Recombinant Human Reactive oxygen species modulator 1 (ROMO1)

What is ROMO1 and what is its cellular localization?

ROMO1 is a novel protein first cloned from head and neck cancer tissue in 2006. It is a highly conserved inner mitochondrial membrane protein comprising 79 amino acids. The protein is encoded by the full sequence: MPVAVGPYGQ SQPSCFDRVK MGFVMGCAVG MAAGALFGTF SCLRIGMRGR ELMGGIGKTM MQSGGTFGTF MAIGMGIRC . Its localization in the mitochondrial membrane is critical for its function as a key modulator of intracellular reactive oxygen species (ROS) . When conducting subcellular fractionation studies, researchers should employ mitochondrial isolation protocols optimized for membrane proteins to ensure accurate localization analysis.

What are the fundamental physiological roles of ROMO1?

ROMO1 serves multiple critical functions in cellular physiology. Primarily, it acts as a key regulator of mitochondrial dynamics and bioenergetics. The protein senses reactive oxygen species and is required for mitochondrial fusion in vitro . ROMO1-induced ROS production is essential for the proliferation of both normal and cancer cells . Knockout studies in mice have demonstrated that ROMO1 is essential for embryonic development, as ROMO1-null mice die before embryonic day 8.5, which occurs earlier than the embryonic lethality observed with knockout of GTPases OPA1 or MFN1/2 that catalyze mitochondrial inner and outer membrane fusion . This suggests a foundational role in early development that precedes other mitochondrial fusion processes.

How can researchers accurately measure ROMO1 expression in experimental samples?

For quantitative assessment of ROMO1 expression in tissue samples, researchers commonly employ the H-score methodology. This scoring system combines the intensity of staining and the percentage of positively stained cells. In clinical studies, the cutoff H score for discriminating between low and high ROMO1 expression is typically defined as the point with the lowest p-value on the log-rank test for all possible H scores . In practical applications, a cut-off value of 200 has been successfully used to classify patients into low and high ROMO1 expression groups . Alternative methods include Western blotting for protein quantification, qPCR for mRNA expression analysis, and immunofluorescence for localization studies, each requiring specific optimization for ROMO1 detection.

What expression systems are optimal for recombinant ROMO1 production?

Multiple expression systems have been successfully employed for recombinant ROMO1 production, each with distinct advantages for different experimental applications:

When selecting an expression system, researchers should consider the downstream application requirements. For functional studies requiring properly folded protein with native post-translational modifications, mammalian expression systems like HEK-293 cells are recommended .

What protein tags are most effective for recombinant ROMO1 purification and detection?

Several fusion tags have been validated for recombinant ROMO1 production:

• GST tag: Commonly used for affinity purification and enhances solubility. Optimal for wheat germ expression systems with elution using reduced glutathione (50 mM Tris-HCl, 10 mM reduced Glutathione, pH 8.0) .

• His tag: Enables metal affinity chromatography purification. Preferred for E. coli expression systems when high purity (>97%) is required .

• Myc-DYKDDDDK Tag: Dual-tagging approach useful for sequential purification steps and provides multiple epitopes for detection. Particularly useful in HEK-293 cell expression systems .

When designing experiments, tag position (N-terminal vs. C-terminal) should be carefully considered, as improper tag placement may interfere with ROMO1's membrane localization or functional domains.

What are the critical storage considerations for maintaining recombinant ROMO1 stability?

Recombinant ROMO1 requires specific storage conditions to maintain structural integrity and functional activity:

• Store at -80°C for long-term preservation

• Aliquot into single-use volumes to avoid repeated freeze-thaw cycles

• Use within three months from the date of receipt for optimal activity

• For buffers, 50 mM Tris-HCl with 10 mM reduced Glutathione at pH 8.0 has been validated as an effective stabilizing solution

Researchers should verify protein stability after storage using analytical techniques such as SDS-PAGE with Coomassie Blue staining before performing functional assays.

How does ROMO1 regulate mitochondrial dynamics and respiratory function?

ROMO1 plays a crucial role in mitochondrial dynamics through several mechanisms. It is required for mitochondrial fusion processes in vitro, and its absence leads to fragmented and morphologically abnormal mitochondria . Research using ROMO1 knockout models has revealed sex-specific effects on mitochondrial morphology. In male mice with beta cell-specific ROMO1 knockout (RABKO), mitochondria appear swollen and fragmented with reduced mtDNA content, while female mice maintain relatively normal mitochondrial morphology .

From a functional perspective, ROMO1 deletion does not affect basal respiration in either sex but significantly reduces spare respiratory capacity (SRC) in both male and female mouse models and in isolated human islets . This reduction in SRC specifically involves the loss of respiratory activity at Complex II/SDH of the electron transport chain. To investigate these effects experimentally, researchers should employ high-resolution respirometry with substrate-inhibitor protocols designed to isolate the activity of individual respiratory complexes.

What methodological approaches are recommended for studying ROMO1's impact on oxidative stress responses?

When investigating ROMO1's role in oxidative stress, multiple complementary approaches are recommended:

• ROS measurement techniques: Use fluorescent probes such as DCFDA for general ROS or MitoSOX for mitochondrial superoxide, with appropriate controls for autofluorescence and probe specificity.

• Antioxidant system analysis: Measure expression and activity of key antioxidant enzymes (SOD, catalase, GPx) to assess adaptive responses to ROMO1-mediated ROS production.

• Oxidative damage assessment: Quantify lipid peroxidation (MDA, 4-HNE), protein carbonylation, and DNA oxidation (8-OHdG) to evaluate the functional consequences of altered ROS levels.

• Adaptation studies: Use time-course experiments with oxidative stressors to differentiate between acute responses and adaptive mechanisms, as ROMO1-induced ROS are implicated in adaptation to oxidative stress, which could be a mechanism underlying chemoresistance in cancer cells .

• Mitochondrial function correlation: Pair ROS measurements with assessment of mitochondrial membrane potential, ATP production, and respiratory chain complex activities to establish mechanistic links.

How does ROMO1 expression correlate with clinical outcomes in cancer patients?

Multiple clinical studies have demonstrated significant correlations between ROMO1 expression and patient outcomes across various cancer types:

These consistent findings across multiple cancer types suggest ROMO1 is a promising biomarker for malignancies with potential applications in treatment selection and prognostication .

What mechanisms link ROMO1 to chemotherapy resistance?

Research indicates several potential mechanisms by which ROMO1 contributes to chemotherapy resistance:

• Adaptation to oxidative stress: ROMO1-induced ROS production enhances expression of various antioxidants, potentially conferring resistance to platinum-related oxidative stress . This adaptive response involves regulation of redox buffering systems that protect cancer cells from therapy-induced damage.

• Altered drug response pathways: ROMO1 overexpression is associated with poor response to treatment in patients receiving platinum-based chemotherapy and EGFR-TKIs , suggesting interference with drug action mechanisms.

• Modulation of cell death pathways: Silencing ROMO1 increases sensitivity to cell death stimuli , indicating its potential role in apoptosis resistance.

• Reduced T790M mutation development: In EGFR-mutant NSCLC, high ROMO1 expression correlates with significantly lower rates of secondary T790M mutation after TKI failure (16.7% vs. 38.3%, p=0.0369) , suggesting altered evolutionary pressure or DNA repair mechanisms.

For experimental investigation of these mechanisms, researchers should combine genetic manipulation approaches (siRNA knockdown, CRISPR-Cas9 deletion, or overexpression) with comprehensive cell death assays, ROS measurement, and drug sensitivity testing.

How should researchers design experiments to evaluate ROMO1 as a predictive biomarker in cancer?

When evaluating ROMO1 as a predictive biomarker, researchers should implement a rigorous experimental design that includes:

• Sample selection and processing:

  • Ensure adequate sample size based on power calculations

  • Use standardized tissue processing protocols

  • Include matched normal tissues when possible

  • Consider microenvironmental factors that may influence ROMO1 expression

• Expression analysis methodology:

  • Establish clear H-score cutoff values using statistical approaches (log-rank test for all possible H scores)

  • Validate cutoffs in independent cohorts

  • Consider multiple detection methods (IHC, qPCR, protein quantification)

• Clinical correlation framework:

  • Define clear clinical endpoints (RR, PFS, OS)

  • Perform both univariate and multivariate analyses

  • Adjust for known prognostic factors

  • Use appropriate statistical methods (Cox proportional hazard regression)

• Validation approaches:

  • Test in multiple cancer types

  • Evaluate in different treatment contexts

  • Consider prospective validation in clinical trials

• Functional validation:

  • Determine whether modulating ROMO1 expression alters treatment response in preclinical models

  • Investigate mechanism of biomarker association

What sex-specific differences should researchers consider when studying ROMO1?

Sex-specific differences in ROMO1 biology represent an important consideration for experimental design and data interpretation. In pancreatic beta cell-specific ROMO1 knockout models, significant sex differences have been observed:

• Glucose homeostasis: Male mice with ROMO1 knockout show impaired glucose homeostasis due to insulin secretion defects, while female mice initially maintain normal function .

• Mitochondrial morphology: Mitochondria from female mice with ROMO1 deletion appear morphologically normal, while male knockout mice exhibit swollen and fragmented mitochondria with reduced mtDNA content .

• Age-dependent effects: While young female mice are initially protected, aging female ROMO1 knockout mice eventually develop loss of spare respiratory capacity and glucose intolerance .

These observations highlight the importance of including both sexes in experimental designs and considering hormonal and metabolic differences that might influence ROMO1 function. Researchers should report sex-stratified data and investigate the mechanisms underlying these differences, potentially including gonadal hormone measurements or gonadectomy studies to determine mediating factors.

What are the methodological challenges in studying ROMO1's role in embryonic development?

Investigating ROMO1's essential role in early embryonic development presents several methodological challenges:

• Early embryonic lethality: ROMO1-null mice die before embryonic day 8.5 , necessitating specialized approaches for studying function:

  • Conditional knockout systems with inducible deletion

  • Embryonic stem cell differentiation models

  • Partial knockdown approaches to maintain minimal function

• Temporal expression patterns: Determining when and where ROMO1 expression becomes critical requires:

  • Stage-specific embryo collection and analysis

  • Single-cell RNA sequencing of early embryos

  • Lineage-specific conditional deletion

• Functional redundancy assessment: Investigating potential compensatory mechanisms involves:

  • Analysis of related family members' expression

  • Pathway analysis to identify alternate routes for key functions

  • Combined deletion of potentially redundant factors

• Mechanistic determination: Distinguishing between direct developmental signaling roles versus secondary effects of mitochondrial dysfunction requires:

  • Rescue experiments with wild-type and mutant constructs

  • Metabolic profiling of embryos prior to developmental arrest

  • Comparative analysis with other mitochondrial fusion protein knockouts that cause later-stage lethality

These approaches can help overcome the fundamental challenge of studying a protein whose complete absence is incompatible with early development.

How can researchers effectively investigate ROMO1's interaction with respiratory complex II/SDH?

To investigate the specific interaction between ROMO1 and Complex II/SDH that affects spare respiratory capacity , researchers should employ a multi-faceted approach:

• Biochemical interaction studies:

  • Co-immunoprecipitation to detect physical association

  • Proximity ligation assays to confirm interaction in intact cells

  • Blue native PAGE to assess integration into respiratory supercomplexes

• Functional respiratory analysis:

  • High-resolution respirometry with substrate-specific protocols

  • Measurement of Complex II activity using spectrophotometric assays

  • Flux analysis with 13C-labeled substrates to track metabolite flow

• Structural biology approaches:

  • Cryo-EM of purified complexes with and without ROMO1

  • Crosslinking mass spectrometry to identify interaction sites

  • Molecular dynamics simulations based on known structures

• Genetic manipulation strategies:

  • CRISPR-mediated introduction of specific mutations in ROMO1 or Complex II subunits

  • Rescue experiments with wild-type or mutant constructs

  • Inducible knockdown systems to monitor temporal aspects of the interaction

These methodologies can help elucidate both the physical and functional relationship between ROMO1 and Complex II/SDH, providing insight into how this interaction regulates spare respiratory capacity and ultimately affects cellular function in both normal physiology and disease states.