The deubiquitinase TRABID stabilizes the K29/K48-specific E3 ubiquitin ligase HECTD1.

Ubiquitin is a versatile posttranslational modification, which is covalently attached to protein targets either as a single moiety or as a ubiquitin chain. In contrast to K48 and K63-linked chains, which have been extensively studied, the regulation and function of most atypical ubiquitin chains are only starting to emerge. The deubiquitinase TRABID/ZRANB1 is tuned for the recognition and cleavage of K29 and K33-linked chains. Yet, substrates of TRABID and the cellular functions of these atypical ubiquitin signals remain unclear. We determined the interactome of two TRABID constructs rendered catalytic dead either through a point mutation in the catalytic cysteine residue or through removal of the OTU catalytic domain. We identified 50 proteins trapped by both constructs and which therefore represent candidate substrates of TRABID. The E3 ubiquitin ligase HECTD1 was then validated as a substrate of TRABID and used UbiCREST and Ub-AQUA proteomics to show that HECTD1 preferentially assembles K29- and K48-linked ubiquitin chains. Further in vitro autoubiquitination assays using ubiquitin mutants established that while HECTD1 can assemble short homotypic K29 and K48-linked chains, it requires branching at K29/K48 in order to achieve its full ubiquitin ligase activity. We next used transient knockdown and genetic knockout of TRABID in mammalian cells in order to determine the functional relationship between TRABID and HECTD1. This revealed that upon TRABID depletion, HECTD1 is readily degraded. Thus, this study identifies HECTD1 as a mammalian E3 ligase that assembles branched K29/K48 chains and also establishes TRABID-HECTD1 as a DUB/E3 pair regulating K29 linkages.

The Ubiquitin System in Alzheimer's Disease.

Alzheimer's disease (AD) is the most common form of dementia, most prevalent in the elderly population and has a significant impact on individuals and their family as well as the health care system and the economy. While the number of patients affected by various forms of dementia including AD is on the increase, there is currently no cure. Although genome-wide association studies have identified genetic markers for familial AD, the molecular mechanisms underlying the initiation and development of both familial and sporadic AD remain poorly understood. Most neurodegenerative diseases and in particular those associated with dementia have been defined as proteinopathies due to the presence of intra- and/or extracellular protein aggregates in the brain of affected individuals. Although loss of proteostasis in AD has been known for decades, it is only in recent years that we have come to appreciate the role of ubiquitin-dependent mechanisms in brain homeostasis and in brain diseases. Ubiquitin is a highly versatile post-translational modification which regulates many aspects of protein fate and function, including protein degradation by the Ubiquitin-Proteasome System (UPS), autophagy-mediated removal of damaged organelles and proteins, lysosomal turnover of membrane proteins and of extracellular molecules brought inside the cell through endocytosis. Amyloid-ß (Aß) fragments as well as hyperphosphorylation of Tau are hallmarks of AD, and these are found in extracellular plaques and intracellular fibrils in the brain of individuals with AD, respectively. Yet, whether it is the oligomeric or the soluble species of Aß and Tau that mediate toxicity is still unclear. These proteins impact on mitochondrial energy metabolism, inflammation, as well as a number of housekeeping processes including protein degradation through the UPS and autophagy. In this chapter, we will discuss the role of ubiquitin in neuronal homeostasis as well as in AD; summarise crosstalks between the enzymes that regulate protein ubiquitination and the toxic proteins Tau and Aß; highlight emerging molecular mechanisms in AD as well as future strategies which aim to exploit the ubiquitin system as a source for next-generation therapeutics.

Activity-Based Probes for HECT E3 Ubiquitin Ligases.

Activity-based probes (ABPs) have been used to dissect the biochemical/structural properties and cellular functions of deubiquitinases. However, their utility in studying cysteine-based E3 ubiquitin ligases has been limited. In this study, we evaluate the use of ubiquitin-ABPs (Ub-VME and Ub-PA) and a novel set of E2-Ub-ABPs on a panel of HECT E3 ubiquitin ligases. Our in vitro data show that ubiquitin-ABPs can label HECT domains. We also provide the first evidence that, in addition to the RBR E3 ubiquitin ligase Parkin, E2-Ub-ABPs can also label the catalytic HECT domains of NEDD4, UBE3C, and HECTD1. Importantly, the endogenous proteasomal E3 ligase UBE3C was also successfully labelled by Ub-PA and His-UBE2D2-Ub-ABP in lysate of cells grown under basal conditions. Our findings provide novel insights into the use of ABPs for the study of HECT E3 ubiquitin ligases.

The E3 ubiquitin ligase HECTD1 contributes to cell proliferation through an effect on mitosis.

The cell cycle is tightly regulated by protein phosphorylation and ubiquitylation events. During mitosis, the multi-subunit cullin-RING E3 ubiquitin ligase APC/c functions as a molecular switch which signals for one cell to divide into two daughter cells, through the ubiquitylation and proteasomal degradation of mitotic cyclins. The contributions of other E3 ligase families during cell cycle progression remain less well understood. Similarly, the roles of ubiquitin chain types beyond homotypic K48 chains in S-phase or branched K11/K48 chains during mitosis, also remain to be fully determined. Our recent findings that HECTD1 ubiquitin ligase activity assembles branched K29/K48 ubiquitin linkages prompted us to evaluate HECTD1 function during the cell cycle. We used transient knockdown and genetic knockout to show that HECTD1 depletion in HEK293T and HeLa cells decreases cell number and we established that this is mediated through loss of ubiquitin ligase activity. Interestingly, we found that HECTD1 depletion increases the proportion of cells with aligned chromosomes (Prometa/Metaphase) and we confirmed this molecularly using phospho-Histone H3 (Ser28) as a marker of mitosis. Time-lapse microscopy of NEBD to anaphase onset established that HECTD1-depleted cells take on average longer to go through mitosis. In line with this data, HECTD1 depletion reduced the activity of the Spindle Assembly Checkpoint, and BUB3, a component of the Mitosis Checkpoint Complex, was identified as novel HECTD1 interactor. BUB3, BUBR1 or MAD2 protein levels remained unchanged in HECTD1-depleted cells. Overall, this study reveals a novel putative role for HECTD1 during mitosis and warrants further work to elucidate the mechanisms involved.

PcG proteins, DNA methylation, and gene repression by chromatin looping.

Many DNA hypermethylated and epigenetically silenced genes in adult cancers are Polycomb group (PcG) marked in embryonic stem (ES) cells. We show that a large region upstream ( approximately 30 kb) of and extending approximately 60 kb around one such gene, GATA-4, is organized-in Tera-2 undifferentiated embryonic carcinoma (EC) cells-in a topologically complex multi-loop conformation that is formed by multiple internal long-range contact regions near areas enriched for EZH2, other PcG proteins, and the signature PcG histone mark, H3K27me3. Small interfering RNA (siRNA)-mediated depletion of EZH2 in undifferentiated Tera-2 cells leads to a significant reduction in the frequency of long-range associations at the GATA-4 locus, seemingly dependent on affecting the H3K27me3 enrichments around those chromatin regions, accompanied by a modest increase in GATA-4 transcription. The chromatin loops completely dissolve, accompanied by loss of PcG proteins and H3K27me3 marks, when Tera-2 cells receive differentiation signals which induce a approximately 60-fold increase in GATA-4 expression. In colon cancer cells, however, the frequency of the long-range interactions are increased in a setting where GATA-4 has no basal transcription and the loops encompass multiple, abnormally DNA hypermethylated CpG islands, and the methyl-cytosine binding protein MBD2 is localized to these CpG islands, including ones near the gene promoter. Removing DNA methylation through genetic disruption of DNA methyltransferases (DKO cells) leads to loss of MBD2 occupancy and to a decrease in the frequency of long-range contacts, such that these now more resemble those in undifferentiated Tera-2 cells. Our findings reveal unexpected similarities in higher order chromatin conformation between stem/precursor cells and adult cancers. We also provide novel insight that PcG-occupied and H3K27me3-enriched regions can form chromatin loops and physically interact in cis around a single gene in mammalian cells. The loops associate with a poised, low transcription state in EC cells and, with the addition of DNA methylation, completely repressed transcription in adult cancer cells.

Molecular discrimination of structurally equivalent Lys 63-linked and linear polyubiquitin chains.

At least eight types of ubiquitin chain exist, and individual linkages affect distinct cellular processes. The only distinguishing feature of differently linked ubiquitin chains is their structure, as polymers of the same unit are chemically identical. Here, we have crystallized Lys 63-linked and linear ubiquitin dimers, revealing that both adopt equivalent open conformations, forming no contacts between ubiquitin molecules and thereby differing significantly from Lys 48-linked ubiquitin chains. We also examined the specificity of various deubiquitinases (DUBs) and ubiquitin-binding domains (UBDs). All analysed DUBs, except CYLD, cleave linear chains less efficiently compared with other chain types, or not at all. Likewise, UBDs can show chain specificity, and are able to select distinct linkages from a ubiquitin chain mixture. We found that the UBAN (ubiquitin binding in ABIN and NEMO) motif of NEMO (NF-kappaB essential modifier) binds to linear chains exclusively, whereas the NZF (Npl4 zinc finger) domain of TAB2 (TAK1 binding protein 2) is Lys 63 specific. Our results highlight remarkable specificity determinants within the ubiquitin system.

Targeting the Ubiquitin System in Glioblastoma.

Glioblastoma is the most common primary brain tumor in adults with poor overall outcome and 5-year survival of less than 5%. Treatment has not changed much in the last decade or so, with surgical resection and radio/chemotherapy being the main options. Glioblastoma is highly heterogeneous and frequently becomes treatment-resistant due to the ability of glioblastoma cells to adopt stem cell states facilitating tumor recurrence. Therefore, there is an urgent need for novel therapeutic strategies. The ubiquitin system, in particular E3 ubiquitin ligases and deubiquitinating enzymes, have emerged as a promising source of novel drug targets. In addition to conventional small molecule drug discovery approaches aimed at modulating enzyme activity, several new and exciting strategies are also being explored. Among these, PROteolysis TArgeting Chimeras (PROTACs) aim to harness the endogenous protein turnover machinery to direct therapeutically relevant targets, including previously considered "undruggable" ones, for proteasomal degradation. PROTAC and other strategies targeting the ubiquitin proteasome system offer new therapeutic avenues which will expand the drug development toolboxes for glioblastoma. This review will provide a comprehensive overview of E3 ubiquitin ligases and deubiquitinating enzymes in the context of glioblastoma and their involvement in core signaling pathways including EGFR, TGF-ß, p53 and stemness-related pathways. Finally, we offer new insights into how these ubiquitin-dependent mechanisms could be exploited therapeutically for glioblastoma.

Methylation-specific PCR.

Methylation-specific polymerase chain reaction (MSP) is a technique that has facilitated the detection of promoter hypermethylation at CpG islands in cell lines and clinical samples, including fresh/frozen tissues. The ability of MSP to differentiate methylated from unmethylated cytosine is dependent upon sodium bisulfite treatment of DNA which retains the methylation marks of cytosines together with the specific amplification of this modified DNA using primer sets complimentary only to the formerly methylated or unmethylated alleles. Nested-MSP (MN-MSP) is an alternative method that overcomes the limitations of MSP, especially when it comes to analyzing samples with low quality/quantity of starting DNA (e.g., paraffin-embedded specimens). MN-MSP includes a first round of amplification using primers unbiased toward the methylation status of a single (MN-MSP) or multiple (multiplex MN-MSP) genes followed by conventional MSP. Although MSP and NM-MSP are simple techniques that can easily be incorporated in most molecular biology laboratories, the ability to accurately determine the promoter methylation status of genes largely depends upon the careful design of MSP primers as well as other steps outlined in this chapter.

Promoter hypermethylation of hallmark cancer genes in atypical adenomatous hyperplasia of the lung.

PURPOSE: According to current models of tumorigenesis, the progression of phenotypic changes culminating in overtly malignant carcinoma is driven by genetic and epigenetic alterations. The recognition of an early form of glandular neoplasia termed atypical adenomatous hyperplasia (AAH), a precursor lesion from which lung adenocarcinomas arise, provides an opportunity for characterizing early epigenetic alterations involved in lung tumorigenesis. EXPERIMENTAL DESIGN: We evaluated AAHs, adjacent normal lung tissue, and synchronous lung adenocarcinomas for promoter hypermethylation of genes implicated in lung tumorigenesis (p16, TIMP3, DAPK, MGMT, RARbeta, RASSF1A, and hTERT). RESULTS: For individual genes and the number of genes methylated, we observed a significant increase in the frequency of promoter hypermethylation in the histologic progression from normal to AAH, with low-grade or high-grade atypia, and finally to adenocarcinoma (P(trend)

Epigenetic alteration of Wnt pathway antagonists in progressive glandular neoplasia of the lung.

BACKGROUND: Atypical adenomatous hyperplasia (AAH) is now recognized as a precursor lesion from which lung adenocarcinomas arise and thus represents an ideal target for studying the early genetic and epigenetic alterations associated with lung tumorigenesis such as alterations of the Wnt pathway. METHODS: We assessed the level of Wnt signaling activity in lung cancer cell lines by determining the level of active beta-catenin and determined the level of expression of Wnt antagonists APC, DKK1, DKK3, LKB1, SFRP1, 2, 4, 5, WIF1 and RUNX3 using reverse transcription-polymerase chain reaction. Using multiplex nested methylation-specific polymerase chain reaction, we analyzed promoter region methylation of these genes in resected lung tissue in the histopathologic sequence of glandular neoplasia (normal lung parenchyma, low-grade and high-grade AAH, adenocarcinoma). RESULTS: The majority of non-small cell lung cancer cell lines (11 of 16, 69%) have evidence of active Wnt signaling and silencing of Wnt antagonists correlated with promoter hypermethylation. Promoter region methylation of Wnt antagonists was common in primary lung adenocarcinoma and there was a significant increase in the frequency of methylation for Wnt antagonist genes and the number of genes methylated with each stage of tumorigenesis (test for rend P

Epigenetic regulation of the Wnt signaling inhibitor DACT2 in human hepatocellular carcinoma.

DACT2 (Dapper, Dishevelled-associated antagonist of ß-catenin homolog 2) is a member of the DACT family involved in the regulation of embryonic development. Human DACT2 is localized on 6q27, a region of frequent loss of heterozygosity in human cancers. However, the regulation of DACT2 expression and function in hepatocellular carcinoma (HCC) remains unclear. In this study, genetic and epigenetic changes of DACT2 were analyzed in HCC cell lines and primary cancer. We found no single-nucleotide polymorphism (SNP) associated with HCC. Promoter region methylation was correlated with loss or reduction of DACT2 expression, and restoration of DACT2 expression was induced by 5-aza-2'-deoxycytidine (5-AZA) in HCC cell lines. Promoter region methylation was found in 54.84% of primary HCC. Reduction of DACT2 expression was associated with promoter hypermethylation, and expression of DACT2 was inversely related to ß-catenin expression in primary HCC. DACT2 suppressed cell proliferation, induced G 2-M arrest in cell lines and inhibited tumor growth in xenograft nude mice. The transcriptional activity of TCF-4 and the expression of Wnt signaling downstream genes were suppressed by DACT2 re-expression and reactivated by depletion of DACT2. In conclusion, DACT2 is frequently methylated in HCC and its expression is regulated by promoter hypermethylation. DACT2 suppresses HCC by inhibiting Wnt signaling in human HCC.

An ankyrin-repeat ubiquitin-binding domain determines TRABID's specificity for atypical ubiquitin chains.

Eight different types of ubiquitin linkages are present in eukaryotic cells that regulate diverse biological processes. Proteins that mediate specific assembly and disassembly of atypical Lys6, Lys27, Lys29 and Lys33 linkages are mainly unknown. We here reveal how the human ovarian tumor (OTU) domain deubiquitinase (DUB) TRABID specifically hydrolyzes both Lys29- and Lys33-linked diubiquitin. A crystal structure of the extended catalytic domain reveals an unpredicted ankyrin repeat domain that precedes an A20-like catalytic core. NMR analysis identifies the ankyrin domain as a new ubiquitin-binding fold, which we have termed AnkUBD, and DUB assays in vitro and in vivo show that this domain is crucial for TRABID efficiency and linkage specificity. Our data are consistent with AnkUBD functioning as an enzymatic S1' ubiquitin-binding site, which orients a ubiquitin chain so that Lys29 and Lys33 linkages are cleaved preferentially.