Disruption in iron homeostasis and impaired activity of iron-sulfur cluster containing proteins in the yeast model of Shwachman-Diamond syndrome

At a Glance

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

Technical Documentation & Analysis: Iron Homeostasis in SDS Yeast Model

This document analyzes the research paper “Disruption in iron homeostasis and impaired activity of iron-sulfur cluster containing proteins in the yeast model of Shwachman-Diamond syndrome” (Jain et al., 2020) to highlight key technical findings and demonstrate how 6CCVD’s advanced MPCVD diamond materials can support and extend this critical biomedical research.

Executive Summary

The study successfully utilized a yeast model ($sdo1\Delta$) of Shwachman-Diamond syndrome (SDS) to elucidate the molecular mechanisms linking $SBDS$ gene deficiency to cellular dysfunction.

Iron Overload: Deletion of $SDO1$ resulted in a significant three-fold over-accumulation of intracellular iron compared to wild-type yeast.
Oxidative Stress Mechanism: The excess iron contributed directly to elevated Reactive Oxygen Species (ROS) and protein oxidation, which was mitigated by treatment with the iron chelator Bathophenanthroline disulfonic acid (BPS).
ISC Pathway Impairment: The activity of key Iron-Sulfur Cluster (ISC) containing enzymes (aconitase and succinate dehydrogenase) was significantly reduced, indicating a defect in ISC biogenesis.
VDAC Mediation: The over-expression of the mitochondrial voltage-dependent anion channel (VDAC) Por1p was identified as a key mediator, linking $SDO1$ loss to iron over-accumulation and subsequent ISC pathway disruption.
Therapeutic Relevance: Genetic deletion of $POR1$ restored regulation of iron metabolism and enhanced ISC enzyme activity in $sdo1\Delta$ strains, suggesting that targeting Por1p or iron overload may be viable therapeutic strategies for SDS.

Technical Specifications

The following table summarizes the key quantifiable parameters and experimental conditions used in the study.

Parameter	Value	Unit	Context
Intracellular Iron Accumulation	3-fold	Increase	$sdo1\Delta$ yeast vs. Wild Type (WT)
Iron Chelation Concentration (BPS)	40 or 120	µM	Used in YPD medium for iron depletion
Hydrogen Peroxide Stress Concentration	3.5	mM	Used to induce protein oxidation
Salt Stress Concentration	600	mM	NaCl
Reductive Stress Concentration	10	mM	$\beta$-ME (Beta-mercaptoethanol)
Culture Temperature (Standard)	30	°C	Standard growth condition
Heat Stress Temperature	37	°C	Used for growth sensitivity assay
FET3 Expression Induction	~4	Times Higher	$sdo1\Delta$ cells vs. WT (indicates impaired iron sensing)
Aconitase Activity	Significantly Reduced	N/A	Observed in $sdo1\Delta$ and $rho^{0}$ strains
SDH Activity Monitoring Wavelength	600	nm	Spectrophotometric assay using dichlorophenol indophenol
Aconitase Activity Monitoring Wavelength	240	nm	Spectrophotometric assay using cis-aconitate conversion

Key Methodologies

The research relied on precise biochemical and analytical techniques to quantify cellular iron, oxidative stress, and enzyme function.

Yeast Culture and Chemical Treatment: Strains were cultured in YPD medium at 30 °C. Iron depletion was achieved by buffering YPD to pH 6.0 (MOPS) and adding the cell-impermeable iron chelator BPS (40 µM or 120 µM).
Intracellular Iron Quantification: Iron levels were measured using graphite furnace atomic absorption spectroscopy (AAS), a technique requiring high-purity sample handling and stable optical components.
ROS Measurement: Intracellular Reactive Oxygen Species (ROS) were quantified using the fluorescent probe 2,7-dichorofluorescein diacetate (H₂DCFDA) and subsequent fluorescence signal measurement, normalized to protein content.
Protein Oxidation Analysis: Protein carbonyls (a marker of oxidative damage) were detected via derivatization with 2,4-dinitrophenylhydrazine (DNPH), followed by immunoblotting.
Enzyme Activity Assays:
- Superoxide Dismutase (SOD) Activity: Analyzed via non-denaturing gel electrophoresis and nitro blue tetrazolium (NBT) staining.
- Aconitase Activity: Monitored spectrophotometrically at 240 nm.
- Succinate Dehydrogenase (SDH) Activity: Monitored spectrophotometrically at 600 nm.

6CCVD Solutions & Capabilities

The precise measurement of metal ions (Fe), reactive species (ROS), and enzyme kinetics (Aconitase, SDH) demands materials with exceptional chemical inertness, thermal stability, and optical purity. 6CCVD’s MPCVD diamond materials are engineered to meet and exceed the requirements for replicating and advancing this complex cellular research, particularly in high-precision analytical setups and next-generation microfluidic platforms.

Research Requirement / Challenge	6CCVD Diamond Solution	Technical Advantage
High-Purity Optical Windows (For AAS and UV-Vis Spectrophotometry at 240 nm/600 nm)	Optical Grade Single Crystal Diamond (SCD)	SCD offers superior transparency across the UV-Vis spectrum, ensuring minimal absorption and scatter, which is critical for accurate quantification of iron (AAS) and enzyme kinetics (Aconitase, SDH). We provide SCD up to 500 µm thick with Ra < 1nm polishing.
Advanced Electrochemical Sensing (Real-time monitoring of Fe²⁺/Fe³⁺ or ROS in complex media)	Heavy Boron-Doped Diamond (BDD) Electrodes	BDD provides an unmatched electrochemical platform due to its wide potential window and extreme chemical inertness, enabling highly sensitive and selective detection of metal ions and reactive species without electrode fouling, ideal for biological buffers.
Custom Microfluidic Platforms (Scaling up cell culture, chelation, and stress exposure assays)	Polycrystalline Diamond (PCD) Wafers	We supply PCD plates up to 125mm in diameter and up to 500 µm thick. Diamond’s high thermal conductivity and inertness make it the ideal substrate for microfluidic chips requiring precise temperature control (e.g., the 37 °C heat stress assay) and resistance to harsh chemicals (e.g., BPS, H₂O₂).
Integrated Sensor Fabrication (Creating on-chip detection systems)	Custom Metalization and Laser Cutting	6CCVD offers in-house metalization (Au, Pt, Ti, W, Cu) for creating integrated contacts or micro-heaters directly on BDD or SCD substrates. We can provide custom dimensions and precise laser cutting services to match unique microfluidic channel designs.

Engineering Support

6CCVD’s in-house PhD team specializes in the application of diamond materials in bio-sensing and high-precision optics. We can assist researchers in selecting the optimal diamond grade (SCD, PCD, or BDD) and surface finish required for similar Iron Homeostasis and Oxidative Stress projects, ensuring material properties enhance experimental reliability and sensitivity.

For custom specifications or material consultation, visit 6ccvd.com or contact our engineering team directly.

Tech Support

Original Source

DOI: https://doi.org/10.1186/s13578-020-00468-2