OCIAD2 Human

What is the evolutionary relationship between OCIAD1 and OCIAD2?

OCIAD2 neighbors OCIAD1/Asrij in most vertebrate genomes, with evidence suggesting these genes emerged through tandem gene duplication somewhere between the Ordovician and Silurian eras. The two genes likely share a common ancestor, with OCIAD2 being a smaller protein sharing homology with the N-terminal region of OCIAD1 . Comparative genomic analyses across vertebrate species can help elucidate conservation patterns of these genes and their potential functional divergence.

What structural elements are critical for OCIAD2 function?

Structure prediction programs, protein disruption studies, biochemical and functional assays have revealed a double helical motif in the OCIA domain that is necessary and sufficient for OCIAD2's proper localization, protein interactions, and STAT3 activation . Research using targeted mutagenesis of this domain can help identify specific amino acid residues critical for these functions.

Where is OCIAD2 predominantly localized within cells?

OCIAD2 demonstrates dual localization, being found in both early endosomes and mitochondria . In lung adenocarcinoma cells, OCIAD2 is predominantly localized at the mitochondrial membrane . Confocal microscopy using fluorescent tags such as FLAG-OCIAD2 co-stained with MitotrackerTM Orange confirms this mitochondrial localization .

What molecular mechanisms facilitate OCIAD2 import into mitochondria?

Despite lacking a typical mitochondrial presequence, OCIAD2 requires the TIM23 complex for import into mitochondria. This unusual import mechanism was demonstrated through in vitro import assays using radioactive [35S]-OCIAD2 synthesized in rabbit reticulocyte lysate. Import was inhibited in mitochondria lacking TIMM23 but unaffected in control cells or those depleted of TIM22 translocase . This suggests OCIAD2 may contain non-canonical internal targeting signals for mitochondrial import.

How does OCIAD2 expression vary across tissues?

OCIAD2 expression is higher in mouse kidney, liver, and brain relative to other tissues . In pathological conditions, OCIAD2 shows higher expression in human lung adenocarcinoma tissues or cell lines compared to normal lung tissue or immortalized normal bronchial epithelial cells . Researchers should consider tissue-specific expression patterns when designing experiments and interpreting results.

What signaling pathways regulate OCIAD2 expression?

TGF-β signaling induces OCIAD2 expression through the pathway TGF-β1→TGF-βR1→SMAD2/3→SMAD4/AR→OCIAD2, with evidence suggesting that androgen receptor (AR) serves as the transcription factor mediating this response . This pathway connection has important implications for understanding OCIAD2's role in cancer development and progression.

How does OCIAD2 contribute to mitochondrial respiratory complex assembly?

OCIAD2 functions as an assembly factor for dimeric complex III (CIII₂) in the mitochondrial inner membrane. Complete knockout of OCIAD2 using CRISPR/Cas9 gene editing in HEK293 cells results in:

• Abnormal mitochondrial morphology

• Substantial decrease of both CIII₂ and supercomplex III₂+IV

• Reduction in CIII enzymatic activity

This identification of OCIAD2 as a protein required for assembly of functional CIII₂ provides new insight into electron transport chain biogenesis.

How does OCIAD2 deficiency affect mitochondrial bioenergetics?

Mitochondria lacking OCIAD2 show:

• Decreased oxygen consumption driven by pyruvate, malate, and glutamate (CI substrate electron donors)

• Decreased oxygen consumption driven by succinate and glycerol-3-phosphate (CIII substrate electron donors)

• No alteration in CIV activity

• Decreased enzymatic activity specifically for CIII

What is OCIAD2's role in lung adenocarcinoma progression?

OCIAD2 shows significantly higher expression in invasive lung adenocarcinoma than in lung adenocarcinoma in situ, with abnormal expression associated with poorer patient prognosis . Experimental suppression of OCIAD2 leads to:

• Downregulation of cellular growth, proliferation, migration, and invasion

• Upregulation of mitochondria-related apoptosis

• Decrease in mitochondrial membrane potential

• Release of cytochrome c

These findings suggest OCIAD2 inhibits mitochondria-initiated apoptosis, thus promoting lung cancer cell survival.

What molecular pathways are altered by OCIAD2 suppression in cancer cells?

Transcriptional profiling using RNA sequencing revealed 137 genes whose expression was commonly altered after OCIAD2 knockdown in three lung adenocarcinoma cell lines (A549, HCC827, and PC9). Pathway enrichment analysis of these genes demonstrated significant enrichment in:

• Apoptotic signaling pathways

• Endoplasmic reticulum (ER) stress pathways

Correlation analysis using LinkedOmics database (515 LUAD cases) showed OCIAD2 was:

• Positively correlated with mitochondrial electron chain components (NDUFA1, SDHB, UQCRQ, ATP5I)

• Positively correlated with mitochondrial translation and homeostasis genes (MRPL54, c19orf70)

• Negatively correlated with proapoptotic initiators (APAF-1, FOXO3, BCL2L11, TP53INP1)

• Negatively correlated with ER stress response genes (ATF6, ERN1)

How does OCIAD2 participate in JAK/STAT signaling?

OCIAD2 interacts with Asrij and STAT3, and knockout/overexpression studies demonstrate that OCIAD2 is essential for STAT3 activation and cell migration, potentially contributing to tumor metastasis . Given the importance of JAK/STAT signaling in development and disease, understanding OCIAD2's regulatory role in this pathway is critical for interpreting its clinical significance.

What protein interaction networks involve OCIAD2?

Proteomic analyses using SILAC (Stable Isotope Labeling by Amino Acids) and subsequent mass spectrometry have identified numerous mitochondrial proteins that interact with OCIAD2 . Among the 180 mitochondrial proteins identified through FLAG-based enrichment, components of respiratory complexes I, III, and IV were significantly represented . These interactions support OCIAD2's role in electron transport chain assembly and function.

What are effective approaches for manipulating OCIAD2 expression in experimental models?

Several validated approaches can be employed:

• siRNA knockdown: Multiple siRNA sequences have been validated for OCIAD2 suppression, with siOCIAD2-I showing the most effective suppression of cellular proliferation

• CRISPR/Cas9 gene editing: Successful OCIAD2-KO has been generated by targeting the second exon of the gene, resulting in a 66-nucleotide deletion (1_66del) encoding the N-terminal part of OCIAD2

• Overexpression studies: FLAG-tagged OCIAD2 constructs have been used successfully for localization and interaction studies

How can researchers effectively assess OCIAD2's impact on mitochondrial function?

Multiple complementary approaches should be employed:

• Oxygen Consumption Rate (OCR) measurements in intact cells to maintain cellular environment

• Analysis of respiratory complex activities using protocols described in Hofhaus et al. (1996) and Doerrier et al. (2018)

• Assessment of enzymatic activities of uncoupled complexes I, III, and IV

• Transmission Electron Microscopy (TEM) for evaluating mitochondrial morphology and cristae formation

• Analysis of mitochondrial membrane potential using appropriate fluorescent probes

What advanced techniques can identify OCIAD2 protein interactions?

For comprehensive identification of OCIAD2 interacting partners:

• Differential SILAC labeling followed by immunoprecipitation and mass spectrometry

• Filtering of identified proteins using the Integrated Mitochondrial Protein Index (IMPI) reference set from MitoMiner database

• Co-immunoprecipitation assays to validate specific interactions

• Proximity labeling approaches such as BioID or APEX2 to identify proteins in close spatial proximity to OCIAD2

How might OCIAD2's mitochondrial function relate to its role in cancer progression?

While OCIAD2 is essential for mitochondrial complex III assembly and function , it also inhibits mitochondria-initiated apoptosis in cancer cells . Research integrating these seemingly divergent functions could provide insights into how cancer cells reprogram mitochondrial metabolism. Experimental approaches might include metabolic flux analysis in OCIAD2-manipulated cancer and non-cancer cells under various stress conditions.

Could OCIAD2 serve as a therapeutic target in cancer?

Given OCIAD2's role in promoting cancer cell survival through inhibition of mitochondria-initiated apoptosis , development of targeted inhibitors might enhance cancer cell sensitivity to treatment. Research should investigate whether OCIAD2 inhibition would have differential effects on normal versus cancer cells, particularly regarding mitochondrial function and cell survival pathways.