Structure, substrate recognition and reactivity of Leishmania major mevalonate kinase

This research was supported by the German Academic Exchange Service (DAAD), the Wellcome Trust (TKS and WNH as Trust Senior Research fellows), the Biotechnology and Biological Science Research Council (Structural Proteomics of Rational Targets) and the European Synchrotron Radiation Facility.Background: Isoprenoid precursor synthesis via the mevalonate route in humans and pathogenic trypanosomatids is an important metabolic pathway. There is however, only limited information available on the structure and reactivity of the component enzymes in trypanosomatids. Since isoprenoid biosynthesis is essential for trypanosomatid viability and may provide new targets for therapeutic intervention it is important to characterize the pathway components. Results: Putative mevalonate kinase encoding genes from Leishmania major (LmMK) and Trypanosoma brucei (TbMK) have been cloned, over-expressed in and proteins isolated from procyclic-form T. brucei. A highly sensitive radioactive assay was developed and shows ATP-dependent phosphorylation of mevalonate. Apo and (R)-mevalonate bound crystal structures of LmMK, from a bacterial expression system, have been determined to high resolution providing, for the first time, information concerning binding of mevalonate to an MK. The mevalonate binds in a deep cavity lined by highly conserved residues. His25 is key for binding and for discrimination of (R)-over (S)-mevalonate, with the main chain amide interacting with the C3 hydroxyl group of ( R)mevalonate, and the side chain contributing, together with Val202 and Thr283, to the construction of a hydrophobic binding site for the C3 methyl substituent. The C5 hydroxyl, where phosphorylation occurs, points towards catalytic residues, Lys18 and Asp155. The activity of LmMK was significantly reduced compared to MK from other species and we were unable to obtain ATP-binding data. Comparisons with the rat MK:ATP complex were used to investigate how this substrate might bind. In LmMK, helix alpha 2 and the preceding polypeptide adopt a conformation, not seen in related kinase structures, impeding access to the nucleotide triphosphate binding site suggesting that a conformational rearrangement is required to allow ATP binding. Conclusion: Our new structural information, consistent with data on homologous enzymes allows a detailed description of how mevalonate is recognized and positioned for catalysis in MK. The mevalonate-binding site is highly conserved yet the ATP-binding site is structurally distinct in LmMK. We are unable to provide a definitive explanation for the low activity of recombinant protein isolated from a bacterial expression system compared to material isolated from procyclic-form Trypanosoma brucei.Publisher PDFPeer reviewe

RosettaBackrub--a web server for flexible backbone protein structure modeling and design.

The RosettaBackrub server (http://kortemmelab.ucsf.edu/backrub) implements the Backrub method, derived from observations of alternative conformations in high-resolution protein crystal structures, for flexible backbone protein modeling. Backrub modeling is applied to three related applications using the Rosetta program for structure prediction and design: (I) modeling of structures of point mutations, (II) generating protein conformational ensembles and designing sequences consistent with these conformations and (III) predicting tolerated sequences at protein-protein interfaces. The three protocols have been validated on experimental data. Starting from a user-provided single input protein structure in PDB format, the server generates near-native conformational ensembles. The predicted conformations and sequences can be used for different applications, such as to guide mutagenesis experiments, for ensemble-docking approaches or to generate sequence libraries for protein design

Methodological challenges in working with Indigenous communities

n their epistemological article, Grenoble and Osipov (2023, henceforth G&O) touch on some of the practical and ideological difficulties in working with Indigenous communities, in particular in relation to their work with the Even communities in northeastern Russia. They mention low speaker numbers and the associated challenge of obtaining sufficient data, the practical difficulties of reaching these communities (both physically and digitally), the existing skepticism in Indigenous communities towards western notions of measurement, and the need to acknowledge the difficult histories that Indigenous people have faced since being colonized. Based on our own work with the Indigenous Sámi populations in northern Norway and Sweden, we fully endorse their experiences and views, despite some differences pointed out below. We also wish to add another aspect, namely the attitudes, values and existing power struggles in our own scientific community, which not only make it hard to do research on small populations in the first place but, ultimately, prevent this research from getting the visibility it deserves

Roma women’s higher education participation: whose responsibility?

There are striking gaps between Roma and non-Roma higher education (HE) participation rates, with less than 1% of Roma possessing a tertiary-level qualification [United Nations Development Programme, World Bank and European Commission. 2011a. “The Situation of Roma in 11 EU Member States.” Accessed 3 April 2015. http://issuu.com/undp_in_europe_cis/docs/_roma_at_a_glance_web/1#download]. As the Decade of Roma Inclusion (2005–2015) closes, this renders the present a salient moment to reflect on Roma students’ HE experiences. Widening educational access for marginalised groups raises specific questions about where responsibility for doing so lies – with tensions between individualised articulations of raising aspiration and notions of collective responsibility framed in a social justice agenda. Drawing on interviews with five Roma women students, this paper unpacks the contradictions between desiring access to HE for individual self-betterment and concurrent pulls towards educating for the wider benefit of ‘improving’ Roma communities. Using Ahmed’s [2012. On Being Included: Racism and Diversity in Institutional Life. Durham, North Carolina: Duke University Press] work on institutional belonging, we explore the specifically gendered nature of these narratives in how ‘doubly’ marginalised bodies are positioned as outsiders, in receipt of an educational gift

Review of methods for the detection of Lawsonia intracellularis infection in pigs

Lawsonia intracellularis is an obligate intracellular bacterium associated with enteric disease in pigs. Clinical signs include weight loss, diarrhea, and, in some cases, sudden death. The hallmark lesion is the thickening of the intestinal mucosa caused by increased epithelial cell replication, known as proliferative enteropathy. The immune response to L. intracellularis is not well defined, and detection of the infection, especially in the early stages, is still a significant challenge. We review here the main approaches used to identify this important but poorly understood pathogen. Detection of L. intracellularis infection as the cause of clinical disease is confounded by the high prevalence of the pathogen in many countries and that several other pathogens can produce similar clinical signs. A single L. intracellularis–specific ELISA and several amplification assays are available commercially to aid detection and surveillance, although histopathology remains the primary way to reach a conclusive diagnosis. There are major gaps in our understanding of L. intracellularis pathogenesis, especially how the host responds to infection and the factors that drive infection toward different clinical outcomes. Knowledge of pathogenesis will increase the predictive value of antemortem tests to guide appropriate interventions, including identification and treatment of subclinically affected pigs in the early stages of disease, given that this important manifestation reduces pig productivity and contributes to the economic burden of L. intracellularis worldwide

Can policies improve language vitality? The Sámi languages in Sweden and Norway

Introduction: Language policies are often aimed at changing language behaviours, yet it is notoriously difficult to assess their effects. This study investigates language use and competence in the Indigenous Sámi populations of Norway and Sweden in light of the national-level policies the two countries have adopted. Methods: We provide a cross-country comparison of relevant educational, linguistic and budgetary policies in Sweden and Norway. Next, we present novel data from a survey with 5,416 Sámi and non-Sámi participants in 20 northern municipalities, examining Sámi language use and proficiencies across generations and contexts. Lexical proficiency in North Sámi was tested in a small subset of participants. Results: Sámi language use has dropped considerably over the past three generations. Only a small proportion of Sámi are highly fluent and use a Sámi language with their children (around 4% in Sweden and 11% in Norway). One fifth of Sámi adults use a Sámi language at least ‘occasionally’, and use is most common in the home context. Sámi language knowledge remains negligible in the majority population. Discussion: The higher levels of language use and proficiency in Norway seem at least in part to reflect the more favourable policies adopted there. In both countries, more work is needed to increase speaker numbers, also in the majority population

Voice Onset Time in multilingual speakers: Italian heritage speakers in Germany with L3 English

This study brings together two previously largely independent fields of multilingual language acquisition: heritage language and third language (L3) acquisition. We investigate the production of fortis and lenis stops in semi-naturalistic speech in the three languages of 20 heritage speakers (HSs) of Italian with German as a majority language and English as L3. The study aims to identify the extent to which the HSs produce distinct values across all three languages, or whether crosslinguistic influence (CLI) occurs. To this end, we compare the HSs’ voice onset time (VOT) values with those of L2 English speakers from Italy and Germany. The language triad exhibits overlapping and distinct VOT realizations, making VOT a potentially vulnerable category. Results indicate CLI from German into Italian, although a systemic difference is maintained. When speaking English, th

Experience of discrimination in egalitarian societies: the Sámi and majority populations in Sweden and Norway

The Sámi people stand out as the only Indigenous minority in an egalitarian European context, namely the Nordic Countries. Therefore, inequalities that they may face are worth closer inspection. Drawing on the distinction between inequalities among individuals (vertical) and between groups (horizontal), we investigate how different types of inequalities affect the Sámi today. We formulate a series of hypotheses on how social, economic, cultural, and political inequalities are linked with discrimination experience, and test these with original data from a population survey conducted in northern Norway and northern Sweden simultaneously in 2021. The findings show that Sámi ethnic background increases the probability of experiencing discrimination. While individual-level economic inequality is also pertinent, this does not directly materialise as between-group inequality. Instead, minority language use is a strong predictor of discrimination experience, revealing the socio-cultural nature of ethnic inequalities. Cross-country differences are only reflected in the effect of minority language use