Mechanism of USP7/HAUSP activation by its C-terminal ubiquitin-like domain and allosteric regulation by GMP-synthetase.

The ubiquitin-specific protease USP7/HAUSP regulates p53 and MDM2 levels, and cellular localization of FOXO4 and PTEN, and hence is critically important for their role in cellular processes. Here we show how the 64 kDa C-terminal region of USP7 can positively regulate deubiquitinating activity. We present the crystal structure of this USP7/HAUSP ubiquitin-like domain (HUBL) comprised of five ubiquitin-like (Ubl) domains organized in 2-1-2 Ubl units. The last di-Ubl unit, HUBL-45, is sufficient to activate USP7, through binding to a "switching" loop in the catalytic domain, which promotes ubiquitin binding and increases activity 100-fold. This activation can be enhanced allosterically by the metabolic enzyme GMPS. It binds to the first three Ubl domains (HUBL-123) and hyperactivates USP7 by stabilization of the HUBL-45-dependent active state.

The translation initiation factor 3f (eIF3f) exhibits a deubiquitinase activity regulating Notch activation.

Activation of the mammalian Notch receptor after ligand binding relies on a succession of events including metalloprotease-cleavage, endocytosis, monoubiquitination, and eventually processing by the gamma-secretase, giving rise to a soluble, transcriptionally active molecule. The Notch1 receptor was proposed to be monoubiquitinated before its gamma-secretase cleavage; the targeted lysine has been localized to its submembrane domain. Investigating how this step might be regulated by a deubiquitinase (DUB) activity will provide new insight for understanding Notch receptor activation and downstream signaling. An immunofluorescence-based screening of an shRNA library allowed us to identify eIF3f, previously known as one of the subunits of the translation initiation factor eIF3, as a DUB targeting the activated Notch receptor. We show that eIF3f has an intrinsic DUB activity. Knocking down eIF3f leads to an accumulation of monoubiquitinated forms of activated Notch, an effect counteracted by murine WT eIF3f but not by a catalytically inactive mutant. We also show that eIF3f is recruited to activated Notch on endocytic vesicles by the putative E3 ubiquitin ligase Deltex1, which serves as a bridging factor. Finally, catalytically inactive forms of eIF3f as well as shRNAs targeting eIF3f repress Notch activation in a coculture assay, showing that eIF3f is a new positive regulator of the Notch pathway. Our results support two new and provocative conclusions: (1) The activated form of Notch needs to be deubiquitinated before being processed by the gamma-secretase activity and entering the nucleus, where it fulfills its transcriptional function. (2) The enzyme accounting for this deubiquitinase activity is eIF3f, known so far as a translation initiation factor. These data improve our knowledge of Notch signaling but also open new avenues of research on the Zomes family and the translation initiation factors.

Identification of F-box only protein 7 as a negative regulator of NF-kappaB signalling.

The nuclear factor κB (NF-κB) signalling pathway controls important cellular events such as cell proliferation, differentiation, apoptosis and immune responses. Pathway activation occurs rapidly upon TNFα stimulation and is highly dependent on ubiquitination events. Using cytoplasmic to nuclear translocation of the NF-κB transcription factor family member p65 as a read-out, we screened a synthetic siRNA library targeting enzymes involved in ubiquitin conjugation and de-conjugation for modifiers of regulatory ubiquitination events in NF-κB signalling. We identified F-box protein only 7 (FBXO7), a component of Skp, Cullin, F-box (SCF)-ubiquitin ligase complexes, as a negative regulator of NF-κB signalling. F-box protein only 7 binds to, and mediates ubiquitin conjugation to cIAP1 and TRAF2, resulting in decreased RIP1 ubiquitination and lowered NF-κB signalling activity.

Loss of the cylindromatosis tumour suppressor inhibits apoptosis by activating NF-kappaB.

Protein modification by the conjugation of ubiquitin moieties--ubiquitination--plays a major part in many biological processes, including cell cycle and apoptosis. The enzymes that mediate ubiquitin-conjugation have been well-studied, but much less is known about the ubiquitin-specific proteases that mediate de-ubiquitination of cellular substrates. To study this gene family, we designed a collection of RNA interference vectors to suppress 50 human de-ubiquitinating enzymes, and used these vectors to identify de-ubiquitinating enzymes in cancer-relevant pathways. We report here that inhibition of one of these enzymes, the familial cylindromatosis tumour suppressor gene (CYLD), having no known function, enhances activation of the transcription factor NF-kappaB. We show that CYLD binds to the NEMO (also known as IKKgamma) component of the IkappaB kinase (IKK) complex, and appears to regulate its activity through de-ubiquitination of TRAF2, as TRAF2 ubiquitination can be modulated by CYLD. Inhibition of CYLD increases resistance to apoptosis, suggesting a mechanism through which loss of CYLD contributes to oncogenesis. We show that this effect can be relieved by aspirin derivatives that inhibit NF-kappaB activity, which suggests a therapeutic intervention strategy to restore growth control in patients suffering from familial cylindromatosis.

A Functional Genomic Screen Identifies the Deubiquitinase USP11 as a Novel Transcriptional Regulator of ERα in Breast Cancer.

Approximately 70% of breast cancers express estrogen receptor α (ERα) and depend on this key transcriptional regulator for proliferation and differentiation. While patients with this disease can be treated with targeted antiendocrine agents, drug resistance remains a significant issue, with almost half of patients ultimately relapsing. Elucidating the mechanisms that control ERα function may further our understanding of breast carcinogenesis and reveal new therapeutic opportunities. Here, we investigated the role of deubiquitinases (DUB) in regulating ERα in breast cancer. An RNAi loss-of-function screen in breast cancer cells targeting all DUBs identified USP11 as a regulator of ERα transcriptional activity, which was further validated by assessment of direct transcriptional targets of ERα. USP11 expression was induced by estradiol, an effect that was blocked by tamoxifen and not observed in ERα-negative cells. Mass spectrometry revealed a significant change to the proteome and ubiquitinome in USP11-knockdown (KD) cells in the presence of estradiol. RNA sequencing in LCC1 USP11-KD cells revealed significant suppression of cell-cycle-associated and ERα target genes, phenotypes that were not observed in LCC9 USP11-KD, antiendocrine-resistant cells. In a breast cancer patient cohort coupled with in silico analysis of publicly available cohorts, high expression of USP11 was significantly associated with poor survival in ERα-positive (ERα+) patients. Overall, this study highlights a novel role for USP11 in the regulation of ERα activity, where USP11 may represent a prognostic marker in ERα+ breast cancer. SIGNIFICANCE: A newly identified role for USP11 in ERα transcriptional activity represents a novel mechanism of ERα regulation and a pathway to be exploited for the management of ER-positive breast cancer.

Regulated HIV-2 RNA dimerization by means of alternative RNA conformations.

The dimer initiation site (DIS) hairpin of the HIV-2 untranslated leader RNA mediates in vitro dimerization through 'loop-loop kissing' of a loop-exposed palindrome sequence. Premature RNA dimerization must be prevented during the retroviral life cycle. A regulatory mechanism has been proposed for the HIV-1 leader RNA that can adopt an alternative conformation in which the DIS motif is effectively masked by long-distance base pairing with upstream leader sequences. We now report that HIV-2 RNA dimerization is also regulated. Sequestering of the DIS motif by base pairing interactions with downstream leader sequences mediates a switch to a dimerization-impaired conformation. The existence of two alternative conformations of the HIV-2 leader RNA is supported by UV melting experiments. Furthermore, the equilibrium between the two conformations can be shifted by annealing of antisense oligonucleotides or by deletion of certain leader regions. These measures have a profound impact on the dimerization properties of the transcript, demonstrating a mutual exclusivity between the alternative conformation and dimerization, similar to what has been described for the HIV-1 leader. The overall resemblance in regulation of HIV-1 and HIV-2 RNA dimerization suggests that a similar mechanism may be operating in other lentiviruses and perhaps all retroviridae.

Functional annotation of deubiquitinating enzymes using RNA interference.

Protein ubiquitination is a dynamic process, depending on a tightly regulated balance between the activity of ubiquitin ligases and their antagonists, the ubiquitin-specific proteases or deubiquitinating enzymes. The family of ubiquitin ligases has been studied intensively and it is well established that their deregulation contributes to diverse disease processes, including cancer. Much less is known about the function and regulation of the large group of deubiquitinating enzymes. This chapter describes how RNA interference against deubiquitinating enzymes can be used to elucidate their function. The application of this technology will greatly improve the functional annotation of this family of proteases.

USP15 stabilizes TGF-ß receptor I and promotes oncogenesis through the activation of TGF-ß signaling in glioblastoma.

In advanced cancer, including glioblastoma, the transforming growth factor ß (TGF-ß) pathway acts as an oncogenic factor and is considered to be a therapeutic target. Using a functional RNAi screen, we identified the deubiquitinating enzyme ubiquitin-specific peptidase 15 (USP15) as a key component of the TGF-ß signaling pathway. USP15 binds to the SMAD7-SMAD specific E3 ubiquitin protein ligase 2 (SMURF2) complex and deubiquitinates and stabilizes type I TGF-ß receptor (TßR-I), leading to an enhanced TGF-ß signal. High expression of USP15 correlates with high TGF-ß activity, and the USP15 gene is found amplified in glioblastoma, breast and ovarian cancer. USP15 amplification confers poor prognosis in individuals with glioblastoma. Downregulation or inhibition of USP15 in a patient-derived orthotopic mouse model of glioblastoma decreases TGF-ß activity. Moreover, depletion of USP15 decreases the oncogenic capacity of patient-derived glioma-initiating cells due to the repression of TGF-ß signaling. Our results show that USP15 regulates the TGF-ß pathway and is a key factor in glioblastoma pathogenesis.

The deubiquitinating enzyme USP26 is a regulator of androgen receptor signaling.

The androgen receptor (AR) is a member of the nuclear receptor superfamily and is essential for male sexual development and maturation, as well as prostate cancer development. Regulation of AR signaling activity depends on several posttranslational modifications, one of these being ubiquitination. We screened a short hairpin library targeting members of the deubiquitination enzyme family and identified the X-linked deubiquitination enzyme USP26 as a novel regulator of AR signaling. USP26 is a nuclear protein that binds to AR via three important nuclear receptor interaction motifs, and modulates AR ubiquitination, consequently influencing AR activity and stability. Our data suggest that USP26 assembles with AR and other cofactors in subnuclear foci, and serves to counteract hormone-induced AR ubiquitination, thereby contributing to the regulation of AR transcriptional activity.

Bile salt-stimulated lipase from human milk binds DC-SIGN and inhibits human immunodeficiency virus type 1 transfer to CD4+ T cells.

A wide range of pathogens, including human immunodeficiency virus type 1 (HIV-1), hepatitis C virus, Ebola virus, cytomegalovirus, dengue virus, Mycobacterium, Leishmania, and Helicobacter pylori, can interact with dendritic cell (DC)-specific ICAM3-grabbing nonintegrin (DC-SIGN), expressed on DCs and a subset of B cells. More specifically, the interaction of the gp120 envelope protein of HIV-1 with DC-SIGN can facilitate the transfer of virus to CD4+ T lymphocytes in trans and enhance infection. We have previously demonstrated that a multimeric LeX component in human milk binds to DC-SIGN, preventing HIV-1 from interacting with this receptor. Biochemical analysis reveals that the compound is heat resistant, trypsin sensitive, and larger than 100 kDa, indicating a specific glycoprotein as the inhibitory compound. By testing human milk from three different mothers, we found the levels of DC-SIGN binding and viral inhibition to vary between samples. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and matrix-assisted laser desorption ionization analysis, we identified bile salt-stimulated lipase (BSSL), a Lewis X (LeX)-containing glycoprotein found in human milk, to be the major variant protein between the samples. BSSL isolated from human milk bound to DC-SIGN and inhibited the transfer of HIV-1 to CD4+ T lymphocytes. Two BSSL isoforms isolated from the same human milk sample showed differences in DC-SIGN binding, illustrating that alterations in the BSSL forms explain the differences observed. These results indicate that variations in BSSL lead to alterations in LeX expression by the protein, which subsequently alters the DC-SIGN binding capacity and the inhibitory effect on HIV-1 transfer. Identifying the specific molecular interaction between the different forms may aid in the future design of antimicrobial agents.

A genomic and functional inventory of deubiquitinating enzymes.

Posttranslational modification of proteins by the small molecule ubiquitin is a key regulatory event, and the enzymes catalyzing these modifications have been the focus of many studies. Deubiquitinating enzymes, which mediate the removal and processing of ubiquitin, may be functionally as important but are less well understood. Here, we present an inventory of the deubiquitinating enzymes encoded in the human genome. In addition, we review the literature concerning these enzymes, with particular emphasis on their function, specificity, and the regulation of their activity.

The deubiquitinating enzyme USP1 regulates the Fanconi anemia pathway.

Protein ubiquitination and deubiquitination are dynamic processes implicated in the regulation of numerous cellular pathways. Monoubiquitination of the Fanconi anemia (FA) protein FANCD2 appears to be critical in the repair of DNA damage because many of the proteins that are mutated in FA are required for FANCD2 ubiquitination. By screening a gene family RNAi library, we identify the deubiquitinating enzyme USP1 as a novel component of the Fanconi anemia pathway. Inhibition of USP1 leads to hyperaccumulation of monoubiquitinated FANCD2. Furthermore, USP1 physically associates with FANCD2, and the proteins colocalize in chromatin after DNA damage. Finally, analysis of crosslinker-induced chromosomal aberrations in USP1 knockdown cells suggests a role in DNA repair. We propose that USP1 deubiquitinates FANCD2 when cells exit S phase or recommence cycling after a DNA damage insult and may play a critical role in the FA pathway by recycling FANCD2.

Reversal of senescence in mouse fibroblasts through lentiviral suppression of p53.

Senescence is generally defined as an irreversible state of G(1) cell cycle arrest in which cells are refractory to growth factor stimulation. In mouse embryo fibroblasts (MEFs), induction of senescence requires the presence of p19(ARF) and p53, as genetic ablation of either of these genes allows escape from senescence and leads to immortalization. We have developed a lentiviral vector that directs the synthesis of a p53-specific short hairpin transcript, which mediates stable suppression of p53 expression through RNA interference. We show that suppression of p53 expression in senescent MEFs leads to rapid cell cycle re-entry, is associated with loss of expression of senescence-associated genes, and leads to immortalization. These data indicate that senescence in MEFs is reversible and demonstrate that both initiation and maintenance of senescence is p53-dependent.

Requirements for RNA heterodimerization of the human immunodeficiency virus type 1 (HIV-1) and HIV-2 genomes.

Retroviruses are prone to recombination because they package two copies of the RNA genome. Whereas recombination is a frequent event within the human immunodeficiency virus type 1 (HIV-1) and HIV-2 groups, no HIV-1/HIV-2 recombinants have been reported thus far. The possibility of forming HIV-1/HIV-2 RNA heterodimers was studied in vitro. In both viruses, the dimer initiation site (DIS) hairpin is used to form dimers, but these motifs appear too dissimilar to allow RNA heterodimer formation. Multiple mutations were introduced into the HIV-2 DIS element to gradually mimic the HIV-1 hairpin. First, the loop-exposed palindrome of HIV-1 was inserted. This self-complementary sequence motif forms the base pair interactions of the kissing-loop (KL) dimer complex, but such a modification is not sufficient to permit RNA heterodimer formation. Next, the HIV-2 DIS loop size was shortened from 11 to 9 nucleotides, as in the HIV-1 DIS motif. This modification also results in the presentation of the palindromes in the same position within the hairpin loop. The change yielded a modest level of RNA heterodimers, which was not significantly improved by additional sequence changes in the loop and top base pair. No isomerization of the KL dimer to the extended duplex dimer form was observed for the heterodimers. These combined results indicate that recombination between HIV-1 and HIV-2 is severely restricted at the level of RNA dimerization.