sirna-screening-to-identify-ubiquitin-ubiquitin-like-system

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

University of Dundee

Huntington's disease (HD) is one of a group of neurodegenerative disorders caused by the pathological expansion of a glutamine tract. A hallmark of these so-called polyglutamine diseases is the presence of ubiquitylated inclusion bodies, which sequester various components of the 19S and 20S proteasomes. In addition, the ubiquitin-proteasome system (UPS) has been shown to be severely impaired in vitro in cells overexpressing mutant huntingtin. Thus, because of its fundamental housekeeping function, impairment of the UPS in neurons could contribute to neurotoxicity. We have recently proposed that the proteasome activator REGgamma could contribute to UPS impairment in polyglutamine diseases by suppressing the proteasomal catalytic sites responsible for cleaving Gln-Gln bonds. Capping of proteasomes with REGgamma could therefore contribute to a potential 'clogging' of the proteasome by pathogenic polyglutamines. We show here that genetic reduction of REGgamma has no effect on the well-defined neurological phenotype of R6/2 HD mice and does not affect inclusion body formation in the R6/2 brain. Surprisingly, we observe increased proteasomal 'chymotrypsin-like' activity in 13-week-old R6/2 brains relative to non-R6/2, irrespective of REGgamma levels. However, assays of 26S proteasome activity in mouse brain extracts reveal no difference in proteolytic activity regardless of R6/2 or REGgamma genotype. These findings suggest that REGgamma is not a viable therapeutic target in polyglutamine disease and that overall proteasome function is not impaired by trapped mutant polyglutamine in R6/2 mice.

Proteasome impairment does not contribute to pathogenesis in R6/2 Huntington's disease mice: exclusion of proteasome activator REGgamma as a therapeutic target.

Molecular chaperones facilitate and regulate protein conformational change within cells. This encompasses many fundamental cellular processes: including the correct folding of nascent chains; protein transport and translocation; signal transduction and protein quality control. Chaperones are, therefore, important in several forms of human disease, including neurodegeneration. Within the retina, the highly specialized photoreceptor cell presents a fascinating paradigm to investigate the specialization of molecular chaperone function and reveals unique chaperone requirements essential to photoreceptor function. Mutations in several photoreceptor proteins lead to protein misfolding mediated neurodegeneration. The best characterized of these are mutations in the molecular light sensor, rhodopsin, which cause autosomal dominant retinitis pigmentosa. Rhodopsin biogenesis is likely to require chaperones, while rhodopsin misfolding involves molecular chaperones in quality control and the cellular response to protein aggregation. Furthermore, the specialization of components of the chaperone machinery to photoreceptor specific roles has been revealed by the identification of mutations in molecular chaperones that cause inherited retinal dysfunction and degeneration. These chaperones are involved in several important cellular pathways and further illuminate the essential and diverse roles of molecular chaperones.

Molecular chaperones and photoreceptor function.

Impairment of the ubiquitin-proteasome system (UPS) has long been considered an attractive hypothesis to explain the selective dysfunction and death of neurons in polyglutamine disorders such as Huntington's disease (HD). The fact that inclusion bodies in HD mouse models and patient brains are rich in ubiquitin and proteasome components suggests that the UPS may be hindered directly or indirectly by inclusion bodies or their misfolded monomeric or oligomeric precursors. However, studies into UPS function in various polyglutamine disease models have yielded conflicting results, suggesting mutant polyglutamine tracts may exert different effects on the UPS depending on protein context, expression level, subcellular localisation and cell-type. To investigate UPS function in a well-characterised mouse model of HD, we have crossed R6/2 HD mice with transgenic UPS reporter mice expressing the GFPu construct. The GFPu construct comprises GFP fused to a constitutive degradation signal (CL-1) that promotes its rapid degradation under conditions of a healthy UPS. Using a combination of immunoblot analysis, fluorescence and immunofluorescence microscopy studies, we found that steady-state GFPu levels were not detectably different between R6/2 and non-R6/2 brain. We observed no correlation between inclusion body formation and GFPu accumulation, suggesting no direct relationship between protein aggregation and global UPS inhibition in R6/2 mice. These findings suggest that while certain branches of the UPS can be impaired by mutant polyglutamine proteins, such proteins do not necessarily cause total blockade of UPS-dependent degradation. It is therefore likely that the relationship between mutant polyglutamine proteins and the UPS is more complex than originally anticipated.

The ubiquitin-proteasome reporter GFPu does not accumulate in neurons of the R6/2 transgenic mouse model of Huntington's disease.

Huntington's disease (HD) is an inherited neurodegenerative disease caused by the expansion of a polyglutamine tract in the protein huntingtin (htt). HD brains are characterized by the presence of ubiquitin-positive neuronal inclusion bodies, suggesting that disturbances in the distribution of cellular ubiquitin may contribute to disease pathology. The fact that several neurodegenerative diseases are caused by mutations in ubiquitin-processing enzymes and that the polyubiquitin genes are required for resistance to cellular stress led us to investigate the effect of perturbing the ubiquitin system in HD. We crossed R6/2 transgenic HD mice with heterozygous polyubiquitin Ubc knockout mice (Ubc+/-) and assessed the effect on the R6/2 neurological phenotype. Although the R6/2 phenotype was largely unaffected, surprisingly we observed some subtle improvements in various behavioural activities correlating with heterozygous Ubc knockout. Interestingly, immunoblot analysis revealed that the levels of monoubiquitylated histone H2A (uH2A), a modification associated with gene repression, were significantly increased in the brains of R6/2 mice. Furthermore, the reduction of Ubc expression in R6/2; Ubc+/- mice largely prevented this increase in uH2A levels. However, we were not able to show by the use of a limited number of quantitative RT-PCR assays that changes in the amount of uH2A in the R6/2-Ubc mice had an effect on disease-associated transcriptional abnormalities. These results suggest that the expression of aggregation-prone mutant htt causes disturbances to the ubiquitin system, which may contribute to disease due to the diverse and important roles of ubiquitin.

The polyubiquitin Ubc gene modulates histone H2A monoubiquitylation in the R6/2 mouse model of Huntington's disease.

Mutations in AIPL1 cause the inherited blindness Leber congenital amaurosis (LCA). AIPL1 has previously been shown to interact with NUB1, which facilitates the proteasomal degradation of proteins modified with the ubiquitin-like protein FAT10. Here we report that AIPL1 binds non-covalently to free FAT10 and FAT10ylated proteins and can form a ternary complex with FAT10 and NUB1. In addition, AIPL1 antagonised the NUB1-mediated degradation of the model FAT10 conjugate, FAT10-DHFR, and pathogenic mutations of AIPL1 were defective in inhibiting this degradation. While all AIPL1 mutants tested still bound FAT10-DHFR, there was a close correlation between the ability of the mutants to interact with NUB1 and their ability to prevent NUB1-mediated degradation. Interestingly, AIPL1 also co-immunoprecipitated the E1 activating enzyme for FAT10, UBA6, suggesting AIPL1 may have a role in directly regulating the FAT10 conjugation machinery. These studies are the first to implicate FAT10 in retinal cell biology and LCA pathogenesis, and reveal a new role of AIPL1 in regulating the FAT10 pathway.

The inherited blindness protein AIPL1 regulates the ubiquitin-like FAT10 pathway.

Protein misfolding and aggregation are associated with many neurodegenerative diseases, including Huntington's disease. The cellular machinery for maintaining proteostasis includes molecular chaperones that facilitate protein folding and reduce proteotoxicity. Increasing the protein folding capacity of cells through manipulation of DNAJ chaperones has been shown to suppress aggregation and ameliorate polyglutamine toxicity in cells and flies. However, to date these promising findings have not been translated to mammalian models of disease. To address this issue, we developed transgenic mice that over-express the neuronal chaperone HSJ1a (DNAJB2a) and crossed them with the R6/2 mouse model of Huntington's disease. Over-expression of HSJ1a significantly reduced mutant huntingtin aggregation and enhanced solubility. Surprisingly, this was mediated through specific association with K63 ubiquitylated, detergent insoluble, higher order mutant huntingtin assemblies that decreased their ability to nucleate further aggregation. This was dependent on HSJ1a client binding ability, ubiquitin interaction and functional co-operation with HSP70. Importantly, these changes in mutant huntingtin solubility and aggregation led to improved neurological performance in R6/2 mice. These data reveal that prevention of further aggregation of detergent insoluble mutant huntingtin is an additional level of quality control for late stage chaperone-mediated neuroprotection. Furthermore, our findings represent an important proof of principle that DNAJ manipulation is a valid therapeutic approach for intervention in Huntington's disease.

Suppression of protein aggregation by chaperone modification of high molecular weight complexes.

HIF1A (hypoxia-inducible factor 1α) is the master regulator of the cellular response to hypoxia and is implicated in cancer progression. Whereas the regulation of HIF1A protein in response to oxygen is well characterized, less is known about the fate of HIF1A mRNA. In the present study, we have identified the pseudo-DUB (deubiquitinating enzyme)/deadenylase USP52 (ubiquitin-specific protease 52)/PAN2 [poly(A) nuclease 2] as an important regulator of the HIF1A-mediated hypoxic response. Depletion of USP52 reduced HIF1A mRNA and protein levels and resulted in reduced expression of HIF1A-regulated hypoxic targets due to a 3'-UTR (untranslated region)-dependent poly(A)-tail-length-independent destabilization in HIF1A mRNA. MS analysis revealed an association of USP52 with several P-body (processing body) components and we confirmed further that USP52 protein and HIF1A mRNA co-localized with cytoplasmic P-bodies. Importantly, P-body dispersal by knockdown of GW182 or LSM1 resulted in a reduction of HIF1A mRNA levels. These data uncover a novel role for P-bodies in regulating HIF1A mRNA stability, and demonstrate that USP52 is a key component of P-bodies required to prevent HIF1A mRNA degradation.

John S. Bett

MRC Protein Phosphorylation and Ubiquitylation Unit,
College of Life Sciences,
MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences

University of Dundee

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

John S. Bett

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University of Dundee

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

MRC Protein Phosphorylation and Ubiquitylation Unit,
College of Life Sciences,
MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences