Recombinant Bovine Reactive oxygen species modulator 1 (ROMO1)

What are the structural and functional characteristics of recombinant bovine ROMO1?

Recombinant bovine ROMO1 retains the key structural and functional characteristics of the native protein when properly expressed. The protein localizes specifically to the inner mitochondrial membrane, where it regulates mitochondrial ROS levels. Studies indicate that ROMO1 is essential for embryonic development, as ROMO1-null mice die before embryonic day 8.5, even earlier than mice lacking established mitochondrial fusion proteins like OPA1 or MFN1/2 . This suggests ROMO1 has fundamental roles beyond those currently characterized. Functionally, ROMO1 influences mitochondrial spare respiratory capacity (SRC), particularly through effects on Complex II/SDH activity, indicating its importance in cellular bioenergetics under stress conditions.

How is ROMO1 expression regulated under normal and stress conditions?

The regulation of ROMO1 expression involves multiple cellular mechanisms responsive to various physiological and stress conditions. Under oxidative stress, ROMO1 expression typically increases, creating a potential feedback loop as ROMO1 itself modulates ROS production. This redox-sensitive regulation appears to be a key aspect of cellular adaptation to stress conditions. Studies have demonstrated that in pathological states like cancer, ROMO1 expression is often dysregulated, with high expression correlating with poor prognosis in certain cancers, including glioblastoma . The precise transcriptional, translational, and post-translational mechanisms controlling ROMO1 levels remain areas requiring further investigation.

What are the most effective techniques for producing and purifying recombinant bovine ROMO1?

For producing high-quality recombinant bovine ROMO1, researchers should consider these methodological approaches:

• Expression system selection: Mammalian expression systems (HEK293 or CHO cells) are preferable for ROMO1 production as they provide appropriate post-translational modifications and proper folding of this mitochondrial protein.

• Vector design considerations:

  • Include an N-terminal mitochondrial targeting sequence if using non-specific expression systems

  • Add an affinity tag (His-tag or FLAG-tag) for purification while ensuring it doesn't interfere with localization

  • Consider inducible expression systems to control toxicity if overexpression affects cell viability

• Purification strategy:

  • Use mild detergents suitable for membrane proteins (e.g., digitonin, DDM)

  • Implement affinity chromatography followed by size-exclusion chromatography

  • Verify functional integrity after purification using ROS modulation assays

When working with recombinant ROMO1, researchers must validate protein activity, as improper folding or loss of function during purification can significantly impact experimental outcomes.

What are the validated methods for measuring ROMO1-mediated ROS production in experimental systems?

Multiple complementary techniques have been validated for measuring ROMO1-mediated ROS production:

When designing experiments to study ROMO1-mediated ROS production, it's crucial to include both gain-of-function (overexpression) and loss-of-function (knockdown/knockout) approaches for comprehensive analysis.

How can researchers effectively manipulate ROMO1 expression in cellular and animal models?

Researchers can employ several strategies to manipulate ROMO1 expression for functional studies:

• Cellular models:

  • Transient approaches: siRNA for short-term knockdown; plasmid transfection for overexpression

  • Stable approaches: shRNA or CRISPR/Cas9 for long-term knockdown/knockout; lentiviral transduction for stable overexpression

  • Validation methods: Western blotting, qRT-PCR, and immunofluorescence to confirm expression changes

• Animal models:

  • Conditional knockout systems using Cre-loxP (such as the RABKO model for beta-cell specific deletion)

  • Bone marrow transplantation with ROMO1-manipulated cells for studying immune function

  • Tissue-specific expression systems to avoid embryonic lethality observed in global knockouts

• Experimental design considerations:

  • Include appropriate controls (scrambled siRNA, empty vectors)

  • Validate functional consequences beyond expression changes

  • Consider temporal aspects, as acute vs. chronic ROMO1 manipulation may have different outcomes

The choice of model should align with specific research questions, as ROMO1's effects can be tissue-specific and context-dependent.

How does ROMO1 influence mitochondrial dynamics and what are the implications for cellular bioenergetics?

ROMO1 plays a critical role in regulating mitochondrial dynamics with significant consequences for cellular bioenergetics:

• Mitochondrial morphology:

  • ROMO1 is required for proper mitochondrial fusion

  • In ROMO1-deficient cells, mitochondria appear swollen and fragmented with reduced mtDNA content

  • These morphological changes directly impact respiratory function

• Bioenergetic effects:

  • ROMO1 is particularly important for maintaining spare respiratory capacity (SRC)

  • Deletion of ROMO1 reduces SRC through specific loss of respiratory activity at Complex II/SDH

  • This impairs cellular ability to respond to increased energy demands

• Metabolic consequences:

  • In pancreatic beta cells, ROMO1 deficiency leads to impaired glucose-stimulated insulin secretion

  • The metabolic effects show sexual dimorphism, with male mice exhibiting more severe phenotypes initially

  • Aging female mice eventually develop similar metabolic deficits

• Mechanistic model:

  Parameter	ROMO1 Present	ROMO1 Deficient
  Mitochondrial Morphology	Fused, networked	Fragmented, swollen
  mtDNA Content	Normal	Reduced
  Basal Respiration	Normal	Normal
  Spare Respiratory Capacity	Intact	Reduced
  Complex II/SDH Activity	Normal	Impaired
  Stress Response Capacity	High	Compromised

These findings highlight ROMO1's importance in maintaining mitochondrial functional reserve, which is critical for cells to meet fluctuating energy demands.

What is the relationship between ROMO1 and macrophage polarization in cancer microenvironments?

ROMO1 exerts profound effects on macrophage polarization within cancer microenvironments, contributing significantly to tumor progression:

• M2 polarization promotion:

  • ROMO1 overexpression in macrophages results in M2 polarization characterized by:

    • Increased expression of CD206, Arginase1, Ym1, IL-10, and TGF-β (M2 markers)

    • Decreased expression of iNOS, IL-6, Nos2, TNF-α, and IL-23 (M1 markers)

  • This polarization creates an immunosuppressive tumor microenvironment

• Molecular mechanism:

  • ROMO1 overexpression inhibits the activation of the mTORC1 pathway

  • Specifically, ROMO1 suppresses phosphorylation of AKT, RAPTOR, S6K1, and 4E-BP1

  • When mTORC1 activation is rescued (using 3-BDO), the effects of ROMO1 overexpression on macrophage polarization are significantly reversed

• Functional consequences in tumors:

  • In glioblastoma models, ROMO1 overexpression in bone marrow cells:

    • Significantly inhibits immune responses within the tumor microenvironment

    • Accelerates tumor growth

    • Shortens survival in mouse models

  • These effects are macrophage-dependent, as macrophage depletion reverses these outcomes

• Clinical correlations:

  • High ROMO1 expression correlates with poor prognosis in glioblastoma patients

  • This likely reflects the immunosuppressive microenvironment created by ROMO1-overexpressing macrophages

This relationship between ROMO1 and macrophage polarization represents an important mechanism of tumor immune evasion and suggests ROMO1 as a potential target for cancer immunotherapy.

How does ROMO1 contribute to the regulation of spare respiratory capacity in mitochondria?

ROMO1 plays a critical role in regulating mitochondrial spare respiratory capacity (SRC), with significant implications for cellular responses to stress:

• Definition and significance of SRC:

  • SRC represents the extra mitochondrial capacity available for energy production under increased demand

  • It serves as a crucial buffer during periods of cellular stress or high energy requirements

  • SRC is calculated as the difference between maximal and basal respiration

• ROMO1's specific impact on SRC:

  • Deletion of ROMO1 in both mouse and human cells reduces spare respiratory capacity

  • This reduction occurs even when basal respiration remains normal

  • The effect involves specific loss of respiratory activity at Complex II/SDH (succinate dehydrogenase)

• Sex-dependent and age-related effects:

  • In young mice, ROMO1 deletion shows more pronounced effects in males

  • Female mice with ROMO1 knockout initially maintain normal function

  • With aging, female ROMO1-deficient mice also develop reduced SRC and metabolic dysfunction

• Functional consequences:

  • In pancreatic beta cells, reduced SRC impairs glucose-stimulated insulin secretion

  • This manifests as glucose intolerance in male mice

  • The findings highlight SRC's importance in specialized cellular functions that require metabolic flexibility

ROMO1's regulation of SRC, particularly through effects on Complex II/SDH activity, represents a crucial aspect of its physiological importance and may explain why ROMO1 deficiency has context-specific effects in different tissues and conditions.

What molecular mechanisms underlie ROMO1's role in ROS production and redox signaling?

The molecular mechanisms underlying ROMO1's control of ROS production and redox signaling involve multiple pathways:

These mechanisms position ROMO1 as a central regulator of mitochondrial ROS production with significant implications for cellular signaling, energy metabolism, and stress responses.

How does ROMO1 contribute to cancer progression and immune evasion?

ROMO1 contributes to cancer progression and immune evasion through several interconnected mechanisms:

• Clinical significance:

  • High ROMO1 expression correlates with poor prognosis in multiple cancers

  • In glioblastoma patients, elevated ROMO1 expression is associated with worse outcomes

• Immune microenvironment modulation:

  • ROMO1 is highly expressed in tumor-associated macrophages

  • ROMO1 overexpression promotes M2 (immunosuppressive) macrophage polarization by:

    • Inhibiting mTORC1 signaling

    • Increasing expression of immunosuppressive cytokines (IL-10, TGF-β)

    • Decreasing pro-inflammatory mediators (TNF-α, IL-6)

  • This creates an immunosuppressive tumor microenvironment that facilitates tumor growth

• Experimental evidence:

  • In glioblastoma mouse models, ROMO1 overexpression in bone marrow cells:

    • Accelerates tumor growth

    • Significantly shortens survival

    • Inhibits anti-tumor immune responses

  • These effects are macrophage-dependent, as demonstrated by reversal with macrophage-depleting agents

• T cell suppression:

  • ROMO1 appears to regulate the crosstalk between tumor-associated macrophages and T cells

  • The immunosuppressive environment created by ROMO1-overexpressing macrophages inhibits T cell function

  • This contributes to tumor immune evasion

These findings position ROMO1 as an important regulator of tumor immune microenvironments and suggest it as a potential target for cancer immunotherapy strategies.

What potential therapeutic approaches target ROMO1 in disease treatment?

Several promising therapeutic approaches targeting ROMO1 are being investigated:

• Cancer immunotherapy strategies:

  • ROMO1 inhibition: Knockdown of ROMO1 in bone marrow cells inhibits glioblastoma growth and extends survival in mouse models

  • Combination therapy: ROMO1 inhibition synergizes with PD-1 blockade, significantly improving survival outcomes in glioblastoma models

  • Mechanism: These approaches work by shifting macrophage polarization from immunosuppressive M2 to anti-tumor M1 phenotypes

• RNA interference approaches:

  • shRNA technologies: Successfully used to knock down ROMO1 expression in experimental models

  • Delivery systems: Various nanoparticle-based and viral delivery methods could be adapted for ROMO1-targeting RNAi

• Metabolic disorder applications:

  • Based on ROMO1's role in beta cell function and glucose homeostasis, ROMO1-targeted therapies might have applications in diabetes

  • Such approaches would aim to enhance or preserve spare respiratory capacity in affected cells

• Combined treatment data:

  Treatment Approach	Effect on Tumor Growth	Effect on Survival	Effect on Immune Response	Reference
  Control	Baseline	Baseline	Baseline
  ROMO1 inhibition	Significant reduction	Extended	Enhanced M1 polarization
  PD-1 blockade alone	Moderate reduction	Moderate extension	Enhanced T cell activity
  Combined ROMO1 inhibition + PD-1 blockade	Maximum reduction	Maximum extension	Enhanced M1 polarization and T cell activity

• Challenges and considerations:

  • Given ROMO1's fundamental roles in mitochondrial function and embryonic development, targeted delivery to specific tissues may be necessary

  • Potential off-target effects must be carefully evaluated

  • Sex-dependent responses might necessitate personalized therapeutic approaches

These therapeutic strategies highlight ROMO1's potential as a target in multiple disease contexts, particularly in combination with established immunotherapies for cancer.

How do ROMO1 expression patterns correlate with disease progression and patient outcomes?

ROMO1 expression patterns show important correlations with disease progression and patient outcomes across several conditions:

• Cancer prognosis:

  • High ROMO1 expression correlates with poor prognosis in glioblastoma patients

  • Similar associations have been reported in non-small cell lung cancer, where ROMO1 overexpression predicts unfavorable outcomes and lymphatic metastasis

  • These correlations suggest ROMO1 expression has potential as a prognostic biomarker

• Mechanistic basis for prognostic correlations:

  • ROMO1 overexpression promotes immunosuppressive microenvironments through macrophage polarization

  • Enhanced ROS production may support cancer cell survival through redox adaptations

  • Effects on mitochondrial dynamics might contribute to metabolic advantages for tumor cells

• Metabolic disorders:

  • ROMO1 dysfunction in pancreatic beta cells contributes to impaired glucose homeostasis

  • Sex differences in these effects suggest hormone-dependent regulation

  • Age-related effects indicate ROMO1's role may become more crucial during aging

• Diagnostic and therapeutic implications:

  • ROMO1 expression assessment could aid in patient stratification for targeted therapies

  • Monitoring ROMO1 levels might help track treatment efficacy or disease progression

  • Combinations with other biomarkers could enhance prognostic accuracy

These correlations between ROMO1 expression and disease outcomes highlight its potential value as both a biomarker and therapeutic target, warranting further clinical investigation across multiple disease contexts.

What is the relationship between ROMO1 function and beta cell dysfunction in metabolic disorders?

ROMO1 plays a critical role in pancreatic beta cell function, with important implications for metabolic disorders:

• Glucose homeostasis effects:

  • Knockout of ROMO1 in adult pancreatic beta cells results in impaired glucose homeostasis

  • This manifests as defective insulin secretion in response to glucose stimulation

  • The phenotype shows sexual dimorphism, with young male mice exhibiting more immediate effects

• Mitochondrial basis of dysfunction:

  • ROMO1 deletion leads to swollen, fragmented mitochondria in beta cells

  • Reduction in mtDNA content is observed

  • Most critically, ROMO1 deficiency reduces spare respiratory capacity (SRC)

  • The specific loss of respiratory activity at Complex II/SDH compromises beta cell function

• Physiological consequences:

  • Beta cells rely heavily on mitochondrial function for glucose sensing and insulin secretion

  • Reduced SRC impairs the ability to respond to metabolic demands

  • This manifests as defective glucose-stimulated insulin secretion

  • Over time, this contributes to glucose intolerance

• Aging and sex-specific effects:

  • Female mice with ROMO1 deletion initially maintain normal function

  • With aging, female ROMO1-deficient mice also develop reduced SRC and glucose intolerance

  • This suggests interactions with age-related factors and potential hormonal influences

These findings position ROMO1 as an important regulator of beta cell mitochondrial function with significant implications for understanding metabolic disorders. The data suggest that ROMO1 dysfunction could contribute to diabetes development, particularly in the context of aging.

How can ROMO1 inhibition enhance the efficacy of immune checkpoint blockade in cancer therapy?

ROMO1 inhibition significantly enhances the efficacy of immune checkpoint blockade through complementary mechanisms:

• Synergistic therapeutic effects:

  • The combination of ROMO1 inhibition with PD-1 blockade significantly improves survival outcomes in glioblastoma mouse models

  • This combination therapy shows greater efficacy than either treatment alone

• Mechanistic basis for synergy:

  • ROMO1 inhibition promotes M1 (anti-tumor) macrophage polarization by:

    • Decreasing ROS levels in macrophages

    • Increasing expression of pro-inflammatory mediators

    • Decreasing immunosuppressive factors

  • PD-1 blockade primarily enhances T cell activity

  • Together, these approaches address both innate and adaptive immune suppression

• Experimental evidence:

  • In glioblastoma models, the combination of ROMO1 inhibition (via shRNA) and anti-PD-1 treatment:

    • Maximally inhibited tumor growth

    • Significantly extended survival

    • Enhanced both macrophage M1 polarization and T cell function

• Translational implications:

  • ROMO1 inhibition could address a major limitation of checkpoint inhibitors in glioblastoma

  • Current checkpoint inhibitor monotherapies show limited efficacy in many glioblastoma patients

  • Targeting the immunosuppressive myeloid compartment through ROMO1 inhibition may overcome resistance mechanisms

• Biomarker potential:

  • ROMO1 expression levels could potentially identify patients most likely to benefit from combination approaches

  • Monitoring macrophage polarization states might serve as a pharmacodynamic marker

This synergistic relationship between ROMO1 inhibition and checkpoint blockade represents a promising strategy for enhancing cancer immunotherapy efficacy, particularly in difficult-to-treat malignancies like glioblastoma.

What are the most significant unresolved questions regarding ROMO1 function and regulation?

Despite recent advances in understanding ROMO1, several critical questions remain unresolved:

• Molecular mechanism questions:

  • What is the precise molecular mechanism by which ROMO1 regulates Complex II/SDH activity?

  • How does ROMO1 influence mTORC1 signaling at the molecular level?

  • What are the direct protein interaction partners of ROMO1 in different cellular contexts?

• Regulatory questions:

  • What factors control ROMO1 expression under normal and pathological conditions?

  • How is ROMO1 activity post-translationally regulated?

  • What explains the sex-specific effects of ROMO1 deficiency observed in metabolic studies?

• Physiological role questions:

  • Why is ROMO1 essential for early embryonic development?

  • How does ROMO1 function differ across various tissues and cell types?

  • What is the evolutionary significance of ROMO1's high conservation across species?

• Disease-related questions:

  • Beyond macrophage polarization, what other mechanisms might explain ROMO1's association with cancer progression?

  • Could ROMO1 play roles in neurodegenerative or cardiovascular diseases through its effects on mitochondrial function?

  • What is the significance of ROMO1 in aging-related cellular dysfunction?

Addressing these questions will require innovative experimental approaches and may yield important insights into fundamental cellular processes and disease mechanisms.

What emerging technologies might advance ROMO1 research in the coming years?

Several emerging technologies hold promise for advancing ROMO1 research:

• Advanced imaging technologies:

  • Super-resolution microscopy for visualizing ROMO1's precise localization and dynamics within mitochondria

  • Live-cell imaging with genetically encoded sensors to monitor ROMO1-associated ROS production in real-time

  • Correlative light and electron microscopy to link ROMO1 localization with mitochondrial ultrastructure

• Proteomic and interactomic approaches:

  • Proximity labeling techniques (BioID, APEX) to identify ROMO1 interaction partners

  • Hydrogen-deuterium exchange mass spectrometry to analyze conformational changes

  • Crosslinking mass spectrometry to map interaction interfaces

• Structural biology methods:

  • Cryo-electron microscopy to determine ROMO1's structure within the mitochondrial membrane

  • Single-particle analysis to understand ROMO1's channel properties

  • Computational structural biology to model ROMO1's interactions with other proteins

• Gene editing and functional genomics:

  • CRISPR-based screens to identify synthetic lethal interactions with ROMO1

  • Base editing or prime editing for subtle modification of ROMO1 sequence

  • Temporal control of ROMO1 expression using optogenetic or chemically inducible systems

• Translational research tools:

  • Patient-derived organoids to study ROMO1 in human disease contexts

  • Humanized mouse models incorporating patient-specific ROMO1 variants

  • AI-driven drug discovery to identify ROMO1 modulators

These technologies will enable researchers to address fundamental questions about ROMO1 function while advancing its potential as a therapeutic target.