Recombinant Bovine Metalloendopeptidase OMA1, mitochondrial (OMA1)

What is OMA1 and what is its primary function in mitochondria?

OMA1 is an ATP-independent zinc metalloprotease located in the inner membrane of mitochondria. It functions as a redox-dependent protein with multiple transmembrane domains and zinc finger binding motifs. OMA1's primary role is maintaining mitochondrial homeostasis by participating in the quality control system that regulates the balance between mitochondrial fusion and fission .

How is OMA1 structurally characterized and where is it expressed in mammals?

In humans, the OMA1 gene is located on chromosome 1p32.2-p32.1 and contains 9 exons. The encoded protein consists of 524 amino acids with a molecular weight of approximately 60.1 kDa. The first 13 amino acids constitute a signal peptide .

Mammalian OMA1 features an extended carboxyl terminal and a positively charged amino-terminal domain, which is particularly important for OMA1 activation. Mutations in this positively charged domain impair activation without affecting the protein's proteolytic function .

Regarding expression, OMA1 is widely distributed throughout the body but shows particularly high expression in metabolically active tissues including the heart, skeletal muscle, kidney, and liver .

What is the relationship between OMA1 and OPA1 in mitochondrial dynamics?

The relationship between OMA1 and OPA1 represents a critical regulatory axis in mitochondrial dynamics. OPA1 is a dynamin-related GTPase that promotes mitochondrial inner membrane fusion. Under normal conditions, OPA1 exists in both long (L-OPA1) and short (S-OPA1) forms, with the long form being essential for fusion .

When cells experience stress (such as loss of membrane potential or ATP depletion), OMA1 becomes activated and cleaves L-OPA1 to S-OPA1, which lacks fusion-promoting activity. This proteolytic conversion tips the balance toward mitochondrial fragmentation, facilitating the isolation and elimination of damaged mitochondrial segments .

Research has established that OMA1 plays a non-redundant role in this process, as demonstrated in OMA1-deficient mouse models where OPA1 processing is significantly altered, resulting in profound perturbations in mitochondrial dynamics .

How is OMA1 activated during cellular stress conditions?

OMA1 activation involves a sophisticated mechanism tied to cellular stress detection. Under basal conditions, OMA1 exists in an inactive form. Various stress stimuli, including loss of mitochondrial membrane potential, ATP depletion, oxidative stress, and hypoxia-reperfusion, can trigger OMA1 activation .

The activation process involves self-cleavage of OMA1, which transforms it from its inactive precursor to an active protease. This autocatalytic processing is a critical step in the stress response pathway. Recent research by Khalimonchuk and colleagues at UNL has focused on clarifying these "murky mechanisms" behind OMA1 activation .

Interestingly, OMA1 contains a redox-sensing switch that participates in its activation. 3D modeling and experimental evidence suggest that cysteine 403 in mammalian OMA1 serves as part of this sensor, allowing the protein to respond directly to oxidative stress conditions .

What is the significance of OMA1's redox-sensing capability?

The redox-sensing capability of OMA1 represents a sophisticated adaptation that allows mitochondria to respond rapidly to oxidative stress. Research has identified cysteine 403 as a critical residue in this redox-sensing switch in mammalian cells .

Using prime editing techniques, researchers developed a mouse sarcoma cell line with OMA1 cysteine 403 mutated to alanine. This mutation significantly impaired mitochondrial responses to stress, including ATP production and mitochondrial fission. Importantly, cells bearing this mutation showed resistance to apoptosis and enhanced mitochondrial DNA release .

The redox-sensing function appears to be evolutionarily conserved, as similar mechanisms have been observed in yeast. This conservation suggests fundamental importance in cellular stress responses across diverse organisms. The direct connection between oxidative status and OMA1 activation provides cells with an immediate response system to mitochondrial stress without requiring additional signaling intermediates .

How does OMA1 inactivation occur after stress resolution?

OMA1 inactivation is a crucial aspect of restoring normal mitochondrial dynamics after stress resolution. Research indicates that OMA1 undergoes further proteolytic processing that leads to its inactivation once its function has been fulfilled .

Two mechanisms have been proposed for this inactivation:

• Autocatalytic processing: OMA1 can cleave itself, resulting in inactivation

• YME1L1-mediated cleavage: Another mitochondrial protease, YME1L1, may target activated OMA1 for degradation

This self-limiting activation ensures that OMA1's effects on mitochondrial dynamics are temporary and proportional to the stress level. Such precise regulation prevents excessive mitochondrial fragmentation, which would be detrimental to cellular energy production and survival .

The balance between OMA1 activation and inactivation represents a critical regulatory node in mitochondrial quality control, with important implications for cellular adaptation to stress conditions.

What are the optimal methods for expressing and purifying recombinant bovine OMA1?

For successful expression and purification of recombinant bovine OMA1, researchers should consider several methodological approaches:

Expression Systems:

• Mammalian expression systems (HEK293 or CHO cells) are recommended for obtaining properly folded OMA1 with appropriate post-translational modifications

• Baculovirus-insect cell systems represent an alternative for higher yield while maintaining proper protein folding

• Bacterial systems (E. coli) may be used for truncated versions or specific domains but often struggle with full-length OMA1 due to its multiple transmembrane domains

Purification Strategy:

• Use a dual-tag approach (e.g., His-tag combined with FLAG or Strep-tag) to enhance specificity and purity

• Include mild detergents (0.1-1% digitonin or DDM) in all buffers to maintain protein solubility

• Employ size exclusion chromatography as a final purification step to ensure homogeneity

Critical Considerations:

• Maintain reducing conditions throughout purification to preserve the native state of cysteine residues, particularly the redox-sensitive Cys403

• Consider co-expression with OMA1 partner proteins to enhance stability

• Use protease inhibitors to prevent autoproteolysis during purification

What assays can be used to evaluate OMA1 proteolytic activity in vitro?

Several robust assays can be employed to measure OMA1 proteolytic activity:

OPA1 Cleavage Assay:

• Incubate purified OMA1 with recombinant OPA1 substrate

• Monitor conversion of long-form OPA1 to short form using western blotting

• Quantify band intensity ratios to determine cleavage efficiency

Fluorogenic Peptide Assays:

• Design peptides containing OMA1 recognition sequences flanked by fluorophore/quencher pairs

• Measure fluorescence increase as peptide cleavage separates quencher from fluorophore

• This approach allows for high-throughput screening of activity modulators

Reconstituted Liposome Systems:

• Incorporate OMA1 into liposomes mimicking the mitochondrial inner membrane composition

• Add fluorescently labeled substrates and monitor cleavage products

• This system better approximates the native environment of OMA1

Redox Sensitivity Assessment:

• Perform activity assays under varying redox conditions (GSH/GSSG ratios)

• Compare wild-type OMA1 activity with cysteine mutants (particularly C403A)

• This approach helps delineate the relationship between redox sensing and enzymatic activity

How can researchers effectively model OMA1 activity in cellular systems?

Cell Line Selection and Modification:

• Create OMA1 knockout cell lines using CRISPR-Cas9 gene editing as experimental controls

• Develop cell lines with tagged OMA1 variants (wild-type and mutants) for localization and functional studies

• Primary cells from OMA1-deficient mice can provide physiologically relevant models

Stress Induction Protocols:

• Mitochondrial membrane potential disruption: CCCP (5-10 μM, 1-4 hours)

• Oxidative stress: H₂O₂ (100-500 μM, 1-6 hours)

• Hypoxia-reperfusion: Culture under 1% O₂ followed by normal oxygen conditions

• ATP depletion: Oligomycin treatment

Activity Assessment Methods:

• Monitor OPA1 processing via western blotting

• Track mitochondrial morphology using fluorescence microscopy and mitochondria-targeted GFP

• Assess mitochondrial function parameters (membrane potential, ATP production, ROS levels)

• Evaluate cell death markers in response to stress conditions with and without OMA1 activity

Data Analysis Table: OMA1 Activity Readouts in Cellular Systems

How does OMA1 function in cancer biology, and what are the implications for therapy?

OMA1's role in cancer appears to be context-dependent, with significant variations across different tumor types. Recent research has revealed several key aspects of OMA1 function in cancer:

Osteosarcoma (OS):

• Elevated OMA1 expression has been observed in OS tumor tissues from patients with advanced disease

• Knockout of OMA1 in OS cells significantly reduces tumor weight, size, and lung metastatic nodules in mouse models

• Mechanistically, OMA1 deficiency increases PINK1 and Parkin levels, inducing excessive mitophagy that leads to increased apoptosis and reduced cell proliferation

• Ciclopirox (CPX), an antifungal drug, has been found to induce OMA1 self-cleavage and degradation in OS cells, reducing tumor development in mice

Soft Tissue Sarcoma:

• Mutation of OMA1's redox-sensing cysteine 403 to alanine prevents tumor development in immunocompetent mice by enhancing anti-tumor immunity

• This mutation impairs mitochondrial responses to stress and promotes mitochondrial DNA release

• High OPA1 expression (the substrate of OMA1) in primary tumors is associated with shorter metastasis-free survival

• Low OPA1 expression correlates with anti-tumor immune signatures

Therapeutic Implications:

• OMA1 inhibition may enhance tumor immunogenicity, particularly in sarcomas

• Targeting OMA1 activity could be more effective in immunocompetent patients, as the anti-tumor effects appear to be partially immune-mediated

• Monitoring OMA1/OPA1 levels might serve as prognostic markers in certain cancers

• Drugs like ciclopirox that modulate OMA1 activity represent potential therapeutic avenues

What is the relationship between OMA1 and neurodegenerative diseases like ALS?

The connection between OMA1 and neurodegenerative diseases is an emerging area of research with significant therapeutic potential:

ALS and Other Late-Onset Neurological Diseases:

• Research at the University of Nebraska-Lincoln has identified OMA1 as a key enzyme that could prove critical in combating ALS and other late-onset neurological diseases

• As part of the intramitochondrial quality control system, OMA1 helps eliminate damaged proteins that might otherwise accumulate and contribute to neurodegeneration

• The stress-responsive nature of OMA1 is particularly relevant in neurodegeneration, where cellular stress is a common feature

Mechanistic Connections:

• Mitochondrial dysfunction is a well-established component of neurodegenerative diseases

• Neurons are particularly vulnerable to defects in mitochondrial quality control due to their high energy demands and limited regenerative capacity

• OMA1's role in regulating mitochondrial dynamics could be crucial for maintaining neuronal health under stress conditions

• The enzyme's ability to eliminate damaged mitochondrial components may prevent the accumulation of dysfunctional mitochondria in neurons

Research Approaches:

• Researchers are using diverse model systems including yeast, mammalian cells, and zebrafish to analyze OMA1 behavior

• The UNL-led team is examining genetic and molecular-level interactions between OMA1 and recently identified "partner proteins" that work together to maintain healthy mitochondrial function

• Clarifying the mechanisms of OMA1 activation could lead to targeted interventions for neurodegenerative diseases

How does OMA1 contribute to cardiac protection against ischemia-reperfusion injury?

OMA1 plays a significant role in cardiac response to ischemia-reperfusion injury (IRI), with important implications for heart disease treatment:

Pathophysiological Mechanism:

• During hypoxia-reperfusion injury (HRI), which mimics cardiac IRI, OMA1 undergoes self-cleavage and activation

• Activated OMA1 cleaves OPA1 from its long form to short form

• This proteolytic conversion leads to mitochondrial fragmentation, cytochrome c release, and apoptosis

• These events contribute significantly to cardiomyocyte death during IRI

Protective Compounds:

• Epigallocatechin gallate (EGCG), a compound found in green tea, has been identified as a potent OMA1 inhibitor

• Using Molecular Operating Environment (MOE) software to simulate binding interactions, researchers found that EGCG directly interacts with OMA1

• EGCG potently inhibits OMA1 self-cleavage, attenuating OPA1 cleavage and subsequent mitochondrial dysfunction

• This inhibition protects cardiomyocytes against hypoxia-reperfusion injury

Research Methodology:

• Mouse embryonic fibroblasts (MEFs) and neonatal mouse cardiomyocytes (NMCMs) subjected to hypoxia-reperfusion injury or H₂O₂ were used as experimental models

• These models effectively mimic the oxidative stress in the heart following ischemia-reperfusion injury

• The protective effects of OMA1 inhibition were assessed by measuring mitochondrial fragmentation, cytochrome c release, and apoptosis

How do OMA1-mediated mitochondrial dynamics influence metabolic homeostasis?

OMA1's impact on whole-body metabolism represents a fascinating and complex area of investigation:

Metabolic Phenotype of OMA1 Deficiency:

• OMA1-deficient mice develop marked obesity with significant metabolic alterations

• These mice exhibit reduced energy expenditure and altered thermogenic response

• The metabolic phenotype is accompanied by transcriptional changes in genes involved in lipid and glucose metabolic pathways

• Substantial alterations in circulating blood parameters have also been observed

Mechanistic Basis:

• OMA1 deficiency causes profound perturbation of the mitochondrial fusion–fission equilibrium

• In brown adipose tissue and primary adipocytes lacking OMA1, mitochondrial functionality is significantly altered

• OMA1 plays an essential and non-redundant role in the proteolytic regulation of OPA1

• The resulting changes in mitochondrial dynamics directly impact energy metabolism and expenditure

Research Implications:

• The OMA1-OPA1 axis represents a potential therapeutic target for metabolic disorders

• Understanding the tissue-specific effects of OMA1 deficiency could provide insights into energy metabolism regulation

• The connection between mitochondrial dynamics and whole-body metabolism suggests new approaches to obesity and related metabolic diseases

What is the relationship between OMA1 and other mitochondrial quality control proteins?

OMA1 functions within a complex network of mitochondrial quality control proteins:

Interactions with Other Proteases:

• OMA1 and m-AAA protease have overlapping activity in mitochondrial protein quality control

• YME1L1, another mitochondrial protease, may be involved in OMA1 regulation through mediating its cleavage and inactivation

• The human lineal homologous gene of OMA1 is MPRP-1, suggesting evolutionarily conserved quality control mechanisms

Integration with Mitophagy Pathways:

• OMA1 deficiency increases PINK1 and Parkin levels, key regulators of mitophagy

• In osteosarcoma cells, OMA1 knockout reduces the amount of cytosolic p53 and p53-associated cytosolic Parkin

• These changes increase mitochondrial p53, leading to enhanced apoptosis

• The integration of OMA1 activity with mitophagy pathways ensures coordinated mitochondrial quality control

Redox Signaling Network:

• OMA1's redox-sensing capability (via Cys403) connects it to cellular redox signaling networks

• This connection allows OMA1 to respond to oxidative stress and coordinate quality control responses

• The mutation of Cys403 reduces mitochondrial ROS levels and increases cytosolic glycogen synthase kinase 3β (GSK3β) levels

• These changes alter downstream signaling cascades including β-catenin pathways

How can structural insights into OMA1 inform the development of specific modulators?

Understanding OMA1's structure provides critical insights for developing targeted modulators:

Structural Determinants of Activation:

• The positively charged amino-terminal domain is crucial for OMA1 activation

• Mutations in this domain impair activation without affecting proteolytic function

• The zinc-binding motifs are essential for catalytic activity

• Structure-function studies of these domains could guide the design of activation modulators

Redox-Sensitive Regions:

• Cysteine 403 has been identified as a critical residue in the redox-sensing mechanism

• 3D modeling of OMA1 has confirmed the importance of this residue

• Compounds targeting this region could modulate OMA1's response to oxidative stress

• Both activators and inhibitors could be designed based on interactions with this region

Small Molecule Binding Pockets:

Potential Applications Table: Structure-Based OMA1 Modulators