Crystal Structure of the Cul2-Rbx1-EloBC-VHL Ubiquitin Ligase Complex

Cullin RING E3 ubiquitin ligases (CRLs) function in the ubiquitin proteasome system to catalyze the transfer of ubiquitin from E2 conjugating enzymes to specific substrate proteins. CRLs are large dynamic complexes and attractive drug targets for the development of small-molecule inhibitors and chemical inducers of protein degradation. The atomic details of whole CRL assembly and interactions that dictate subunit specificity remain elusive. Here we present the crystal structure of a pentameric CRL2VHL complex, composed of Cul2, Rbx1, Elongin B, Elongin C, and pVHL. The structure traps a closed state of full-length Cul2 and a new pose of Rbx1 in a trajectory from closed to open conformation. We characterize hotspots and binding thermodynamics at the interface between Cul2 and pVHL-EloBC and identify mutations that contribute toward a selectivity switch for Cul2 versus Cul5 recognition. Our findings provide structural and biophysical insights into the whole Cul2 complex that could aid future drug targeting

Opposing effects of cancer-type-specific SPOP mutants on BET protein degradation and sensitivity to BET inhibitors.

It is generally assumed that recurrent mutations within a given cancer driver gene elicit similar drug responses. Cancer genome studies have identified recurrent but divergent missense mutations affecting the substrate-recognition domain of the ubiquitin ligase adaptor SPOP in endometrial and prostate cancers. The therapeutic implications of these mutations remain incompletely understood. Here we analyzed changes in the ubiquitin landscape induced by endometrial cancer-associated SPOP mutations and identified BRD2, BRD3 and BRD4 proteins (BETs) as SPOP-CUL3 substrates that are preferentially degraded by endometrial cancer-associated SPOP mutants. The resulting reduction of BET protein levels sensitized cancer cells to BET inhibitors. Conversely, prostate cancer-specific SPOP mutations resulted in impaired degradation of BETs, promoting their resistance to pharmacologic inhibition. These results uncover an oncogenomics paradox, whereby mutations mapping to the same domain evoke opposing drug susceptibilities. Specifically, we provide a molecular rationale for the use of BET inhibitors to treat patients with endometrial but not prostate cancer who harbor SPOP mutations

Cullin3-KLHL15 ubiquitin ligase mediates CtIP protein turnover to fine-tune DNA-end resection

Human CtIP is a decisive factor in DNA double-strand break repair pathway choice by enabling DNA-end resection, the first step that differentiates homologous recombination (HR) from non-homologous end-joining (NHEJ). To coordinate appropriate and timely execution of DNA-end resection, CtIP function is tightly controlled by multiple protein-protein interactions and post-translational modifications. Here, we identify the Cullin3 E3 ligase substrate adaptor Kelch-like protein 15 (KLHL15) as a new interaction partner of CtIP and show that KLHL15 promotes CtIP protein turnover via the ubiquitin-proteasome pathway. A tripeptide motif (FRY) conserved across vertebrate CtIP proteins is essential for KLHL15-binding; its mutation blocks KLHL15-dependent CtIP ubiquitination and degradation. Consequently, DNA-end resection is strongly attenuated in cells overexpressing KLHL15 but amplified in cells either expressing a CtIP-FRY mutant or lacking KLHL15, thus impacting the balance between HR and NHEJ. Collectively, our findings underline the key importance and high complexity of CtIP modulation for genome integrity

Identification and developmental expression of the full complement of Cytochrome P450 genes in Zebrafish

© The Authors, 2010. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in BMC Genomics 11 (2010): 643, doi:10.1186/1471-2164-11-643.Increasing use of zebrafish in drug discovery and mechanistic toxicology demands knowledge of cytochrome P450 (CYP) gene regulation and function. CYP enzymes catalyze oxidative transformation leading to activation or inactivation of many endogenous and exogenous chemicals, with consequences for normal physiology and disease processes. Many CYPs potentially have roles in developmental specification, and many chemicals that cause developmental abnormalities are substrates for CYPs. Here we identify and annotate the full suite of CYP genes in zebrafish, compare these to the human CYP gene complement, and determine the expression of CYP genes during normal development. Zebrafish have a total of 94 CYP genes, distributed among 18 gene families found also in mammals. There are 32 genes in CYP families 5 to 51, most of which are direct orthologs of human CYPs that are involved in endogenous functions including synthesis or inactivation of regulatory molecules. The high degree of sequence similarity suggests conservation of enzyme activities for these CYPs, confirmed in reports for some steroidogenic enzymes (e.g. CYP19, aromatase; CYP11A, P450scc; CYP17, steroid 17a-hydroxylase), and the CYP26 retinoic acid hydroxylases. Complexity is much greater in gene families 1, 2, and 3, which include CYPs prominent in metabolism of drugs and pollutants, as well as of endogenous substrates. There are orthologous relationships for some CYP1 s and some CYP3 s between zebrafish and human. In contrast, zebrafish have 47 CYP2 genes, compared to 16 in human, with only two (CYP2R1 and CYP2U1) recognized as orthologous based on sequence. Analysis of shared synteny identified CYP2 gene clusters evolutionarily related to mammalian CYP2 s, as well as unique clusters. Transcript profiling by microarray and quantitative PCR revealed that the majority of zebrafish CYP genes are expressed in embryos, with waves of expression of different sets of genes over the course of development. Transcripts of some CYP occur also in oocytes. The results provide a foundation for the use of zebrafish as a model in toxicological, pharmacological and chemical disease research.This work was supported by NIH grants R01ES015912 and P42ES007381 (Superfund Basic Research Program at Boston University) (to JJS). MEJ was a Guest Investigator at the Woods Hole Oceanographic Institution (WHOI) and was supported by grants from the Swedish research council Formas and Carl Trygger's foundation. AK was a Post-doctoral Fellow at WHOI, and was supported by a fellowship from the Japanese Society for Promotion of Science (JSPS). JZ and TP were Guest Students at the WHOI and were supported by a CAPES Ph.D. Fellowship and CNPq Ph.D. Sandwich Fellowship (JZ), and by a CNPq Ph.D. Fellowship (TP), from Brazil

Mobile health (mHealth) for headache disorders: a review of the evidence base

The aim of this review was to explore and summarise the evidence base for using mobile apps for the management of headache disorders. PubMed, Web of Science, Embase and Scopus were searched for studies reporting the use of mobile health applications (apps) for managing headache disorders. Different combinations of keywords for mobile health, smartphone and headache were used for electronic search. Six studies (two journal papers and four conference papers) met the inclusion criteria and were reviewed. All of the reviewed studies were categorised as the lowest level (level IV) of evidence according to the National Health and Medical Research Council (NHMRC) hierarchy of evidence. The results of the studies demonstrated the feasibility and acceptability of a few mobile apps for headache in very limited settings, and indicated that these apps can be effective tools for identifying trigger factors of migraine, improving self-management of headache disorders, and mediating the interactions between headache sufferers and their treating doctors. The role of participatory research and involvement of patients and clinicians in the development of such apps was also highlighted. Despite the availability of numerous mobile apps for headache disorders, the evidence base to support their effectiveness and clinical safety is very weak. The current literature indicates that mobile apps have the potential for improving the care of headache sufferers, but this is yet to be proven by adequately designed studies

Effect of Preceding Crop Type and Different Amounts of Nitrogen Fertilizer on the Yield and Quality of Rapeseed Oil

Rapeseed needs a sufficient amount of nitrogen for optimal seed yield, but the efficiency of this plant in using nitrogenous (N) fertilizers is low, which causes an increase in N consumption in the cultivation of this plant. The need to maintain a high grain yield necessitates the use of a suitable timely program and manage the consumption of N fertilizers. Thus, a field experiment was conducted at the Research Station of Agriculture and Natural Resources of Khuzestan University during 2020-2021 growing season to evaluate the effect of preceding crop and N fertilizer on grain yield of rapeseed. The experiment was carried out as split plots based on a RCBD with three replications. Preceding crops (clover-barley mixture, rapeseed, corn, fallow, mung bean, rice and wheat) were allocated to the main plots and N levels (0.0, 100, 160 and 220 kg ha-1 N from the source of urea fertilizer) were assigned to the sub plots. Results indicated that the grain yield of rapeseed depends mostly on grain number, but the grain yield components (including pods number per plant, grain number per plant, grain number per pods) were mainly affected by the main effect of preceding crop and nitrogen. With the application of 100, 160 and 220 kg ha-1 N the number of grains per pod increased by 34%, 39% and 33%, respectively. The number of grains per plant was also increased by the application of nitrogen fertilizer, where all three levels of nitrogen fertilizer resulted in more than 200% increases in this grain yield attribute. The highest grain yield of rapeseed (3877 kg ha-1) was observed when mung bean was used as preceding crop and 220 kg ha-1 N fertilizer was applied, though it was not significantly different from the treatments of mung bean preceding crop and application of 160 kg ha-1 nitrogen fertilizer (with the production of 3105 kg ha-1 grain) and fallow preceding and 160 kg ha-1 N fertilizer. The lowest grain yield of rapeseed was obtained when corn and rapeseed were used as preceding crops and no N fertilizer was applied, as these preceding crops resulted in only 488 and 497 kg ha-1 grain of rapeseed, respectively. Rapeseed had the highest oil yield (1645 kg ha-1) with mung bean being the preceding crop and 220 kg ha-1 N application. It seems that for achieving the highest oil yield of rapeseed, mung bean preceding crop and 220 kg ha-1 N can be focused on for the next researches

Digital health interventions for chronic diseases: A scoping review of evaluation frameworks

Background Monitoring and evaluations of digital health (DH) solutions for the management of chronic diseases are quite heterogeneous and evidences around evaluating frameworks are inconsistent. An evidenced-based framework is needed to inform the evaluation process and rationale of such interventions. We aimed to explore the nature, extent and components of existing DH frameworks for chronic diseases. Methods This review was conducted based on the five steps of Arksey and O'Malley's scoping review methodology. Out of 172 studies identified from, PubMed, Embase and Web of Science, 11 met our inclusion criteria. The reviewed studies developed DH frameworks for chronic diseases and published between 2010 and 2018. Results According to WHO guidelines for monitoring and evaluation of DH interventions, we identified seven Conceptual frameworks, two Results frameworks, one Logical framework and one Theory of change. The frameworks developed for providing interventions such as self-management, achieving personal goals and reducing relapse for cardiovascular disease, diabetes, chronic obstructive pulmonary disease and severe mental health. A few studies reported evaluation of the frameworks using randomised clinical trials (n=3) and feasibility testing via Likert scale survey (n=2). A wide range of outcomes were reported including access to care, cost-effectiveness, behavioural outcomes, patient-provider communications, technology acceptance and user experience. Conclusion There is a lack of evidence on the application of consistent DH frameworks. Future research should address the use of evidence-based frameworks into the research design, monitoring and evaluation process. This review explores the nature of DH frameworks for the management of chronic diseases and provides examples to guide monitoring and evaluation of interventions. Author(s) (or their employer(s)) 2020