Development of a live cell assay for the zinc transporter ZnT8.

Zinc is an essential trace element that is involved in many biological processes and in cellular homeostasis. In pancreatic ß-cells, zinc is crucial for the synthesis, processing, and secretion of insulin, which plays a key role in glucose homeostasis and which deficiency is the cause of diabetes. The accumulation of zinc in pancreatic cells is regulated by the solute carrier transporter SLC30A8 (or Zinc Transporter 8, ZnT8), which transports zinc from cytoplasm in intracellular vesicles. Allelic variants of SLC30A8 gene have been linked to diabetes. Given the physiological intracellular localization of SLC30A8 in pancreatic ß-cells and the ubiquitous endogenous expression of other Zinc transporters in different cell lines that could be used as cellular model for SLC30A8 recombinant over-expression, it is challenging to develop a functional assay to measure SLC30A8 activity. To achieve this goal, we have firstly generated a HEK293 cell line stably overexpressing SLC30A8, where the over-expression favors the partial localization of SLC30A8 on the plasma membrane. Then, we used the combination of this cell model, commercial FluoZin-3 cell permeant zinc dye and live cell imaging approach to follow zinc flux across SLC30A8 over-expressed on plasma membrane, thus developing a novel functional imaging- based assay specific for SLC30A8. Our novel approach can be further explored and optimized, paving the way for future small molecule medium-throughput screening.

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.

AspSnFR: A genetically encoded biosensor for real-time monitoring of aspartate in live cells.

Aspartate is crucial for nucleotide synthesis, ammonia detoxification, and maintaining redox balance via the malate-aspartate-shuttle (MAS). To disentangle these multiple roles of aspartate metabolism, tools are required that measure aspartate concentrations in real time and in live cells. We introduce AspSnFR, a genetically encoded green fluorescent biosensor for intracellular aspartate, engineered through displaying and screening biosensor libraries on mammalian cells. In live cells, AspSnFR is able to precisely and quantitatively measure cytosolic aspartate concentrations and dissect its production from glutamine. Combining high-content imaging of AspSnFR with pharmacological perturbations exposes differences in metabolic vulnerabilities of aspartate levels based on nutrient availability. Further, AspSnFR facilitates tracking of aspartate export from mitochondria through SLC25A12, the MAS' key transporter. We show that SLC25A12 is a rapidly responding and direct route to couple Ca2+ signaling with mitochondrial aspartate export. This establishes SLC25A12 as a crucial link between cellular signaling, mitochondrial respiration, and metabolism.

Image-based quantification of mitochondrial iron uptake via Mitoferrin-2.

Iron is a trace element that is critical for most living organisms and plays a key role in a wide variety of metabolic processes. In the mitochondrion, iron is involved in producing iron-sulfur clusters and synthesis of heme and kept within physiological ranges by concerted activity of multiple molecules. Mitochondrial iron uptake is mediated by the solute carrier transporters Mitoferrin-1 (SLC25A37) and Mitoferrin-2 (SLC25A28). While Mitoferrin-1 is mainly involved in erythropoiesis, the cellular function of the ubiquitously expressed Mitoferrin-2 remains less well defined. Furthermore, Mitoferrin-2 is associated with several human diseases, including cancer, cardiovascular and metabolic diseases, hence representing a potential therapeutic target. Here, we developed a robust approach to quantify mitochondrial iron uptake mediated by Mitoferrin-2 in living cells. We utilize HEK293 cells with inducible expression of Mitoferrin-2 and measure iron-induced quenching of rhodamine B[(1,10-phenanthroline-5-yl)-aminocarbonyl]benzyl ester (RPA) fluorescence and validate this assay for medium-throughput screening. This assay may allow identification and characterization of Mitoferrin-2 modulators and could enable drug discovery for this target.

Experimental and Computational Analysis of Newly Identified Pathogenic Mutations in the Creatine Transporter SLC6A8.

Creatine is an essential metabolite for the storage and rapid supply of energy in muscle and nerve cells. In humans, impaired metabolism, transport, and distribution of creatine throughout tissues can cause varying forms of mental disability, also known as creatine deficiency syndrome (CDS). So far, 80 mutations in the creatine transporter (SLC6A8) have been associated to CDS. To better understand the effect of human genetic variants on the physiology of SLC6A8 and their possible impact on CDS, we studied 30 missense variants including 15 variants of unknown significance, two of which are reported here for the first time. We expressed these variants in HEK293 cells and explored their subcellular localization and transport activity. We also applied computational methods to predict variant effect and estimate site-specific changes in thermodynamic stability. To explore variants that might have a differential effect on the transporter's conformers along the transport cycle, we constructed homology models of the inward facing, and outward facing conformations. In addition, we used mass-spectrometry to study proteins that interact with wild type SLC6A8 and five selected variants in HEK293 cells. In silico models of the protein complexes revealed how two variants impact the interaction interface of SLC6A8 with other proteins and how pathogenic variants lead to an enrichment of ER protein partners. Overall, our integrated analysis disambiguates the pathogenicity of 15 variants of unknown significance revealing diverse mechanisms of pathogenicity, including two previously unreported variants obtained from patients suffering from the creatine deficiency syndrome.

Gain-of-function genetic screens in human cells identify SLC transporters overcoming environmental nutrient restrictions.

Solute carrier (SLC) transporters control fluxes of nutrients and metabolites across membranes and thereby represent a critical interface between the microenvironment and cellular and subcellular metabolism. Because of substantial functional overlap, the interplay and relative contributions of SLCs in response to environmental stresses remain poorly elucidated. To infer functional relationships between SLCs and metabolites, we developed a strategy to identify SLCs able to sustain cell viability and proliferation under growth-limiting concentrations of essential nutrients. One-by-one depletion of 13 amino acids required for cell proliferation enabled gain-of-function genetic screens using a SLC-focused CRISPR/Cas9-based transcriptional activation approach to uncover transporters relieving cells from growth-limiting metabolic bottlenecks. Among the transporters identified, we characterized the cationic amino acid transporter SLC7A3 as a gene that, when up-regulated, overcame low availability of arginine and lysine by increasing their uptake, whereas SLC7A5 was able to sustain cellular fitness upon deprivation of several neutral amino acids. Moreover, we identified metabolic compensation mediated by the glutamate/aspartate transporters SLC1A2 and SLC1A3 under glutamine-limiting conditions. Overall, this gain-of-function approach using human cells uncovered functional transporter-nutrient relationships and revealed that transport activity up-regulation may be sufficient to overcome environmental metabolic restrictions.

Impedance-Based Phenotypic Readout of Transporter Function: A Case for Glutamate Transporters.

Excitatory amino acid transporters (EAAT/SLC1) mediate Na+-dependent uptake of extracellular glutamate and are potential drug targets for neurological disorders. Conventional methods to assess glutamate transport in vitro are based on radiolabels, fluorescent dyes or electrophysiology, which potentially compromise the cell's physiology and are generally less suited for primary drug screens. Here, we describe a novel label-free method to assess human EAAT function in living cells, i.e., without the use of chemical modifications to the substrate or cellular environment. In adherent HEK293 cells overexpressing EAAT1, stimulation with glutamate or aspartate induced cell spreading, which was detected in real-time using an impedance-based biosensor. This change in cell morphology was prevented in the presence of the Na+/K+-ATPase inhibitor ouabain and EAAT inhibitors, which suggests the substrate-induced response was ion-dependent and transporter-specific. A mechanistic explanation for the phenotypic response was substantiated by actin cytoskeleton remodeling and changes in the intracellular levels of the osmolyte taurine, which suggests that the response involves cell swelling. In addition, substrate-induced cellular responses were observed for cells expressing other EAAT subtypes, as well as in a breast cancer cell line (MDA-MB-468) with endogenous EAAT1 expression. These findings allowed the development of a label-free high-throughput screening assay, which could be beneficial in early drug discovery for EAATs and holds potential for the study of other transport proteins that modulate cell shape.

An Overview of Cell-Based Assay Platforms for the Solute Carrier Family of Transporters.

The solute carrier (SLC) superfamily represents the biggest family of transporters with important roles in health and disease. Despite being attractive and druggable targets, the majority of SLCs remains understudied. One major hurdle in research on SLCs is the lack of tools, such as cell-based assays to investigate their biological role and for drug discovery. Another challenge is the disperse and anecdotal information on assay strategies that are suitable for SLCs. This review provides a comprehensive overview of state-of-the-art cellular assay technologies for SLC research and discusses relevant SLC characteristics enabling the choice of an optimal assay technology. The Innovative Medicines Initiative consortium RESOLUTE intends to accelerate research on SLCs by providing the scientific community with high-quality reagents, assay technologies and data sets, and to ultimately unlock SLCs for drug discovery.

Three-Dimensional Primary Cell Culture: A Novel Preclinical Model for Pancreatic Neuroendocrine Tumors.

Molecular mechanisms underlying the development and progression of pancreatic neuroendocrine tumors (PanNETs) are still insufficiently understood. Efficacy of currently approved PanNET therapies is limited. While novel treatment options are being developed, patient stratification permitting more personalized treatment selection in PanNET is yet not feasible since no predictive markers are established. The lack of representative in vitro and in vivo models as well as the rarity and heterogeneity of PanNET are prevailing reasons for this. In this study, we describe an in vitro 3-dimensional (3-D) human primary PanNET culture system as a novel preclinical model for more personalized therapy selection. We present a screening platform allowing multicenter sample collection and drug screening in 3-D cultures of human primary PanNET cells. We demonstrate that primary cells isolated from PanNET patients and cultured in vitro form islet-like tumoroids. Islet-like tumoroids retain a neuroendocrine phenotype and are viable for at least 2 weeks in culture with a high success rate (86%). Viability can be monitored continuously allowing for a per-well normalization. In a proof-of-concept study, islet-like tumoroids were screened with three clinically approved therapies for PanNET: sunitinib, everolimus and temozolomide. Islet-like tumoroids display varying in vitro response profiles to distinct therapeutic regimes. Treatment response of islet-like tumoroids differs also between patient samples. We believe that the presented human PanNET screening platform is suitable for personalized drug testing in a larger patient cohort, and a broader application will help in identifying novel markers predicting treatment response and in refining PanNET therapy.

Epistasis-driven identification of SLC25A51 as a regulator of human mitochondrial NAD import.

About a thousand genes in the human genome encode for membrane transporters. Among these, several solute carrier proteins (SLCs), representing the largest group of transporters, are still orphan and lack functional characterization. We reasoned that assessing genetic interactions among SLCs may be an efficient way to obtain functional information allowing their deorphanization. Here we describe a network of strong genetic interactions indicating a contribution to mitochondrial respiration and redox metabolism for SLC25A51/MCART1, an uncharacterized member of the SLC25 family of transporters. Through a combination of metabolomics, genomics and genetics approaches, we demonstrate a role for SLC25A51 as enabler of mitochondrial import of NAD, showcasing the potential of genetic interaction-driven functional gene deorphanization.

Assessing Autophagy in Archived Tissue or How to Capture Autophagic Flux from a Tissue Snapshot.

Autophagy is a highly conserved degradation mechanism that is essential for maintaining cellular homeostasis. In human disease, autophagy pathways are frequently deregulated and there is immense interest in targeting autophagy for therapeutic approaches. Accordingly, there is a need to determine autophagic activity in human tissues, an endeavor that is hampered by the fact that autophagy is characterized by the flux of substrates whereas histology informs only about amounts and localization of substrates and regulators at a single timepoint. Despite this challenging task, considerable progress in establishing markers of autophagy has been made in recent years. The importance of establishing clear-cut autophagy markers that can be used for tissue analysis cannot be underestimated. In this review, we attempt to summarize known techniques to quantify autophagy in human tissue and their drawbacks. Furthermore, we provide some recommendations that should be taken into consideration to improve the reliability and the interpretation of autophagy biomarkers in human tissue samples.

Genetic and epigenetic drivers of neuroendocrine tumours (NET).

Neuroendocrine tumours (NET) of the gastrointestinal tract and the lung are a rare and heterogeneous group of tumours. The molecular characterization and the clinical classification of these tumours have been evolving slowly and show differences according to organs of origin. Novel technologies such as next-generation sequencing revealed new molecular aspects of NET over the last years. Notably, whole-exome/genome sequencing (WES/WGS) approaches underlined the very low mutation rate of well-differentiated NET of all organs compared to other malignancies, while the engagement of epigenetic changes in driving NET evolution is emerging. Indeed, mutations in genes encoding for proteins directly involved in chromatin remodelling, such as DAXX and ATRX are a frequent event in NET. Epigenetic changes are reversible and targetable; therefore, an attractive target for treatment. The discovery of the mechanisms underlying the epigenetic changes and the implication on gene and miRNA expression in the different subgroups of NET may represent a crucial change in the diagnosis of this disease, reveal new therapy targets and identify predictive markers. Molecular profiles derived from omics data including DNA mutation, methylation, gene and miRNA expression have already shown promising results in distinguishing clinically and molecularly different subtypes of NET. In this review, we recapitulate the major genetic and epigenetic characteristics of pancreatic, lung and small intestinal NET and the affected pathways. We also discuss potential epigenetic mechanisms leading to NET development.

Autophagy Inhibition Improves Sunitinib Efficacy in Pancreatic Neuroendocrine Tumors via a Lysosome-dependent Mechanism.

Increasing the efficacy of approved systemic treatments in metastasized pancreatic neuroendocrine tumors (PanNET) is an unmet medical need. The antiangiogenic tyrosine kinase inhibitor sunitinib is approved for PanNET treatment. In addition, sunitinib is a lysosomotropic drug and such drugs can induce lysosomal membrane permeabilization as well as autophagy. We investigated sunitinib-induced autophagy as a possible mechanism of PanNET therapy resistance. Sunitinib accumulated in lysosomes and induced autophagy in PanNET cell lines. Adding the autophagy inhibitor chloroquine reduced cell viability in cell lines and in primary cells isolated from PanNET patients. The same treatment combination reduced tumor burden in the Rip1Tag2 transgenic PanNET mouse model. The combination of sunitinib and chloroquine reduced recovery and induced apoptosis in vitro, whereas single treatments did not. Knockdown of key autophagy proteins in combination with sunitinib showed similar effect as chloroquine. Sunitinib also induced lysosomal membrane permeabilization, which further increased in the presence of chloroquine or knockdown of lysosome-associated membrane protein (LAMP2). Both combinations led to cell death. Our data indicate that chloroquine increases sunitinib efficacy in PanNET treatment via autophagy inhibition and lysosomal membrane permeabilization. We suggest that adding chloroquine to sunitinib treatment will increase efficacy of PanNET treatment and that such patients should be included in respective ongoing clinical trials. Mol Cancer Ther; 16(11); 2502-15. ©2017 AACR.