Disruption in iron homeostasis and impaired activity of iron-sulfur cluster containing proteins in the yeast model of Shwachman-Diamond syndrome
At a Glance
Section titled âAt a Glanceâ| Metadata | Details |
|---|---|
| Publication Date | 2020-09-11 |
| Journal | Cell & Bioscience |
| Authors | Ayushi Jain, Phubed Nilatawong, Narinrat Mamak, Laran T. Jensen, Amornrat Naranuntarat Jensen |
| Institutions | Mahidol University |
| Citations | 3 |
| Analysis | Full AI Review Included |
Executive Summary
Section titled âExecutive SummaryâThis research utilizes a Saccharomyces cerevisiae model (lacking the SDO1 gene, the ortholog of human SBDS) to investigate the molecular pathogenesis of Shwachman-Diamond Syndrome (SDS), focusing on iron homeostasis and mitochondrial function.
- Core Defect: Deletion of SDO1 results in a three-fold over-accumulation of intracellular iron, mimicking iron overload conditions seen in other mitochondrial diseases.
- Functional Impairment: This iron overload leads to impaired activity of critical Iron-Sulfur Cluster (ISC) containing enzymes, specifically Aconitase and Succinate Dehydrogenase (SDH), and reduces the activity of Manganese Superoxide Dismutase (Sod2p).
- Oxidative Stress Mechanism: The excess iron contributes to elevated Reactive Oxygen Species (ROS) and protein oxidation, which can be mitigated by using the cell-impermeable iron chelator, Bathophenanthroline Disulfonic Acid (BPS).
- VDAC Linkage: The iron accumulation and subsequent ISC enzyme impairment are linked to the over-expression of the mitochondrial Voltage-Dependent Anion Channel (VDAC), Por1p.
- Mitigation Strategy: Deletion of POR1 in the sdo1Î strain successfully limits iron accumulation and restores the activity of Aconitase and SDH, suggesting Por1p over-expression mediates the disease phenotype.
- Proposed Pathway: Oxidative stress resulting from POR1 over-expression disrupts ISC protein activity and iron homeostasis, contributing to SDS pathogenesis.
Technical Specifications
Section titled âTechnical SpecificationsâThe following table summarizes key quantitative data and experimental parameters derived from the study of the Saccharomyces cerevisiae model.
| Parameter | Value | Unit | Context |
|---|---|---|---|
| Intracellular Iron Overload | 3-fold higher | Ratio | Iron content in sdo1Î yeast vs. Wild-Type (WT) |
| Iron Chelation Concentration (BPS) | 40 or 120 | ”M | Bathophenanthroline Disulfonic Acid (BPS) used in YPD medium |
| Culture Temperature | 30 | °C | Standard yeast growth conditions |
| Stationary Phase Culture Time | 40 | hours | Used for Aconitase and SDH activity assays |
| Hydrogen Peroxide Stress | 2 | mmol/L | Used to induce oxidative damage for protein carbonylation analysis |
| Aconitase Activity | P < 0.001 | Statistical Significance | Reduction observed in sdo1Î cells vs. WT |
| Succinate Dehydrogenase (SDH) Activity | P < 0.001 | Statistical Significance | Reduction observed in sdo1Î cells vs. WT |
| Sod2p Activity Enhancement | Significant (P < 0.01) | Ratio | Improvement in sdo1Î cells following BPS treatment |
| ROS Level Reduction | Significant (P < 0.01) | Ratio | Reduction in sdo1Î cells following BPS treatment |
Key Methodologies
Section titled âKey MethodologiesâThe study employed standard yeast genetics and biochemical assays, focusing on precise measurement of metal content, enzyme function, and oxidative stress markers.
- Strain Generation and Culture: Saccharomyces cerevisiae strains (BY4741/4742 background) were used, including deletion mutants (sdo1Î, por1Î, rhoÂș, and double mutants). Cells were cultured in enriched YPD medium at 30 °C.
- Iron Depletion: Iron chelation was achieved by supplementing the medium (adjusted to pH 6.0 with MOPS) with 40 ”M or 120 ”M BPS (Bathophenanthroline Disulfonic Acid).
- Intracellular Iron Measurement: Iron concentration was quantified using Graphite Furnace Atomic Absorption Spectroscopy (GFAAS) on washed cell pellets, normalized to cell count (nmole Fe/109 cells).
- Oxidative Stress Analysis (ROS): Intracellular ROS levels were measured using the fluorescent probe 2,7-dichorofluorescein diacetate (H2DCFDA), with fluorescence normalized to protein concentration.
- Protein Carbonylation: Oxidative damage to proteins was assessed by derivatization with 2,4-dinitrophenylhydrazine (DNPH), followed by immunoblotting using an anti-DNP antibody.
- Enzyme Activity Assays:
- Sod2p (Superoxide Dismutase): Activity was analyzed by non-denaturing gel electrophoresis and staining with nitro blue tetrazolium (NBT).
- Aconitase: Activity was monitored spectrophotometrically at 240 nm, tracking the conversion of cis-aconitate to isocitrate.
- Succinate Dehydrogenase (SDH): Activity was measured in isolated mitochondria by monitoring the reduction of dichlorophenol indophenol at 600 nm.
- Gene Expression Monitoring: The induction of the high-affinity iron uptake gene FET3 was monitored using a FET3-lacZ reporter plasmid and subsequent ÎČ-galactosidase assays.
Commercial Applications
Section titled âCommercial ApplicationsâWhile this research is fundamentally biomedical, the underlying principles of precise trace metal control, management of reactive species, and monitoring of enzyme function are relevant to advanced materials engineering, particularly in high-purity synthesis environments.
- High-Purity Precursor Management: The study highlights the extreme sensitivity of biological systems (and by extension, catalytic processes) to trace metal contamination (iron overload). This reinforces the need for rigorous control and purification of precursors used in CVD processes (e.g., those supplied by 6ccvd.com) to prevent catalytic poisoning or defect formation.
- Environmental Control in Synthesis: The mechanism involves Reactive Oxygen Species (ROS) generated by misregulated iron, leading to material (protein) damage. In CVD and materials synthesis (e.g., 2D materials, graphene), managing the oxidative environment (analogous to ROS) and preventing unwanted side reactions caused by trace contaminants is critical for achieving high-quality, defect-free films.
- Biosensor and Bioreactor Design: The use of enzyme activity (Aconitase, SDH) as a highly sensitive readout for cellular stress and metal status provides a model for developing advanced biosensors capable of detecting subtle changes in environmental toxicity or metal availability.
- Drug/Inhibitor Screening: The successful mitigation of defects using BPS (a chelator) and POR1 deletion demonstrates a pathway for screening small molecules that target VDACs or metal homeostasis, potentially leading to new therapeutic agents or industrial inhibitors for metal-induced degradation.