Recombinant Human Protein FAM210B (FAM210B)

What is FAM210B and where is it localized in cells?

FAM210B (Family with sequence similarity 210 member B, also known as C20orf108) is a protein that belongs to the domain of unknown function 1279 (DUF1279) family. Cellular localization studies have confirmed that FAM210B is a mitochondrial membrane protein, although there have been conflicting reports about its precise location. Some studies indicate it is an outer mitochondrial membrane protein , while others have identified it as an inner mitochondrial membrane protein . The protein contains an N-terminal 47 amino acid mitochondrial targeting sequence (MTS) . FAM210B is expressed in various cell types but has particularly high expression in erythroid cells during the later stages of differentiation .

What are the key biological functions of FAM210B?

FAM210B serves multiple critical biological functions:

• Tumor Suppression: FAM210B acts as a cancer progression suppressor in various cancer types including lung adenocarcinoma, ovarian cancer, and breast cancer .

• Erythroid Differentiation: FAM210B plays a crucial role in erythrocyte development and differentiation as a target of GATA-1 transcription factor .

• Metabolic Regulation: FAM210B regulates mitochondrial metabolism, particularly affecting the balance between oxidative phosphorylation and glycolysis .

• Immune Modulation: Recent evidence suggests FAM210B affects immune system function, with its deletion associated with lupus-like symptoms in experimental models .

What experimental models are available for studying FAM210B?

Several experimental models have been developed for FAM210B research:

How does FAM210B regulate mitochondrial metabolism and energy homeostasis?

FAM210B significantly impacts cellular metabolism through multiple mechanisms:

FAM210B interacts with mitochondrial ATP synthase components, particularly subunits alpha (ATP5A) and beta (ATP5B), as demonstrated through affinity purification and co-immunoprecipitation studies . This interaction suggests a direct role in regulating oxidative phosphorylation efficiency.

The loss of FAM210B leads to a distinct metabolic reprogramming characterized by:

• Increased mitochondrial respiratory capacity

• Reduced glycolytic activity

• Downregulation of pyruvate dehydrogenase kinase 4 (PDK4)

This metabolic shift is particularly significant in cancer cells, where FAM210B deficiency promotes a phenotype that enhances invasive properties. Mechanistically, FAM210B appears to regulate the balance between oxidative phosphorylation and glycolysis, with its loss causing cells to favor mitochondrial respiration—a metabolic feature that can promote metastatic potential in certain cancers .

Restoration of PDK4 expression or pharmacological inhibition of mitochondrial respiration in FAM210B-knockdown cancer cells partially reverses their invasive phenotype, confirming the causal relationship between FAM210B-mediated metabolic changes and cellular behavior .

What is the role of FAM210B in cancer progression and how can it be targeted therapeutically?

FAM210B functions as a tumor suppressor in multiple cancer types. The mechanisms through which FAM210B suppresses cancer progression include:

• Activation of IFN-α/β signaling: In lung adenocarcinoma, FAM210B upregulates type I interferon expression, which activates the STAT1/IRF9/IFIT3 axis, leading to inhibition of cancer cell proliferation and migration .

• Metabolic regulation: FAM210B loss promotes metabolic reprogramming that favors cancer metastasis, particularly through the downregulation of PDK4 .

• TGF-β pathway modulation: Reduced FAM210B expression activates TGF-β signaling and subsequent epithelial-mesenchymal transition (EMT)-associated gene expression, promoting invasiveness .

• Interaction with TOM70 (TOMM70): FAM210B functionally interacts with TOM70, a translocase of the outer mitochondrial membrane, which affects its regulatory functions in cancer progression .

Clinical analyses have consistently shown that decreased FAM210B expression correlates with poor survival in cancer patients . This makes FAM210B a potential prognostic marker and therapeutic target.

Potential therapeutic approaches could include:

• Restoring FAM210B expression in cancers where it is downregulated

• Targeting downstream pathways affected by FAM210B loss, particularly PDK4-mediated metabolic effects

• Combining FAM210B-targeted therapies with immune checkpoint inhibitors, given its connection to interferon signaling

What methodologies are most effective for studying FAM210B protein-protein interactions?

Effective methodologies for studying FAM210B protein interactions include:

• Affinity purification coupled with mass spectrometry: This approach successfully identified multiple subunits of mitochondrial ATP synthases as FAM210B-interacting partners in K562 cells expressing His/biotin-tagged FAM210B .

• Co-immunoprecipitation with mitochondrial fractionation: This method confirmed the interaction between FAM210B and endogenous ATP5A and ATP5B in both whole-cell lysates and isolated mitochondrial fractions .

• Proximity labeling techniques: Though not explicitly mentioned in the search results, BioID or APEX2-based proximity labeling would be appropriate for identifying FAM210B's interactome within the mitochondrial membrane environment.

• Yeast two-hybrid screening: This could be used as a complementary approach to identify potential interacting partners.

For researchers studying FAM210B interactions, it is recommended to include both whole-cell and mitochondrial fraction analyses, as FAM210B interactions may occur during protein transit or within the mitochondria. Additionally, using both N- and C-terminal tagged versions of FAM210B can help ensure that tagging does not interfere with specific interactions.

How does FAM210B influence erythroid differentiation and what are the implications for hematological disorders?

FAM210B plays a crucial role in erythroid differentiation:

• FAM210B is identified as a target of GATA-1, a key transcription factor in erythropoiesis, and is induced by erythropoietin .

• Knockout and knockdown studies in human induced pluripotent stem-derived erythroid progenitor (HiDEP) cells showed that FAM210B depletion affects erythroid differentiation, leading to increased frequency of orthochromatic erythroblasts and decreased frequencies of basophilic/polychromatic erythroblasts .

• FAM210B promotes the formation of mitochondrial iron transport complexes by binding with protoporphyrinogen oxidase as a cohesive protein in terminal heme enzyme oligomers, making it necessary for terminal erythrocyte differentiation and mitochondrial iron input .

• FAM210B interacts with multiple subunits of mitochondrial ATP synthetase to regulate erythrocyte differentiation and development by promoting mitochondrial energy metabolism .

The implications for hematological disorders are significant:

• In Fam210b knockout mice, abnormal erythrocyte differentiation and development were observed, with increased CD71+ erythroid cells and elevated levels of reactive oxygen species (ROS) in erythrocytes .

• These changes were associated with the development of lupus-like autoimmunity in approximately 15.68% of knockout mice .

• The connection between FAM210B, erythroid development, and autoimmunity suggests potential roles in various hematological and autoimmune disorders.

Therapeutic targeting of FAM210B could be explored for conditions involving abnormal erythropoiesis or ROS-mediated autoimmunity.

What are the optimal conditions for expressing and purifying recombinant FAM210B protein?

Based on the available research, the following approach is recommended for successful expression and purification of recombinant FAM210B:

• Expression System Selection: Human cell lines such as HEK293T cells are preferred over bacterial systems due to the need for proper folding and potential post-translational modifications of FAM210B as a mitochondrial membrane protein.

• Vector Design Considerations:

  • Include His/biotin tagging for efficient purification as demonstrated in K562 cells

  • Consider the impact of N-terminal vs. C-terminal tags, as the N-terminal contains the mitochondrial targeting sequence

  • For structural studies, construct truncated versions that exclude the mitochondrial targeting sequence (first 47 amino acids)

• Purification Strategy:

  • Two-step purification using affinity chromatography followed by size exclusion

  • For membrane protein purification, include appropriate detergents (e.g., n-dodecyl β-D-maltoside)

  • Consider native conditions to preserve protein-protein interactions

• Verification Methods:

  • Western blotting with anti-FAM210B antibodies

  • Mass spectrometry confirmation

  • Functional assays to verify biological activity

How can researchers effectively measure FAM210B's effects on mitochondrial function?

To comprehensively assess FAM210B's impact on mitochondrial function, researchers should employ multiple complementary approaches:

• Respiratory Analysis:

  • Measure oxygen consumption rate (OCR) using Seahorse XF Analyzer to quantify changes in mitochondrial respiratory capacity

  • Assess extracellular acidification rate (EACR) to determine glycolytic activity

  • Compare basal respiration, maximal respiration, and spare respiratory capacity between FAM210B-modulated and control cells

• Mitochondrial Membrane Potential:

  • Use fluorescent probes like JC-1 or TMRM to assess changes in mitochondrial membrane potential

  • Flow cytometry or confocal microscopy for visualization and quantification

• ROS Detection:

  • Measure reactive oxygen species levels, particularly in erythroid cells where FAM210B knockout increases ROS production

  • Use probes such as DCFDA or MitoSOX Red for detection

• ATP Production:

  • Quantify ATP levels using luminescence-based assays

  • Assess the impact of FAM210B on ATP synthase activity, given its interaction with ATP5A and ATP5B subunits

• Metabolic Profiling:

  • Perform targeted metabolomics focusing on TCA cycle intermediates

  • Measure PDK4 expression and activity as a downstream target of FAM210B

  • Analyze pyruvate dehydrogenase complex activity

• Mitochondrial Dynamics:

  • Evaluate potential changes in mitochondrial morphology, fusion, and fission

  • Assess mitochondrial mass and distribution

What experimental designs best demonstrate FAM210B's role in cancer progression?

Based on the literature, effective experimental designs to investigate FAM210B's role in cancer progression include:

• In Vitro Functional Assays:

  • Cell proliferation assays comparing FAM210B knockdown/knockout vs. control cells

  • Colony formation assays to assess clonogenic potential

  • Migration and invasion assays (Transwell, wound healing) to evaluate metastatic potential

  • 3D spheroid cultures to model tumor growth in a more physiologically relevant environment

• Gene Expression Profiling:

  • RNA-seq analysis comparing FAM210B-modulated cells to identify affected pathways

  • Focus on innate immune-related signaling, particularly IFN-α/β pathways in lung adenocarcinoma

  • Validation of key targets by RT-qPCR and Western blotting

• In Vivo Models:

  • Xenograft models using FAM210B-manipulated cancer cell lines

  • Metastasis models (e.g., tail vein injection) to assess impact on cancer spread

  • Patient-derived xenografts to validate findings in more clinically relevant models

• Rescue Experiments:

  • Restoration of FAM210B expression in knockdown cells to confirm phenotype reversibility

  • PDK4 restoration in FAM210B-depleted cells to assess metabolic rescue

  • Pharmacological inhibition of mitochondrial respiration to reverse invasive phenotypes caused by FAM210B loss

• Clinical Correlation:

  • Analysis of FAM210B expression in patient tumor samples correlated with survival outcomes

  • Immunohistochemistry to assess FAM210B protein levels in primary tumors vs. metastatic sites

What is the relationship between FAM210B and autoimmunity, particularly in systemic lupus erythematosus?

Recent research has revealed an unexpected connection between FAM210B and autoimmunity:

• Knockout Phenotype: Approximately 15.68% of Fam210b knockout mice spontaneously developed lupus-like autoimmunity characterized by skin ulcerations, splenomegaly, increased anti-double-stranded DNA (anti-dsDNA) IgG antibodies, and anti-nuclear antibodies (ANA) .

• Cellular Mechanism: Single-cell sequencing showed that Fam210b is mainly expressed in erythroid cells, and its knockout resulted in abnormal erythrocyte differentiation and development in mouse spleens .

• Immunological Effects:

  • Fam210b knockout led to increased numbers of CD71+ erythroid cells

  • Elevated levels of reactive oxygen species (ROS) were observed in erythrocytes

  • Co-culture of CD71+ erythroid cells with lymphocytes resulted in lymphocyte activation and promoted dsDNA and IgG production

• Potential Therapeutic Implications: FAM210B reduction may serve as a novel key marker that triggers the development of SLE, suggesting it could be a potential therapeutic target for autoimmune conditions .

This emerging research direction suggests FAM210B may have broader physiological roles beyond cancer suppression and erythroid differentiation, potentially linking mitochondrial function to immune regulation.

How does FAM210B interact with the interferon signaling pathway and what are the implications for cancer immunotherapy?

FAM210B has been shown to interact with interferon signaling in several important ways:

• Activation of Type I Interferon Signaling: In lung adenocarcinoma, FAM210B upregulates IFN-α/β expression, leading to activation of the STAT1/IRF9/IFIT3 axis .

• Innate Immune Pathway Regulation: RNA-seq analysis indicates that FAM210B plays a role in regulating innate immune-related signaling pathways in LUAD cells, particularly involving the production of type I interferon .

• Functional Mechanism: FAM210B appears to work in conjunction with TOM70 (TOMM70) to modulate the expression of IFN-α/β and influence proliferative and metastatic phenotypes of cancer cells .

The implications for cancer immunotherapy are significant:

• FAM210B could potentially enhance the efficacy of existing immunotherapies by promoting a more immunogenic tumor microenvironment through interferon signaling

• Restoration of FAM210B expression or function could represent a novel therapeutic approach to boost anti-tumor immune responses

• Combination strategies targeting both FAM210B and immune checkpoint inhibitors might yield synergistic effects

Further research should explore the precise mechanisms by which FAM210B influences the tumor immune microenvironment and whether these effects vary across different cancer types.

What novel detection methods can accurately quantify FAM210B expression in clinical samples?

For clinical applications, accurate quantification of FAM210B is essential. Several methodological approaches show promise:

• Digital Droplet PCR (ddPCR):

  • Provides absolute quantification of FAM210B mRNA

  • Higher sensitivity than traditional qPCR for detecting low-abundance transcripts

  • Particularly valuable for analyzing clinical samples with limited material

• Multiplex Immunohistochemistry (mIHC):

  • Allows simultaneous detection of FAM210B with other markers (e.g., mitochondrial markers, cell-type specific markers)

  • Provides spatial context within tissue architecture

  • Can be automated for high-throughput clinical sample analysis

• Mass Cytometry (CyTOF):

  • Enables single-cell analysis of FAM210B in conjunction with dozens of other proteins

  • Particularly useful for analyzing heterogeneous samples like bone marrow for erythroid lineage studies

  • Can identify rare cell populations with altered FAM210B expression

• Liquid Biopsy Approaches:

  • Analysis of circulating tumor cells or extracellular vesicles for FAM210B expression

  • Potential for non-invasive monitoring of FAM210B status in cancer patients

  • Development of FAM210B as a biomarker for treatment response or disease progression

• Single-cell RNA Sequencing:

  • Provides detailed analysis of FAM210B expression across different cell types

  • Particularly valuable for understanding cellular heterogeneity in complex tissues

  • Has been successfully used to demonstrate FAM210B expression primarily in erythroid cells

How can FAM210B be utilized as a biomarker in cancer prognosis and treatment response?

FAM210B shows significant potential as a biomarker in clinical oncology:

• Prognostic Value:

  • Multiple studies have demonstrated that decreased FAM210B expression correlates with poor survival in cancer patients

  • Low FAM210B expression is associated with enhanced metastasis, making it a potential marker for identifying patients at higher risk for disease progression

• Predictive Biomarker Development:

  • Expression levels could potentially predict response to therapies targeting metabolic pathways

  • Given its role in interferon signaling, FAM210B might predict response to immunotherapies

• Implementation Approaches:

  • Inclusion of FAM210B in multi-gene expression panels for cancer prognosis

  • Development of immunohistochemistry scoring systems for routine pathological assessment

  • Integration with other clinical and molecular parameters for more accurate risk stratification

• Monitoring Strategies:

  • Serial assessment of FAM210B expression to track treatment response

  • Potential for liquid biopsy applications to enable non-invasive monitoring

The clinical utility of FAM210B as a biomarker would benefit from standardized quantification methods and prospective validation in larger patient cohorts across multiple cancer types.

What strategies can enhance FAM210B expression or activity for therapeutic purposes?

Several approaches could potentially enhance FAM210B expression or activity for therapeutic purposes:

• Epigenetic Modulation:

  • FAM210B expression is selectively upregulated by trichostatin A (TSA), a histone deacetylase inhibitor

  • Other epigenetic modulators could be screened for their ability to upregulate FAM210B expression

• Gene Therapy Approaches:

  • Viral vector-mediated delivery of FAM210B to restore expression in cancers where it is downregulated

  • CRISPR activation (CRISPRa) systems targeting the FAM210B promoter to enhance endogenous expression

• Small Molecule Development:

  • Screening for compounds that stabilize FAM210B protein or enhance its activity

  • Design of mimetic peptides that could reproduce key functional domains of FAM210B

• Targeting Upstream Regulators:

  • Enhancing GATA-1 activity in erythroid cells could increase FAM210B expression

  • Identifying and modulating other transcription factors that regulate FAM210B

• Combination Strategies:

  • Combining FAM210B-enhancing therapies with metabolic inhibitors to synergistically affect cancer cell metabolism

  • Using FAM210B enhancement in conjunction with immunotherapies to boost interferon signaling effects

When developing such strategies, researchers should consider cell type-specific effects, as FAM210B functions may vary between cancer cells and normal cells, particularly those of the erythroid lineage.