The solute carrier SPNS2 recruits PI(4,5)P2 to synergistically regulate transport of sphingosine-1-phosphate.

Solute carrier spinster homolog 2 (SPNS2), one of only four known major facilitator superfamily (MFS) lysolipid transporters in humans, exports sphingosine-1-phosphate (S1P) across cell membranes. Here, we explore the synergistic effects of lipid binding and conformational dynamics on SPNS2's transport mechanism. Using mass spectrometry, we discovered that SPNS2 interacts preferentially with PI(4,5)P2. Together with functional studies and molecular dynamics (MD) simulations, we identified potential PI(4,5)P2 binding sites. Mutagenesis of proposed lipid binding sites and inhibition of PI(4,5)P2 synthesis reduce S1P transport, whereas the absence of the N terminus renders the transporter essentially inactive. Probing the conformational dynamics of SPNS2, we show how synergistic binding of PI(4,5)P2 and S1P facilitates transport, increases dynamics of the extracellular gate, and stabilizes the intracellular gate. Given that SPNS2 transports a key signaling lipid, our results have implications for therapeutic targeting and also illustrate a regulatory mechanism for MFS transporters.

Structure and inhibition mechanism of the human citrate transporter NaCT.

Citrate is best known as an intermediate in the tricarboxylic acid cycle of the cell. In addition to this essential role in energy metabolism, the tricarboxylate anion also acts as both a precursor and a regulator of fatty acid synthesis1-3. Thus, the rate of fatty acid synthesis correlates directly with the cytosolic concentration of citrate4,5. Liver cells import citrate through the sodium-dependent citrate transporter NaCT (encoded by SLC13A5) and, as a consequence, this protein is a potential target for anti-obesity drugs. Here, to understand the structural basis of its inhibition mechanism, we determined cryo-electron microscopy structures of human NaCT in complexes with citrate or a small-molecule inhibitor. These structures reveal how the inhibitor-which binds to the same site as citrate-arrests the transport cycle of NaCT. The NaCT-inhibitor structure also explains why the compound selectively inhibits NaCT over two homologous human dicarboxylate transporters, and suggests ways to further improve the affinity and selectivity. Finally, the NaCT structures provide a framework for understanding how various mutations abolish the transport activity of NaCT in the brain and thereby cause epilepsy associated with mutations in SLC13A5 in newborns (which is known as SLC13A5-epilepsy)6-8.

Structural basis for inhibition of the drug efflux pump NorA from Staphylococcus aureus.

Membrane protein efflux pumps confer antibiotic resistance by extruding structurally distinct compounds and lowering their intracellular concentration. Yet, there are no clinically approved drugs to inhibit efflux pumps, which would potentiate the efficacy of existing antibiotics rendered ineffective by drug efflux. Here we identified synthetic antigen-binding fragments (Fabs) that inhibit the quinolone transporter NorA from methicillin-resistant Staphylococcus aureus (MRSA). Structures of two NorA-Fab complexes determined using cryo-electron microscopy reveal a Fab loop deeply inserted in the substrate-binding pocket of NorA. An arginine residue on this loop interacts with two neighboring aspartate and glutamate residues essential for NorA-mediated antibiotic resistance in MRSA. Peptide mimics of the Fab loop inhibit NorA with submicromolar potency and ablate MRSA growth in combination with the antibiotic norfloxacin. These findings establish a class of peptide inhibitors that block antibiotic efflux in MRSA by targeting indispensable residues in NorA without the need for membrane permeability.

An eight centre, retrospective, clinical practice data analysis of algorithm-based treatment with follitropin delta.

RESEARCH QUESTION: Can efficacy and success rates of the first recombinant FSH expressed in a human cell line with an individualized dosing algorithm based on body weight and anti-Müllerian hormone (AMH) as shown in the ESTHER-1 trial be confirmed in routine clinical practice? DESIGN: In eight reproductive medicine centres in Germany, observational data of 360 women who underwent ovarian stimulation with follitropin delta were evaluated as part of the quality control from January 2018 to June 2019. The data were analysed retrospectively. RESULTS: Mean age of patients was 33.5 (±3.8) years. Pretreatment AMH concentrations ranged from <0.5 ng/ml or 3.6 pmol/l (2.5%) to >5.6 ng/ml or 40 pmol/l (19.7%), with 79.7% of all AMH measurements above 2.0 ng/ml or 14.5 pmol/l. The mean number of oocytes obtained in n = 359 first follitropin delta cycles was 11.2 (±6.7) oocytes with 42.1% of patients having between eight and 14 oocytes retrieved at oocyte retrieval. The average clinical pregnancy rate in the first cycle with a fresh embryo transfer was 38.2% with a mean of 1.4 embryos per transfer. The cumulative pregnancy rate was 49.4% for the first stimulation cycle (including cryopreservation cycles generated from the first stimulation cycle). CONCLUSION: The goal of obtaining an adequate number of oocytes (8-14 oocytes) using the follitropin delta dosing algorithm was reached in 42.1% of patients despite a wide range of pretreatment AMH values, while achieving very good clinical pregnancy rates. Hence, algorithm-based ovarian stimulation with follitropin delta remains highly effective in clinical practice.

Thoracic Endemic Fungi in the United States: Importance of Patient Location.

The digitization of radiographic studies along with high-speed transmission of images has formed the basis of teleradiology, which has become an integral component in the workflow of a contemporary radiology practice. It is with this advent and growing utilization of teleradiology that the significance of the source location of images has gained importance. Specifically, the importance of where the patient resides and what endemic fungi occur in that location cannot be underestimated. In the United States, histoplasmosis, coccidioidomycosis, blastomycosis, and cryptococcosis are caused by endemic fungi occurring in the Ohio and Mississippi river valleys, the Southwest, the Upper Midwest, and the Pacific Northwest, respectively. All of these organisms enter the body through the respiratory system and have the potential to cause significant morbidity and mortality. Patients infected with these fungi are often asymptomatic but may present with acute flulike symptoms such as fever, cough, or dyspnea. Patients may also present with vague chronic symptoms including cough, fever, malaise, and weight loss. Thoracic manifestations at radiography and CT include consolidation, nodules, cavities, lymphadenopathy, and pleural disease. PET may show fluorine 18-fluorodeoxyglucose uptake with active acute or chronic infections, and it is difficult to distinguish infections from malignancy. Imaging findings may be nonspecific and can be confused with other disease processes, including malignancy. The patient demographics, clinical history, and location are clues that may lead to a proper diagnosis of endemic fungal disease. The radiologist should be cognizant of the patient location to provide a correct and timely radiologic diagnosis that helps guide the clinician to initiate appropriate therapy. ©RSNA, 2021.

Predicting the optimal growth temperatures of prokaryotes using only genome derived features.

MOTIVATION: Optimal growth temperature is a fundamental characteristic of all living organisms. Knowledge of this temperature is central to the study of a prokaryote, the thermal stability and temperature dependent activity of its genes, and the bioprospecting of its genome for thermally adapted proteins. While high throughput sequencing methods have dramatically increased the availability of genomic information, the growth temperatures of the source organisms are often unknown. This limits the study and technological application of these species and their genomes. Here, we present a novel method for the prediction of growth temperatures of prokaryotes using only genomic sequences. RESULTS: By applying the reverse ecology principle that an organism's genome includes identifiable adaptations to its native environment, we can predict a species' optimal growth temperature with an accuracy of 5.17°C root-mean-square error and a coefficient of determination of 0.835. The accuracy can be further improved for specific taxonomic clades or by excluding psychrophiles. This method provides a valuable tool for the rapid calculation of organism growth temperature when only the genome sequence is known. AVAILABILITY AND IMPLEMENTATION: Source code, genomes analyzed and features calculated are available at: https://github.com/DavidBSauer/OGT_prediction. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Rapid Bioinformatic Identification of Thermostabilizing Mutations.

Ex vivo stability is a valuable protein characteristic but is laborious to improve experimentally. In addition to biopharmaceutical and industrial applications, stable protein is important for biochemical and structural studies. Taking advantage of the large number of available genomic sequences and growth temperature data, we present two bioinformatic methods to identify a limited set of amino acids or positions that likely underlie thermostability. Because these methods allow thousands of homologs to be examined in silico, they have the advantage of providing both speed and statistical power. Using these methods, we introduced, via mutation, amino acids from thermoadapted homologs into an exemplar mesophilic membrane protein, and demonstrated significantly increased thermostability while preserving protein activity.

Protein interactions central to stabilizing the K+ channel selectivity filter in a four-sited configuration for selective K+ permeation.

The structural and functional conversion of the nonselective NaK channel to a K(+) selective channel (NaK2K) allows us to identify two key residues, Tyr and Asp in the filter sequence of TVGYGD, that participate in interactions central to stabilizing the K(+) channel selectivity filter. By using protein crystallography and channel electrophysiology, we demonstrate that the K(+) channel filter exists as an energetically strained structure and requires these key protein interactions working in concert to hold the filter in the precisely defined four-sited configuration that is essential for selective K(+) permeation. Disruption of either interaction, as tested on both the NaK2K and eukaryotic K(v)1.6 channels, can reduce or completely abolish K(+) selectivity and in some cases may also lead to channel inactivation due to conformational changes at the filter. Additionally, on the scaffold of NaK we recapitulate the protein interactions found in the filter of the Kir channel family, which uses a distinct interaction network to achieve similar stabilization of the filter.

Tuning the ion selectivity of tetrameric cation channels by changing the number of ion binding sites.

Selective ion conduction across ion channel pores is central to cellular physiology. To understand the underlying principles of ion selectivity in tetrameric cation channels, we engineered a set of cation channel pores based on the nonselective NaK channel and determined their structures to high resolution. These structures showcase an ensemble of selectivity filters with a various number of contiguous ion binding sites ranging from 2 to 4, with each individual site maintaining a geometry and ligand environment virtually identical to that of equivalent sites in K(+) channel selectivity filters. Combined with single channel electrophysiology, we show that only the channel with four ion binding sites is K(+) selective, whereas those with two or three are nonselective and permeate Na(+) and K(+) equally well. These observations strongly suggest that the number of contiguous ion binding sites in a single file is the key determinant of the channel's selectivity properties and the presence of four sites in K(+) channels is essential for highly selective and efficient permeation of K(+) ions.

Pho pictures provide powerful perspectives of phosphate importing proteins.

In this issue of Structure, Schneider et al.1 report multiple structures of the low-affinity inorganic-phosphate transporter Pho90 from Saccharomyces cerevisiae. With remarkable resolution of the Divalent Anion Sodium Symporter family member, their cryo-EM studies of this fungal protein reveal new modes of sodium, substrate, and lipid binding.

The binding and mechanism of a positive allosteric modulator of Kv3 channels.

Small-molecule modulators of diverse voltage-gated K+ (Kv) channels may help treat a wide range of neurological disorders. However, developing effective modulators requires understanding of their mechanism of action. We apply an orthogonal approach to elucidate the mechanism of action of an imidazolidinedione derivative (AUT5), a highly selective positive allosteric modulator of Kv3.1 and Kv3.2 channels. AUT5 modulation involves positive cooperativity and preferential stabilization of the open state. The cryo-EM structure of the Kv3.1/AUT5 complex at a resolution of 2.5 Å reveals four equivalent AUT5 binding sites at the extracellular inter-subunit interface between the voltage-sensing and pore domains of the channel's tetrameric assembly. Furthermore, we show that the unique extracellular turret regions of Kv3.1 and Kv3.2 essentially govern the selective positive modulation by AUT5. High-resolution apo and bound structures of Kv3.1 demonstrate how AUT5 binding promotes turret rearrangements and interactions with the voltage-sensing domain to favor the open conformation.

Still in Search for an EAAT Activator: GT949 Does Not Activate EAAT2, nor EAAT3 in Impedance and Radioligand Uptake Assays.

Excitatory amino acid transporters (EAATs) are important regulators of amino acid transport and in particular glutamate. Recently, more interest has arisen in these transporters in the context of neurodegenerative diseases. This calls for ways to modulate these targets to drive glutamate transport, EAAT2 and EAAT3 in particular. Several inhibitors (competitive and noncompetitive) exist to block glutamate transport; however, activators remain scarce. Recently, GT949 was proposed as a selective activator of EAAT2, as tested in a radioligand uptake assay. In the presented research, we aimed to validate the use of GT949 to activate EAAT2-driven glutamate transport by applying an innovative, impedance-based, whole-cell assay (xCELLigence). A broad range of GT949 concentrations in a variety of cellular environments were tested in this assay. As expected, no activation of EAAT3 could be detected. Yet, surprisingly, no biological activation of GT949 on EAAT2 could be observed in this assay either. To validate whether the impedance-based assay was not suited to pick up increased glutamate uptake or if the compound might not induce activation in this setup, we performed radioligand uptake assays. Two setups were utilized; a novel method compared to previously published research, and in a reproducible fashion copying the methods used in the existing literature. Nonetheless, activation of neither EAAT2 nor EAAT3 could be observed in these assays. Furthermore, no evidence of GT949 binding or stabilization of purified EAAT2 could be observed in a thermal shift assay. To conclude, based on experimental evidence in the present study GT949 requires specific assay conditions, which are difficult to reproduce, and the compound cannot simply be classified as an activator of EAAT2 based on the presented evidence. Hence, further research is required to develop the tools needed to identify new EAAT modulators and use their potential as a therapeutic target.

Protein Binder Toolbox for Studies of Solute Carrier Transporters.

Transporters of the solute carrier superfamily (SLCs) are responsible for the transmembrane traffic of the majority of chemical substances in cells and tissues and are therefore of fundamental biological importance. As is often the case with membrane proteins that can be heavily glycosylated, a lack of reliable high-affinity binders hinders their functional analysis. Purifying and reconstituting transmembrane proteins in their lipidic environments remains challenging and standard approaches to generate binders for multi-transmembrane proteins, such as SLCs, channels or G protein-coupled receptors (GPCRs) are lacking. While generating protein binders to 27 SLCs, we produced full length protein or cell lines as input material for binder generation by selected binder generation platforms. As a result, we obtained 525 binders for 22 SLCs. We validated the binders with a cell-based validation workflow using immunofluorescent and immunoprecipitation methods to process all obtained binders. Finally, we demonstrated the potential applications of the binders that passed our validation pipeline in structural, biochemical, and biological applications using the exemplary protein SLC12A6, an ion transporter relevant in human disease. With this work, we were able to generate easily renewable and highly specific binders against SLCs, which will greatly facilitate the study of this neglected protein family. We hope that the process will serve as blueprint for the generation of binders against the entire superfamily of SLC transporters.

Structure and function of the SIT1 proline transporter in complex with the COVID-19 receptor ACE2.

Proline is widely known as the only proteogenic amino acid with a secondary amine. In addition to its crucial role in protein structure, the secondary amino acid modulates neurotransmission and regulates the kinetics of signaling proteins. To understand the structural basis of proline import, we solved the structure of the proline transporter SIT1 in complex with the COVID-19 viral receptor ACE2 by cryo-electron microscopy. The structure of pipecolate-bound SIT1 reveals the specific sequence requirements for proline transport in the SLC6 family and how this protein excludes amino acids with extended side chains. By comparing apo and substrate-bound SIT1 states, we also identify the structural changes that link substrate release and opening of the cytoplasmic gate and provide an explanation for how a missense mutation in the transporter causes iminoglycinuria.

Advancing drug discovery through assay development: a survey of tool compounds within the human solute carrier superfamily.

With over 450 genes, solute carriers (SLCs) constitute the largest transporter superfamily responsible for the uptake and efflux of nutrients, metabolites, and xenobiotics in human cells. SLCs are associated with a wide variety of human diseases, including cancer, diabetes, and metabolic and neurological disorders. They represent an important therapeutic target class that remains only partly exploited as therapeutics that target SLCs are scarce. Additionally, many small molecules reported in the literature to target SLCs are poorly characterized. Both features may be due to the difficulty of developing SLC transport assays that fulfill the quality criteria for high-throughput screening. Here, we report one of the main limitations hampering assay development within the RESOLUTE consortium: the lack of a resource providing high-quality information on SLC tool compounds. To address this, we provide a systematic annotation of tool compounds targeting SLCs. We first provide an overview on RESOLUTE assays. Next, we present a list of SLC-targeting compounds collected from the literature and public databases; we found that most data sources lacked specificity data. Finally, we report on experimental tests of 19 selected compounds against a panel of 13 SLCs from seven different families. Except for a few inhibitors, which were active on unrelated SLCs, the tested inhibitors demonstrated high selectivity for their reported targets. To make this knowledge easily accessible to the scientific community, we created an interactive dashboard displaying the collected data in the RESOLUTE web portal (https://re-solute.eu). We anticipate that our open-access resources on assays and compounds will support the development of future drug discovery campaigns for SLCs.

A cross-sectional analysis of the prevalence of Barrett esophagus in otolaryngology patients with laryngeal symptoms.

BACKGROUND: Populations at risk for esophageal adenocarcinoma remain poorly defined. Laryngeal symptoms can be secondary to laryngopharyngeal reflux (LPR) and can occur without associated gastroesophageal reflux symptoms such as heartburn and regurgitation. GOAL: We sought to determine the prevalence of Barrett esophagus (BE) in otolaryngology patients with laryngeal symptoms±typical gastroesophageal reflux disease (GERD) symptoms. STUDY: We performed a cross-sectional study of otolaryngology clinic patients who reported laryngeal symptoms. Symptoms, medications, and exposure histories were obtained. Unsedated transnasal endoscopy was performed. Suspected BE was biopsied and confirmed histologically. Risk factors and prevalence of BE were assessed. RESULTS: Two hundred ninety-five patients were enrolled [73% male, median age 60 y (interquartile range 51 to 68 y)]. The overall prevalence of BE was 11.8% (n=33). Antisecretory medication use was present in 56% (n=156) of patients at enrollment. Compared with patients without BE, patients with BE were more likely to be male (P=0.01) and to report occupational lung injury (P=0.001). Duration, but not severity of laryngeal symptoms, significantly increased the odds of BE (odds ratio, 5.64; 95% confidence interval, 1.28-24.83; for a duration of symptoms >5 y). Of patients with BE, 58% (n=19) had coexisting LPR and GERD symptoms and 30% (n=10) had only LPR symptoms. Presence and size of hiatal hernia and length of columnar-lined esophagus were significant risk factors for BE. CONCLUSIONS: Long-standing laryngeal symptoms are associated with the presence of BE in otolaryngology patients. Patients with chronic laryngeal symptoms and no identifiable ear, nose, or throat etiology for those symptoms may benefit from endoscopic screening regardless of whether typical GERD symptoms are present.

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.

The role of cyclooxygenase-2 in mechanical ventilation-induced lung injury.

Mechanical ventilation is necessary for patients with acute respiratory failure, but can cause or propagate lung injury. We previously identified cyclooxygenase-2 as a candidate gene in mechanical ventilation-induced lung injury. Our objective was to determine the role of cyclooxygenase-2 in mechanical ventilation-induced lung injury and the effects of cyclooxygenase-2 inhibition on lung inflammation and barrier disruption. Mice were mechanically ventilated at low and high tidal volumes, in the presence or absence of pharmacologic cyclooxygenase-2-specific inhibition with 3-(4-methylsulphonylphenyl)-4-phenyl-5-trifluoromethylisoxazole (CAY10404). Lung injury was assessed using markers of alveolar-capillary leakage and lung inflammation. Cyclooxygenase-2 expression and activity were measured by Western blotting, real-time PCR, and lung/plasma prostanoid analysis, and tissue sections were analyzed for cyclooxygenase-2 staining by immunohistochemistry. High tidal volume ventilation induced lung injury, significantly increasing both lung leakage and lung inflammation relative to control and low tidal volume ventilation. High tidal volume mechanical ventilation significantly induced cyclooxygenase-2 expression and activity, both in the lungs and systemically, compared with control mice and low tidal volume mice. The immunohistochemical analysis of lung sections localized cyclooxygenase-2 expression to monocytes and macrophages in the alveoli. The pharmacologic inhibition of cyclooxygenase-2 with CAY10404 significantly decreased cyclooxygenase activity and attenuated lung injury in mice ventilated at high tidal volume, attenuating barrier disruption, tissue inflammation, and inflammatory cell signaling. This study demonstrates the induction of cyclooxygenase-2 by mechanical ventilation, and suggests that the therapeutic inhibition of cyclooxygenase-2 may attenuate ventilator-induced acute lung injury.

Mucoepidermoid Carcinomas of the Larynx.

OBJECTIVES: Discussions regarding the specific management and outcomes for laryngeal MEC are limited to very small, single-institution case series. To look further into the diagnosis and management of these uncommon non-squamous cell carcinomas of the larynx, we present 3 recent cases of laryngeal MEC treated at our institution. METHODS: Patients at a tertiary hospital treated for MEC between October 2019 and December 2020 were retrospectively identified. Chart review, imaging analysis, and histologic slide creation were completed for all patients. RESULTS: We identified and treated 2 patients with high-grade supraglottic and 1 patient with intermediate-grade glottic MEC. These patients presented to our clinic with a primary complaint of either gradual, worsening dysphonia, dysphagia, or both. All patients underwent laryngovideostroboscopy as well as panendoscopy with directed submucosal biopsy, which was consistent with MEC. MRI was performed in 2 of the cases further elucidating the extent of submucosal spread. PET-CT was performed in all 3 cases, and none demonstrated evidence of regional or distal metastases. Surgically, high-grade MEC lesions were treated with a total laryngectomy. The intermediate MEC lesion was managed with a supracricoid partial laryngectomy (SCPL). Surgical margins were free of tumor in all cases with no nodal metastases by modified radical neck dissection. Radiation therapy was offered to both high-grade MEC patients and declined by one. Radiation was not recommended to the patient with intermediate-grade MEC as we believed that the risk of additional treatment outweighed the benefit. CONCLUSION: We believe that MEC of the larynx should be considered in patients with atypical submucosal laryngeal masses. Laryngovideostroboscopy, MRI, and PET imaging may be valuable in determining the extent of the lesions and planning appropriate surgery. Postoperative radiation therapy should be considered a per tumor grade in other more studied sites, as there is no data on efficacy in laryngeal MEC.