Extracellular modulation of TREK-2 activity with nanobodies provides insight into the mechanisms of K2P channel regulation.

Potassium channels of the Two-Pore Domain (K2P) subfamily, KCNK1-KCNK18, play crucial roles in controlling the electrical activity of many different cell types and represent attractive therapeutic targets. However, the identification of highly selective small molecule drugs against these channels has been challenging due to the high degree of structural and functional conservation that exists not only between K2P channels, but across the whole K+ channel superfamily. To address the issue of selectivity, here we generate camelid antibody fragments (nanobodies) against the TREK-2 (KCNK10) K2P K+ channel and identify selective binders including several that directly modulate channel activity. X-ray crystallography and CryoEM data of these nanobodies in complex with TREK-2 also reveal insights into their mechanisms of activation and inhibition via binding to the extracellular loops and Cap domain, as well as their suitability for immunodetection. These structures facilitate design of a biparatropic inhibitory nanobody with markedly improved sensitivity. Together, these results provide important insights into TREK channel gating and provide an alternative, more selective approach to modulation of K2P channel activity via their extracellular domains.

Molecular basis for pH sensing in the KDEL trafficking receptor.

Trafficking receptors control protein localization through the recognition of specific signal sequences that specify unique cellular locations. Differences in luminal pH are important for the vectorial trafficking of cargo receptors. The KDEL receptor is responsible for maintaining the integrity of the ER by retrieving luminally localized folding chaperones in a pH-dependent mechanism. Structural studies have revealed the end states of KDEL receptor activation and the mechanism of selective cargo binding. However, precisely how the KDEL receptor responds to changes in luminal pH remains unclear. To explain the mechanism of pH sensing, we combine analysis of X-ray crystal structures of the KDEL receptor at neutral and acidic pH with advanced computational methods and cell-based assays. We show a critical role for ordered water molecules that allows us to infer a direct connection between protonation in different cellular compartments and the consequent changes in the affinity of the receptor for cargo.

Future opportunities in solute carrier structural biology.

Solute carriers (SLCs) control the flow of small molecules and ions across biological membranes. Over the last 20 years, the pace of research in SLC biology has accelerated markedly, opening new opportunities to treat metabolic diseases, cancer and neurological disorders. Recently, new families of atypical SLCs, with roles in organelle biology, metabolite signaling and trafficking, have expanded their roles in the cell. This Perspective discusses work leading to current advances and the emerging opportunities to target and modulate SLCs to uncover new biology and treat human disease.

Speaking the same language - a scoping review to identify the terminology associated with social prescribing.

AIM: To identify the social prescribing-related terminology within the peer-reviewed literature of the UK and the grey literature from Wales. BACKGROUND: Social prescribing has seen a period of development that has been accompanied by a proliferation of related terminology and a lack of standardisation in the manner in which it is employed. This creates barriers to engagement and impairs communication, both between professionals and members of the public. The Wales School for Social Prescribing Research and Public Health Wales committed to the development of a glossary of terms for social prescribing, to facilitate the clarification and standardisation of the associated terminology. Here, we describe the first step in that process. METHOD: A scoping review of the peer-reviewed UK literature and Welsh grey literature was conducted. The titles and abstracts of 46,242 documents and the full text of 738 documents were screened. Data were charted from 205 documents. Data capture included terminology, the location within the UK of the research or intervention described in the article, and the perspective from which the article was authored. A general inductive approach was used to categorise the terms by theme. FINDINGS: This research serves to highlight the breadth and diversity of the terminology associated with social prescribing. Results demonstrate aspects of shared commonality and clear distinction between the terminology from the two literature sources. The greatest contributions of terms were from articles that examined research and/or interventions in England and that were authored from the perspective of health or health and social care. The research indicates that nation- and sector-specific terms may not be adequately represented in the literature at large. Looking forward, it will be important to ensure that social prescribing terminology within the UK literature is culturally relevant and accurately reflects the terminology used by the workforce who encounter and deliver social prescribing.

Structural basis for triacylglyceride extraction from mycobacterial inner membrane by MFS transporter Rv1410.

Mycobacterium tuberculosis is protected from antibiotic therapy by a multi-layered hydrophobic cell envelope. Major facilitator superfamily (MFS) transporter Rv1410 and the periplasmic lipoprotein LprG are involved in transport of triacylglycerides (TAGs) that seal the mycomembrane. Here, we report a 2.7 Å structure of a mycobacterial Rv1410 homologue, which adopts an outward-facing conformation and exhibits unusual transmembrane helix 11 and 12 extensions that protrude ~20 Å into the periplasm. A small, very hydrophobic cavity suitable for lipid transport is constricted by a functionally important ion-lock likely involved in proton coupling. Combining mutational analyses and MD simulations, we propose that TAGs are extracted from the core of the inner membrane into the central cavity via lateral clefts present in the inward-facing conformation. The functional role of the periplasmic helix extensions is to channel the extracted TAG into the lipid binding pocket of LprG.

Molecular basis for selective uptake and elimination of organic anions in the kidney by OAT1.

In mammals, the kidney plays an essential role in maintaining blood homeostasis through the selective uptake, retention or elimination of toxins, drugs and metabolites. Organic anion transporters (OATs) are responsible for the recognition of metabolites and toxins in the nephron and their eventual urinary excretion. Inhibition of OATs is used therapeutically to improve drug efficacy and reduce nephrotoxicity. The founding member of the renal organic anion transporter family, OAT1 (also known as SLC22A6), uses the export of α-ketoglutarate (α-KG), a key intermediate in the Krebs cycle, to drive selective transport and is allosterically regulated by intracellular chloride. However, the mechanisms linking metabolite cycling, drug transport and intracellular chloride remain obscure. Here, we present cryogenic-electron microscopy structures of OAT1 bound to α-KG, the antiviral tenofovir and clinical inhibitor probenecid, used in the treatment of Gout. Complementary in vivo cellular assays explain the molecular basis for α-KG driven drug elimination and the allosteric regulation of organic anion transport in the kidney by chloride.

Alcohol-related brain damage: A mixed-method evaluation of an online awareness-raising programme for frontline care and support practitioners.

INTRODUCTION: Alcohol-related brain damage (ARBD) is an umbrella term referring to the neurocognitive impairments caused by excessive and prolonged alcohol use and the associated nutritional deficiencies. This study evaluated the outcomes of an online research-informed training program for ARBD which aimed to improve client outcomes by promoting support staff's awareness and confidence in working with clients who may have (or who are at risk of developing) the condition. METHODS: Staff working within a large non-governmental non-profit housing organisation (n = 883) enrolled in the training program. Questionnaires were used pre- and post-training to collect self-reported awareness of ARBD and confidence in supporting individuals with the condition. Semi-structured interviews were conducted with 27 staff members approximately 10 weeks post-completion of the program. Interviews were audio-recorded, transcribed verbatim and analysed by employing qualitative content analysis. RESULTS: Findings from the questionnaires indicated a significant increase in all measures after completing the training program. Three main themes were developed based on the interview data: changes to awareness and understanding; professional practice; and training-specific characteristics. Participants reported changes in their ability to identify potential service users with ARBD and confidence in doing so. DISCUSSION AND CONCLUSION: Our findings demonstrate that online training programs can be effective in improving support staff's ability to identify ARBD, potentially leading an increase in signposting service users to relevant services. The research-informed nature of the training demonstrates that translating research findings directly to frontline workers can have a substantial impact and may improve outcomes for this client group.

Structural basis for proton coupled cystine transport by cystinosin.

Amino acid transporters play a key role controlling the flow of nutrients across the lysosomal membrane and regulating metabolism in the cell. Mutations in the gene encoding the transporter cystinosin result in cystinosis, an autosomal recessive metabolic disorder characterised by the accumulation of cystine crystals in the lysosome. Cystinosin is a member of the PQ-loop family of solute carrier (SLC) transporters and uses the proton gradient to drive cystine export into the cytoplasm. However, the molecular basis for cystinosin function remains elusive, hampering efforts to develop novel treatments for cystinosis and understand the mechanisms of ion driven transport in the PQ-loop family. To address these questions, we present the crystal structures of cystinosin from Arabidopsis thaliana in both apo and cystine bound states. Using a combination of in vitro and in vivo based assays, we establish a mechanism for cystine recognition and proton coupled transport. Mutational mapping and functional characterisation of human cystinosin further provide a framework for understanding the molecular impact of disease-causing mutations.

The Paradox of the Frontal Lobe Paradox. A Scoping Review.

The "frontal lobe paradox" highlights a phenomenon in which a subset of patients who possess frontal lobe damage and exhibit marked impairments in everyday life are still able to able to verbally describe a logical course of action relating to a task and perform well in interview and test settings. Such cases pose a challenge with regard to the assessment of mental capacity within clinical settings. Recent position articles state that the frontal lobe paradox is a well-known phenomenon within the field of neuropsychology, anecdotal reports from clinicians in the UK suggest this is not the case. Consequently, we conducted a scoping review to examine the breadth and depth of literature relating to the frontal lobe paradox. Searches were conducted using electronic databases and search engines, which were supplemented with a snowball search of the references used within relevant literature. We identified and reviewed 28 documents specifically related to the frontal lobe paradox. Nearly 50% of all identified academic texts published since 2000 were position articles that cited a handful of case studies published between 1936 and 1986 as evidence for the phenomenon. We also observed instances of articles citing position articles as evidence of the frontal lobe paradox. Overall, our findings indicate a lack of readily accessible research specific to the frontal lobe paradox. In particular, there is a lack of contemporary research specific to the subject and an absence of clarification as to which syndromes and disorders are included within the term.

Patched 1 regulates Smoothened by controlling sterol binding to its extracellular cysteine-rich domain.

Smoothened (SMO) transduces the Hedgehog (Hh) signal across the plasma membrane in response to accessible cholesterol. Cholesterol binds SMO at two sites: one in the extracellular cysteine-rich domain (CRD) and a second in the transmembrane domain (TMD). How these two sterol-binding sites mediate SMO activation in response to the ligand Sonic Hedgehog (SHH) remains unknown. We find that mutations in the CRD (but not the TMD) reduce the fold increase in SMO activity triggered by SHH. SHH also promotes the photocrosslinking of a sterol analog to the CRD in intact cells. In contrast, sterol binding to the TMD site boosts SMO activity regardless of SHH exposure. Mutational and computational analyses show that these sites are in allosteric communication despite being 45 angstroms apart. Hence, sterols function as both SHH-regulated orthosteric ligands at the CRD and allosteric ligands at the TMD to regulate SMO activity and Hh signaling.

Proton coupling and the multiscale kinetic mechanism of a peptide transporter.

Proton-coupled peptide transporters (POTs) are crucial for the uptake of di- and tripeptides as well as drug and prodrug molecules in prokaryotes and eukaryotic cells. We illustrate from multiscale modeling how transmembrane proton flux couples within a POT protein to drive essential steps of the full functional cycle: 1) protonation of a glutamate on transmembrane helix 7 (TM7) opens the extracellular gate, allowing ligand entry; 2) inward proton flow induces the cytosolic release of ligand by varying the protonation state of a second conserved glutamate on TM10; 3) proton movement between TM7 and TM10 is thermodynamically driven and kinetically permissible via water proton shuttling without the participation of ligand. Our results, for the first time, give direct computational confirmation for the alternating access model of POTs, and point to a quantitative multiscale kinetic picture of the functioning protein mechanism.

Structural basis for recognition and transport of folic acid in mammalian cells.

Structural studies on mammalian vitamin transport lag behind other metabolites. Folates, also known as B9 vitamins, are essential cofactors in one-carbon transfer reactions in biology. Three different systems control folate uptake in the human body; folate receptors function to capture and internalise extracellular folates via endocytosis, whereas two major facilitator superfamily transporters, the reduced folate carrier (RFC; SLC19A1) and proton-coupled folate transporter (PCFT; SLC46A1) control the transport of folates across cellular membranes. Targeting specific folate transporters is being pursued as a route to developing new antifolates with improved pharmacology. Recent structures of the proton-coupled folate transporter, PCFT, revealed key insights into antifolate recognition and the mechanism of proton-coupled transport. Combined with previously determined structures of folate receptors and new predictions for the structure of the RFC, we are now able to develop a structure-based understanding of folate and antifolate recognition to accelerate efforts in antifolate drug development.

Kynurenine importation by SLC7A11 propagates anti-ferroptotic signaling.

IDO1 oxidizes tryptophan (TRP) to generate kynurenine (KYN), the substrate for 1-carbon and NAD metabolism, and is implicated in pro-cancer pathophysiology and infection biology. However, the mechanistic relationships between IDO1 in amino acid depletion versus product generation have remained a longstanding mystery. We found an unrecognized link between IDO1 and cell survival mediated by KYN that serves as the source for molecules that inhibit ferroptotic cell death. We show that this effect requires KYN export from IDO1-expressing cells, which is then available for non-IDO1-expressing cells via SLC7A11, the central transporter involved in ferroptosis suppression. Whether inside the "producer" IDO1+ cell or the "receiver" cell, KYN is converted into downstream metabolites, suppressing ferroptosis by ROS scavenging and activating an NRF2-dependent, AHR-independent cell-protective pathway, including SLC7A11, propagating anti-ferroptotic signaling. IDO1, therefore, controls a multi-pronged protection pathway from ferroptotic cell death, underscoring the need to re-evaluate the use of IDO1 inhibitors in cancer treatment.

Weaker connectivity in resting state networks is associated with disinhibited eating in older adults.

BACKGROUND/OBJECTIVES: Obesity affects more than forty percent of adults over the age of sixty. Aberrant eating styles such as disinhibition have been associated with the engagement of brain networks underlying executive functioning, attentional control, and interoception. However, these effects have been exclusively studied in young samples overlooking those most at risk of obesity related harm. METHODS: Here we assessed associations between resting-state functional connectivity and disinhibited eating (using the Three Factor Eating Questionnaire) in twenty-one younger (aged 19-34 years, BMI range: 18-31) and twenty older (aged 60-73 years, BMI range: 19-32) adults matched for BMI. The Alternative Healthy Eating Index was used to quantify diet quality. RESULTS: Older, compared to younger, individuals reported lower levels of disinhibited eating, consumed a healthier diet, and had weaker connectivity in the frontoparietal (FPN) and default mode (DMN) networks. In addition, associations between functional connectivity and eating behaviour differed between the two age groups. In older adults, disinhibited eating was associated with weaker connectivity in the FPN and DMN--effects that were absent in the younger sample. Importantly, these effects could not be explained by differences in habitual diet. CONCLUSIONS: These findings point to a change in interoceptive signalling as part of the ageing process, which may contribute to behavioural changes in energy intake, and highlight the importance of studying this under researched population.

Molecular basis for redox control by the human cystine/glutamate antiporter system xc.

Cysteine plays an essential role in cellular redox homoeostasis as a key constituent of the tripeptide glutathione (GSH). A rate limiting step in cellular GSH synthesis is the availability of cysteine. However, circulating cysteine exists in the blood as the oxidised di-peptide cystine, requiring specialised transport systems for its import into the cell. System xc- is a dedicated cystine transporter, importing cystine in exchange for intracellular glutamate. To counteract elevated levels of reactive oxygen species in cancerous cells system xc- is frequently upregulated, making it an attractive target for anticancer therapies. However, the molecular basis for ligand recognition remains elusive, hampering efforts to specifically target this transport system. Here we present the cryo-EM structure of system xc- in both the apo and glutamate bound states. Structural comparisons reveal an allosteric mechanism for ligand discrimination, supported by molecular dynamics and cell-based assays, establishing a mechanism for cystine transport in human cells.

Cryo-EM structure of PepT2 reveals structural basis for proton-coupled peptide and prodrug transport in mammals.

The SLC15 family of proton-coupled solute carriers PepT1 and PepT2 play a central role in human physiology as the principal route for acquiring and retaining dietary nitrogen. A remarkable feature of the SLC15 family is their extreme substrate promiscuity, which has enabled the targeting of these transporters for the improvement of oral bioavailability for several prodrug molecules. Although recent structural and biochemical studies on bacterial homologs have identified conserved sites of proton and peptide binding, the mechanism of peptide capture and ligand promiscuity remains unclear for mammalian family members. Here, we present the cryo-electron microscopy structure of the outward open conformation of the rat peptide transporter PepT2 in complex with an inhibitory nanobody. Our structure, combined with molecular dynamics simulations and biochemical and cell-based assays, establishes a framework for understanding peptide and prodrug recognition within this pharmaceutically important transporter family.

Cryo-EM structure and resistance landscape of M. tuberculosis MmpL3: An emergent therapeutic target.

Tuberculosis (TB) is the leading cause of death from a single infectious agent and in 2019 an estimated 10 million people worldwide contracted the disease. Although treatments for TB exist, continual emergence of drug-resistant variants necessitates urgent development of novel antituberculars. An important new target is the lipid transporter MmpL3, which is required for construction of the unique cell envelope that shields Mycobacterium tuberculosis (Mtb) from the immune system. However, a structural understanding of the mutations in Mtb MmpL3 that confer resistance to the many preclinical leads is lacking, hampering efforts to circumvent resistance mechanisms. Here, we present the cryoelectron microscopy structure of Mtb MmpL3 and use it to comprehensively analyze the mutational landscape of drug resistance. Our data provide a rational explanation for resistance variants local to the central drug binding site, and also highlight a potential alternative route to resistance operating within the periplasmic domain.

A signal capture and proofreading mechanism for the KDEL-receptor explains selectivity and dynamic range in ER retrieval.

ER proteins of widely differing abundance are retrieved from the Golgi by the KDEL-receptor. Abundant ER proteins tend to have KDEL rather than HDEL signals, whereas ADEL and DDEL are not used in most organisms. Here, we explore the mechanism of selective retrieval signal capture by the KDEL-receptor and how HDEL binds with 10-fold higher affinity than KDEL. Our results show the carboxyl-terminus of the retrieval signal moves along a ladder of arginine residues as it enters the binding pocket of the receptor. Gatekeeper residues D50 and E117 at the entrance of this pocket exclude ADEL and DDEL sequences. D50N/E117Q mutation of human KDEL-receptors changes the selectivity to ADEL and DDEL. However, further analysis of HDEL, KDEL, and RDEL-bound receptor structures shows that affinity differences are explained by interactions between the variable -4 H/K/R position of the signal and W120, rather than D50 or E117. Together, these findings explain KDEL-receptor selectivity, and how signal variants increase dynamic range to support efficient ER retrieval of low and high abundance proteins.

Structural basis of antifolate recognition and transport by PCFT.

Folates (also known as vitamin B9) have a critical role in cellular metabolism as the starting point in the synthesis of nucleic acids, amino acids and the universal methylating agent S-adenylsmethionine1,2. Folate deficiency is associated with a number of developmental, immune and neurological disorders3-5. Mammals cannot synthesize folates de novo; several systems have therefore evolved to take up folates from the diet and distribute them within the body3,6. The proton-coupled folate transporter (PCFT) (also known as SLC46A1) mediates folate uptake across the intestinal brush border membrane and the choroid plexus4,7, and is an important route for the delivery of antifolate drugs in cancer chemotherapy8-10. How PCFT recognizes folates or antifolate agents is currently unclear. Here we present cryo-electron microscopy structures of PCFT in a substrate-free state and in complex with a new-generation antifolate drug (pemetrexed). Our results provide a structural basis for understanding antifolate recognition and provide insights into the pH-regulated mechanism of folate transport mediated by PCFT.