Identifizierung und Charakterisierung neuer Interaktoren des von Hippel-Lindau Tumorsuppressors

Die Inaktivierung des von Hippel-Lindau (VHL) Tumorsuppressors spielt eine Rolle in der Entstehung von verschiedenen gut- und bösartigen Tumoren mit hoher Gewebespezifität. Als substraterkennende Untereinheit des CBCVHL Ubiquitin Ligase Komplexes steuert VHL den sauerstoffabhängigen Abbau des Transkriptionsfaktors HIF1/2α. HIF1/2α aktiviert die Transkription einer Vielzahl von Faktoren, die für den Energiehaushalt der Zelle und die Blutgefäßneubildung von entscheidender Bedeutung sind. Die Akkumulation von HIF1/2α führt zu deren konstitutiver Expression und fördert somit das Wachstum von Tumoren durch eine verbesserte Nährstoffversorgung. Der sauerstoffabhängige Mechanismus der HIF-Erkennung wird durch die Aktivität einer neuen Familie von Prolylhydroxylasen reguliert, die möglicherweise ihrerseits eine Reihe von zellulären Substraten haben. Trotz der guten Korrelation zwischen bestimmten, den HIF-Abbau beeinflussenden VHL-Mutationen und dem Auftreten von verschiedenen Krankheitssubtypen sind noch nicht alle Phänotypen im Zusammenhang mit VHL erklärbar. Vor allem die Identifizierung neuer Substrate für den CBCVHL Komplex ist für ein umfassendes Verständnis der VHL-Krankheit von Interesse. In dieser Arbeit wurden unterschiedliche Methoden zur Identifizierung neuer Substrate von VHL angewendet. Durch Affinitätschromatographie mit einem rekombinanten Komplex aus VHL, Elongin B und Elongin C (VCB) konnte Daxx als neuer Interaktor von VHL identifiziert werden. Daxx bindet Elongin B/C-unabhängig an VHL, und seine Stabilität wird nicht durch VHL reguliert. Zudem bildet Daxx einen Komplex mit dem VHL-Substrat HIF1α. Dies weist auf eine mögliche Funktion von VHL neben seiner Rolle als Ubiquitin Ligase hin, z.B. in der Regulation von Daxx als transkriptionellem Repressor. In einem funktionalisierten „TwoHybrid“-Screen konnte der Mechanismus der HIF-Regulation in S. cerevisiae rekonstituiert werden. Dies ermöglichte die Identifizierung weiterer potentieller VHL-Substrate, unter anderem Diacylglycerol Kinase iota (DGKι). DGKι weist zwei Erkennungsmotive für Prolylhydroxylasen auf und wird in Gehirn und Retina exprimiert. In diesem Organen kommt es bei VHL-Patienten zur Entstehung von Hämangioblastomen. DGKι wird in vivo ubiquityliert und bindet sowohl an VHL, als auch an zwei der drei bekannten Prolylhydroxylasen. Mit Mutanten von DGKι konnte allerdings gezeigt werden, dass Bindung und Ubiquitylierung nicht über den gleichen Mechanismus erfolgen wie bei HIF1α. Möglicherweise spielen Ubiquitylierung und VHL-Bindung getrennte Rollen in unterschiedlichen zellulären Prozessen. Es wird zunehmend deutlicher, dass VHL nicht nur eine Komponente des CBCVHL Komplexes bildet, sondern weitere Funktionen in der Zelle erfüllt. VHL spielt eine Rolle in der Assemblierung der Fibronektinmatrix, der Regulation von Mikrotubulistabilität und –dynamik und der Transkriptionskontrolle. Eine weitere Charakterisierung des nicht-degradativen Einflusses von VHL auf die in dieser Arbeit beschriebenen Bindungspartner ist nötig, um die zelluläre Wirkungsweise von VHL vollständig zu verstehen

A yeast two-hybrid system reconstituting substrate recognition of the von Hippel-Lindau tumor suppressor protein

The von Hippel-Lindau tumor suppressor protein (pVHL) is inactivated in the hereditary cancer syndrome von Hippel-Lindau disease and in the majority of sporadic renal carcinomas. pVHL is the substrate-binding subunit of the CBCVHL ubiquitin ligase complex that negatively regulates cell growth by promoting the degradation of hypoxia-inducible transcription factor subunits (HIF1/2α). Proteomics-based identification of novel pVHL substrates is hampered by their short half-life and low abundancy in mammalian cells. The usefulness of yeast two-hybrid (Y2H) approaches, on the other hand, has been limited by the failure of pVHL to adopt its native structure and by the absence of prolylhydroxylase activity critical for pVHL substrate recognition. Therefore, we modified the Y2H system to faithfully reconstitute the physical interaction between pVHL and its substrates. Our approach relies on the coexpression of pVHL with the cofactors Elongin B and Elongin C and with HIF1/2α prolylhydroxylases. In a proof-of-principle Y2H screen, we identified the known substrates HIF1/2α and new candidate substrates including diacylglycerol kinase iota, demonstrating that our strategy allows detection of stable interactions between pVHL and otherwise elusive cellular targets. Additional future applications may include structure/function analyses of pVHL-HIF1/2α binding and screens for therapeutically relevant compounds that either stabilize or disrupt this interaction

Dialect, interaction and class positioning at school: from deficit to difference to repertoire.

Sociolinguists have been fighting dialect prejudice since the 1960s, but deficit views of non-standard English are regaining currency in educational discourse. In this paper I argue that the traditional sociolinguistic response – stressing dialect systematicity and tolerance of ‘difference’ – may no longer be effective by questioning a key assumption that both deficit and difference approaches share, namely that there exist discrete varieties of English. Based on an empirical study of the language of working-class children in north-east England, I demonstrate that non-standard dialects of English do not have a discrete system of grammar that is isolated from other varieties; rather local dialect forms interact with a range of semiotic resources (including standard forms) within speakers’ repertoires. Interactional analyses of the children’s spontaneous speech highlight this hybridity, as well as the social meanings behind the linguistic choices children make. I conclude by addressing educational responses to non-standard dialect in the classroom, suggesting that it is not the presence or absence of non-standard forms in children’s speech that raises educational issues; rather, educational responses which problematise non-standard voices risk marginalising working-class speech, and may contribute to the alienation of working-class children, or significant groups of them, within the school system

A cyclic GMP signalling module that regulates gliding motility in a malaria parasite.

The ookinete is a motile stage in the malaria life cycle which forms in the mosquito blood meal from the zygote. Ookinetes use an acto-myosin motor to glide towards and penetrate the midgut wall to establish infection in the vector. The regulation of gliding motility is poorly understood. Through genetic interaction studies we here describe a signalling module that identifies guanosine 3', 5'-cyclic monophosphate (cGMP) as an important second messenger regulating ookinete differentiation and motility. In ookinetes lacking the cyclic nucleotide degrading phosphodiesterase delta (PDEdelta), unregulated signalling through cGMP results in rounding up of the normally banana-shaped cells. This phenotype is suppressed in a double mutant additionally lacking guanylyl cyclase beta (GCbeta), showing that in ookinetes GCbeta is an important source for cGMP, and that PDEdelta is the relevant cGMP degrading enzyme. Inhibition of the cGMP-dependent protein kinase, PKG, blocks gliding, whereas enhanced signalling through cGMP restores normal gliding speed in a mutant lacking calcium dependent protein kinase 3, suggesting at least a partial overlap between calcium and cGMP dependent pathways. These data demonstrate an important function for signalling through cGMP, and most likely PKG, in dynamically regulating ookinete gliding during the transmission of malaria to the mosquito

Adherence to Treatment Guidelines and Associated Survival in Older Patients with Prostate Cancer: A Prospective Multicentre Cohort Study

International audienceThe guidelines on prostate cancer treatment in older men recommend evaluating the patient’s underlying health status before treatment selection. We aimed to evaluate the frequency of a guideline–discordant treatment (GDT), identify factors associated with GDT, and assess the relationship between GDT and overall survival. We studied patients with prostate cancer aged 70 or older included in the ELCAPA cohort between 2010 and 2019. Multivariable logistic regression assessed GDT-associated factors. The restricted mean survival time (RMST) assessed the 24- and 36-month OS using stabilized inverse probability of treatment weighting of propensity scores. We included 356 patients (median age: 81 years), and 164 (46%) received a GDT (95% confidence interval (CI) = (41–51%)). Patients with metastases were less likely to receive a GDT (adjusted odds ratio (95% CI) = 0.34 (0.17–0.69); p = 0.003). After weighting, the RMST at 24 months was shorter in the GDT group (13.9 months, vs. 17 months for compliant treatments; difference (95% CI): −3.1 months (−5.3, −1.0); p = 0.004). RMST at 36 months was 18.5 months, vs. 21.8 months (difference: −3.3 months (−6.7, 0.0); p = 0.053). GDT is common in older patients with prostate cancer and especially those with non-metastatic disease. GDT was associated with worse survival, independently of health status and tumour characteristics

Cognitive rehabilitation and aerobic exercise for cognitive impairment in people with progressive multiple sclerosis (CogEx): a randomised, blinded, sham-controlled trial

Background Cognitive dysfunction in people with relapsing-remitting multiple sclerosis can improve with cognitive rehabilitation or exercise. Similar effects have not been clearly shown in people with progressive multiple sclerosis. We aimed to investigate the individual and synergistic effects of cognitive rehabilitation and exercise in patients with progressive multiple sclerosis.Methods CogEx was a randomised, sham-controlled trial completed in 11 hospital clinics, universities, and rehabilitation centres in Belgium, Canada, Denmark, Italy, UK, and USA. Patients with progressive multiple sclerosis were eligible for inclusion if they were aged 25-65 years and had an Expanded Disability Status Scale (EDSS) score of less than 7. All had impaired processing speed defined as a performance of 1 center dot 282 SD or greater below normative data on the Symbol Digit modalities Tests (SDMT). Participants were randomly assigned (1:1:1:1), using an interactive web-response system accessed online from each centre, to cognitive rehabilitation plus exercise, cognitive rehabilitation plus sham exercise, exercise plus sham cognitive rehabilitation, or sham exercise plus sham cognitive rehabilitation. The study statistician created the randomisation sequence that was stratified by centre. Participants, outcome assessors, and investigators were blinded to group allocation. The study statistician was masked to treatment during analysis only. Interventions were conducted two times per week for 12 weeks: cognitive rehabilitation used an individualised, computer-based, incremental approach to improve processing speed; sham cognitive rehabilitation consisted of internet training provided individually; the exercise intervention involved individualised aerobic training using a recumbent arm-leg stepper; and the sham exercise involved stretching and balance tasks without inducing cardiovascular strain. The primary outcome measure was processing speed measured by SDMT at 12 weeks; least squares mean differences were compared between groups using linear mixed model in all participants who had a 12-week assessment. The trial is registered with ClinicalTrials.gov, NCT03679468, and is completed.Findings Between Dec 14, 2018, and April 2, 2022, 311 people with progressive multiple sclerosis were enrolled and 284 (91%) completed the 12-week assessment (117/311 [38%] male and 194/311 [62%] female). The least squares mean group differences in SDMT at 12 weeks did not differ between groups (p=0 center dot 85). Compared with the sham cognitive rehabilitation and sham exercise group (n=67), differences were -1 center dot 30 (95% CI -3 center dot 75 to 1 center dot 16) for the cognitive rehabilitation plus exercise group (n=70); -2 center dot 78 (-5 center dot 23 to -0 center dot 33) for the sham cognitive rehabilitation plus exercise group (n=71); and -0 center dot 71 (-3 center dot 11 to 1 center dot 70) for the cognitive rehabilitation plus sham exercise group (n=76). 11 adverse events possibly related to the interventions occurred, six in the exercise plus sham cognitive rehabilitation group (pain, dizziness, and falls), two in the cognitive rehabilitation plus sham exercise group (headache and pain), two in the cognitive rehabilitation and exercise group (increased fatigue and pain), and one in the dual sham group (fall).Interpretation Combined cognitive rehabilitation plus exercise does not seem to improve processing speed in people with progressive multiple sclerosis. However, our sham interventions were not inactive.Studies comparing interventions with a non-intervention group are needed to investigate whether clinically meaningful improvements in processing speed might be attainable in people with progressive multiple sclerosis.Copyright (c) 2023 Elsevier Ltd. All rights reserved