Relocation of active site carboxylates in major facilitator superfamily multidrug transporter LmrP reveals plasticity in proton interactions

The expression of polyspecific membrane transporters is one important mechanism by which cells can obtain resistance to structurally different antibiotics and cytotoxic agents. These transporters reduce intracellular drug concentrations to subtoxic levels by mediating drug efflux across the cell envelope. The major facilitator superfamily multidrug transporter LmrP from $\textit{Lactococcus}$ lactis catalyses drug efflux in a membrane potential and chemical proton gradient-dependent fashion. To enable the interaction with protons and cationic substrates, LmrP contains catalytic carboxyl residues on the surface of a large interior chamber that is formed by transmembrane helices. These residues co-localise together with polar and aromatic residues, and are predicted to be present in two clusters. To investigate the functional role of the catalytic carboxylates, we generated mutant proteins catalysing membrane potential-independent dye efflux by removing one of the carboxyl residues in Cluster 1. We then relocated this carboxyl residue to six positions on the surface of the interior chamber, and tested for restoration of wildtype energetics. The reinsertion at positions towards Cluster 2 reinstated the membrane potential dependence of dye efflux. Our data uncover a remarkable plasticity in proton interactions in LmrP, which is a consequence of the flexibility in the location of key residues that are responsible for proton/multidrug antiport.A.V.N. is a research associate funded by the Biotechnology and Biological Sciences Research Council (BBSRC). H.S., S.R. and Z.T. received scholarships from the Cambridge Commonwealth, European and International Trust. A.N. is the recipient of a Herchel-Smith Scholarship. K.A. is funded through a programme grant from the Human Frontier Science Program

Decentralizing energy for a high - demand, low - Carbon world

Decentralization can play a role in achieving an affordable, clean, and resilient power system. Yet, transitioning away from traditional centralized energy networks requires large-scale changes across sectors. This Voices asks: what are the challenges and potential solutions associated with realizing the rapid and effective decentralization of global energy

Role of mitochondrial raft-like microdomains in the regulation of cell apoptosis

Lipid rafts are envisaged as lateral assemblies of specific lipids and proteins that dissociate and associate rapidly and form functional clusters in cell membranes. These structural platforms are not confined to the plasma membrane; indeed lipid microdomains are similarly formed at subcellular organelles, which include endoplasmic reticulum, Golgi and mitochondria, named raft-like microdomains. In addition, some components of raft-like microdomains are present within ER-mitochondria associated membranes. This review is focused on the role of mitochondrial raft-like microdomains in the regulation of cell apoptosis, since these microdomains may represent preferential sites where key reactions take place, regulating mitochondria hyperpolarization, fission-associated changes, megapore formation and release of apoptogenic factors. These structural platforms appear to modulate cytoplasmic pathways switching cell fate towards cell survival or death. Main insights on this issue derive from some pathological conditions in which alterations of microdomains structure or function can lead to severe alterations of cell activity and life span. In the light of the role played by raft-like microdomains to integrate apoptotic signals and in regulating mitochondrial dynamics, it is conceivable that these membrane structures may play a role in the mitochondrial alterations observed in some of the most common human neurodegenerative diseases, such as Amyotrophic lateral sclerosis, Huntington's chorea and prion-related diseases. These findings introduce an additional task for identifying new molecular target(s) of pharmacological agents in these pathologies

Inequalities in higher education in low‐ and middle‐income countries:A scoping review of the literature

Motivation:  Higher  education  is  regarded  as  a  key  instrument  to  enhance  socioeconomic  mobility  andreduce inequalities. Recent literature reviews have examined inequalities in the higher education systemsof  high-income  countries,  but  less  is  known  about  the  situation  in  low-  and  middle-income  countries,where higher education is expanding fast.Purpose:  The  article  reviews  the  academic  literature  on  higher  education  in  low-  and  middle-incomecountries using a research framework inspired by social justice and capability approaches. It considers the financial,  socio-cultural,  human,  and  political  resource  domains  on  which  people  draw,  and  how  they relate to access, participation, and outcomes in higher education.Methods: A literature search for studies explicitly discussing in-country  inequalities  in  higher  education revealed  22  publications. Substantial  knowledge  gaps remain,  especially  regarding  the  political  (and decision-making)  side  of  inequalities;  the  ideologies  and  philosophies  underpinning  higher  education systems; and the linkages between resource domains, both micro and macro.Findings:  The  review  highlights  key  elements  for  policy-makers  and  researchers:  (1)  the  financial  lens alone  is  insufficient  to  understand  and  tackle  inequalities,  since  these  are  also  shaped  by  human  and other non-financial factors; (2) socio-cultural constructs are central in explaining unequal outcomes; and (3) inequalities develop throughout one’s life and need to be considered during, but also before and afterhigher education.  The scope  of  inequalities  is  wide, and  the literature  offers a  few ideas  for short-term fixes such as part-time and online education.Policy implications: Inclusive policy frameworks for higher education should include explicit goals related to (in)equality,  which  are  best  measured in  terms  of  the  extent  to  which  certain  actions  or  choices are feasible for all. Policies in these frameworks, we argue, should go beyond providing financial support, and also address socio-cultural and human resource constraints and challenges in retention, performance, and labour market outcomes. Finally, they should consider relevant contextual determinants of inequalities.</p

High-throughput expression and purification of human solute carriers for structural and biochemical studies

Solute carriers (SLCs) are membrane transporters that import and export a range of endogenous and exogenous substrates, including ions, nutrients, metabolites, neurotransmitters, and pharmaceuticals. Despite having emerged as attractive therapeutic targets and markers of disease, this group of proteins is still relatively underdrugged by current pharmaceuticals. Drug discovery projects for these transporters are impeded by limited structural, functional, and physiological knowledge, ultimately due to the difficulties in the expression and purification of this class of membrane-embedded proteins. Here, we demonstrate methods to obtain high-purity, milligram quantities of human SLC transporter proteins using codon-optimized gene sequences. In conjunction with a systematic exploration of construct design and high-throughput expression, these protocols ensure the preservation of the structural integrity and biochemical activity of the target proteins. We also highlight critical steps in the eukaryotic cell expression, affinity purification, and size-exclusion chromatography of these proteins. Ultimately, this workflow yields pure, functionally active, and stable protein preparations suitable for high-resolution structure determination, transport studies, small-molecule engagement assays, and high-throughput in vitro screening

Post-Streptococcal Antibodies Are Associated with Metabolic Syndrome in a Population-Based Cohort

Background: Streptococcal infections are known to trigger autoimmune disorders, affecting millions worldwide. Recently, we found an association between post-streptococcal autoantibodies against Protein Disulphide Isomerase (PDI), an enzyme involved in insulin degradation and insulin resistance. This led us to evaluate associations between post-streptococcal antibodies and metabolic syndrome, as defined by the updated National Cholesterol Education Program definition, 2005. Methods and Findings: Metabolic data (HDL, triglycerides, fasting glucose, blood pressure, waist circumference, BMI, smoking), post-streptococcal antibodies (anti-Streptolysin O (ASO) and anti-PDI), and C-reactive protein (CRP, as a general inflammatory marker), were assessed in 1156 participants of the Wisconsin Sleep Cohort Study. Anti-PDI antibodies were found in 308 participants (26.6%), ASO$100 in 258 (22.3%), and 482 (41.7%) met diagnostic criteria for metabolic syndrome. Anti-PDI antibodies but not ASO were significantly associated with metabolic syndrome [n = 1156, OR 1.463 (95 % CI 1.114, 1.920), p = 0.0062; adjusted for age, gender, education, smoking]. Importantly, the anti-PDI- metabolic syndrome association remained significant after adjusting for CRP and fasting insulin. Conclusions: Post-streptococcal anti-PDI antibodies are associated with metabolic syndrome regardless of fasting insulin and CRP levels. Whereas these data are in line with a growing body of evidence linking infections, immunity an

The role of the mitochondria and the endoplasmic reticulum contact sites in the development of the immune responses

Abstract Mitochondria and endoplasmic reticulum (ER) contact sites (MERCs) are dynamic modules enriched in subset of lipids and specialized proteins that determine their structure and functions. The MERCs regulate lipid transfer, autophagosome formation, mitochondrial fission, Ca2+ homeostasis and apoptosis. Since these functions are essential for cell biology, it is therefore not surprising that MERCs also play a critical role in organ physiology among which the immune system stands by its critical host defense function. This defense system must discriminate and tolerate host cells and beneficial commensal microorganisms while eliminating pathogenic ones in order to preserve normal homeostasis. To meet this goal, the immune system has two lines of defense. First, the fast acting but unspecific innate immune system relies on anatomical physical barriers and subsets of hematopoietically derived cells expressing germline-encoded receptors called pattern recognition receptors (PRR) recognizing conserved motifs on the pathogens. Second, the slower but very specific adaptive immune response is added to complement innate immunity. Adaptive immunity relies on another set of specialized cells, the lymphocytes, harboring receptors requiring somatic recombination to be expressed. Both innate and adaptive immune cells must be activated to phagocytose and process pathogens, migrate, proliferate, release soluble factors and destroy infected cells. Some of these functions are strongly dependent on lipid transfer, autophagosome formation, mitochondrial fission, and Ca2+ flux; this indicates that MERCs could regulate immunity

Protein Co-Expression Analysis as a Strategy to Complement a Standard Quantitative Proteomics Approach:Case of a Glioblastoma Multiforme Study

Although correlation network studies from co-expression analysis are increasingly popular, they are rarely applied to proteomics datasets. Protein co-expression analysis provides a complementary view of underlying trends, which can be overlooked by conventional data analysis. The core of the present study is based on Weighted Gene Co-expression Network Analysis applied to a glioblastoma multiforme proteomic dataset. Using this method, we have identified three main modules which are associated with three different membrane associated groups; mitochondrial, endoplasmic reticulum, and a vesicle fraction. The three networks based on protein co-expression were assessed against a publicly available database (STRING) and show a statistically significant overlap. Each of the three main modules were de-clustered into smaller networks using different strategies based on the identification of highly connected networks, hierarchical clustering and enrichment of Gene Ontology functional terms. Most of the highly connected proteins found in the endoplasmic reticulum module were associated with redox activity while a core of the unfolded protein response was identified in addition to proteins involved in oxidative stress pathways. The proteins composing the electron transfer chain were found differently affected with proteins from mitochondrial Complex I being more down-regulated than proteins from Complex III. Finally, the two pyruvate kinases isoforms show major differences in their co-expressed protein networks suggesting roles in different cellular locations