The mechanism of cell death in prion disease is unknown but is associated with the production of a misfolded conformer of the prion protein. We report that disease-associated prion protein specifically inhibits the proteolytic beta subunits of the 26S proteasome. Using reporter substrates, fluorogenic peptides, and an activity probe for the beta subunits, this inhibitory effect was demonstrated in pure 26S proteasome and three different cell lines. By challenge with recombinant prion and other amyloidogenic proteins, we demonstrate that only the prion protein in a nonnative beta sheet conformation inhibits the 26S proteasome at stoichiometric concentrations. Preincubation with an antibody specific for aggregation intermediates abrogates this inhibition, consistent with an oligomeric species mediating this effect. We also present evidence for a direct relationship between prion neuropathology and impairment of the ubiquitin-proteasome system (UPS) in prion-infected UPS-reporter mice. Together, these data suggest a mechanism for intracellular neurotoxicity mediated by oligomers of misfolded prion protein.

The proteasome is an essential evolutionary conserved protease involved in many regulatory systems. Here, we describe the synthesis and characterization of the activity-based, fluorescent, and cell-permeable inhibitor Bodipy TMR-Ahx(3)L(3)VS (MV151), which specifically targets all active subunits of the proteasome and immunoproteasome in living cells, allowing for rapid and sensitive in-gel detection. The inhibition profile of a panel of commonly used proteasome inhibitors could be readily determined by MV151 labeling. Administration of MV151 to mice allowed for in vivo labeling of proteasomes, which correlated with inhibition of proteasomal degradation in the affected tissues. This probe can be used for many applications ranging from clinical profiling of proteasome activity, to biochemical analysis of subunit specificity of inhibitors, and to cell biological analysis of the proteasome function and dynamics in living cells.

Impairment of the ubiquitin/proteasome system has been proposed to play a role in neurodegenerative disorders such as Alzheimer and Parkinson diseases. Although recent studies confirmed that some disease-related proteins block proteasomal degradation, and despite the existence of excellent animal models of both diseases, in vivo data about the system are lacking. We have developed a model for in vivo analysis of the ubiquitin/proteasome system by generating mouse strains transgenic for a green fluorescent protein (GFP) reporter carrying a constitutively active degradation signal. Administration of proteasome inhibitors to the transgenic animals resulted in a substantial accumulation of GFP in multiple tissues, confirming the in vivo functionality of the reporter. Moreover, accumulation of the reporter was induced in primary neurons by UBB+1, an aberrant ubiquitin found in Alzheimer disease. These transgenic animals provide a tool for monitoring the status of the ubiquitin/proteasome system in physiologic or pathologic conditions.

The presence of endoplasmic reticulum (ER) stress and impaired ubiquitin-proteasome system (UPS) activity has been independently implicated in the pathophysiology of conformational diseases. Here, we reveal a link between ER stress and the functionality of the UPS. Treatment of cells with different ER stressors delayed the degradation of an ER reporter substrate and caused a subtle but consistent accumulation of three independent nuclear/cytosolic UPS reporter substrates. A similar signature increase was observed upon induction of ER stress in transgenic mice expressing a reporter substrate. Cells undergoing ER stress failed to clear efficiently UBB+1, an aberrant ubiquitin found in conformational diseases, which in turn caused general impairment of the UPS. We conclude that ER stress has a general inhibitory effect on the UPS. The compromised UPS during ER stress may explain the long-term gradual accumulation of misfolded proteins as well as the selective vulnerability of particular cell populations in conformational diseases.

The dynamic behavior of proteins is critical for cellular homeostasis. However, analyzing dynamics of proteins and protein complexes in vivo has been difficult. Here we describe recombination-induced tag exchange (RITE), a genetic method that induces a permanent epitope-tag switch in the coding sequence after a hormone-induced activation of Cre recombinase. The time-controlled tag switch provides a unique ability to detect and separate old and new proteins in time and space, which opens up opportunities to investigate the dynamic behavior of proteins. We validated the technology by determining exchange of endogenous histones in chromatin by biochemical methods and by visualizing and quantifying replacement of old by new proteasomes in single cells by microscopy. RITE is widely applicable and allows probing spatiotemporal changes in protein properties by multiple methods.chromatin | histone | proteasome | protein dynamics | turnover P roteins are dynamic molecules. Their abundance is controlled by synthesis and degradation and they can be subject to posttranslational processing, modification, and demodification. In addition, most proteins are very mobile and undergo interactions with multiple other protein partners (1-4). However, little is known about the dynamics of proteins within macromolecular complexes in vivo (2, 4). Studying time-dependent changes in physical properties of proteins or protein turnover requires methods to distinguish resident (old) proteins from new proteins. Current methods that do so are usually based on fluorescent reporters or differential chemical labeling. For example, fluorescence recovery after photo bleaching relies on exchange of the old bleached protein by nonbleached proteins (1, 3, 4). Alternative methods involve time-dependent changes in fluorescence, nonspecific pulse-chase labeling of proteins with labeled amino acids, or labeling with chemical dyes that specifically bind to short tags (5-7). Although suitable for detection of proteins by microscopy or mass spectrometry, a limitation of these methods is that they do not provide a handle for biochemical analysis of old and new proteins and their complexes. To solve this problem and to eliminate the requirement for chemical labels or UV light we developed recombination-induced tag exchange (RITE), a method in which a genetic epitope tag is switched by transient induction of a site-specific recombinase. As a consequence, old and newly synthesized proteins are differentially tagged, which enables monitoring of protein dynamics by multiple techniques, as illustrated here. In contrast to inducible expression strategies (8-12), differential tagging by a time-controlled site-specific protease (13), or the labeling methods described above, RITE allows parallel detection and purification of old and new proteins under physiological conditions and over long periods of time.We used RITE to probe the stability of chromatin. Photobleaching experiments using histones tagged with fluorescent reporters suggest that chromatin is a static comp...

Conditions causing an increase in misfolded or aberrant proteins can impair the activity of the ubiquitin/ proteasome system (UPS). This observation is of particular interest, given the fact that proteotoxic stress is closely associated with a large variety of disorders. Although impairment of the UPS appears to be a general consequence of proteotoxic insults, the underlying mechanisms remain enigmatic. Here, we show that heat shock-induced proteotoxic stress resulted in conjugation of ubiquitin to detergent-insoluble protein aggregates, which coincided with reduced levels of free ubiquitin and impediment of ubiquitin-dependent proteasomal degradation. Interestingly, whereas soluble proteasome substrates returned to normal levels after a transient accumulation, the levels of an aggregation-prone substrate remained high even when the free ubiquitin levels were restored. Consistently, overexpression of ubiquitin prevented accumulation of soluble but not aggregation-prone substrates in thermally stressed cells. Notably, cells were also unable to resume degradation of aggregation-prone substrates after treatment with the translation inhibitor puromycin, indicating that selective accumulation of aggregation-prone proteins is a consistent feature of proteotoxic stress. Our data suggest that the failure of the UPS to clear aggregated proteins in the aftermath of proteotoxic stress episodes may contribute to the selective deposition of aggregation-prone proteins in conformational diseases.

Nucleosome composition actively contributes to chromatin structure and accessibility. Cells have developed mechanisms to remove or recycle histones, generating a landscape of differentially aged nucleosomes. This study aimed to create a highresolution, genome-wide map of nucleosome turnover in Schizosaccharomyces pombe. The recombination-induced tag exchange (RITE) method was used to study replication-independent nucleosome turnover through the appearance of new histone H3 and the disappearance or preservation of old histone H3. The genome-wide location of histones was determined by chromatin immunoprecipitation-exonuclease methodology (ChIP-exo). The findings were compared with diverse chromatin marks, including histone variant H2A.Z, post-translational histone modifications, and Pol II binding. Finally, genomewide mapping of the methylation states of H4K20 was performed to determine the relationship between methylation (mono, di, and tri) of this residue and nucleosome turnover. Our analysis showed that histone recycling resulted in low nucleosome turnover in the coding regions of active genes, stably expressed at intermediate levels. High levels of transcription resulted in the incorporation of new histones primarily at the end of transcribed units. H4K20 was methylated in lowturnover nucleosomes in euchromatic regions, notably in the coding regions of long genes that were expressed at low levels. This transcription-dependent accumulation of histone methylation was dependent on the histone chaperone complex FACT. Our data showed that nucleosome turnover is highly dynamic in the genome and that several mechanisms are at play to either maintain or suppress stability. In particular, we found that FACT-associated transcription conserves histones by recycling them and is required for progressive H4K20 methylation.

Following mitosis, daughter cells must inherit a functional set of essential proteins and organelles. We applied a genetic tool to simultaneously monitor the kinetics and distribution of old and new proteins marking all intracellular compartments in budding yeasts. Most organelles followed a general pattern whereby preexisting proteins are symmetrically partitioned followed by template-based incorporation of new proteins. Peroxisomes belong to this group, supporting a model of biogenesis by growth and division from preexisting peroxisomes. We detected two exceptions: the nuclear pore complex (NPC) and the spindle pole body (SPB). Old NPCs are stably inherited during successive generations but remained separated from new NPCs, which are incorporated de novo in mother and daughter cells. Only the SPB displayed asymmetrical distribution, with old components primarily inherited by daughter cells and new proteins equally incorporated in both cells. Our analysis resolves conflicting models (peroxisomes, NPC) and reveals unique patterns (NPC, SPB) of organelle inheritance.Saccharomyces cerevisiae | protein dynamics | nuclear envelope | centrosome | live-cell imaging