MOAP1 Human

What is MOAP1 and what is its primary function in human cells?

MOAP1, initially named MAP-1, was first identified as a binding partner of the proapoptotic protein Bax through a yeast two-hybrid screen. It contains a BH3-like motif that is essential for its proapoptotic function . MOAP1 primarily functions as an effector for facilitating Bax function in mitochondria during apoptosis. When MOAP1 levels are reduced through RNAi knockdown, cells show significant inhibition of apoptotic signaling triggered by multiple apoptotic stimuli and promote anchorage-independent growth of tumor cells . Remarkably, isolated mitochondria from MOAP1 knockdown cells are highly resistant to the cytochrome c-releasing effect of recombinant Bax, suggesting MOAP1's crucial role in Bax-mediated apoptosis .

MOAP1 is also known as PNMA4 (Paraneoplastic Ma antigen 4), suggesting a potential role in paraneoplastic neurological syndromes . Its involvement in both cancer progression and neurological diseases makes it a significant target for diverse research fields in human cellular biology .

How is MOAP1 regulated at the protein level?

MOAP1 is a short-lived protein with a half-life of approximately 25 minutes that undergoes rapid turnover in cells . It is constitutively degraded by the ubiquitin-proteasome system (UPS), which plays a major role in its fast turnover across multiple cell lines, including human primary cells .

When cells are exposed to apoptotic stimuli such as TRAIL (tumor necrosis factor-related apoptosis-inducing ligand), DNA-damaging agents, or endoplasmic reticulum stress inducers like thapsigargin, MOAP1 protein levels are rapidly up-regulated . This up-regulation occurs through inhibition of its polyubiquitination process rather than increased mRNA expression . The center region of MOAP1 (amino acids 141-190) appears to contain a functional domain sufficient for mediating the stabilization effect induced by apoptotic stimuli .

Interestingly, while proteasome inhibitors (MG132, LLnL, epoxomycin, and lactacystin) dramatically elevate endogenous MOAP1 protein levels, combining proteasome inhibitors with apoptotic stimuli does not further increase MOAP1 levels, supporting the hypothesis that apoptotic stimuli primarily work through inhibiting MOAP1 degradation .

What is the relationship between MOAP1 and cancer development?

MOAP1 plays a critical role in cancer development primarily through its function in regulating apoptosis . Several lines of evidence support its tumor-suppressive properties:

• Knockdown of MOAP1 promotes anchorage-independent growth of tumor cells, suggesting its role in preventing tumorigenic transformation .

• MOAP1 links the tumor suppressor RASSF1A to Bax activation, connecting death receptor- and activated K-Ras-mediated apoptotic signaling to the intrinsic apoptotic pathway .

• Cells with higher levels of MOAP1 show increased sensitivity to apoptotic stimuli. For example, HCT116 and MCF-7 clonal lines stably expressing exogenous MOAP1 display enhanced sensitivity to TRAIL and thapsigargin-induced apoptosis .

• In cells stably expressing higher levels of MOAP1, greater activation of Bax is observed upon treatment with apoptotic stimuli, and their mitochondria are more sensitive to the cytochrome c-releasing effect of recombinant Bax .

The reduced expression of MOAP1 in certain cancers may contribute to apoptosis resistance and tumor progression, making it a potential target for cancer therapeutics.

What experimental approaches are recommended for studying MOAP1 protein degradation?

Studying MOAP1 protein degradation requires specialized techniques due to its low abundance and rapid turnover. Based on the research literature, the following methodological approaches are recommended:

• Cycloheximide (CHX) chase assays: This approach allows researchers to measure MOAP1's half-life by blocking new protein synthesis with CHX and tracking the degradation of existing MOAP1 over time. This technique revealed MOAP1's short half-life of approximately 25 minutes .

• Combined immunoprecipitation (IP)/Western blot analysis: Due to MOAP1's low abundance, a combined IP/Western blot approach significantly improves detection sensitivity. This method effectively demonstrated the rapid up-regulation of MOAP1 in response to various apoptotic stimuli across multiple cell lines .

• Ubiquitination assays: To study MOAP1 ubiquitination, researchers can:

  • Express HA-tagged ubiquitin alongside MOAP1 in cells

  • Treat cells with proteasome inhibitors to allow accumulation of ubiquitinated MOAP1

  • Immunoprecipitate MOAP1 and detect ubiquitinated forms using anti-HA antibodies

  • Assess the effect of apoptotic stimuli on MOAP1 ubiquitination by pretreating cells before adding proteasome inhibitors

• Domain mapping experiments: Using deletion mutants to identify regions of MOAP1 critical for its ubiquitination and degradation. This approach identified the center portion (amino acids 141-190) as essential for coupling MOAP1 to the UPS .

• Heterologous protein fusion analysis: Testing whether MOAP1 degradation signals can confer instability to stable proteins like GST. This approach confirmed that the degradable property within MOAP1 is transferable to heterologous proteins that are not normally regulated by UPS .

These techniques should be combined with appropriate controls, including global ubiquitination analysis and parallel study of other UPS-regulated proteins to ensure the specificity of observed effects.

How can researchers effectively measure MOAP1 protein levels given its low abundance?

MOAP1's low abundance in mammalian cells presents a significant challenge for accurate quantification. Based on established protocols, the following methodological approaches are recommended:

• Combined immunoprecipitation/Western blot analysis: This method significantly enhances detection sensitivity for low-abundance proteins like MOAP1. Researchers should first immunoprecipitate MOAP1 from cell lysates using specific antibodies, followed by Western blot analysis for detection .

• Subcellular fractionation: Since MOAP1 is primarily localized to mitochondria, enriching mitochondrial fractions before analysis can improve detection. Heavy membrane fractions containing mitochondria show clear accumulation of MOAP1 upon treatment with apoptotic stimuli .

• Stable cell line models: Generating stable cell lines expressing tagged versions of MOAP1 (e.g., myc-tagged MOAP1) can facilitate detection while maintaining physiologically relevant expression levels. HCT116 and MCF-7 clonal lines stably expressing myc-MOAP1 have been successfully used to study MOAP1 function .

• Proteasome inhibitor treatment: Since MOAP1 is rapidly degraded by the proteasome, short-term treatment with proteasome inhibitors like MG132 can increase MOAP1 levels to improve detection without significantly altering cellular physiology .

• Quantitative real-time PCR: Although protein regulation occurs primarily post-translationally, qRT-PCR can be used as a complementary approach to confirm stable mRNA levels during apoptotic stimuli exposure .

When designing experiments to detect MOAP1, researchers should be aware that expression levels vary between cell types, with MCF-7 cells showing higher basal levels compared to other mammalian cell types .

What are the mechanisms of MOAP1 stabilization by different apoptotic stimuli?

• TRAIL-induced stabilization: TRAIL treatment rapidly up-regulates MOAP1 protein levels in multiple cell lines, including HCT116, H1299, SY5Y, and HeLa cells. The kinetics of MOAP1 induction by TRAIL is similar to Bax activation in most cell lines . Importantly, TRAIL can effectively trigger MOAP1 up-regulation even in cells like SY5Y where it fails to induce Bax activation and apoptosis .

• DNA damage-induced stabilization: DNA-damaging agents such as etoposide and camptothecin effectively stabilize MOAP1. This process appears to be p53-independent, as similar effects are observed in p53 wild-type cells (SY5Y, A2780), p53 mutant cells (293T), and p53 null cells (H1299) .

• Endoplasmic reticulum stress: The endoplasmic reticulum stress inducer thapsigargin rapidly enhances MOAP1 levels in multiple cell lines .

• Serum withdrawal: Serum deprivation also leads to MOAP1 stabilization, suggesting a role in nutrient deprivation-induced apoptosis .

• Cell type-specific responses: MCF-7 cells, which have higher basal levels of MOAP1, show differential responses to apoptotic stimuli. Only etoposide and camptothecin effectively induce MOAP1 up-regulation in these cells .

The stabilization of MOAP1 is not dependent on caspase activation, as the broad-spectrum caspase inhibitor z-VAD fails to inhibit MOAP1 up-regulation by apoptotic stimuli . The precise molecular mechanisms by which individual stimuli inhibit MOAP1 ubiquitination may involve posttranslational modifications (phosphorylation, sumoylation), regulation by specific deubiquitination enzymes, or association with regulatory partners .

Understanding these stimulus-specific mechanisms provides opportunities for targeted approaches to modulate MOAP1 stability in research and therapeutic contexts.

What experimental models are most suitable for studying MOAP1 in mitochondrial function?

To effectively study MOAP1's role in mitochondrial function and Bax-mediated cytochrome c release, several experimental models have proven particularly valuable:

• Isolated mitochondria systems: Heavy membrane fractions containing mitochondria can be isolated from cells and used to directly assess the effect of recombinant Bax on cytochrome c release. This system demonstrated that mitochondria from MOAP1 knockdown cells are highly resistant to Bax-mediated cytochrome c release .

• Protein depletion and reconstitution assays: A sophisticated approach involves:

  • Treating cells with cycloheximide to deplete short-lived proteins including MOAP1

  • Isolating mitochondria from these cells

  • Testing whether these mitochondria have impaired response to recombinant Bax

  • Reconstituting the system with in vitro-translated MOAP1 to restore functionality

  This approach demonstrated that MOAP1 is a key short-lived mitochondrial protein required for facilitating Bax function .

• Stable expression cell lines: HCT116 and MCF-7 clonal lines stably expressing myc-tagged MOAP1 at levels higher than endogenous MOAP1 but lower than transient overexpression provide a useful model for studying how elevated MOAP1 levels affect mitochondrial sensitivity to apoptotic stimuli .

• Subcellular fractionation: Fractionation studies confirm that MOAP1 accumulation upon apoptotic stimuli treatment occurs primarily in mitochondria-enriched heavy membrane fractions, supporting its function in this organelle .

When designing experiments using these models, researchers should consider that:

• MOAP1 expression levels vary between cell types

• Different apoptotic stimuli may have cell type-specific effects on MOAP1 stabilization

• The interplay between MOAP1 and other mitochondrial proteins involved in apoptosis regulation should be accounted for

These experimental systems offer complementary approaches to understand MOAP1's precise role in regulating mitochondrial functions during apoptosis.

How can researchers distinguish between MOAP1-dependent and MOAP1-independent Bax activation?

Distinguishing between MOAP1-dependent and MOAP1-independent Bax activation requires careful experimental design. The following methodological approaches are recommended:

• RNAi-mediated knockdown with complementation:

  • Knockdown endogenous MOAP1 using siRNA or shRNA

  • Complement with siRNA-resistant MOAP1 variants (wild-type or mutants)

  • Compare Bax activation patterns across different apoptotic stimuli

• Stimulus-specific analysis: Different apoptotic stimuli may rely on MOAP1 to varying degrees. For example, while TRAIL failed to induce Bax activation in SY5Y cells, it effectively triggered MOAP1 up-regulation . Comparative analysis across multiple stimuli can reveal MOAP1-dependent and independent pathways.

• Direct binding assays: Use co-immunoprecipitation or proximity ligation assays to directly measure MOAP1-Bax interaction in response to different stimuli or in different cellular contexts .

• Bax conformational change detection: Employ conformation-specific antibodies (like 6A7) that recognize the activated form of Bax to quantify activation independent of MOAP1 levels .

• Cell-free reconstitution systems: Using purified components to reconstitute Bax activation in vitro with or without MOAP1 can help determine the direct contribution of MOAP1 to Bax activation .

• Mitochondrial assays with isolated components:

  • Isolate mitochondria from MOAP1-depleted cells

  • Test sensitivity to recombinant Bax-mediated cytochrome c release

  • Reconstitute with in vitro-translated MOAP1 or MOAP1 mutants

These approaches, used in combination, can effectively distinguish the specific contribution of MOAP1 to Bax activation in different cellular contexts and in response to various apoptotic stimuli.

What are the current experimental challenges in studying MOAP1-Bax interactions?

Studying MOAP1-Bax interactions presents several experimental challenges that researchers should consider when designing their studies:

• Low abundance of endogenous MOAP1: MOAP1 is a low-abundance protein in most mammalian cells, making detection of endogenous interactions difficult. Sensitive detection methods like combined immunoprecipitation/Western blot analysis are necessary .

• Rapid turnover rate: With a half-life of approximately 25 minutes, MOAP1 levels fluctuate rapidly, potentially affecting interaction dynamics with Bax. Time-course studies need careful synchronization and rapid processing .

• Transient nature of interactions: The MOAP1-Bax interaction may be transient during the apoptotic process, making it challenging to capture consistently. Crosslinking approaches or real-time imaging of tagged proteins may help address this challenge.

• Subcellular localization constraints: While Bax translocates to mitochondria during activation, capturing this dynamic process alongside MOAP1 interaction requires sophisticated live-cell imaging or fractionation approaches .

• Potential conformational requirements: The interaction between MOAP1 and Bax likely requires specific conformational states of both proteins, which may be lost in certain experimental conditions or detection methods.

• Multiple regulatory inputs: Both MOAP1 and Bax are regulated by multiple factors, including other proteins like RASSF1A, making it difficult to isolate direct MOAP1-Bax interactions from the broader regulatory network .

• Cell type variations: Different cell types show varying levels of MOAP1 and potentially different regulatory mechanisms for MOAP1-Bax interactions. MCF-7 cells appear to have higher basal levels of MOAP1 than other mammalian cell types, which may affect interaction studies .

Addressing these challenges requires combining multiple complementary approaches, careful selection of cell models, and consideration of the dynamic nature of the apoptotic process.

What methodologies can be used to study MOAP1's role in cancer progression?

To effectively investigate MOAP1's role in cancer progression, researchers can employ the following methodological approaches:

• Expression analysis in clinical samples:

  • Compare MOAP1 expression levels between normal and tumor tissues

  • Correlate expression with patient outcomes and tumor stages

  • Assess correlation with other apoptotic regulators and known oncogenes/tumor suppressors

• Stable expression and knockdown models:

  • Generate cell lines with stable MOAP1 overexpression or knockdown

  • Compare sensitivity to apoptotic stimuli, as demonstrated with HCT116 and MCF-7 cell lines

  • Assess effects on cell proliferation, migration, invasion, and anchorage-independent growth

• Xenograft tumor models:

  • Implant MOAP1-modulated cancer cells into immunodeficient mice

  • Monitor tumor growth, metastasis, and response to chemotherapeutic agents

  • Analyze tumor samples for apoptotic markers and MOAP1 expression/stability

• Pathway analysis:

  • Investigate the relationship between MOAP1 and the tumor suppressor RASSF1A in cell death receptor and K-Ras-mediated apoptotic signaling

  • Examine MOAP1's interaction with the TRAIL pathway, which is important in cancer therapy

• Drug sensitivity profiling:

  • Test whether MOAP1 expression levels affect sensitivity to chemotherapeutic agents

  • Screen for compounds that stabilize MOAP1 by inhibiting its ubiquitination as potential therapeutic agents

• Genetic correlation studies:

  • Analyze cancer genomics databases for correlations between MOAP1 alterations and cancer progression

  • Examine associations with specific cancer subtypes or clinical features

• MOAP1 regulatory mechanism investigation:

  • Identify E3 ligases responsible for MOAP1 ubiquitination in cancer cells

  • Determine whether these regulatory mechanisms are altered in cancer contexts

By integrating these approaches, researchers can comprehensively assess MOAP1's role in cancer progression and identify potential therapeutic strategies targeting this pathway in cancer treatment.

What are the potential therapeutic applications of targeting MOAP1 stability?

Targeting MOAP1 stability represents a promising therapeutic approach for various diseases, particularly cancers. Several strategic interventions can be considered:

How does MOAP1 interact with other apoptotic regulators beyond Bax?

MOAP1's interactions with the apoptotic machinery extend beyond its well-established relationship with Bax. Current research suggests several important interactions:

• RASSF1A pathway interaction: MOAP1 links the tumor suppressor RASSF1A to Bax activation. RASSF1A specifically connects cell death receptor- and activated K-Ras-mediated apoptotic signaling to Bax activation through binding to MOAP1 . This interaction establishes MOAP1 as a bridge between extrinsic apoptotic signals and the intrinsic mitochondrial pathway.

• TRAIL signaling: MOAP1 is rapidly up-regulated in response to TRAIL treatment in multiple cell lines, suggesting a role in death receptor-mediated apoptosis . The specific molecular interactions between MOAP1 and components of the TRAIL signaling pathway warrant further investigation.

• Mitochondrial protein network: As a mitochondria-enriched protein, MOAP1 likely interacts with other mitochondrial proteins involved in apoptosis regulation. The observation that isolated mitochondria depleted of short-lived proteins become resistant to Bax-mediated cytochrome c release suggests MOAP1 may function within a network of rapidly turned-over mitochondrial proteins .

• Ubiquitin-proteasome system components: MOAP1's rapid degradation involves specific recognition by UPS components. Identifying these interactions could reveal additional regulatory layers for apoptotic signaling .

• BH3-only protein interactions: Since MOAP1 contains a BH3-like motif, it may interact with other Bcl-2 family proteins beyond Bax, potentially influencing the broader apoptotic balance .

Understanding these interactions is methodologically challenging due to MOAP1's low abundance and rapid turnover. Advanced proteomics approaches including proximity labeling techniques, protein crosslinking followed by mass spectrometry, and high-sensitivity co-immunoprecipitation studies would be valuable for mapping MOAP1's complete interactome in the apoptotic pathway.

What is known about the role of MOAP1 in neurological diseases?

While the search results provide limited information on MOAP1's specific role in neurological diseases, several important connections can be highlighted:

• Paraneoplastic neurological syndromes: MOAP1 is also known as PNMA4 (Paraneoplastic Ma antigen 4), suggesting a potential connection to paraneoplastic neurological disorders . These conditions involve immune responses against neuronal antigens that are abnormally expressed in tumors, leading to neurological symptoms.

• Apoptotic regulation in neurological contexts: Given MOAP1's critical role in apoptosis regulation, its dysfunction could contribute to inappropriate neuronal cell death in neurodegenerative diseases or resistance to apoptosis in brain tumors .

• Relationship to other PNMA family members: As part of the Paraneoplastic Ma antigens (PNMA) family , MOAP1/PNMA4 may share functional characteristics with other family members that have been implicated in neurological disorders.

• Expression in neural tissues: The Allen Brain Atlas may contain information about MOAP1 expression patterns in neural tissues, potentially highlighting specific brain regions where MOAP1 function is particularly important .

To advance understanding of MOAP1's role in neurological diseases, researchers should consider:

• Conducting comprehensive expression analyses of MOAP1 in various neurological disease models and patient samples

• Investigating MOAP1 regulation in neuron-specific contexts, considering potential differences from its regulation in other cell types

• Exploring interactions between MOAP1 and neurological disease-associated proteins

• Developing neuron-specific MOAP1 knockout or knockin models to assess behavioral and pathological consequences

This represents an emerging research area that could provide valuable insights into both neurological disease mechanisms and novel therapeutic approaches.

What are the recommended antibodies and reagents for studying MOAP1?

Based on the research methodologies described in the search results, the following technical resources are recommended for MOAP1 research:

Antibodies for MOAP1 detection:

• For sensitive detection of endogenous MOAP1, antibodies suitable for both immunoprecipitation and Western blot analysis are essential due to MOAP1's low abundance

• Antibodies recognizing both native and tagged (e.g., myc-tagged, HA-tagged) versions of MOAP1 for studies using exogenous expression systems

Reagents for studying MOAP1 degradation:

• Proteasome inhibitors: MG132, LLnL, epoxomycin, and lactacystin have all been shown to effectively block MOAP1 degradation

• Cycloheximide for protein stability assays to determine MOAP1's half-life

• HA-tagged ubiquitin expression constructs for ubiquitination assays

Cell models:

• HCT116 and MCF-7, which have been successfully used to generate stable MOAP1-expressing cell lines

• SY5Y cells, which show MOAP1 up-regulation by TRAIL even without Bax activation, providing a useful model for studying MOAP1 regulation independent of apoptotic execution

• H1299 (p53 null) and 293T (p53 mutant) cells for studying p53-independent MOAP1 regulation

Apoptotic stimuli for studying MOAP1 regulation:

• TRAIL: Effective in rapidly upregulating MOAP1 in multiple cell lines

• Thapsigargin: Endoplasmic reticulum stress inducer that enhances MOAP1 levels

• DNA-damaging agents: Etoposide and camptothecin effectively stabilize MOAP1

• Serum withdrawal: Another effective stimulus for MOAP1 upregulation

Expression constructs:

• MOAP1 deletion mutants (particularly the M5 mutant containing amino acids 115-190) for domain mapping studies

• GST-MOAP1 fusion constructs for studying the transferability of degradation signals

• siRNA-resistant MOAP1 expression constructs for knockdown/rescue experiments

Researchers should validate these reagents in their specific experimental systems and consider the rapid turnover of MOAP1 when designing experimental protocols, particularly regarding sample collection timing and processing.

What bioinformatic tools are useful for analyzing MOAP1 in different datasets?

For comprehensive analysis of MOAP1 across different research contexts, the following bioinformatic tools and resources are particularly valuable:

• Ma'ayan Laboratory Harmonizome: This resource indicates that MOAP1 has 4,719 functional associations with biological entities spanning 8 categories, offering a rich dataset for exploring MOAP1's functional relationships .

• Allen Brain Atlas: Contains data on MOAP1 expression patterns in neural tissues, which could be particularly relevant for neurological research .

• Gene expression databases: Resources like GEO (Gene Expression Omnibus) contain datasets that can be analyzed for MOAP1 expression patterns across different tissues, disease states, and experimental conditions.

• Protein interaction databases:

  • STRING (Search Tool for the Retrieval of Interacting Genes/Proteins)

  • BioGRID (Biological General Repository for Interaction Datasets)

  • IntAct molecular interaction database
    These can help identify known and predicted MOAP1 interaction partners.

• Cancer genomics resources:

  • The Cancer Genome Atlas (TCGA)

  • Cancer Cell Line Encyclopedia (CCLE)

  • cBioPortal for Cancer Genomics
    These provide data on MOAP1 alterations, expression, and correlations with clinical outcomes across cancer types .

• Pathway analysis tools:

  • KEGG (Kyoto Encyclopedia of Genes and Genomes)

  • Reactome

  • Gene Ontology enrichment analysis tools
    These help place MOAP1 in broader biological pathways and processes.

• Protein structure prediction tools:

  • AlphaFold or RoseTTAFold for predicting MOAP1 structure

  • Protein-protein docking tools for modeling MOAP1-Bax interactions

• Sequence analysis tools:

  • BLAST for identifying homologs across species

  • Multiple sequence alignment tools for evolutionary conservation analysis

  • Tools for identifying functional motifs, including ubiquitination sites

• Single-cell RNA sequencing analysis platforms:

  • Seurat

  • Scanpy
    These can reveal cell type-specific expression patterns of MOAP1.

When using these tools, researchers should consider MOAP1's alternative names (MAP-1, PNMA4) to ensure comprehensive data retrieval across databases that may use different nomenclature .