Identification of essential transporter genes in the colorectal carcinoma cell model HCT116 by focused CRISPR/Cas9 KO screens.
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ABSTRACT:
ORGANISM(S): blank sample Homo sapiens
PROVIDER: GSE277685 | GEO | 2024/09/25
REPOSITORIES: GEO
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Project description:Transporters mediate and control the flux of molecules across compartmental membranes. The human genome encodes 1500 genes with transport functions, of which the solute carriers (SLCs) form the largest superfamily with more than 450 members. Over 250 different SLCs are expressed in a typical human cell, many exhibiting overlapping expression patterns and substrate specificities. The collective role of these often seemingly redundant transporters in defining cellular outcomes, such as cell survival, remains unclear. Here, we performed pooled combinatorial KO screens to identify genetic interactions between 258 expressed SLCs, and between a subset of SLCs and selected metabolic enzymes under different growth conditions using both CRISPR-Cas12a and -Cas9 double knockout systems in the colorectal carcinoma cell line HCT116.
2024-06-19 | GSE269905 | GEO
Project description:Objectives: The aim of this study was to investigate the prognostic significance of thirteen anticancer drug-relevant solute carrier transporters (SLCs) in pancreatic cancer in the context of clinical-pathological characteristics and the KRAS mutation status of tumors. Methods: Tumors and non-neoplastic pancreatic tissues were obtained from 32 histologically verified patients with pancreatic ductal adenocarcinoma. The transcript profile of SLCs was assessed using quantitative real-time PCR. KRAS mutations in exon 2 were assessed by high resolution melting analysis and confirmed by sequencing. Results: SLC22A3 was upregulated and SLC22A1, SLC22A2, SLC22A11, SLC28A1, SLC28A3 and SLC29A1 were downregulated when compared with non-neoplastic pancreatic tissues. Moreover, significantly lower levels of SLC22A1, SLC22A11 and SLC29A1 were found in tumors with angioinvasion. There was also a significantly higher transcript level of SLC28A1 in tumors with regional lymph nodes affected by metastasis. The study found that a high expression of SLC22A1 or SLC28A1was significantly associated with poor overall survival in unselected patients. In contrast, a high expression of SLC22A3 or SLC29A3 was significantly associated with longer overall survival in patients treated with nucleoside analogs. Finally, SLC levels were not found to be associated with KRAS mutation status in exon 2. Conclusions: This study identified a number of associations of SLCs with prognosis of pancreatic cancer patients. Transcript levels of thirteen anticancer drug-relevant solute carrier transporters (SLCs) were determined by qPCR in pancreatic ductal adenocarcinomas and non-neoplastic tissue samples from 32 pancreatic cancer patients. MRPL19, ELF1 and POLR2A were used as reference genes for data normalization.
2013-06-30 | E-GEOD-47368 | biostudies-arrayexpress
Project description:Objectives: The aim of this study was to investigate the prognostic significance of thirteen anticancer drug-relevant solute carrier transporters (SLCs) in pancreatic cancer in the context of clinical-pathological characteristics and the KRAS mutation status of tumors. Methods: Tumors and non-neoplastic pancreatic tissues were obtained from 32 histologically verified patients with pancreatic ductal adenocarcinoma. The transcript profile of SLCs was assessed using quantitative real-time PCR. KRAS mutations in exon 2 were assessed by high resolution melting analysis and confirmed by sequencing. Results: SLC22A3 was upregulated and SLC22A1, SLC22A2, SLC22A11, SLC28A1, SLC28A3 and SLC29A1 were downregulated when compared with non-neoplastic pancreatic tissues. Moreover, significantly lower levels of SLC22A1, SLC22A11 and SLC29A1 were found in tumors with angioinvasion. There was also a significantly higher transcript level of SLC28A1 in tumors with regional lymph nodes affected by metastasis. The study found that a high expression of SLC22A1 or SLC28A1was significantly associated with poor overall survival in unselected patients. In contrast, a high expression of SLC22A3 or SLC29A3 was significantly associated with longer overall survival in patients treated with nucleoside analogs. Finally, SLC levels were not found to be associated with KRAS mutation status in exon 2. Conclusions: This study identified a number of associations of SLCs with prognosis of pancreatic cancer patients.
2013-06-30 | GSE47368 | GEO
Project description:Creatine is an essential metabolite for the storage and rapid supply of energy in muscle and nerve cells. In humans, the creatine deficiency syndrome (CDS) is manifested by impaired metabolism, transport, and distribution of creatine throughout the body leading to severe forms of mental disabilities. One of the common cause of CDS is mutations that impact the function of the creatine transporter (SLC6A8). This study characterized 30 missense variants of SLC6A8, which include 15 variants of unknown significance and two which were not reported before. These variants were expressed in HEK293 cells, and their subcellular localization and transport activity was assessed. Further, for the first time the interactome of SLC6A8 WT was characterized by quantitative interaction proteomics. Computational methods were then used to first assess the impact of mutations upon the thermodynamic stability of SLC6A8. This was expanded by modeling the impact of mutations upon the inward and outward facing transporter conformation. Next, identified SLC6A8 protein interactions binary complexes were modelled and used to characterize the impact of variants upon the thermodynamic stability of the complexes. This was followed up, by performing for a subset of the variants the interaction proteomics analysis to study changes upon the identified SLC6A8 WT interactome.
2024-01-03 | PXD047111 | Pride
Project description:Mutations in the solute carrier family 6-member 8 (Slc6a8) gene, encoding the protein responsible for cellular creatine (Cr) uptake, cause Creatine Transporter Deficiency (CTD), an X-linked neurometabolic disorder presenting with intellectual disability, autistic-like features, and epilepsy. The pathological determinants of CTD are still poorly understood, hindering the development of therapies. In this study, we generated an extensive transcriptomic profile of CTD showing that Cr deficiency causes perturbations of gene expression in excitatory neurons, inhibitory cells, and oligodendrocytes which result in remodeling of circuit excitability and synaptic wiring. We also identified specific alterations of parvalbumin-expressing (PV+) interneurons, exhibiting a reduction in cellular and synaptic density, and a hypofunctional electrophysiological phenotype. Mice lacking Slc6a8 only in PV+ interneurons recapitulated numerous CTD features, including cognitive deterioration, impaired cortical processing and hyperexcitability of brain circuits, demonstrating that Cr deficit in PV+ interneurons is sufficient to determine the neurological phenotype of CTD. Moreover, a pharmacological treatment targeted to restore the efficiency of PV+ synapses significantly improved cortical activity in Slc6a8 knock-out animals. Altogether, these data demonstrate that Slc6a8 is critical for the normal function of PV+ interneurons and that impairment of these cells is central in the disease pathogenesis, suggesting a novel therapeutic venue for CTD.
2023-03-14 | GSE216766 | GEO
Project description:Mutations in the X-linked solute carrier family 6-member (Slc6a8) gene, encoding the protein responsible for cellular creatine (Cr) uptake, cause Creatine Transporter Deficiency (CTD), an X-linked neurometabolic disorder presenting with cognitive dysfunction, autistic-like features, and epilepsy. The pathological determinants of CTD are still poorly understood and this lack of knowledge might hinder the development of therapeutic strategies. Here, we show that Cr deficiency perturbs gene expression in excitatory neurons, inhibitory cells and oligodendrocytes, inducing a remodeling of circuit excitability and synaptic wiring, while no effects are present in astrocytes and endothelial cells. We also describe specific alterations of high-energy demanding, Parvalbumin-expressing (PV+) interneurons, exhibiting a reduction in cellular and synaptic density, and a hypofunctional phenotype, affecting initiation, kinetics and frequency of action potentials. Mice lacking Slc6a8 only in PV+ neurons recapitulate numerous CTD features, such as cognitive deterioration, impaired cortical processing and hyperexcitability of brain circuits, demonstrating that Cr deficit in PV+ cells is sufficient to determine the CTD neurological endophenotype. Moreover, a pharmacological treatment targeted to restore the efficiency of PV+ synapses induces a significant improvement of cortical activity in Slc6a8 knock-out animals. Altogether, these data establish that Slc6a8 is critical for the normal function of PV+ interneurons and that a subtle dysfunction of these cells is critical for the disease pathogenesis, suggesting a novel therapeutic venue for CTD.
2023-06-01 | GSE218797 | GEO
Project description:Specific protein binders with high affinity, such as antibodies or nanobodies, are scarce for most solute carrier (SLCs) transporters. Moreover, general approaches that can systematically generate binders for multi-transmembrane proteins, such as SLCs, channels or GPCRs, are still missing. Purifying and reconstituting transmembrane proteins in their lipidic environments remain challenging, hence membrane proteins normally act as poor input material for binder generation. To overcome this challenge, we first identified state-of-the-art methods to generate protein binders targeting membrane transporters. Next, we produced full length protein or cell lines as input material for 27 SLCs, which were then processed in the selected binder generation platforms. As a result, we obtained >300 binders for 23 SLCs, 1 SLC failed and 3 SLCs are still in process. Then we established a cell-based validation workflow using immunofluorescent and immunoprecipitation methods to process all obtained binders. To further highlight the suitability of high-performance binders as an important tool for the biomolecular characterization of SLCs, we tested whether three SLC12A6 binders could be used in a quantitative co-immunoprecipitation followed by mass spectrometry (IP-MS) approach. This initial analysis of the IP-MS dataset showed that SLC12A6 specific binders are capable of strongly enriching SLC12A6. We also showed that despite high sequence similarity, the different binders showed different binder to target ratios, indicating different affinities to SLC12A6 in cell lysates. 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.
2024-06-17 | PXD046995 | Pride
Project description:<p>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.</p>
2022-09-20 | MTBLS4268 | MetaboLights
Project description:In the present study, the transcriptional analysis of CD biopsies reveals profound alterations in the ileum transportome profile. More than 60 SLC transporters showed different expression pattern compared with the healthy donors, being mostly decreased. Changes were confirmed in almost all the eighteen altered SLCs analyzed by RT-PCR. The results obtained display alterations in amino acid transporters, purinome members, Zn transporters and metallothioneins. All together, these alterations which mainly involve transporters localized at the apical membrane of the enterocyte anticipate impaired amino acid uptake and purinergic responses. Remarkably, incubation of explants with specific commensal bacteria restored almost all CD transportome alterations. Genome wide transcriptomic profile of Ileal mucosa from five healthy and six Chron's disease samples .
2016-03-29 | E-GEOD-68570 | biostudies-arrayexpress
Project description:In the present study, the transcriptional analysis of CD biopsies reveals profound alterations in the ileum transportome profile. More than 60 SLC transporters showed different expression pattern compared with the healthy donors, being mostly decreased. Changes were confirmed in almost all the eighteen altered SLCs analyzed by RT-PCR. The results obtained display alterations in amino acid transporters, purinome members, Zn transporters and metallothioneins. All together, these alterations which mainly involve transporters localized at the apical membrane of the enterocyte anticipate impaired amino acid uptake and purinergic responses. Remarkably, incubation of explants with specific commensal bacteria restored almost all CD transportome alterations.
2016-03-29 | GSE68570 | GEO