-Like Inclusions Resembling Human Pale Bodies

Ubiquitin-positive intraneuronal inclusions are a consistent feature of the major human neurodegenerative diseases, suggesting that dysfunction of the ubiquitin proteasome system is central to disease etiology. Research using inhibitors of the 20S proteasome to model Parkinsons disease is controversial. We report for the first time that specifically 26S proteasomal dysfunction is sufficient to trigger neurodegenerative disease. Here, we describe novel conditional genetic mouse models using the Cre/loxP system to spatially restrict inactivation of Psmc1 (Rpt2/S4) to neurons of either the substantia nigra or forebrain (e.g., cortex, hippocampus, and striatum). PSMC1 is an essential subunit of the 26S proteasome and Psmc1 conditional knock-out mice display 26S proteasome depletion in targeted neurons, in which the 20S proteasome is not affected. Impairment of specifically ubiquitin-mediated protein degradation caused intraneuronal Lewy-like inclusions and extensive neurodegeneration in the nigrostriatal pathway and forebrain regions. Ubiquitin and α-synuclein neuropathology was evident, similar to human Lewy bodies, but interestingly, inclusion bodies contained mitochondria. We support this observation by demonstrating mitochondria in an early form of Lewy body (pale body) from Parkinsons disease patients. The results directly confirm that 26S dysfunction in neurons is involved in the pathology of neurodegenerative disease. The model demonstrates that 26S proteasomes are necessary for normal neuronal homeostasis and that 20S proteasome activity is insufficient for neuronal survival. Finally, we are providing the first reproducible genetic platform for identifying new therapeutic targets to slow or prevent neurodegeneration.

Ubiquitin in Neurodegenerative Diseases

Immunochemical staining to detect ubiquitin has become an essential technique in evaluating neurodegenerative processes. Age related staining is seen in myelin, in nerve processes in lysosome‐related dense bodies, and in corpora amylacea. There is a constant association between filamentous inclusions and the presence of ubiquitin. Intermediate filaments associated with ubiquitin, α B crystallin and enzymes of the ubiquitin pathway are the basis of Lewy bodies and Rosenthal fibres, as well as related bodies outside the nervous system. Neurofibrillary tangles in diverse diseases are associated with ubiquitin as are several other tau containing inclusions in both neurones and glia. Inclusions in motor neurones and non‐motor cortex characterizing amyotrophic lateral sclerosis (ALS) and certain related forms of frontal lobe dementia can only be readily detected by anti‐ubiquitin. Anti‐ubiquitin also identifies both filamentous and lysosomal structures in neuronal processes as well as in some swollen neurones. Involvement of ubiquitin‐containing elements of the lysosomal system appears important in pathogenesis of prion encephalopathies. Despite great advances in understanding cell biology of the ubiquitin pathway there are as yet few insights into the precise role played by ubiquitin in neuronal disease.

Neurofibrillary tangles of Alzheimer's disease brains contain 14-3-3 proteins☆

The localisation of 14-3-3 proteins compared to that of tau and ubiquitin-protein conjugates in sections of hippocampus from Alzheimers disease (AD) brains was examined by immunohistochemistry. In all cases (n = 10), anti-14-3-3 stained a proportion of neurofibrillary tangles (NFT). In general, NFT stained by anti-14-3-3 were smaller than those stained by anti-tau or anti-ubiquitin-protein conjugates and were more confined to the neuronal cell body. Occasionally, cortical Lewy bodies in cases of Lewy body dementia were also found to be 14-3-3-positive. Since 14-3-3 proteins are central to MAP kinase signalling, the results support the proposal that this pathway is in part responsible for the hyperphosphorylation of tau, which leads to the formation of the paired helical filaments seen in AD brains.

Assembly, structure, and function of the 26S proteasome

The 26S proteasome is a large multiprotein complex involved in the regulated degradation of ubiquitinated proteins in the cell. The 26S proteasome has been shown to control an increasing number of essential biochemical mechanisms of the cellular lifecycle including DNA synthesis, repair, transcription, translation, and cell signal transduction. Concurrently, it is increasingly seen that malfunction of the ubiquitin proteasome system contributes to the pathogenesis of disease. The recent identification of four molecular chaperones, in addition to five previously identified chaperones, have provided mechanistic insight into how this cellular megastructure is assembled in the cell. These data, together with new insights into the structure and function of the proteasome, provide a much better understanding of this complex protease.

Gankyrin Is an Ankyrin-repeat Oncoprotein That Interacts with CDK4 Kinase and the S6 ATPase of the 26 S Proteasome

A yeast two-hybrid screen with the human S6 (TBP7, RPT3) ATPase of the 26 S proteasome has identified gankyrin, a liver oncoprotein, as an interacting protein. Gankyrin interacts with both free and regulatory complex-associated S6 ATPase and is not stably associated with the 26 S particle. Deletional mutagenesis shows that the C-terminal 78 amino acids of the S6 ATPase are necessary and sufficient to mediate the interaction with gankyrin. Deletion of an orthologous gene in Saccharomyces cerevisiae suggests that it is dispensable for cell growth and viability. Overexpression and precipitation of tagged gankyrin from cultured cells detects a complex containing co-transfected tagged S6 ATPase (or endogenous S6) and endogenous cyclin D-dependent kinase CDK4. The proteasomal ATPases are part of the AAA (ATPases associated with diverse cellular activities) family, members of which are molecular chaperones; gankyrin complexes may therefore influence CDK4 function during oncogenesis.

Ubiquitinated protein conjugates are specifically enriched in the lysosomal system of fibroblasts

Ubiquitin‐protein conjugates are found by imniunogold electron microscopy to be enriched (12‐fold) in the lysosomal compartment of 3T3‐L1 fibroblasts. Treatment of fibroblasts with the cysteine protease inhibitor E‐64 leads to an expansion of the lysosomal compartment and as a result an increase in the cellular content of ubiquitin‐protein conjugates. There is no change in the specific enrichment of ubiquitin‐protein conjugates in the lysosomal compartment following E‐64 treatment. The results suggest that some ubiquitin‐protein conjugates may normally be degraded lysosomally following sequestration by microautophagy and imply that protein ubiquitination may be one of the signals for protein uptake into lysosomes.

The ubiquitin protein catabolic disorders

The ubiquitin–proteasome system of intracellular proteolysis is essential for cell viability. We propose the concept that neurodegenerative diseases such as Alzheimers and Parkinsons, as well as other conditions including some types of cancer, collectively represent a raft of ‘ubiquitin protein catabolic disorders’ in which altered function of the ubiquitin–proteasome system can cause or directly contribute to disease pathogenesis. Genetic abnormalities within the ubiquitin pathway, either in ubiquitin‐ligase (E3) enzymes or in deubiquitinating enzymes, cause disease because of problems associated with substrate recognition or supply of free ubiquitin, respectively. In some cases, mutations in protein substrates of the ubiquitin–proteasome system may directly contribute to disease progression because of inefficient substrate recognition. Mutations in transcripts for the ubiquitin protein itself (as a result of ‘molecular misreading’) also affect ubiquitin‐dependent proteolysis with catastrophic consequences. This has been shown in Alzheimers disease and could apply to other age‐associated neurodegenerative conditions. Within the nervous system, accumulation of unwanted proteins as a result of defective ubiquitin‐dependent proteolysis may contribute to aggregation events, which underlie the pathogenesis of several major human neurodegenerative diseases.