ENOPH1 Human

What is ENOPH1 and what is its primary function in human cells?

ENOPH1 is a newly identified enzyme of the methionine salvage pathway that plays important roles in stress responses and cell proliferation . It is widely expressed in the brain and has been implicated in neurodevelopmental disorders and anxiety responses . The protein functions as part of metabolic pathways that help cells respond to various stressors, with its dysregulation being associated with pathological conditions including ischemic injury and potentially cancer . Recent research has revealed that ENOPH1's enzymatic activity contributes to cellular stress management systems, particularly in endothelial cells where it appears to regulate apoptotic processes under stress conditions .

What experimental models are commonly used to study ENOPH1 function?

Several experimental models have been established to investigate ENOPH1 function:

• In vivo models: ENOPH1 knockout mice (ENOPH1 KO) compared with wild type (WT) mice, typically subjected to transient middle cerebral artery occlusion (tMCAO) to simulate ischemic conditions .

• In vitro models: Brain microvascular endothelial cell lines (bEND3 cells) exposed to oxygen-glucose deprivation (OGD), which effectively simulates ischemic conditions at the cellular level .

• Genetic manipulation approaches:

  • ENOPH1 knockdown using siRNA techniques

  • ENOPH1 overexpression using CRISPR-activation plasmids

  • These approaches enable researchers to directly assess the functional consequences of altered ENOPH1 expression .

The combination of these models allows for comprehensive investigation of ENOPH1's role in normal and pathological conditions, particularly in cerebrovascular function .

How is ENOPH1 expression measured in experimental systems?

Researchers employ multiple complementary techniques to measure ENOPH1 expression:

• mRNA expression analysis:

  • Real-time RT-PCR to quantify ENOPH1 mRNA levels in isolated microvessels or cultured cells .

• Protein expression assessment:

  • Western blotting for quantification of ENOPH1 protein levels .

  • Immunofluorescence staining for visualization of cellular localization.

• Temporal expression patterns:

  • Time-course experiments reveal that ENOPH1 mRNA expression increases rapidly (within 1 hour) after OGD exposure and remains elevated through 6 hours of OGD treatment .

These methodological approaches provide a thorough characterization of ENOPH1 expression under various experimental conditions, enabling researchers to correlate expression changes with functional outcomes .

What is the role of ENOPH1 in blood-brain barrier dysfunction during cerebral ischemia?

ENOPH1 plays a critical role in blood-brain barrier (BBB) dysfunction during cerebral ischemia through several interconnected mechanisms:

• BBB permeability regulation: Knockout of ENOPH1 significantly decreases BBB permeability after ischemic injury, suggesting that ENOPH1 activation contributes to BBB breakdown . This has been demonstrated using FITC-dextran staining methods to assess vascular leakage .

• Extracellular matrix (ECM) integrity: ENOPH1 increases the activity of matrix metalloproteinases MMP-2/9, which promote extracellular matrix degradation and tight junction protein breakdown . This ultimately compromises BBB structural integrity.

• Tight junction and adherens junction modulation: ENOPH1 knockout upregulates the expression of tight junction and adherens junction proteins after ischemia, preserving BBB structure . Specifically, ENOPH1 appears to negatively regulate proteins critical for maintaining endothelial cell-to-cell contacts.

• Endothelial cell survival: ENOPH1 mediates cerebral microvascular endothelial cell apoptosis under ischemic conditions, contributing to BBB disruption . Knockdown of ENOPH1 significantly reduces OGD-induced endothelial monolayer permeability increase, further supporting its role in BBB integrity .

These findings collectively demonstrate that ENOPH1 exacerbates BBB dysfunction during cerebral ischemia, making it a potential therapeutic target for preventing secondary injury after stroke .

How does ENOPH1 regulate endothelial cell death pathways under ischemic conditions?

ENOPH1 regulates endothelial cell death through multiple pathways under ischemic conditions:

• Oxidative stress modulation: ENOPH1 promotes reactive oxygen species (ROS) generation in OGD-treated endothelial cells. Knockdown of ENOPH1 significantly attenuates OGD-induced ROS production, while overexpression enhances it .

• Apoptotic pathway activation: ENOPH1 influences key apoptosis-associated proteins:

  • Increases cleaved caspase-3/caspase-3 ratio

  • Enhances PARP cleavage

  • Elevates Bax/Bcl-2 ratio

• Endoplasmic reticulum (ER) stress response: ENOPH1 modulates ER stress proteins including Ire-1, Calnexin, GRP78, and PERK in OGD-treated endothelial cells. Knockdown of ENOPH1 attenuates the activation of these stress response proteins .

• Experimental validation: Cell viability assays (MTT formation), cell death assessments (lactate dehydrogenase release), and apoptosis detection (TUNEL staining) consistently demonstrate that ENOPH1 knockdown attenuates OGD-induced endothelial cell death, while overexpression potentiates it .

The methodological approach of both knockdown and overexpression studies provides strong evidence that ENOPH1 is a pro-apoptotic factor in endothelial cells under ischemic conditions, contributing significantly to cell death processes .

What is the relationship between ENOPH1 and ADI1 in cellular stress responses?

The interaction between ENOPH1 and aci-reductone dioxygenase 1 (ADI1) represents a key regulatory mechanism in cellular stress responses:

• Protein-protein interaction: OGD treatment enhances the interaction between ENOPH1 and ADI1, as demonstrated by co-immunoprecipitation assays and co-immunofluorescence staining .

• Expression regulation: While OGD upregulates both ENOPH1 and ADI1 expression, knockdown of ENOPH1 has no effect on OGD-induced ADI1 upregulation, suggesting these are parallel responses to stress .

• Subcellular localization changes:

  • OGD enhances ADI1 translocation from the nucleus to the cytoplasm

  • ENOPH1 knockdown potentiates this translocation, indicating that ENOPH1 normally restrains ADI1 cytoplasmic localization

• Functional outcomes: ENOPH1 silencing enhances the interaction between ADI1 and MT1-MMP (membrane type 1 matrix metalloproteinase) by promoting the nuclear translocation of ADI1, which inhibits MT1-MMP activity in endothelial cells after OGD .

This regulatory relationship ultimately affects extracellular matrix integrity, as evidenced by decreased expression of Tenascin C (Tnc) and Fibronectin 1 (Fn1) when ENOPH1 is silenced, which inhibits ECM degradation . The methodological approaches combining protein interaction studies with subcellular localization analysis provide a comprehensive understanding of this complex regulatory network .

What experimental approaches are most effective for studying ENOPH1's mechanism in BBB breakdown?

Based on the research literature, the most effective experimental approaches for investigating ENOPH1's role in BBB breakdown include:

• Complementary in vivo and in vitro models:

  • In vivo: ENOPH1 knockout mice subjected to transient middle cerebral artery occlusion (tMCAO)

  • In vitro: Brain microvascular endothelial cells (bEND3) exposed to oxygen-glucose deprivation (OGD)

• Comprehensive BBB integrity assessment:

  • FITC-dextran staining to measure BBB permeability

  • Western blotting and co-immunofluorescence to analyze tight junction (TJ) and adherens junction (AJ) protein expression

  • Gelatin zymography to analyze MMP-2/9 activity

• Advanced protein interaction studies:

  • Co-immunoprecipitation assays to measure interactions between ENOPH1, ADI1, and MT1-MMP

  • Co-immunofluorescence to visualize protein localization and interactions

• Quantitative proteomics:

  • Differential protein expression analysis to identify pathways affected by ENOPH1 modulation

  • This approach revealed key ECM proteins (Tnc and Fn1) regulated by ENOPH1

• Cellular manipulation techniques:

  • siRNA knockdown of ENOPH1

  • CRISPR activation plasmids for ENOPH1 overexpression

  • These complementary approaches provide gain- and loss-of-function evidence

The integration of these methodologies has proven most effective for elucidating the complex mechanisms by which ENOPH1 contributes to BBB breakdown during ischemic injury .

How can ENOPH1 be targeted therapeutically in ischemic stroke models?

Based on the research data, several therapeutic approaches targeting ENOPH1 show promise for ischemic stroke treatment:

• Genetic knockdown strategies:

  • siRNA-mediated ENOPH1 silencing has demonstrated significant neuroprotective effects by:

    • Ameliorating cerebral ischemic injury

    • Decreasing BBB permeability

    • Inhibiting MMP-2/9 activity

    • Upregulating tight junction/adherens junction proteins

    • Reversing extracellular matrix destruction

• Molecular pathway targeting:

  • Inhibiting the downstream effects of ENOPH1 on MMP-2/9 activity could preserve BBB integrity

  • Promoting ADI1-MT1-MMP interaction, which is normally suppressed by ENOPH1, represents another potential intervention point

• Anti-apoptotic approaches:

  • Targeting ENOPH1's effects on apoptosis-associated proteins (caspase-3, PARP, Bax/Bcl-2)

  • Reducing ENOPH1-mediated ROS generation and ER stress

• Translational considerations:

  • The rapid upregulation of ENOPH1 after ischemia (within 1-3 hours) suggests a critical early intervention window

  • Any therapeutic approach would need to act quickly to prevent the cascade of BBB breakdown

The research suggests that ENOPH1 represents "a new therapeutic target for ischemic stroke" with multiple potential intervention strategies that could preserve BBB integrity and reduce secondary damage after cerebral ischemia .

What are the key experimental controls needed when studying ENOPH1 function?

When studying ENOPH1 function, several critical experimental controls should be implemented:

• Genetic manipulation controls:

  • For ENOPH1 knockdown: Non-targeting siRNA control

  • For ENOPH1 overexpression: Empty vector CRISPR activation plasmid control

  • Verification of knockdown/overexpression efficiency by both mRNA and protein quantification

• In vivo experimental controls:

  • Wild-type mice compared to ENOPH1 knockout mice

  • Sham-operated animals versus tMCAO-subjected animals

  • Contralateral (non-ischemic) hemisphere versus ipsilateral (ischemic) hemisphere within the same animal

• In vitro experimental controls:

  • Normoxic conditions versus OGD conditions

  • Time-course experiments (1h, 3h, 6h OGD) to distinguish early versus late effects

  • Cell viability assays using multiple complementary methods (MTT, LDH release, TUNEL)

• Protein interaction studies:

  • Input controls for co-immunoprecipitation experiments

  • IgG controls for non-specific binding

  • Reciprocal co-immunoprecipitation (pulling down with anti-ENOPH1 versus anti-ADI1)

• BBB integrity assessment:

  • Multiple complementary techniques (FITC-dextran leakage, tight junction protein expression, gelatin zymography for MMP activity)

These methodological controls collectively ensure the reliability and reproducibility of findings related to ENOPH1 function in both normal and pathological conditions .

How can researchers distinguish between ENOPH1's direct effects and secondary consequences?

Distinguishing between direct ENOPH1 effects and secondary consequences requires sophisticated experimental approaches:

• Temporal sequencing studies:

  • Time-course experiments measuring ENOPH1 expression, downstream protein activation, and functional outcomes

  • Evidence shows ENOPH1 upregulation begins early (1h after OGD), preceding significant cell death (observed at 6h)

• Pathway inhibition approaches:

  • Using specific inhibitors of downstream effectors (e.g., MMP inhibitors, ROS scavengers) in combination with ENOPH1 manipulation

  • If inhibiting a downstream pathway blocks ENOPH1's effects, this suggests a direct mechanistic relationship

• Protein interaction studies:

  • Direct protein-protein interactions (ENOPH1-ADI1) identified through co-immunoprecipitation

  • Subcellular localization changes (nuclear-cytoplasmic translocation) monitored through immunofluorescence

  • These approaches help establish proximal versus distal effects

• In vitro reconstitution experiments:

  • Using purified proteins to test direct enzymatic activities and interactions

  • This approach can definitively establish direct versus indirect effects

• Complementary gain- and loss-of-function studies:

  • ENOPH1 knockdown and overexpression producing opposite effects on the same parameters (ROS generation, apoptosis markers, ER stress proteins)

  • This bidirectional manipulation strongly supports direct causality rather than coincidental association

These methodological approaches collectively help distinguish between ENOPH1's primary effects and downstream consequences in complex biological systems .

What are the priority research questions regarding ENOPH1 in human disease?

Based on current knowledge, several priority research questions emerge for further investigation:

• Broader disease relevance:

  • Beyond cerebral ischemia, what roles does ENOPH1 play in other neurological disorders?

  • How does ENOPH1 contribute to hepatocellular carcinoma progression, as suggested by metabolomic data?

  • Are there connections between ENOPH1 and neurodegenerative diseases?

• Mechanistic details:

  • What is the precise enzymatic activity of ENOPH1 in the context of BBB dysfunction?

  • How does nuclear-cytoplasmic shuttling of ADI1 mechanistically inhibit MT1-MMP activity?

  • What are the upstream regulators of ENOPH1 expression during stress conditions?

• Therapeutic development:

  • Can small molecule inhibitors of ENOPH1 be developed for stroke therapy?

  • What is the therapeutic window for ENOPH1 targeting after ischemic stroke?

  • Are there potential adverse effects of ENOPH1 inhibition given its role in normal cellular metabolism?

• Clinical translation:

  • Can ENOPH1 serve as a biomarker for BBB dysfunction or stroke severity?

  • Are there human polymorphisms in ENOPH1 that correlate with stroke outcomes?

  • How do age, sex, and comorbidities influence ENOPH1 expression and function?

• Methodological advances needed:

  • Development of specific ENOPH1 inhibitors

  • Non-invasive imaging approaches to monitor ENOPH1 activity in vivo

  • High-throughput screening methods to identify ENOPH1 modulators

Addressing these research priorities will significantly advance our understanding of ENOPH1's role in human disease and potential therapeutic applications .

What technical challenges exist in studying ENOPH1 function and expression?

Researchers face several significant technical challenges when investigating ENOPH1:

• Specificity of tools and reagents:

  • Limited availability of highly specific antibodies for ENOPH1 detection

  • Potential cross-reactivity with related enzymes in the methionine salvage pathway

  • Need for validated tools to distinguish between active and inactive forms of ENOPH1

• Temporal dynamics:

  • ENOPH1 expression changes rapidly after stress (within hours)

  • Capturing these dynamic changes requires precise timing in experimental protocols

  • Different downstream effects may occur at different time points after ENOPH1 activation

• Cell type specificity:

  • ENOPH1 may have different functions in different cell types (endothelial cells vs. neurons vs. glial cells)

  • Current research has focused primarily on endothelial cells

  • Developing cell-type specific knockout models presents technical challenges

• Translating in vitro findings to in vivo models:

  • OGD in cultured cells may not fully recapitulate the complexity of in vivo ischemia

  • The multicellular nature of the BBB makes it difficult to isolate endothelial-specific effects

• Distinguishing enzymatic from non-enzymatic functions:

  • ENOPH1 has both catalytic activity and protein-protein interaction functions

  • Separating these functions experimentally requires sophisticated mutagenesis approaches

Addressing these technical challenges will require development of new tools and methodologies, as well as collaborative approaches combining expertise in biochemistry, cell biology, and neuroscience .