Biogenesis Of Yeast Proteasomes

The question is how 2 x 14 different subunits incorporate into the right position of such a complex structure as the eukaryotic 20S proteasome. 20S pro-teasomes with low complexity exist in archaeons and eubacteria, which harbour one or two different a and P subunits. Their subunits could be expressed as recombinant proteins in E. coli. This approach allowed to identify assembly intermediates and finally the reconstitution of the mature particle. Based on these studies two models arose which...

Introduction

In January 1998, a news summary in the journal Science proclaimed Possible New Cause of Alzheimer's Disease Found. The headline referred to a report by a team led by Fred van Leeuwen, at the Netherlands Institute for Brain Research, that aberrant forms of ubiquitin and amyloid precursor protein, so called +1 proteins, could be detected in the pathological lesions that characterise the Alzheimer's disease brain1. The mutant proteins appeared to result from mistakes during protein synthesis, by a...

The Fate Of Inclusions In Cultured Neurons

Equally important as the question of how and why inclusions are formed, is the question of their fate within the cell, or neuron, once they are formed. Once formed, are inclusions permanent structures Does their ultrastructure change If inclusions are, in fact, removed from the cell, what is the mechanism for their removal This is a relatively new area of research, but recent data suggest that the autophagic lysosomal system, the other major cellular system devoted to the removal of proteins,...

Benefits of Studies in Yeast for Neurodegenerative Diseases

Until now, we are far away from understanding how proteasome biogenesis is regulated in different compartments. To elucidate the structure, the assembly and activation of 26S proteasomes in different compartments will be a challenge in the future. The understanding of disfunctions in proteasome activation may be a prerequisite to improve the design of a new generation of drugs that cure neurodegenerative diseases. Combined therapies selective for several decisive events that characterize these...

Oxidative Stress and Lipoxygenases

Oxidative Stress

Another source of oxidative stress associated with arachidonic acid signaling in the CNS is the lypoxygenase (LOX) pathway. Although eicosanoid Figure 10. Synthesis pathway of cyclopentenone prostaglandins of the J2 series. Reproduced with permission from (29). synthesis in the CNS involves cyclooxygenase as well as lypoxygenase pathways, much less is known about the contribution of the latter to oxidative stress induced by neuroinflammation. LOXs are a family of monomeric non-heme, non-sulfur...

The Preferred Peptide Bond Cleavage Sites and Inhibition

The peptide cleavage preference of the P5 subunit descends from the archetype proteasome P-subunit which confers chymotrypsin-like activity to ancestral 20S proteasomes (7). Biochemical studies using chromogenic or fluoro-genic peptide substrates and specific inhibitors revealed the cleavage preference of eukaryotic proteasomes at the carboxyl group of large hydrophobic amino acid residues. In these assays the endopeptidase hydrolyses the amide bond between the carbonyl group of an amino acid...

Genetic Activators of Protein Degradation by the UPS

Although the focus of this section is on inhibitors of the UPS we want to discuss a very interesting chemical genetic approach to selectively increase the degradation of UPS substrates. This strategy enables the design and synthesis of molecules that will specifically bind to selected proteins in vivo and target them for degradation by the UPS. (16). PROteolysis TArgeting Chimeric molecule S (PROTACS) are heterobifunctional molecules that comprise a ligand for the target protein, a linker...

The 19S Regulatory Complex

The 19S regulatory complex is required for recognition of the bulk polyu-biquitylated substrates. It is composed of about eighteen subunits, which were assigned to two subcomplexes, the base and the lid. With few exceptions protea-somal subunits are encoded by essential genes in yeast. Thus, each subunit plays an important role in the proteolytic scenario. However, the functions of most subunits of the regulatory complex are still unknown (30)(Fig. 3). Figure 3. The 26S proteasome is formed by...

Clinical And Neuropathological Hallmarks Of Huntingtons Disease

Huntington's disease (HD) is a devastating autosomal dominant neurodegenerative disorder caused by a CAG triplet-repeat expansion coding for a poly-glutamine (polyQ) sequence in the N-terminal region of the huntingtin (htt) protein (1). The causing mutation is fully penetrating but age of onset and clinical manifestations may vary considerably amongst mutation carriers. Typically, patients suffer from motor dysfunction, cognitive decline and psychological disturbances striking at about age 40...

Parallels Between The Aggresome And Lewy Bodies

Several similarities have been pointed out between aggresomes and Lewy bodies (LB), the hallmark pathologic features of Parkinson's disease and of dementia with LBs. In cellular models, the aggresomes that result from the overexpression of parkin or synphilin-1, for example, accompanied with proteasomal inhibition, have both morphologic and immunocytochemical characteristics of LBs, including the core and halo organization and the presence of vimentin, y-tubulin, a-synuclein, synphilin-1,...

Characteristics Of Aggresomes Formed By aSynuclein And Synphilin1

Aggresomes can form efficiently in 293 cells upon the over-expression of synphilin-1 (9,27). This protein, originally isolated by virtue of its interaction with a-synuclein (28), is a presynaptic molecule associated with synaptic vesicles (29) and is present in LBs, especially in the core region (30). Co-expression of a-synuclein and synphilin-1 in cellular models gives rise to eosinophilic cytoplasmic inclusions (28), and over-expression of synphilin-1 alone can also produce inclusions in...

Inhibitors Of The Proteasome

The available proteasome inhibitors and their properties will be briefly discussed because they are important not only as probes for the study of the UPS but also as potential therapeutic agents for human disease. A detailed report of proteasome inhibitors is available in three excellent reviews (9-11). The three peptidase activities associated with the proteasome belong to the class of N-terminal nucleophilic hydrolases which, in the case of the protea-some, use the side chains of their...

UCHL1 and Ubiquitin Stabilization

Uchl1 Inhibitor

UCH-L1 is a small but abundant protein in neurons. Although the role of UCH-L1 in vivo remains unclear, its abundance and neuron-specific expression suggest a role in neuronal function. Although it has been established that deletion of Uchll in mice causes gracile axonal dystrophy, Osaka (2003) recently reported a novel in vivo role for UCH-L1 in Ub homeostasis, namely that UCH-L1 functions as a Ub carrier protein (42). These data show that UCH-L1 is associated with Ub in neurons and suggest...

Parkin In The Genetics Of Parkinsons Disease

Until about a decade ago, PD was thought to have little or no genetic component. However, the identification of several genes for monogenically inherited forms of the disease, although rare, and accounting for only 5-10 of the cases, has accelerated the study of molecular pathways leading to PD (13). Mutations in at least five genes have been strongly linked to autosomal dominant or recessive forms of PD, including a-synuclein, parkin, DJ-1, PTEN-induced kinase 1 (PINK1), and Leucine-rich...

Inflammation Regulator with Deubiquitinating and Ubiquitin Ligase Properties

The zinc-f nger protein A20 is a regulator of inflammation and cell survival by preventing NFkB activation and apoptosis. A20-deficient mice exhibit chronic inflammation and cell death because of failure to inhibit NFkB transcriptional activity (55). A20 was recently shown to be a de-ubiquitinating enzyme (56). The amino-terminal domain of A20, which is a de-ubiquitinating enzyme of the OTU (ovarian tumor) family, removes K63-linked ubiquitin chains from the receptor interacting protein (RIP),...

The Role Of The Proteasome

In non-dividing cells, such as the great majority of neurons in the adult brain, the protein content of cells is approximately constant. Since protein synthesis is continuous, it must be matched by an equal rate of protein degradation. Cellular proteins can be degraded by the lysosomal system, but a system of greater importance to the normal functioning of nervous system is the proteasome, which is described in detail elsewhere in this volume. The ubiquitin-proteasome system is essential to the...

Inflammation and UCHL1 Activity

Some of the products of inflammation, such as the neurotoxic PGD2 and its metabolites PGJ2, A12-PGJ2 and 15d-PGJ2, directly affect the activity of components of the UPS leading to a raise in the levels of ubiquitinated proteins in neuronal cells (27). Although one study reported the binding of biotinylated 15d-PGJ2 to the proteasome (44), PGD2 and its metabolites appear to not affect directly the activities of the 26S or 20S proteasomes (42 45). Instead, A12-PGJ2 was found to inhibit the...

The Relation between Ubiquitin Ligase Activity of UCHL1 and PD

The simplest explanation for the genetic association of UCH-L1 variants to PD is that S18Y is protective because its effect on UCH-L1 hydrolytic activity is opposite to that of the I93M mutation. However, this explanation is inconsistent with the predicted location of residue 18 on the protein surface, distal to the active site and the Ub binding site (Figure 2) (30,31). Furthermore, the fact that position 18 is one of only a few residues that are not conserved between human and other mammals...

References

Imanishi, T., Itoh, T., Suzuki, Y., O'Donovan, C., Fukuchi, S., Koyanagi, K. O., Barrero, R. A., Tamura, T., Yamaguchi-Kabata, Y., Tanino, M., Yura, K., Miyazaki, S., Ikeo, K., Homma, K., Kasprzyk, A., Nishikawa, T., Hirakawa, M., Thierry-Mieg, J., Thierry-Mieg, D., Ashurst, J., Jia, L., Nakao, M., Thomas, M. A., Mulder, N., Karavidopoulou, Y, Jin, L., Kim, S., Yasuda, T., Lenhard, B., Eveno, E., Suzuki, Y., Yamasaki, C., Takeda, J., Gough, C., Hilton, P., Fujii, Y., Sakai, H., Tanaka, S.,...

Oxidative Modifications and Reduction of UCHL1 Hydrolase Activity in PD

Oxidative stress is another important factor that has been implicated in the pathogenesis of a number of age-related neurodegenerative diseases, including PD and AD. UCH-L1 is a member of the papain-like cysteine protease family, having conserved Cys and His residues within the active site. Certain cysteine proteases are known targets for covalent modification during cellular oxidative stress (32), and several lines of evidence implicate this phenomenon in sporadic PD (33). One of the...

The Use of Proteasome Inhibitors in Animal Models

The effect of proteasome inhibitors on the brain of intact animals has recently been explored. Initial studies with the administration of the tea polyphenol epigallocatechin-3gallate into the stomach of male and female mice, demonstrated a wide organ distribution including the brain (24). Acute systemic administration of PSI Z-Ile-Glu(OtBu)-Ala-Leu-al to ovariectomized adult female rats increased progesterone receptor levels in the preoptic area and hippocampus but not in the frontal cortex...

Biogenesis Of PrPC

In order to understand the replication and pathogenic potential of PrPSc, much attention has been focused on PrPC, the immediate substrate of PrPSc. At the outset, the biogenesis of PrPC appears quite mundane. PrPC is a glycoprotein linked to the cell surface by a glycosylphosphatidyl inositol (GPI) anchor. Like other secretory proteins, PrPC is synthesized in the endoplasmic reticulum, where the N-terminal signal peptide is cleaved co-translationally, and the C-terminal GPI signal peptide is...

Genetic Manipulation of Proteasome Activity in Animal Models

To investigate changes in UPS activity in in vivo paradigms, several transgenic mice were developed expressing a variety of reporter proteins such as ubiquitin-luciferase (34) and GFP with a constitutively active degradation signal (35). These reporter proteins are rapidly degraded under homeostatic conditions and stabilized in a time- and dose-dependent manner in response to proteasome inhibitors. A hexahistidine-tagged ubiquitin-GFP transgenic mouse was developed to facilitate the analysis of...

Proteasome Function In Human Lewy Body Diseases

There is also evidence of proteasome dysfunction in human diseases that prominently include a-synuclein pathology. McNaught and Jenner were the first to demonstrate that proteasome activity is lower in the substantia nigra of postmortem tissue taken from PD patients compared to controls (37). A difficulty with this study is that tissue from the substantia nigra was used. One can argue that this is the most relevant tissue as it is the region of the brain that is most prominently affected in PD....