Deubiquitylating enzymes and drug discovery: emerging opportunities

More than a decade after a Nobel Prize was awarded for the discovery of the ubiquitin-proteasome system and clinical approval of proteasome and ubiquitin E3 ligase inhibitors, first-generation deubiquitylating enzyme (DUB) inhibitors are now approaching clinical trials. However, although our knowledge of the physiological and pathophysiological roles of DUBs has evolved tremendously, the clinical development of selective DUB inhibitors has been challenging. In this Review, we discuss these issues and highlight recent advances in our understanding of DUB enzymology and biology as well as technological improvements that have contributed to the current interest in DUBs as therapeutic targets in diseases ranging from oncology to neurodegeneration.The authors thank L. Greger for the generation of the phylogenic tree. Research in the S.P.J. laboratory is funded by the Cancer Research UK (CRUK) Program Grant C6/A18796 and a Wellcome Trust Investigator Award (206388/Z/17/Z), and institute core infrastructure funding is provided by the CRUK (C6946/A24843) and the Wellcome Trust (WT203144)

A population-based analysis of germline BAP1 mutations in melanoma

Germline mutation of the BRCA1 associated protein-1 (BAP1) gene has been linked to uveal melanoma, mesothelioma, meningioma, renal cell carcinoma and basal cell carcinoma. Germline variants have also been found in familial cutaneous melanoma pedigrees, but their contribution to sporadic melanoma has not been fully assessed. We sequenced BAP1 in 1,977 melanoma cases and 754 controls and used deubiquitinase assays, a pedigree analysis, and a histopathological review to assess the consequences of the mutations found. Sequencing revealed 30 BAP1 variants in total, of which 27 were rare (ExAc allele frequency <0.002). Of the 27 rare variants, 22 were present in cases (18 missense, one splice acceptor, one frameshift and two near splice regions) and 5 in controls (all missense). A missense change (S98R) in a case that completely abolished BAP1 deubiquitinase activity was identified. Analysis of cancers in the pedigree of the proband carrying the S98R variant and in two other pedigrees carrying clear loss-of-function alleles showed the presence of BAP1-associated cancers such as renal cell carcinoma, mesothelioma and meningioma, but not uveal melanoma. Two of these three probands carrying BAP1 loss-of-function variants also had melanomas with histopathological features suggestive of a germline BAP1 mutation. The remaining cases with germline mutations, which were predominantly missense mutations, were associated with less typical pedigrees and tumours lacking a characteristic BAP1-associated histopathological appearances, but may still represent less penetrant variants. Germline BAP1 alleles defined as loss-of-function or predicted to be deleterious/damaging are rare in melanoma

Nitrogen hydrides in the cold envelope of IRAS16293-2422

Nitrogen is the fifth most abundant element in the Universe, yet the gas-phase chemistry of N-bearing species remains poorly understood. Nitrogen hydrides are key molecules of nitrogen chemistry. Their abundance ratios place strong constraints on the production pathways and reaction rates of nitrogen-bearing molecules. We observed the class 0 protostar IRAS16293-2422 with the heterodyne instrument HIFI, covering most of the frequency range from 0.48 to 1.78~THz at high spectral resolution. The hyperfine structure of the amidogen radical o-NH2 is resolved and seen in absorption against the continuum of the protostar. Several transitions of ammonia from 1.2 to 1.8~THz are also seen in absorption. These lines trace the low-density envelope of the protostar. Column densities and abundances are estimated for each hydride. We find that NH:NH2:NH3=5:1:300. {Dark clouds chemical models predict steady-state abundances of NH2 and NH3 in reasonable agreement with the present observations, whilst that of NH is underpredicted by more than one order of magnitude, even using updated kinetic rates. Additional modelling of the nitrogen gas-phase chemistry in dark-cloud conditions is necessary before having recourse to heterogen processes

The distribution of water in the high-mass star-forming region NGC 6334I

We present observations of twelve rotational transitions of H2O-16, H2O-18, and H2O-17 toward the massive star-forming region NGC 6334 I, carried out with Herschel/HIFI as part of the guaranteed time key program Chemical HErschel Surveys of Star forming regions (CHESS). We analyze these observations to obtain insights into physical processes in this region. We identify three main gas components (hot core, cold foreground, and outflow) in NGC 6334 I and derive the physical conditions in these components. The hot core, identified by the emission in highly excited lines, shows a high excitation temperature of 200 K, whereas water in the foreground component is predominantly in the ortho- and para- ground states. The abundance of water varies between 4 10^-5 (outflow) and 10^-8 (cold foreground gas). This variation is most likely due to the freeze-out of water molecules onto dust grains. The H2O-18/H2O-17 abundance ratio is 3.2, which is consistent with the O-18/O-17 ratio determined from CO isotopologues. The ortho/para ratio in water appears to be relatively low 1.6(1) in the cold, quiescent gas, but close to the equilibrium value of three in the warmer outflow material (2.5(0.8)).Comment: 7 pages, 3 figures, accepted by A&

The CHESS chemical Herschel surveys of star forming regions: Peering into the protostellar shock L1157-B1. I. Shock chemical complexity

We present the first results of the unbiased survey of the L1157-B1 bow shock, obtained with HIFI in the framework of the key program Chemical Herschel surveys of star forming regions (CHESS). The L1157 outflow is driven by a low-mass Class 0 protostar and is considered the prototype of the so-called chemically active outflows. The bright blue-shifted bow shock B1 is the ideal laboratory for studying the link between the hot (around 1000-2000 K) component traced by H2 IR-emission and the cold (around 10-20 K) swept-up material. The main aim is to trace the warm gas chemically enriched by the passage of a shock and to infer the excitation conditions in L1157-B1. A total of 27 lines are identified in the 555-636 GHz region, down to an average 3 sigma level of 30 mK. The emission is dominated by CO(5-4) and H2O(110-101) transitions, as discussed by Lefloch et al. (2010). Here we report on the identification of lines from NH3, H2CO, CH3OH, CS, HCN, and HCO+. The comparison between the profiles produced by molecules released from dust mantles (NH3, H2CO, CH3OH) and that of H2O is consistent with a scenario in which water is also formed in the gas-phase in high-temperature regions where sputtering or grain-grain collisions are not efficient. The high excitation range of the observed tracers allows us to infer, for the first time for these species, the existence of a warm (> 200 K) gas component coexisting in the B1 bow structure with the cold and hot gas detected from ground

First detection of ND in the solar-mass protostar IRAS16293-2422

In the past decade, much progress has been made in characterising the processes leading to the enhanced deuterium fractionation observed in the ISM and in particular in the cold, dense parts of star forming regions such as protostellar envelopes. Very high molecular D/H ratios have been found for saturated molecules and ions. However, little is known about the deuterium fractionation in radicals, even though simple radicals often represent an intermediate stage in the formation of more complex, saturated molecules. The imidogen radical NH is such an intermediate species for the ammonia synthesis in the gas phase. Herschel/HIFI represents a unique opportunity to study the deuteration and formation mechanisms of such species, which are not observable from the ground. We searched here for the deuterated radical ND in order to determine the deuterium fractionation of imidogen and constrain the deuteration mechanism of this species. We observed the solar-mass Class 0 protostar IRAS16293-2422 with the heterodyne instrument HIFI as part of the Herschel key programme CHESS (Chemical HErschel Surveys of Star forming regions). The deuterated form of the imidogen radical ND was detected and securely identified with 2 hyperfine component groups of its fundamental transition in absorption against the continuum background emitted from the nascent protostar. The 3 groups of hyperfine components of its hydrogenated counterpart NH were also detected in absorption. We derive a very high deuterium fractionation with an [ND]/[NH] ratio of between 30 and 100%. The deuterium fractionation of imidogen is of the same order of magnitude as that in other molecules, which suggests that an efficient deuterium fractionation mechanism is at play. We discuss two possible formation pathways for ND, by means of either the reaction of N+ with HD, or deuteron/proton exchange with NH.Comment: Accepted; To appear in A&A Herschel/HIFI Special Issu

Ortho-to-para ratio of interstellar heavy water

Despite the low elemental deuterium abundance in the Galaxy, enhanced molecular D/H ratios have been found in the environments of low-mass star forming regions, and in particular the Class 0 protostar IRAS 16293-2422. The CHESS (Chemical HErschel Surveys of Star forming regions) Key Program aims at studying the molecular complexity of the interstellar medium. The high sensitivity and spectral resolution of the HIFI instrument provide a unique opportunity to observe the fundamental 1,1,1 - 0,0,0 transition of the ortho-D2O molecule, inaccessible from the ground, and to determine the ortho-to-para D2O ratio. We have detected the fundamental transition of the ortho-D2O molecule at 607.35 GHz towards IRAS 16293-2422. The line is seen in absorption with a line opacity of 0.62 +/- 0.11 (1 sigma). From the previous ground-based observations of the fundamental 1,1,0 - 1,0,1 transition of para-D2O seen in absorption at 316.80 GHz we estimate a line opacity of 0.26 +/- 0.05 (1 sigma). We show that the observed absorption is caused by the cold gas in the envelope of the protostar. Using these new observations, we estimate for the first time the ortho to para D2O ratio to be lower than 2.6 at a 3 sigma level of uncertainty, to be compared with the thermal equilibrium value of 2:1.Comment: 5 pages, 5 figures, accepted the A&A HIFI Special Issue as a lette

CHESS, Chemical Herschel surveys of star forming regions:Peering into the protostellar shock L1157-B1

The outflow driven by the low-mass class 0 protostar L1157 is the prototype of the so-called chemically active outflows. The bright bowshock B1 in the southern outflow lobe is a privileged testbed of magneto-hydrodynamical (MHD) shock models, for which dynamical and chemical processes are strongly interdependent. We present the first results of the unbiased spectral survey of the L1157-B1 bowshock, obtained in the framework of the key program "Chemical Herschel Surveys of Star Forming Regions" (CHESS). The main aim is to trace the warm and chemically enriched gas and to infer the excitation conditions in the shock region. The CO 5-4 and H2O lines have been detected at high-spectral resolution in the unbiased spectral survey of the HIFI-Band 1b spectral window (555-636 GHz), presented by Codella et al. in this volume. Complementary ground-based observations in the submm window help establish the origin of the emission detected in the main-beam of HIFI, and the physical conditions in the shock.}{Both lines exhibit broad wings, which extend to velocities much higher than reported up to now. We find that the molecular emission arises from two regions with distinct physical conditions: an extended, warm (100K), dense (3e5 cm-3) component at low-velocity, which dominates the water line flux in Band~1; a secondary component in a small region of B1 (a few arcsec) associated with high-velocity, hot (> 400 K) gas of moderate density ((1.0-3.0)e4 cm-3), which appears to dominate the flux of the water line at 179mu observed with PACS. The water abundance is enhanced by two orders of magnitude between the low- and the high-velocity component, from 8e-7 up to 8e-5. The properties of the high-velocity component agree well with the predictions of steady-state C-shock models.Comment: Accepted for Publication in Astronomy and Astrophysics (Herschel Special Issue

Identification of a Small TAF Complex and Its Role in the Assembly of TAF-Containing Complexes

TFIID plays a role in nucleating RNA polymerase II preinitiation complex assembly on protein-coding genes. TFIID is a multisubunit complex comprised of the TATA box binding protein (TBP) and 14 TBP-associated factors (TAFs). Another class of multiprotein transcriptional regulatory complexes having histone acetyl transferase (HAT) activity, and containing TAFs, includes TFTC, STAGA and the PCAF/GCN5 complex. Looking for as yet undiscovered subunits by a proteomic approach, we had identified TAF8 and SPT7L in human TFTC preparations. Subsequently, however, we demonstrated that TAF8 was not a stable component of TFTC, but that it is present in a small TAF complex (SMAT), containing TAF8, TAF10 and SPT7L, that co-purified with TFTC. Thus, TAF8 is a subunit of both TFIID and SMAT. The latter has to be involved in a pathway of complex formation distinct from the other known TAF complexes, since these three histone fold (HF)-containing proteins (TAF8, TAF10 and SPT7L) can never be found together either in TFIID or in STAGA/TFTC HAT complexes. Here we show that TAF8 is absolutely necessary for the integration of TAF10 in a higher order TFIID core complex containing seven TAFs. TAF8 forms a heterodimer with TAF10 through its HF and proline rich domains, and also interacts with SPT7L through its C-terminal region, and the three proteins form a complex in vitro and in vivo. Thus, the TAF8-TAF10 and TAF10-SPT7L HF pairs, and also the SMAT complex, seem to be important regulators of the composition of different TFIID and/or STAGA/TFTC complexes in the nucleus and consequently may play a role in gene regulation

Electron Transport Properties of Single-Molecule-Bearing Multiple Redox Levels Studied by EC-STM/STS

Multielectron systems as possible components of molecular electronics devices are attracting compelling experimental and theoretical interest. Here we studied by electrochemical scanning tunneling techniques (EC-STMicroscopy and EC-STSpectroscopy) the electron transport properties of a redox molecule endowed with two redox levels, namely, the hydroquinone/quinone (H2Q/Q) couple. By forming self-assembled monolayers on Au(111) of oligo-phenylene-vinylene (OPV) derivatized H2Q/Q moieties, we were able to explore the features of the tunneling current/overpotential relation in the EC-STS setup. The behavior of the tunneling current sheds light onto the mechanism of electron transport involving the redox levels of the H2Q/Q redox pair coupled to tip and substrate electrodes