Ubiquitin-activating enzyme activity contributes to differential accumulation of mutant huntingtin in brain and peripheral tissues.

Huntington's disease (HD) belongs to a family of neurodegenerative diseases caused by misfolded proteins and shares the pathological hallmark of selective accumulation of misfolded proteins in neuronal cells. Polyglutamine expansion in the HD protein, huntingtin (Htt), causes selective neurodegeneration that is more severe in the striatum and cortex than in other brain regions, but the mechanism behind this selectivity is unknown. Here we report that in HD knock-in mice, the expression levels of mutant Htt (mHtt) are higher in brain tissues than in peripheral tissues. However, the expression of N-terminal mHtt via stereotaxic injection of viral vectors in mice also results in greater accumulation of mHtt in the striatum than in muscle. We developed an in vitro assay that revealed that extracts from the striatum and cortex promote the formation of high-molecular weight (HMW) mHtt compared with the relatively unaffected cerebellar and peripheral tissue extracts. Inhibition of ubiquitin-activating enzyme E1 (Ube1) increased the levels of HMW mHtt in the relatively unaffected tissues. Importantly, the expression levels of Ube1 are lower in brain tissues than peripheral tissues and decline in the nuclear fraction with age, which is correlated with the increased accumulation of mHtt in the brain and neuronal nuclei during aging. Our findings suggest that decreased targeting of misfolded Htt to the proteasome for degradation via Ube1 may underlie the preferential accumulation of toxic forms of mHtt in the brain and its selective neurodegeneration.

Differential ubiquitination and degradation of huntingtin fragments modulated by ubiquitin-protein ligase E3A.

Ubiquitination of misfolded proteins, a common feature of many neurodegenerative diseases, is mediated by different lysine (K) residues in ubiquitin and alters the levels of toxic proteins. In Huntington disease, polyglutamine expansion causes N-terminal huntingtin (Htt) to misfold, inducing neurodegeneration. Here we report that shorter N-terminal Htt fragments are more stable than longer fragments and find differential ubiquitination via K63 of ubiquitin. Aging decreases proteasome-mediated Htt degradation, at the same time increasing K63-mediated ubiquitination and subsequent Htt aggregation in HD knock-in mice. The association of Htt with the K48-specific E3 ligase, Ube3a, is decreased in aged mouse brain. Overexpression of Ube3a in HD mouse brain reduces K63-mediated ubiquitination and Htt aggregation, enhancing its degradation via the K48 ubiquitin-proteasome system. Our findings suggest that aging-dependent Ube3a levels result in differential ubiquitination and degradation of Htt fragments, thereby contributing to the age-related neurotoxicity of mutant Htt.

Proteolytic and non-proteolytic roles of ubiquitin and the ubiquitin proteasome system in transcriptional regulation.

The ubiquitin proteasome system (UPS) regulates perhaps the most intriguing balance in all of biology: how cells control protein function and malfunction in order to regulate, and eventually eliminate, the old and error prone while simultaneously synthesizing and orchestrating the new. In light of the growing notion that ubiquitination and the 26S proteasome are central to a multiplicity of diverse cellular functions, we discuss here the proteolytic and non-proteolytic roles of the UPS in regulating pathways ultimately involved in protein synthesis and activity including roles in epigenetics, transcription, and post-translational modifications. This article is part of a Special Issue entitled The 26S Proteasome: When degradation is just not enough!

Phosphorylation and ubiquitination of degron proximal residues are essential for class II transactivator (CIITA) transactivation and major histocompatibility class II expression.

Major histocompatibility (MHC) class II molecules are cell surface glycoproteins that present extracellular antigens to CD4(+) T cells and are essential for initiation of the adaptive immune response. MHC class II expression requires recruitment of a master regulator, the class II transactivator (CIITA), to the MHC class II promoter. Post-translational modifications to CIITA play important roles in modulating CIITA mediated transcription of various genes in different cell types. We have previously linked regulation of CIITA to the Ubiquitin Proteasome System (UPS), and we and others have demonstrated that mono-ubiquitination of CIITA dramatically increases its transactivity whereas poly-ubiquitination leads to CIITA degradation. Here we identify three degron proximal lysine residues, Lys-315, Lys-330, and Lys-333, and a phosphorylation site, Ser-280, located within the CIITA degron, that regulate CIITA ubiquitination, stability, and MHC class II expression. Together, these findings contribute to the developing post-translational modification code for CIITA.

Association of the 19S proteasomal ATPases with the ATPase-binding domain of CIITA is essential for CIITA stability and MHC class II expression.

Major histocompatibility class II (MHC class II) molecules are glycoproteins that present extracellular antigens to CD4(+) T cells and are essential for initiation of adaptive immune responses. MHC class II expression requires recruitment of a master regulator, the class II transactivator (CIITA), to the MHC class II promoter. Others and we have earlier linked CIITA to the ubiquitin-proteasome system by showing that mono-ubiquitination of CIITA increases its transactivity, whereas poly-ubiquitination of CIITA leads to its degradation. We have further shown that the 26S proteasome also has non-proteolytic functions in MHC class II transcription, as 19S ATPase subunits of the 26S proteasome positively regulate MHC class II transcription and are necessary for stable promoter binding of CIITA. Although these basic requirements of the proteasome to initiate MHC class II transcription are known, how CIITA is recruited, stabilized, and degraded remains unclear. Here, we identify a novel N-terminal 19S ATPase-binding domain of CIITA. The ATPase-binding domain lies within the proline/serine/threonine-rich region of CIITA and encompasses a majority of the CIITA degron sequence. Absence of the ATPase-binding domain increases the half-life of CIITA, but blocks MHC class II surface expression, indicating that CIITA requires interaction with the 19S ATPases for both appropriate deployment and destruction.

Regulation of acetylation at the major histocompatibility complex class II proximal promoter by the 19S proteasomal ATPase Sug1.

Recent studies have made evident the fact that the 19S regulatory component of the proteasome has functions that extend beyond degradation, particularly in the regulation of transcription. Although 19S ATPases facilitate chromatin remodeling and acetylation events in yeast (Saccharomyces cerevisiae), it is unclear if they play similar roles in mammalian cells. We have recently shown that the 19S ATPase Sug1 positively regulates the transcription of the critical inflammatory gene for major histocompatibility complex class II (MHC-II) by stabilizing enhanceosome assembly at the proximal promoter. We now show that Sug1 is crucial for regulating histone H3 acetylation at the MHC-II proximal promoter. Sug1 binds to acetylated histone H3 and, in the absence of Sug1, histone H3 acetylation is dramatically decreased at the proximal promoter, with a preferential loss of acetylation at H3 lysine 18. Sug1 also binds to the MHC-II histone acetyltransferase CREB-binding protein (CBP) and is critical for the recruitment of CBP to the MHC-II proximal promoter. Our current study strongly implicates the 19S ATPase Sug1 in modifying histones to initiate MHC-II transcription and provides novel insights into the role of the proteasome in the regulation of mammalian transcription.

The 19S proteasome ATPase Sug1 plays a critical role in regulating MHC class II transcription.

Emerging evidence in yeast suggests roles for ATPases of the 19S proteasome as mediators of transcriptional systems through their association with actively transcribed promoters, facilitation of clearance of paused elongation complexes and recruitment of coactivators. Although 19S subunits also regulate mammalian transcription, their role in recruiting transcription factors remains unclear. Here, we demonstrate for the first time a role for the 19S proteasome ATPase Sug1 in regulating transcription of the critical adaptive immune gene, MHC class II. Sug1 associates with the class II transactivator, CIITA, and with the MHC class II proximal promoter. In the absence of Sug1, HLA-DR promoter activity and MHC class II transcription are decreased. Critically, CIITA association with the MHC II promoter is dramatically decreased when Sug1 expression is reduced, even under conditions of proteasome inhibition. In contrast to the rapid promoter association of the 19S subunit, a 20S proteasome subunit associates with the MHC class II proximal promoter following prolonged cytokine stimulation and its association corresponds with pronounced promoter disassociation of CIITA. Taken together, these data demonstrate that both 19S and 20S subunits of the 26S proteasome play specific and critical roles in regulating CIITA activity and MHC class II transcription.