Faculty Opinions recommendation of Optimization of a dicarboxylic series for in vivo inhibition of citrate transport by the solute carrier 13 (SLC13) family.

2016 ◽  
Author(s):  
John Lowe
Keyword(s):  
Citrate Transport ◽  
Solute Carrier

Optimization of a Dicarboxylic Series for in Vivo Inhibition of Citrate Transport by the Solute Carrier 13 (SLC13) Family

2016 ◽  
Vol 59 (3) ◽  
pp. 1165-1175 ◽  
Author(s):  
Kim Huard ◽  
James R. Gosset ◽  
Justin I. Montgomery ◽  
Adam Gilbert ◽  
Matthew M. Hayward ◽  
...  
Keyword(s):  
Citrate Transport ◽  
Solute Carrier

scholarly journals In Vivo Evidence for TonB Dimerization

2003 ◽  
Vol 185 (19) ◽  
pp. 5747-5754 ◽  
Author(s):  
Annette Sauter ◽  
S. Peter Howard ◽  
Volkmar Braun
Keyword(s):  
Single Point ◽  
Point Mutations ◽  
Ferric Citrate ◽  
Site Directed Mutagenesis ◽  
Toxin Gene ◽  
Transmembrane Region ◽  
Dimer Formation ◽  
Citrate Transport ◽  
Hybrid Proteins

ABSTRACT TonB, in complex with ExbB and ExbD, is required for the energy-dependent transport of ferric siderophores across the outer membrane of Escherichia coli, the killing of cells by group B colicins, and infection by phages T1 and φ80. To gain insights into the protein complex, TonB dimerization was studied by constructing hybrid proteins from complete TonB (containing amino acids 1 to 239) [TonB(1-239)] and the cytoplasmic fragment of ToxR which, when dimerized, activates the transcription of the cholera toxin gene ctx. ToxR(1-182)-TonB(1-239) activated the transcription of lacZ under the control of the ctx promoter (P ctx ::lacZ). Replacement of the TonB transmembrane region by the ToxR transmembrane region resulted in the hybrid proteins ToxR(1-210)-TonB(33-239) and ToxR(1-210)-TonB(164-239), of which only the latter activated P ctx ::lacZ transcription. Dimer formation was reduced but not abolished in a mutant lacking ExbB and ExbD, suggesting that these complex components may influence dimerization but are not strictly required and that the N-terminal cytoplasmic membrane anchor and the C-terminal region are important for dimer formation. The periplasmic TonB fragment, TonB(33-239), inhibits ferrichrome and ferric citrate transport and induction of the ferric citrate transport system. This competition provided a means to positively screen for TonB(33-239) mutants which displayed no inhibition. Single point mutations of inactive fragments selected in this manner were introduced into complete TonB, and the phenotypes of the TonB mutant strains were determined. The mutations located in the C-terminal half of TonB, three of which (Y163C, V188E, and R204C) were obtained separately by site-directed mutagenesis, as was the isolated F230V mutation, were studied in more detail. They displayed different activity levels for various TonB-dependent functions, suggesting function-related specificities which reflect differences in the interactions of TonB with various transporters and receptors.

scholarly journals SLC36A1-mTORC1 signaling drives acquired resistance to CDK4/6 inhibitors

2019 ◽  
Vol 5 (9) ◽  
pp. eaax6352 ◽  
Author(s):  
Akihiro Yoshida ◽  
Yiwen Bu ◽  
Shuo Qie ◽  
John Wrangle ◽  
E. Ramsay Camp ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Fragile X ◽  
Acquired Resistance ◽  
Mammalian Target Of Rapamycin ◽  
Cyclin Dependent Kinase ◽  
Solute Carrier Family ◽  
Potential Therapeutic Target ◽  
Mtorc1 Signaling ◽  
Solute Carrier

The cyclin-dependent kinase 4/6 (CDK4/6) kinase is dysregulated in melanoma, highlighting it as a potential therapeutic target. CDK4/6 inhibitors are being evaluated in trials for melanoma and additional cancers. While beneficial, resistance to therapy is a concern, and the molecular mechanisms of such resistance remain undefined. We demonstrate that reactivation of mammalian target of rapamycin 1 (mTORC1) signaling through increased expression of the amino acid transporter, solute carrier family 36 member 1 (SLC36A1), drives resistance to CDK4/6 inhibitors. Increased expression of SLC36A1 reflects two distinct mechanisms: (i) Rb loss, which drives SLC36A1 via reduced suppression of E2f; (ii) fragile X mental retardation syndrome–associated protein 1 overexpression, which promotes SLC36A1 translation and subsequently mTORC1. Last, we demonstrate that a combination of a CDK4/6 inhibitor with an mTORC1 inhibitor has increased therapeutic efficacy in vivo, providing an important avenue for improved therapeutic intervention in aggressive melanoma.

scholarly journals Surface Signaling in Ferric Citrate Transport Gene Induction: Interaction of the FecA, FecR, and FecI Regulatory Proteins

2000 ◽  
Vol 182 (3) ◽  
pp. 637-646 ◽  
Author(s):  
Sabine Enz ◽  
Susanne Mahren ◽  
Uwe H. Stroeher ◽  
Volkmar Braun
Keyword(s):  
Nitrilotriacetic Acid ◽  
Ferric Citrate ◽  
Binding Assays ◽  
Surface Binding ◽  
Citrate Transport ◽  
C Terminus ◽  
N Terminus ◽  
Surface Signaling

ABSTRACT In Escherichia coli, transcription of the ferric citrate transport genes fecABCDE is controlled by a novel signal transduction mechanism that starts at the cell surface. Binding of ferric citrate to the outer membrane protein FecA initiates a signal that is transmitted by FecR across the cytoplasmic membrane into the cytoplasm where FecI, the sigma factor, is activated. Interaction between the signaling proteins was demonstrated by utilizing two methods. In in vitro binding assays, FecR that was His tagged at the N terminus [(His)10-FecR] and bound to a Ni-nitrilotriacetic acid agarose column was able to retain FecA, and FecR that was His tagged at the C terminus [FecR-(His)6] retained FecI on the column. An N-terminally truncated, induction-negative but transport-active FecA protein did not bind to (His)10-FecR. The in vivo assay involved the determination of the FecA, FecR, and FecI interacting domains with the bacterial two-hybrid Lex-based system. FecA1–79 interacts with FecR101–317 and FecR1–85 interacts with FecI1–173. These data clearly support a model that proposes interaction of the periplasmic N terminus of FecA with the periplasmic C-terminal portion of FecR and interaction of the cytoplasmic N terminus of FecR with FecI, which results in FecI activation.

Homo- and heterodimerization is a common feature of the solute carrier family SLC10 members

2019 ◽  
Vol 400 (10) ◽  
pp. 1371-1384 ◽  
Author(s):  
Saskia Noppes ◽  
Simon Franz Müller ◽  
Josefine Bennien ◽  
Matthias Holtemeyer ◽  
Massimo Palatini ◽  
...  
Keyword(s):  
Bile Acid ◽  
Transport Function ◽  
Solute Carrier Family ◽  
Bile Acid Transporters ◽  
Bile Acid Transporter ◽  
Solute Carrier ◽  
Steroid Sulfate ◽  
Functional Relevance ◽  
Two Hybrid

AbstractThe solute carrier family SLC10 consists of seven members, including the bile acid transporters Na+/taurocholate co-transporting polypeptide (NTCP) and apical sodium-dependent bile acid transporter (ASBT), the steroid sulfate transporter SOAT as well as four orphan carriers (SLC10A3, SLC10A4, SLC10A5 and SLC10A7). Previously, homodimerization of NTCP, ASBT and SOAT was described and there is increasing evidence that carrier oligomerization is an important regulatory factor for protein sorting and transport function. In the present study, homo- and heterodimerization were systematically analyzed among all SLC10 carriers (except for SLC10A3) using the yeast-two-hybrid membrane protein system. Strong homodimerization occurred for NTCP/NTCP, ASBT/ASBT and SLC10A7/SLC10A7. Heterodimerization was observed for most of the SLC10 carrier combinations. Heterodimerization of NTCP was additionally investigated by co-localization of NTCP-GFP and NTCP-mScarlet with respective SLC10 carrier constructs. NTCP co-localized with SLC10A4, SLC10A5, SOAT and SLC10A7. This co-localization was most pronounced for SLC10A4 and was additionally confirmed by co-immunoprecipitation. Interestingly, SLC10 carrier co-expression decreased the taurocholate transport function of NTCP for most of the analyzed constructs, indicating that SLC10 carrier heterodimerization is of functional relevance. In conclusion, homo- and heterodimerization is a common feature of the SLC10 carriers. The relevance of this finding for regulation and transport function of the SLC10 carriersin vivoneeds further investigation.

scholarly journals Targeting SLC3A2 Subunit of System XC- is Essential for m6A Reader YTHDC2 to be an Endogenous Ferroptosis Inducer in Lung Adenocarcinoma

2021 ◽  
Author(s):  
Lifang Ma ◽  
Xiao Zhang ◽  
Keke Yu ◽  
Xin Xu ◽  
Tianxiang Chen ◽  
...  
Keyword(s):  
Lung Adenocarcinoma ◽  
Cell Model ◽  
Rna Stability ◽  
Luciferase Reporter ◽  
Enzyme Linked Immunosorbent Assay ◽  
Ros Generation ◽  
Gene Expressions ◽  
Mobility Shift ◽  
Solute Carrier

Abstract Background: The m6A reader YT521-B homology containing 2 (YTHDC2) inhibits lung adenocarcinoma (LUAD) tumorigenesis by suppressing solute carrier 7A11 (SLC7A11)-dependent antioxidant function. SLC7A11 is a major functional subunit of system XC-. Inhibition of system XC- can induce ferroptosis. However, whether suppressing SLC7A11 is sufficient for YTHDC2 to be an endogenous ferroptosis inducer in LUAD is unknown.Methods: Bioinformatics were used to predict overall survival (OS) and possible correlations. Gene expressions were examined by immunoblotting (IB), reverse transcription-quantitative PCR (RT-qPCR), immunohistochemistry (IHC) and enzyme-linked immunosorbent assay (ELISA). The doxycycline (Dox)-inducible YTHDC2 cell model, spontaneous LUAD and xenograft mouse models were used to study the function of YTHDC2. SYTOX green and C11-BODIPY581/591 staining following flow cytometry were used to evaluate cell death and lipid reactive oxygen species (ROS) generation. Luciferase reporter assay, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) were performed to study promoter. RNA immunoprecipitation (RIP), RNA pull down and photoactivatable ribonucleotide crosslinking and immunoprecipitation (PAR-CLIP) were used to determine protein-RNA interactions. Actinomycin D chase experiments were used for RNA stability and four independent LUAD cohorts with a total volume of 384 were recruited to evaluate clinical significance.Results: Induction of YTHDC2 to a high level can induce ferroptosis in LUAD cells but not in lung and bronchus epithelial cells. In addition to SLC7A11, solute carrier 3A2 (SLC3A2), another subunit of system XC- was equally important for YTHDC2-induced ferroptosis. YTHDC2 m6A-dependently destabilized Homeo box A13 (HOXA13) mRNA because a potential m6A recognition site was identified within its 3’ untranslated region (3’UTR). Interestingly, HOXA13 acted as a transcription factor to stimulate SLC3A2 expression. Thereby, YTHDC2 suppressed SLC3A2 via inhibiting HOXA13 in an m6A-indirect manner. Mouse experiments further confirmed the associations among YTHDC2, SLC3A2 and HOXA13, and demonstrated that SLC3A2 and SLC7A11 were both important for YTHDC2-impaired tumor growth and –induced lipid peroxidation in vivo. Moreover, higher expression of SLC7A11, SLC3A2 and HOXA13 indicate poorer clinical outcome in YTHDC2-suppressed LUAD patients.Conclusion: YTHDC2 is a powerful endogenous ferroptosis inducer and targeting SLC3A2 subunit of system XC- is essential for this process. Increasing YTHDC2 is an alternative ferroptosis-based therapy to treat LUAD.

Transcriptional regulation of urate transportosome member SLC2A9 by nuclear receptor HNF4α

2014 ◽  
Vol 307 (9) ◽  
pp. F1041-F1051 ◽  
Author(s):  
Katharina Prestin ◽  
Stephanie Wolf ◽  
Rico Feldtmann ◽  
Janine Hussner ◽  
Ingrid Geissler ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
Binding Site ◽  
Nuclear Receptor ◽  
In Silico Analysis ◽  
Human Kidney ◽  
Renal Clear Cell Carcinoma ◽  
Key Factor ◽  
Solute Carrier

Renal tubular handling of urate is realized by a network of uptake and efflux transporters, including members of drug transporter families such as solute carrier proteins and ATP-binding cassette transporters. Solute carrier family 2, member 9 (SLC2A9), is one key factor of this so called “urate transportosome.” The aim of the present study was to understand the transcriptional regulation of SLC2A9 and to test whether identified factors might contribute to a coordinated transcriptional regulation of the transporters involved in urate handling. In silico analysis and cell-based reporter gene assays identified a hepatocyte nuclear factor (HNF)4α-binding site in the promoter of SLC2A9 isoform 1, whose activity was enhanced by transient HNF4α overexpression, whereas mutation of the binding site diminished activation. HNF4α overexpression induced endogenous SLC2A9 expression in vitro. The in vivo role of HNF4α in the modulation of renal SLC2A9 gene expression was supported by findings of quantitative real-time RT-PCR analyses and chromatin immunoprecipitation assays. Indeed, mRNA expression of SLC2A9 and HNF4α in human kidney samples was significantly correlated. We also showed that in renal clear cell carcinoma, downregulation of HNF4α mRNA and protein expression was associated with a significant decline in expression of the transporter. Taken together, our data suggest that nuclear receptor family member HNF4α contributes to the transcriptional regulation of SLC2A9 isoform 1. Since HNF4α has previously been assumed to be a modulator of several urate transporters, our findings support the notion that there could be a transcriptional network providing synchronized regulation of the functional network of the urate transportosome.

scholarly journals IMCA Induces Ferroptosis Mediated by SLC7A11 through the AMPK/mTOR Pathway in Colorectal Cancer

2020 ◽  
Vol 2020 ◽  
pp. 1-14 ◽  
Author(s):  
Lei Zhang ◽  
Wen Liu ◽  
Fangyan Liu ◽  
Qun Wang ◽  
Mengjiao Song ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cancer Cells ◽  
Screening Tests ◽  
Therapeutic Drug ◽  
Solute Carrier Family ◽  
Colorectal Cancer Cells ◽  
Solute Carrier ◽  
The Impact

Ferroptosis, implicated in several diseases, is a new form of programmed and nonapoptotic cell death triggered by iron-dependent lipid peroxidation after inactivation of the cystine/glutamate antiporter system xc–, which is composed of solute carrier family 7 membrane 11 (SLC7A11) and solute carrier family 3 membrane 2 (SLC3A2). Therefore, inducing ferroptosis through inhibiting the cystine/glutamate antiporter system xc– may be an effective way to treat cancer. In previous screening tests, we found that the benzopyran derivative 2-imino-6-methoxy-2H-chromene-3-carbothioamide (IMCA) significantly inhibited the viability of colorectal cancer cells. However, the impact of IMCA on ferroptosis remains unknown. Hence, this study investigated the effect of IMCA on ferroptosis and elucidated the underlying molecular mechanism. Results showed that IMCA significantly inhibited the cell viability of colorectal cancer cells in vitro and inhibited tumor growth with negligible organ toxicity in vivo. Further studies showed that IMCA significantly induced the ferroptosis of colorectal cancer cells. Mechanistically, IMCA downregulated the expression of SLC7A11 and decreased the contents of cysteine and glutathione, which resulted in reactive oxygen species accumulation and ferroptosis. Furthermore, overexpression of SLC7A11 significantly attenuated the ferroptosis caused by IMCA. In addition, IMCA regulated the activity of the AMPK/mTOR/p70S6k signaling pathway, which is related to the activity of SLC7A11 and ferroptosis. Collectively, our research provided experimental evidences on the activity and mechanism of ferroptosis induced by IMCA and revealed that IMCA might be a promising therapeutic drug for colorectal cancer.

scholarly journals Functional Interaction of Region 4 of the Extracytoplasmic Function Sigma Factor FecI with the Cytoplasmic Portion of the FecR Transmembrane Protein of the Escherichia coli Ferric Citrate Transport System

2002 ◽  
Vol 184 (13) ◽  
pp. 3704-3711 ◽  
Author(s):  
Susanne Mahren ◽  
Sabine Enz ◽  
Volkmar Braun
Keyword(s):  
Escherichia Coli ◽  
Sigma Factor ◽  
Transmembrane Protein ◽  
Ferric Citrate ◽  
Missense Mutations ◽  
E Coli ◽  
Citrate Transport ◽  
Extracytoplasmic Function Sigma Factor ◽  
Cognate Protein

ABSTRACT Transcriptional regulation of the ferric citrate transport genes of Escherichia coli is initiated by the binding of ferric citrate to the outer membrane protein FecA. This binding elicits a signal that is transmitted by FecR across the cytoplasmic membrane into the cytoplasm, where the sigma factor FecI directs the RNA polymerase to the promoter upstream of the fecABCDE genes. An in vivo deletion analysis using a bacterial two-hybrid system assigned the interaction of the FecR and FecI proteins to the cytoplasmic portion of the FecR transmembrane protein and region 4 of FecI. Missense mutations randomly generated by PCR were localized to region 4 of FecI, and the mutants were impaired with regard to the interaction of FecR with FecI and fecB-lacZ transcription. The cloned region 4 of FecI interfered with fecB-lacZ transcription. Interaction of N-proximal regions of predicted FecR homologs with region 4 of predicted FecI homologs of Pseudomonas aeruginosa was demonstrated. The interaction was specific in that only cognate protein pairs interacted with each other; no interactions occurred between heterologous combinations of the P. aeruginosa proteins and between a P. aeruginosa FecI homolog and E. coli FecR. The results demonstrate that region 4 of FecI specifically binds FecR and that this binding is necessary for FecI to function as a sigma factor.

scholarly journals Comprehensive Substrate Characterization of 22 Antituberculosis Drugs for Multiple Solute Carrier (SLC) Uptake TransportersIn Vitro

2018 ◽  
Vol 62 (9) ◽  
Author(s):  
M. M. Parvez ◽  
Nazia Kaisar ◽  
Ho Jung Shin ◽  
Yoon Jae Lee ◽  
Jae-Gook Shin
Keyword(s):  
Drug Interactions ◽  
Organic Anion ◽  
Organic Anion Transporter ◽  
Antituberculosis Drugs ◽  
Hek 293 ◽  
Slc Transporters ◽  
Anion Transporter ◽  
Solute Carrier

ABSTRACTThe substrate potentials of antituberculosis drugs on solute carrier (SLC) transporters are not well characterized to date, despite a well-established understanding of their drug dispositions and pharmacokinetics. In this study, we investigated comprehensively the substrate potentials of the 22 currently available antituberculosis drugs for SLC family transporter-mediated uptake, usingXenopus laevisoocytes and stably transfected HEK-293 cellsin vitro. The result suggested that ethambutol, isoniazid, amoxicillin, and prothionamide act as novel substrates for the SLC transporters. In addition, in the presence of representative transporter inhibitors, the uptake of the antituberculosis drugs was markedly decreased compared with the uptake in the absence of inhibitor, suggesting involvement of the corresponding transporters. A cellular uptake study was performed, and theKmvalues of ethambutol were found to be 526.1 ± 15.6, 212.0 ± 20.1, 336.8 ± 20.1, and 455.0 ± 28 μM for organic cation transporter 1 (OCT1), OCT2, OCTN1, and OCTN2, respectively. Similarly, theKmof prothionamide was 805.8 ± 23.4 μM for OCT1, while theKmvalues of isoniazid and amoxicillin for organic anion transporter 3 (OAT3) were 233.7 ± 14.1 and 161.4 ± 10.6 μM, respectively. The estimatedin vivodrug-drug interaction indexes fromin vitrotransporter inhibition kinetics for verapamil, probenecid, and ibuprofen against ethambutol, prothionamide, isoniazid, and amoxicillin were found to show potential for clinical drug interactions. In conclusion, this is the first study that demonstrated 22 antituberculosis drug interactions with transporters. This study will be helpful for mechanistic understanding of the disposition, drug-drug interactions, and pharmacokinetics of these antituberculosis drugs.

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