Judy M. Coulson

Deubiquitylases From Genes to Organism

Ubiquitylation is a major posttranslational modification that controls most complex aspects of cell physiology. It is reversed through the action of a large family of deubiquitylating enzymes (DUBs) that are emerging as attractive therapeutic targets for a number of disease conditions. Here, we provide a comprehensive analysis of the complement of human DUBs, indicating structural motifs, typical cellular copy numbers, and tissue expression profiles. We discuss the means by which specificity is achieved and how DUB activity may be regulated. Generically DUB catalytic activity may be used to 1) maintain free ubiquitin levels, 2) rescue proteins from ubiquitin-mediated degradation, and 3) control the dynamics of ubiquitin-mediated signaling events. Functional roles of individual DUBs from each of five subfamilies in specific cellular processes are highlighted with an emphasis on those linked to pathological conditions where the association is supported by whole organism models. We then specifically consider the role of DUBs associated with protein degradative machineries and the influence of specific DUBs upon expression of receptors and channels at the plasma membrane.

Cellular functions of the DUBs

Ubiquitylation is a reversible post-translational modification that has emerged as a key regulator of most complex cellular processes. It may rival phosphorylation in scope and exceed it in complexity. The dynamic nature of ubiquitylation events is important for governing protein stability, maintaining ubiquitin homeostasis and controlling ubiquitin-dependent signalling pathways. The human genome encodes ~80 active deubiquitylating enzymes (DUBs, also referred to as deubiquitinases), which exhibit distinct specificity profiles towards the various ubiquitin chain topologies. As a result of their ability to reverse ubiquitylation, these enzymes control a broad range of key cellular processes. In this Commentary we discuss the cellular functions of DUBs, such as their role in governing membrane traffic and protein quality control. We highlight two key signalling pathways – the Wnt and transforming growth factor β (TGF-β) pathways, for which dynamic ubiquitylation has emerged as a key regulator. We also discuss the roles of DUBs in the nucleus, where they govern transcriptional activity and DNA repair pathways.

Transcriptional regulation: cancer, neurons and the REST.

REST/NRSF was first identified as a transcriptional repressor of neuronal genes in non-neuronal cells. Recent studies have now revealed seemingly paradoxical roles for REST/NRSF in neurogenesis, neural plasticity, tumour suppression and cancer progression.

Emerging roles of deubiquitinases in cancer‐associated pathways

Deubiquitinases (DUBs) are emerging as important regulators of many pathways germane to cancer. They may regulate the stability of key oncogenes, exemplified by USP28 stabilisation of c‐Myc. Alternatively they can negatively regulate ubiquitin‐dependent signalling cascades such as the NF‐κB activation pathway. We review the current literature that associates DUBs with cancer and discuss their suitability as drug targets of the future.

Regulation and role of REST and REST4 variants in modulation of gene expression in in vivo and in vitro in epilepsy models.

Repressor element-1 silencing transcription factor (REST) is a candidate modulator of gene expression during status epilepticus in the rodent. In such models, full-length REST and the truncated REST4 variant are induced and can potentially direct differential gene expression patterns. We have addressed the regulation of these REST variants in rodent hippocampal seizure models and correlated this with expression of the proconvulsant, substance P encoding, PPT-A gene. REST and REST4 were differentially regulated following kainic acid stimulus both in in vitro and in vivo models. REST4 was more tightly regulated than REST in both models and its transient expression correlated with that of the differential regulation of PPT-A. Consistent with this, overexpression of a truncated REST protein (HZ4, lacking the C-terminal repression domain) increased expression of the endogenous PPT-A gene. Similarly the proximal PPT-A promoter reporter gene construct was differentially regulated by the distinct REST isoforms in hippocampal cells with HZ4 being the major inducer of increased reporter expression. Furthermore, REST and REST4 proteins were differentially expressed and compartmentalized within rat hippocampal cells in vitro following noxious stimuli. This differential localization of the REST isoforms was confirmed in the CA1 region following perforant path and kainic acid induction of status epilepticus in vivo. We propose that the interplay between REST and REST4 alter the expression of proconvulsant genes, as exemplified by the PPT-A gene, and may therefore regulate the progression of epileptogenesis.

SCG3 Transcript in Peripheral Blood Is a Prognostic Biomarker for REST-Deficient Small Cell Lung Cancer

Purpose: Specific markers of circulating tumor cells may be informative in managing lung cancer. Because the RE-1 silencing transcription factor (REST/NRSF) is a transcriptional repressor that is inactivated in neuroendocrine lung cancer, we identified REST-regulated transcripts (CHGA, CHGB, SCG3, VGF, and PCSK1) for evaluation as biomarkers in peripheral blood. Experimental Design: Transcripts were screened across lung cancer and normal cell lines. Candidates were assessed by reverse transcription-PCR and hybridization of RNA extracted from the peripheral blood of 111 lung cancer patients obtained at clinical presentation and from 27 cancer-free individuals. Results: Expression profiling revealed multiple chromogranin transcripts were readily induced on REST depletion, most notably SCG3 was induced >500-fold. The SCG3 transcript was also overexpressed by 12,000-fold in neuroendocrine compared with nonneuroendocrine lung cancer cells. In peripheral blood of lung cancer patients and cancer-free individuals, we found that SCG3 was more tumor-specific and more sensitive than other chromogranin transcripts as a biomarker of circulating tumor cells. Overall, 36% of small cell lung cancer (SCLC) and 16% of non-SCLC patients scored positively for normalized SCG3 transcript. This correlated with worse survival among SCLC patients with limited disease (n = 33; P = 0.022) but not extensive disease (n = 29; P = 0.459). Interestingly, the subcohort of 6 SCLC patients with resistance to platinum/etoposide chemotherapy all scored positively for peripheral blood SCG3 transcript (P = 0.022). Conclusions: SCG3 mRNA, a component of the REST-dependent neurosecretory transcriptional profile, provides a sensitive prognostic biomarker for noninvasive monitoring of neuroendocrine lung cancer.

Use of RT-PCR to detect co-expression of neuropeptides and their receptors in lung cancer

Small-cell lung cancer (SCLC) synthesises a wide range of neuropeptides and their corresponding receptors. Together, these can form autocrine growth loops. Non-small-cell lung cancer (NSCLC) does not generally share this neuroendocrine phenotype. In this study, we tested the hypothesis that multiple neuropeptides and their receptors are co-expressed in SCLC, constituting potential autocrine loops. Expression of mRNA for arginine vasopressin, gastrin, cholecystokinin, gastrin-releasing peptide, endothelin and neurotensin, together with their cognate receptors, was evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR) in a panel of human lung cancer cell lines. We have assessed those neuropeptides and neuropeptide receptors that could be used as potential early markers to detect lung cancer cells both as micrometastases in blood and within dysplasia in bronchial biopsies. We establish that although no cell line expressed all neuropeptides, co-expression of neuropeptides and their receptors is common in SCLC but not in NSCLC. We conclude that mRNA for the neuropeptides gastrin-releasing peptide and arginine vasopressin and the cholecystokinin receptor B were most SCLC-specific and RT-PCR for these markers could be used to distinguish between SCLC and NSCLC.

Direct and indirect control of mitogen-activated protein kinase pathway-associated components, BRAP/IMP E3 ubiquitin ligase and CRAF/RAF1 kinase, by the deubiquitylating enzyme USP15.

Background: Deubiquitylases (DUBs) oppose the action of E3-ligases and influence key signalling pathways. Results: USP15 stabilizes the E3 ligase BRAP/IMP, regulates CRAF expression, and is a positive regulator of MEK. Conclusion: USP15 is a positive regulator of the MAPK pathway while stabilizing the E3 ligase BRAP/IMP. Significance: Evidence is provided for novel modes of MAPK pathway regulation by DUBs. The opposing regulators of ubiquitylation status, E3 ligases and deubiquitylases, are often found to be associated in complexes. Here we report on a novel interaction between the E3 ligase BRAP (also referred to as IMP), a negative regulator of the MAPK scaffold protein KSR, and two closely related deubiquitylases, USP15 and USP4. We map the interaction to the N-terminal DUSP-UBL domain of USP15 and the coiled coil region of BRAP. USP15 as well as USP4 oppose the autoubiquitylation of BRAP, whereas BRAP promotes the ubiquitylation of USP15. Importantly, USP15 but not USP4 depletion destabilizes BRAP by promoting its proteasomal degradation, and BRAP-protein levels can be rescued by reintroducing catalytically active but not inactive mutant USP15. Unexpectedly, USP15 depletion results in a decrease in amplitude of MAPK signaling in response to EGF and PDGF. We provide evidence for a model in which the dominant effect of prolonged USP15 depletion upon signal amplitude is due to a decrease in CRAF levels while allowing for the possibility that USP15 may also function to dampen MAPK signaling through direct stabilization of a negative regulator, the E3 ligase BRAP.

Neuron restrictive silencer factor as a modulator of neuropeptide gene expression

We hypothesize that the transcription factor neuron restrictive silencer factor (NRSF) is an important determinant of the expression of the preprotachykinin (PPTA) gene (encoding substance P and Neurokinin A) and arginine vasopressin (AVP) both in neuronal and nonneuronal cells. NRSF, a zinc finger repressor protein, binds the NRSE motif found in many neuronal specific genes at a variety of promoter locations. However, it is found in a similar location at the major transcriptional start site, within both PPTA and AVP peptide promoters. We have correlated modulation of NRSF activity with expression of AVP and PPTA in a variety of cell types, indicating the general mechanism by which this protein may regulate expression. Specifically, they are as follows:(1). Expression of NRSF dramatically represses PPTA promoter activity in reporter gene constructs in primary cultures of DRG neurons.(2). The PPTA promoter activity is regulated differentially in osteoarthritic compared to normal chondrocytes. This regulation correlates with the region containing the NRSE site.(3). We have correlated a splice variant of NRSF with the establishment and progression of small cell lung carcinoma (SCLC) and demonstrated that NRSF variants can directly affect the activity of the AVP promoter in reporter gene constructs. If the deregulated expression of peptides in these diseases point to the mechanism determining the pathology, then perhaps targeting protocols that correct this deregulation may also reverse the specific disease phenotypes. Our data would indicate that modulation of NRSF activity would be a target for such intervention.

Tumour-specific arginine vasopressin promoter activation in small-cell lung cancer

SummarySmall-cell lung cancer (SCLC) can produce numerous mitogenic neuropeptides, which are not found in normal respiratory epithelium. Arginine vasopressin is detected in up to two-thirds of SCLC tumours whereas normal physiological expression is essentially restricted to the hypothalamus. This presents the opportunity to identify elements of the gene promoter which could be exploited for SCLC-specific targeting. A series of human vasopressin 5′ promoter fragments (1048 bp, 468 bp and 199 bp) were isolated and cloned upstream of a reporter gene. These were transfected into a panel of ten cell lines, including SCLC with high or low endogenous vasopressin transcription, non-SCLC and bronchial epithelium. All these fragments directed reporter gene expression in the five SCLC cell lines, but had negligible activity in the control lines. The level of reporter gene expression reflected the level of endogenous vasopressin production, with up to 4.9-fold (s.d. 0.34) higher activity than an SV40 promoter. The elements required for this strong, restricted, SCLC-specific promoter activity are contained within the 199-bp fragment. Further analysis of this region indicated involvement of E-box transcription factor binding sites, although tumour-specificity was retained by a 65-bp minimal promoter fragment. These data show that a short region of the vasopressin promoter will drive strong expression in SCLC in vitro and raise the possibility of targeting gene therapy to these tumours.