The Alzheimer's disease (AD)-associated ubiquilin-1 regulates proteasomal degradation of proteins, including presenilin (PS). PS-dependent γ-secretase generates β-amyloid (Aβ) peptides, which excessively accumulate in AD brain. Here, we have characterized the effects of naturally occurring ubiquilin-1 transcript variants (TVs) on the levels and subcellular localization of PS1 and other γ-secretase complex components and subsequent γ-secretase function in human embryonic kidney 293, human neuroblastoma SH-SY5Y and mouse primary cortical cells. Full-length ubiquilin-1 TV1 and TV3 that lacks the proteasome-interaction domain increased fulllength PS1 levels as well as induced accumulation of high-molecular-weight PS1 and aggresome formation. Accumulated PS1 colocalized with TV1 or TV3 in the aggresomes. Electron microscopy indicated that aggresomes containing TV1 or TV3 were targeted to autophagosomes. TV1-and TV3-expressing cells did not accumulate other unrelated proteasome substrates, suggesting that the increase in PS1 levels was not because of a general impairment of the ubiquitinproteasome system. Furthermore, PS1 accumulation and aggresome formation coincided with alterations in Aβ levels, particularly in cells overexpressing TV3. These effects were not related to altered γ-secretase activity or PS1 binding to TV3. Collectively, our results indicate that specific ubiquilin-1 TVs can cause PS1 accumulation and aggresome formation, which may impact AD pathogenesis or susceptibility.

Aggregation-prone proteins have been suggested to overwhelm and impair the ubiquitin/proteasome system (UPS) in polyglutamine (polyQ) disorders, such as Huntington's disease (HD). Overexpression of an N-terminal fragment of mutant huntingtin (NmutHtt), an aggregation-prone polyQ protein responsible for HD, obstructs the UPS in cellular models. Furthermore, based on the accumulation of polyubiquitin conjugates in brains of R6/2 mice, which express human N-mutHtt and are one of the most severe polyQ disorder models, it has been proposed that UPS dysfunction is a consistent feature of this pathology, occurring in both in vitro and in vivo models. Here, we have exploited transgenic mice that ubiquitously express a ubiquitin fusion degradation proteasome substrate to directly assess the functionality of the UPS in R6/2 mice or the slower onset R6/1 mice. Although expression of N-mutHtt caused a general inhibition of the UPS in PC12 cells, we did not observe an increase in the levels of proteasome reporter substrate in the brains of R6/2 and R6/1 mice. We show that the increase in ubiquitin conjugates in R6/2 mice can be primarily attributed to an accumulation of large ubiquitin conjugates that are different from the conjugates observed upon UPS inhibition. Together our data show that polyubiquitylated proteins accumulate in R6/2 brain despite a largely operative UPS, and suggest that neurons are able to avoid or compensate for the inhibitory effects of N-mutHtt.Huntington ͉ neurodegeneration ͉ protein degradation T he primary proteolytic machinery responsible for the turnover of proteins in the cytosol and nuclei of cells, including the destruction of misfolded or otherwise abnormal proteins, is the ubiquitin/proteasome system (UPS) (1). The UPS is in essence a two-step process: the targeting of proteins through the covalent linkage of polyubiquitin chains (2), and the destruction of ubiquitylated proteins by the proteasome (3). A number of studies have implicated UPS dysfunction in a range of polyglutamine (polyQ) neurodegenerative diseases (4). The aggregation-prone polyQ proteins are postulated to impair the UPS in these diseases, either by overloading the capacity of the cell's UPS machinery (5), by sequestration of essential components of the UPS into inclusions (6), or by obstruction of the proteasome (7). If polyQ proteins themselves inhibit the system crucial to their own degradation, this could elicit a self-perpetuating pathogenic cascade of events, both accelerating the accumulation of the toxic protein, and impairing essential regulatory functions of the UPS (4). With the help of specifically designed reporter substrates, it has been shown that polyQ proteins can cause UPS impairment in cell lines (5,8,9). However, since these experiments rely on acute overexpression of the polyQ protein in cells with limited physiological relevance, it is difficult to extrapolate these findings to the status of the UPS during the progression of the disease in patients.In contrast to the observation with in vitro models, ...

The SUMO‐targeted ubiquitin ligase RNF4 functions at the crossroads of the SUMO and ubiquitin systems. Here, we report that the deubiquitylation enzyme (DUB) ataxin‐3 counteracts RNF4 activity during the DNA double‐strand break (DSB) response. We find that ataxin‐3 negatively regulates ubiquitylation of the checkpoint mediator MDC1, a known RNF4 substrate. Loss of ataxin‐3 markedly decreases the chromatin dwell time of MDC1 at DSBs, which can be fully reversed by co‐depletion of RNF4. Ataxin‐3 is recruited to DSBs in a SUMOylation‐dependent fashion, and in vitro it directly interacts with and is stimulated by recombinant SUMO, defining a SUMO‐dependent mechanism for DUB activity toward MDC1. Loss of ataxin‐3 results in reduced DNA damage‐induced ubiquitylation due to impaired MDC1‐dependent recruitment of the ubiquitin ligases RNF8 and RNF168, and reduced recruitment of 53BP1 and BRCA1. Finally, ataxin‐3 is required for efficient MDC1‐dependent DSB repair by non‐homologous end‐joining and homologous recombination. Consequently, loss of ataxin‐3 sensitizes cells to ionizing radiation and poly(ADP‐ribose) polymerase inhibitor. We propose that the opposing activities of RNF4 and ataxin‐3 consolidate robust MDC1‐dependent signaling and repair of DSBs.

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.

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