A dynamic anchor domain in slc13 transporters controls metabolite transport.

Metabolite transport across cellular membranes is required for bioenergetic processes and metabolic signaling. The solute carrier family 13 (slc13) transporters mediate transport of the metabolites succinate and citrate and hence are of paramount physiological importance. Nevertheless, the mechanisms of slc13 transport and regulation are poorly understood. Here, a dynamic structural slc13 model suggested that an interfacial helix, H4c, which is common to all slc13s, stabilizes the stationary scaffold domain by anchoring it to the membrane, thereby facilitating movement of the SLC13 catalytic domain. Moreover, we found that intracellular determinants interact with the H4c anchor domain to modulate transport. This dual function is achieved by basic residues that alternately face either the membrane phospholipids or the intracellular milieu. This mechanism was supported by several experimental findings obtained using biochemical methods, electrophysiological measurements in Xenopus oocytes, and fluorescent microscopy of mammalian cells. First, a positively charged and highly conserved H4c residue, Arg108, was indispensable and crucial for metabolite transport. Furthermore, neutralization of other H4c basic residues inhibited slc13 transport function, thus mimicking the inhibitory effect of the slc13 inhibitor, slc26a6. Our findings suggest that the positive charge distribution across H4c domain controls slc13 transporter function and is utilized by slc13-interacting proteins in the regulation of metabolite transport.

Systemic Succinate Homeostasis and Local Succinate Signaling Affect Blood Pressure and Modify Risks for Calcium Oxalate Lithogenesis.

BACKGROUND: In the kidney, low urinary citrate increases the risk for developing kidney stones, and elevation of luminal succinate in the juxtaglomerular apparatus increases renin secretion, causing hypertension. Although the association between stone formation and hypertension is well established, the molecular mechanism linking these pathophysiologies has been elusive. METHODS: To investigate the relationship between succinate and citrate/oxalate levels, we assessed blood and urine levels of metabolites, renal protein expression, and BP (using 24-hour telemetric monitoring) in male mice lacking slc26a6 (a transporter that inhibits the succinate transporter NaDC-1 to control citrate absorption from the urinary lumen). We also explored the mechanism underlying this metabolic association, using coimmunoprecipitation, electrophysiologic measurements, and flux assays to study protein interaction and transport activity. RESULTS: Compared with control mice, slc26a6-/- mice (previously shown to have low urinary citrate and to develop calcium oxalate stones) had a 40% decrease in urinary excretion of succinate, a 35% increase in serum succinate, and elevated plasma renin. Slc26a6-/- mice also showed activity-dependent hypertension that was unaffected by dietary salt intake. Structural modeling, confirmed by mutational analysis, identified slc26a6 and NaDC-1 residues that interact and mediate slc26a6's inhibition of NaDC-1. This interaction is regulated by the scaffolding protein IRBIT, which is released by stimulation of the succinate receptor SUCNR1 and interacts with the NaDC-1/slc26a6 complex to inhibit succinate transport by NaDC-1. CONCLUSIONS: These findings reveal a succinate/citrate homeostatic pathway regulated by IRBIT that affects BP and biochemical risk of calcium oxalate stone formation, thus providing a potential molecular link between hypertension and lithogenesis.

A clinical evaluation of an ex vivo organ culture system to predict patient response to cancer therapy.

Introduction: Ex vivo organ cultures (EVOC) were recently optimized to sustain cancer tissue for 5 days with its complete microenvironment. We examined the ability of an EVOC platform to predict patient response to cancer therapy. Methods: A multicenter, prospective, single-arm observational trial. Samples were obtained from patients with newly diagnosed bladder cancer who underwent transurethral resection of bladder tumor and from core needle biopsies of patients with metastatic cancer. The tumors were cut into 250 µM slices and cultured within 24 h, then incubated for 96 h with vehicle or intended to treat drug. The cultures were then fixed and stained to analyze their morphology and cell viability. Each EVOC was given a score based on cell viability, level of damage, and Ki67 proliferation, and the scores were correlated with the patients' clinical response assessed by pathology or Response Evaluation Criteria in Solid Tumors (RECIST). Results: The cancer tissue and microenvironment, including endothelial and immune cells, were preserved at high viability with continued cell division for 5 days, demonstrating active cell signaling dynamics. A total of 34 cancer samples were tested by the platform and were correlated with clinical results. A higher EVOC score was correlated with better clinical response. The EVOC system showed a predictive specificity of 77.7% (7/9, 95% CI 0.4-0.97) and a sensitivity of 96% (24/25, 95% CI 0.80-0.99). Conclusion: EVOC cultured for 5 days showed high sensitivity and specificity for predicting clinical response to therapy among patients with muscle-invasive bladder cancer and other solid tumors.

Comparing Intraperitoneal and Intravenous Personalized ErbB2CAR-T for the Treatment of Epithelial Ovarian Cancer.

High-grade serous ovarian carcinoma (HGSOC) is the most common type of epithelial ovarian cancer. The majority of cases are diagnosed at advanced stages, when intraperitoneal (IP) spread has already occurred. Despite significant surgical and chemotherapeutic advances in HGSOC treatment over the past decades, survival rates with HGSOC have only modestly improved. Chimeric antigen receptor (CAR)-T cells enable T cells to directly bind to tumor-associated antigens in a major histocompatibility complex-independent manner, thereby inducing tumor rejection. While CAR-T cell therapy shows great promise in hematological malignancies, its use in solid tumors is limited. Therefore, innovative approaches are needed to increase the specificity of CAR-modified T cells against solid tumors. The aim of this study was to assess the efficacy and safety of intraperitoneal (IP) versus intravenous (IV) CAR-T cell therapy in the treatment of HGSOC. We constructed a CAR that targets the ErbB2/HER2 protein (ErbB2CAR), which is overexpressed in HGSOC, and evaluated the functionality of ErbB2CAR on ovarian cancer cell lines (OVCAR8, SKOV3, and NAR). Our findings show that an IP injection of ErbB2CAR-T cells to tumor-bearing mice led to disease remission and increased survival compared to the IV route. Moreover, we found that IP-injected ErbB2CART cells circulate to a lesser extent, making them safer for non-tumor tissues than IV-injected cells. Further supporting our findings, we show that the effect of ErbB2CAR-T cells on primary HGSOC tumors is correlated with ErbB2 expression. Together, these data demonstrate the advantages of an IP administration of CAR-T cells over IV administration, offering not only a safer strategy but also the potential for counteracting the effect of ErbB2CAR in HGSOC. Significance: IP-injected ErbB2CAR-T cells led to disease remission and increased survival compared to the IV route. These findings demonstrate the advantages of IP administration, offering a safe treatment strategy with the potential for counteracting the effect of ErbB2CAR in HGSOC.

Novel Human Polymorphisms Define a Key Role for the SLC26A6-STAS Domain in Protection From Ca2+-Oxalate Lithogenesis.

Impaired homeostasis of the carboxylic acids oxalate and citrate, dramatically increases the risk for the formation of Ca2+-oxalate kidney stones, which is the most common form of kidney stones in humans. Renal homeostasis of oxalate and citrate is controlled by complex mechanisms including epithelial transport proteins such as the oxalate transporter, SLC26A6, and the citrate transporters, the SLC13's. These transporters interact via the SLC26A6-STAS domain in vitro, however, the role of the Sulfate Transporter and Anti-Sigma factor antagonist (STAS) domain in Ca2+-oxalate stone formation was not investigated in humans. Here, we report two novel human SLC26A6 polymorphisms identified in the STAS domain of SLC26A6 in two heterozygous carriers. Intriguingly, these individuals have low urinary citrate, but different clinical manifestations. Our in vitro experiments indicate that the homolog mutations of SLC26A6(D23H/D673N) and SLC26A6(D673N) alone abolished the expression and function of SLC26A6, and impaired the regulation of SLC13-mediated citrate transport by SLC26A6. On the other hand, the SLC26A6(R621G) variant showed reduced SLC26A6 protein expression and membrane trafficking, retained full transport activity, but impaired the regulation of the citrate transporter. Accordingly, the human SLC26A6(D23H/D673N) carrier showed a dramatic reduction in urinary citrate concentrations which resulted in Ca2+-oxalate stones formation, as opposed to the carrier of SLC26A6(R621G). Our findings indicate that the human SLC26A6-STAS domain mutations differentially impair SLC26A6 expression, function, and regulation of citrate transporters. This interferes with citrate and oxalate homeostasis thus potentially predisposes to Ca2+-oxalate kidney stones.