Structural and biophysical analysis of the proteasomal deubiquitinase, UCH37

Ubiquitin carboxyl-terminal hydrolase 37, or UCH37, is a deubiquitinating enzyme associated with the 26S proteasome, the primary protein degradation machinery in eukaryotic cells. UCH37 is responsible for the disassembly of polymeric ubiquitin chains, or polyubiquitin, which have been ligated onto proteins in order to target them for degradation. The 26S utilizes two associated deubiquitinating enzymes, UCH37 and USP14, and one intrinsic, Rpn11, to remove polyubiquitin chains from substrate proteins as they are unfolded and translocated into the proteolytic core of the proteasome, where proteins are cleaved into small peptides and then released for recycling by the cell. UCH37 associates with the proteasome via binding of its C-terminal KEKE motif to the C-terminus of Rpn13, a proteasomal ubiquitin receptor which ensnares polyubiquitinated prey for degradation. UCH37 is known to be catalytically activated upon binding to Rpn13, allowing cleavage of Lys48-linked polyubiquitin chains from their distal end, an exo-specific deubiquitination. However, free UCH37 cleaves polyubiquitin poorly and is believed to be autoinhibited by its C-terminal UCHL5-like domain, or ULD, which may also be responsible for its oligomerization in solution. This work examines the structural, biophysical, and catalytic characteristics of UCH37 in order to elucidate its mechanism of activation by Rpn13, assess its biophysical assembly with Rpn13 within the greater proteasomal context, and ascertain its mechanism of exo-specificity despite the proteasome\u27s processing of a variety of polyubiquitinated substrates.^ To this end, a 1.7 Å resolution x-ray crystal structure was solved of the catalytic domain of a UCH37 homolog from Trichinella spiralisin complex with ubiquitin vinyl methyl ester (UbVME), a suicide inhibitor substrate. Our structure, in combination with another solved of a longer construct of TsUCH37 in complex with UbVME, provided structural insights into the ability of UCH37 to process polyubiquitin, namely that its C-terminal UCHL5-like domain (ULD) is responsible for its exo-specific activity due to a network of interactions with ubiquitin\u27s Lys48.^ Through biophysical and kinetic characterization, we have affirmed the poor activity of UCH37 alone, but do not ascribe it to autoinhibition because it does not oligomerize as previously thought, rather we find that it sediments in a monomer-dimer equilibrium in analytical ultracentrifugation experiments. We have characterized its binding and activation by Rpn13, finding that UCH37 binds to Rpn13 with a 22 nM dissociation constant and that mutations to UCH37\u27s ULD render it unable to be activated by Rpn13. Interestingly, we have found that while Rpn13 activates UCH37 for ubiquitin-AMC cleavage, a monoubiquitin fluorogenic substrate, it appears to slow the enzyme\u27s processing of Lys48-linked polyubiquitin chains in our assays.^ Altogether, we have confirmed that UCH37 exists primarily as a monomer which binds tightly to its proteasomal subunit, Rpn13, and can exo-specifically cleave Lys48-linked polyubiquitin chains. However, UCH37 may not be activated as was previously thought, by Rpn13 alone, and likely requires full association with the 26S proteasome

Delivering research impact that is aligned to social priorities requires public participation throughout the process

The notion that increased public participation is a key component of research impact has developed and gained traction. Indeed, recent analysis has shown that public and user participation does play a key role in delivering impact. However, how does this participation work in practice? Steven Hill, Elizabeth Morrow and Fiona Ross note that the majority of public engagement focuses on the dissemination of findings. Consultation and collaboration remain uncommon, with public participation rarely extending to the framing and development of research questions. Such narrow use of participation risks missing opportunities to align impact more closely with social priorities

A Study of the New Zealand Mathematics Curriculum

Given the profound and uncritiqued changes that have been implemented in Aotearoa New Zealand education since the 1990s, this paper provides a critical commentary on the characterising features of the New Zealand mathematics' curriculum in the context of the first stage of a study. The emphasis is on the importance of research design that begins with an explicit, evidence-based hypothesis. To that end, we describe evidence that informs and identifies the study's hypothesised problem and causes. The study itself will show whether or not the hypothesis is justified; that is, is the absence of standardised prescribed content in New Zealand mathematics' curriculum the reason for the country's declining mathematics rankings? The study aims to increase understanding in the field of mathematics education by exploring the effects on New Zealand year 7 public school teachers' mathematics curriculum selection and design practices, teaching practices, and subsequently student achievement.Comment: Submitted to the New Zealand Journal of Educational Studies, 12 June 202

The role of gamma delta T lymphocytes in breast cancer: a review

Gammadelta T (γδT) lymphocytes have provoked interest in oncology, particularly as regards their potential use in immunotherapy, because of their unique ability to recognise antigens without a requirement for major histocompatibility complex antigen presentation, and to quickly activate an anti-tumour response. However, work in some cancers has suggested that they also have pro-tumourigenic activity. Their role in breast cancer is unclear. This review outlines the evidence to date in in vitro studies, in vivo mouse models and in human studies regarding the role of γδT lymphocytes in breast cancer. We describe the seemingly opposing roles of the predominantly circulating Vγ9Vδ2 subtype, which can suppress tumour growth through direct cytotoxicity, induction of apoptosis and inhibition of angiogenesis, and the predominantly tumour-infiltrating γδ1 subtype which can promote tumour growth and spread through immunosuppressant effects. We summarise the evidence in breast cancer for the mechanisms of action of γδT lymphocytes and describe how factors in the tumour microenvironment may affect their function, polarising them towards a pro-tumourigenic, immune-suppressing role. We also describe the experience to date of γδT lymphocytes in immunotherapy for breast cancer and suggest the direction of work going forward, particularly as regards different breast cancer subtypes

Faith no more? The divergence of political trust between urban and rural Europe

Events such as Brexit and the Gilet Jaunes protests have highlighted the spatial nature of populism. In particular, there has been increasing political divergence between urban and rural areas, with rural areas apparently having lost faith in national governments. We investigate this divergence using data on over 125,000 from the European Social Survey from 2008-2018. We show that people in rural areas have lower political trust than urban or peri-urban residents, with this difference clear for six different forms of political institutions, including politicans, political parties, and national parliaments. There has been divergence of political trust between urban and rural Europe since 2008, although this is primarily driven by Southern Europe. While these results can be primarily explained by demographic differences between cities and the countryside, divergent economic experiences, differences in values and perspectives that public services are less effective outside of urban areas, there is a residual 'rural effect' beyond this. We argue that the polarization of urban-rural political trust has important implications for the functioning of political democracies

4 priorities to reaffirm patient voice in the coming era of AI healthcare

Healthcare is becoming both increasingly data driven and automated. Drawing on a largescale review of artificial intelligence developments in the field of mental health and wellbeing, Elizabeth Morrow, Teodor Zidaru-Bărbulescu and Rich Stockley, find that opportunities for patients to influence and inform these future technologies are often lacking, which in turn may heighten disillusionment and lack of trust in them. As such, they propose four priorities for new data driven technologies to ensure they are ethical, effective and equitable for diverse patient groups

The role of systemic and local inflammation, the tumour microenvironment and the IL6/JAK/STAT3 pathway in primary operable breast cancer

Breast cancer is the second most common cause of cancer death in females in the UK. It is a heterogenous disease with subtypes which behave differently. There are targeted treatments available for luminal and HER2+ cancers but treatment resistance and cancer recurrences occur. There are currently no targeted treatments for triple negative breast cancer (TNBC). New prognostic tools to stratify risk to guide use of the most aggressive treatments, and new therapeutic targets are desirable. The role of the tumour microenvironment in tumour progression is increasingly recognised. Features of the tumour such as necrosis and budding have been reported to have a prognostic role in cancer and may be influenced by the tumour microenvironment. Cell signalling pathways such as the IL6/JAK/STAT3 pathway provide a link between the tumour microenvironment and tumour cells. Better understanding of these features and pathways may lead to identification of new prognostic and predictive tools and of new therapeutic targets. The work of this thesis is carried out in two cohorts of patients with primary operable breast cancer with mature follow up. Data was available from clinical records for both regarding patient age, tumour pathology, treatment details and survival. Full section slides from surplus tissue were available from both cohorts and a tissue microarray (TMA) had been previously constructed for the largest cohort. The majority of the work in this thesis is carried out using haematoxylin and eosin (H&E)-stained full section slides and TMA slides stained using immunohistochemistry (IHC) techniques for various proteins. Staining for IL6 expression was carried out using RNA scope. Transcriptomics was carried out using TempOSeq to identify genes associated with tumour budding. The work of this thesis describes a new combined score of tumour necrosis, budding and tumour-stroma percentage (TSP) which has prognostic value in primary operable breast cancer. It identifies a poor prognostic group in oestrogen receptor positive (ER+) disease which could be targeted for more aggressive treatment, and stratifies risk in ER- disease. 9 genes of potential interest for further investigation are identified as being associated with the high budding phenotype in ER- cancers. For the first time in the literature, this work will describe, in luminal A cancers, an association between tumour IL6 and membranous IL6R expression and worse cancer specific survival (CSS), and an association between pSTAT3(Ser727) and improved CSS, indicating potential roles as prognostic markers in this subtype. It will describe the expression and associations with survival of other members of the pathway, informing further research regarding in which subtypes inhibiting targets in the pathway may be of clinical value