Summary of Study ST004015
This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR002513. The data can be accessed directly via it's Project DOI: 10.21228/M8T536 This work is supported by NIH grant, U2C- DK119886. See: https://www.metabolomicsworkbench.org/about/howtocite.php
| Study ID | ST004015 |
| Study Title | SLC45A4 encodes a peroxisomal putrescine transporter that promotes GABA de novo synthesis |
| Study Summary | We found that the expression of SLC45A4 has a strong positive correlation with the cellular level of γ-aminobutyric acid (GABA). Using mass spectrometry and the stable isotope tracing approach, we demonstrated that SLC45A4 promotes GABA de novo synthesis through the Arginine/Ornithine/Putrescine (AOP) pathway. SLC45A4 functions as a putrescine transporter localized to the peroxisome membrane to facilitate GABA production. Taken together, our results revealed a new biochemical mechanism where SLC45A4 controls GABA production. We fed stable isotope-labeled GABA (13C4-GABA) to the control and the SLC45A4 Knock Down (KD) A549 and H1299 cells. In both cell lines, we observed decreases in total GABA levels when SLC45A4 was knocked down. However, we found that the decrease in GABA level was mainly due to a decline in endogenous, unlabeled GABA (12C4-GABA) rather than a decline in the uptake of 13C4-GABA. The 13C4-GABA, which indicates GABA uptake, was significantly but slightly changed in A549 KD cells but not in H1299 KD cells. Our untargeted metabolomics analysis picked up 2,676 features from this dataset. The top 4 hits that are most significantly changed in the KD cells are all related to unlabeled GABA (the protonated ion [M+H]+, the protonated ion with a water loss [M+H-H2O]+, 13C isotope natural abundance ion [13C1-M+H]+, and 13C isotope natural abundance ion with a water loss [13C1-M+H-H2O]+). These results indicate that although it regulates cellular GABA levels, SLC45A4 does not function directly as a GABA transporter. From these results, we inferred that SLC45A4 is required for the de novo synthesis of GABA. |
| Institute | Rutgers University |
| Department | Department of Medicine |
| Last Name | Su |
| First Name | Xiaoyang |
| Address | 7118 Clinical Academic Building |
| xs137@rwjms.rutgers.edu | |
| Phone | 17322355447 |
| Submit Date | 2025-06-19 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | mzXML |
| Analysis Type Detail | LC-MS |
| Release Date | 2025-07-07 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR002513 |
| Project DOI: | doi: 10.21228/M8T536 |
| Project Title: | SLC45A4 encodes a peroxisomal putrescine transporter that promotes GABA de novo synthesis |
| Project Type: | Cellular polar metabolomics |
| Project Summary: | Solute carriers (SLC) are membrane proteins that facilitate the transportation of ions and metabolites across either the plasma membrane or the membrane of intracellular organelles. With more than 450 human genes annotated as SLCs, many of them are still orphan transporters without known biochemical functions. We developed a metabolomic-transcriptomic association analysis, and we found that the expression of SLC45A4 has a strong positive correlation with the cellular level of γ-aminobutyric acid (GABA). Using mass spectrometry and the stable isotope tracing approach, we demonstrated that SLC45A4 promotes GABA de novo synthesis through the Arginine/Ornithine/Putrescine (AOP) pathway. SLC45A4 functions as a putrescine transporter localized to the peroxisome membrane to facilitate GABA production. Taken together, our results revealed a new biochemical mechanism where SLC45A4 controls GABA production. |
| Institute: | Rutgers University |
| Department: | Department of Medicine |
| Last Name: | Su |
| First Name: | Xiaoyang |
| Address: | 7118 Clinical Academic Building |
| Email: | xs137@rwjms.rutgers.edu |
| Phone: | 7322355447 |
| Funding Source: | R01 GM149664 |
Subject:
| Subject ID: | SU004153 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Cell Strain Details: | A549, H1299 |
Factors:
Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)
| mb_sample_id | local_sample_id | Sample source | Cell line | Knockdown |
|---|---|---|---|---|
| SA461946 | lysate_A549_siCtr_1 | Lysate | A549 | Ctrl |
| SA461947 | lysate_A549_siCtr_2 | Lysate | A549 | Ctrl |
| SA461948 | lysate_A549_siCtr_3 | Lysate | A549 | Ctrl |
| SA461949 | lysate_A549_KD45A4_1 | Lysate | A549 | SLC45A4 |
| SA461950 | lysate_A549_KD45A4_2 | Lysate | A549 | SLC45A4 |
| SA461951 | lysate_A549_KD45A4_3 | Lysate | A549 | SLC45A4 |
| SA461952 | lysate_H1299_siCtr_1 | Lysate | H1299 | Ctrl |
| SA461953 | lysate_H1299_siCtr_2 | Lysate | H1299 | Ctrl |
| SA461954 | lysate_H1299_siCtr_3 | Lysate | H1299 | Ctrl |
| SA461955 | lysate_H1299_KD45A4_1 | Lysate | H1299 | SLC45A4 |
| SA461956 | lysate_H1299_KD45A4_2 | Lysate | H1299 | SLC45A4 |
| SA461957 | lysate_H1299_KD45A4_3 | Lysate | H1299 | SLC45A4 |
| Showing results 1 to 12 of 12 |
Collection:
| Collection ID: | CO004146 |
| Collection Summary: | 0.5×10^6 cells in the culture were washed twice with PBS and extracted with 0.5 ml ice-cold 40:40:20 (methanol:acetonitrile:water) solution with 0.5% formic acid. The cells were scraped off the plate followed by incubation on ice for 10 min, and neutralized by the addition of 25 μL 15% (m/v) NH4HCO3 solution. The extracts were transferred to a 1.5 mL tube. For conditioned media metabolite extraction, 5 µL of culture media was mixed with 500 µL of ice-cold 40:40:20 (methanol:acetonitrile:water) solution with 0.5% formic acid and neutralized by the addition of 25 μL 15% (m/v) NH4HCO3 solution. The whole cell or media extracts were then centrifuged at 4°C and 16,000 × g for 10 min and transferred to a clean tube and stored at -80°C until analysis. |
| Sample Type: | Cultured cells |
Treatment:
| Treatment ID: | TR004162 |
| Treatment Summary: | A549 or H1299 cells were seeded in 6-well plates at the density of 2×105 / mL. When the cells reached about 70% confluence, cells were transfected with siRNA Transfection Reagent (Santa Cruz Biotechnology, sc-29528) according to manufacturer’s instructions. siRNAs targeting human SLC45A4 (SCBT, sc-77846) and non-targeting Scramble controls (SCBT, sc-37007) were obtained from SCBT and re-suspended at 10 μM in RNAse free water. 4 μL siRNA (20 nM) was first mixed with 100 μL Opti-MEM (Gibco, 31985070), then mixed with 4 μL siRNA Transfection Reagent contained in another 100 μL Opti-MEM. The mixture was incubated at room temperature for 30 min and mixed with 800 μL Opti-MEM. Cells were washed once with 2 mL Opti-MEM, then covered with 1 ml siRNA mixture and incubated for 6 hours at 37°C, 5% CO2. After the incubation, 1 mL of RPMI 1640 media with 20% FBS and 2× Penicillin-Streptomycin were added. |
Sample Preparation:
| Sampleprep ID: | SP004159 |
| Sampleprep Summary: | The whole cell extracts were then centrifuged at 4°C and 16,000 × g for 10 min and transferred to a clean tube and stored at -80°C until analysis. |
Combined analysis:
| Analysis ID | AN006620 |
|---|---|
| Chromatography ID | CH005030 |
| MS ID | MS006319 |
| Analysis type | MS |
| Chromatography type | HILIC |
| Chromatography system | Thermo Vanquish |
| Column | Waters XBridge BEH Amide (150 mm × 2.1 mm, 2.5 μm) |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Q Exactive Plus Orbitrap |
| Ion Mode | POSITIVE |
| Units | Ion counts |
Chromatography:
| Chromatography ID: | CH005030 |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Waters XBridge BEH Amide (150 mm × 2.1 mm, 2.5 μm) |
| Column Temperature: | 25°C |
| Flow Gradient: | 0 min, 100% B; 3 min, 100% B; 3.2 min, 90% B; 6.2 min, 90% B; 6.5 min, 80% B; 10.5 min, 80% B; 10.7 min, 70% B; 13.5 min, 70% B; 13.7 min, 45% B; 16 min, 45% B; 16.5 min, 100% B; and 22 min, 100% B |
| Flow Rate: | 0.3 mL/min |
| Solvent A: | 95% Water/5% Acetonitrile; 20 mM Acetic acid; 40 mM Ammonium hydroxide, pH 9.4 |
| Solvent B: | 20% Water/80% Acetonitrile; 20 mM Acetic acid; 40 mM Ammonium hydroxide, pH 9.4 |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS006319 |
| Analysis ID: | AN006620 |
| Instrument Name: | Thermo Q Exactive Plus Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | The mass spectrometry analysis was performed on Thermo Q Exactive PLUS instrument with a HESI source, which was set to a spray voltage of −2.7 kV in negative mode and 3.5 kV in positive mode. The sheath, auxiliary, and sweep gas flow rates were 40, 10, and 2 (arbitrary units), respectively. The capillary temperature was set to 300°C, and the aux gas heater was set to 360°C. The S-lens RF level was 45. The m/z scan range was set to 72 to 1000 m/z in either positive or negative ionization mode. The AGC target was set to 3e6, and the maximum IT was 200 ms. Metabolite data were obtained using the El-MAVEN software package (mass accuracy window: 5 ppm). All isotope natural abundance corrections were done using AccuCor (single-isotope labeling) or AccuCor2 (dual-isotope labeling). |
| Ion Mode: | POSITIVE |
| Capillary Temperature: | 300C |
| Ion Spray Voltage: | 3.5kV |
