Recombinant Human Fetal and adult testis-expressed transcript protein (FATE1)

What is the tissue distribution pattern of FATE1?

FATE1 (Fetal and Adult Testis Expressed 1) shows a highly specific expression pattern. It is predominantly expressed in testis, with significant expression also detected in adrenal gland. Lower expression levels can be found in lung, heart, kidney, and whole brain . This restricted expression pattern in normal tissues contrasts with its overexpression in various cancer types, making it an important cancer-testis antigen (CTA) . Researchers investigating FATE1 should consider this tissue distribution when designing experiments, particularly when selecting appropriate control tissues for expression studies.

What are the key molecular characteristics of FATE1?

FATE1 is a 21-kDa protein encoded by a gene mapped to chromosome Xq28 . The protein consists of 183 amino acids and contains specific structural domains including:

• A C-terminal domain (amino acids 125-183) that directs its mitochondrial localization

• A predicted transmembrane segment in the C-terminal region

• Coiled-coil domains that mediate protein-protein interactions

• Multiple stretches of basic residues that contribute to mitochondrial targeting

FATE1 belongs to the Miff protein family and shares structural similarity with Mff (mitochondrial fission factor) in its C-terminal domain, although it lacks the N-terminal domain necessary for Drp1 interaction that is present in Mff .

What are the recommended protocols for detecting FATE1 protein expression?

Detection of FATE1 requires specific methodology due to its restricted expression pattern and subcellular localization. Based on validated approaches, researchers should consider:

Western Blot Analysis:

• Recommended dilution: 1:500-1:1000

• Expected molecular weight: 21 kDa

• Positive controls: Human testis tissue, mouse testis tissue

Immunofluorescence:

• Use paraformaldehyde fixation (typically 4%) followed by permeabilization

• Co-staining with mitochondrial markers (TOM20, HSP60) and ER markers is recommended for localization studies

• Confocal microscopy is preferred for precise subcellular localization analysis

Subcellular Fractionation:

• The Wieckowski procedure is recommended for separating crude mitochondria, ER, pure mitochondria, and MAM fractions

• FATE1 should be detectable in crude mitochondria, ER, and MAM fractions but not in pure mitochondrial fractions

How can recombinant FATE1 be optimally produced and purified?

Production of recombinant FATE1 requires careful consideration of expression systems and purification strategies:

Expression Systems:

• Cell-free protein synthesis (CFPS) systems have been successfully used

• HEK-293 cells expression system provides proper folding and post-translational modifications

• Wheat germ-based systems can be used for smaller fragments

Purification Tags:

• Strep-Tag, His-tag, and GST fusion systems have all been validated

• One-step Strep-tag purification has been effective for CFPS-expressed FATE1

Purification Quality:

• Expected purity: >70-80% as determined by SDS-PAGE, Western Blot, and analytical SEC (HPLC)

• Storage recommendations: Aliquot and store at -20°C in buffer containing glycerol (typically 50%) to maintain stability

How can researchers accurately determine FATE1's subcellular localization?

FATE1's precise subcellular localization is critical for understanding its function. Methodological approaches should include:

Immunofluorescence Co-localization:

• Co-stain with established markers including:

  • Mitochondrial markers: TOM20, HSP60

  • ER markers: SERCA2, calnexin

  • MAM markers: VDAC1, Sigma-1 receptor (S1R)

• Calculate Pearson's correlation coefficient for quantitative assessment of co-localization

Subcellular Fractionation:

• Use the Wieckowski method to isolate:

  • Crude mitochondria

  • ER fraction

  • Pure mitochondria

  • MAM fraction

• Confirm fraction purity using established markers for each compartment

Electron Microscopy:

• Immunoelectron microscopy has confirmed that FATE1 is associated with the mitochondrial surface

• This technique can provide nanometer-scale resolution of protein localization

What methodologies are used to study FATE1's role in ER-mitochondria communication?

FATE1 functions at the interface between ER and mitochondria, regulating organelle communication. Key methodological approaches include:

ER-Mitochondria Distance Measurement:

• Confocal microscopy with ER and mitochondrial markers

• Electron microscopy for high-resolution analysis

• Quantitative measurement of the distance between organelles using image analysis software

Calcium Transfer Assays:

• Measure mitochondrial calcium uptake following ER calcium release using:

  • Genetically encoded calcium indicators targeted to mitochondria

  • Histamine stimulation to induce ER calcium release

  • Fluorescent calcium indicators (Fluo-4, Rhod-2)

Protein-Protein Interaction Studies:

• Co-immunoprecipitation to identify FATE1 interactors at the ER-mitochondria interface

• Proximity ligation assays to visualize and quantify interactions in situ

• Mass spectrometry to identify novel interaction partners

How does FATE1 mechanistically regulate mitochondrial morphology and fusion?

FATE1 has been shown to promote mitochondrial hyperfusion, a phenomenon with implications for cancer cell survival. Research methodologies should include:

Mitochondrial Morphology Assessment:

• Live-cell imaging of mitochondrial networks using mitochondria-targeted fluorescent proteins

• Quantitative analysis of mitochondrial parameters (length, interconnectivity, aspect ratio)

• Time-lapse imaging to capture dynamic changes in mitochondrial morphology

Fusion Protein Interaction Studies:

• Co-immunoprecipitation with mitochondrial fusion proteins (Mfn1, Mfn2)

• Reconstitution experiments in Mfn1/Mfn2 knockout cells

• Analysis suggests a specific role for Mfn2, but not Mfn1, in FATE1-mediated effects

Response to Fusion/Fission Stimuli:

• Compare mitochondrial fragmentation in response to TNF and valinomycin between FATE1-expressing and control cells

• Measure fusion/fission events in real-time using photoactivatable mitochondrial markers

What experimental approaches can measure FATE1's impact on cancer cell apoptosis resistance?

FATE1 contributes to cancer cell survival through multiple mechanisms. Experimental strategies include:

Apoptosis Assays:

• Caspase-3/7 activity measurements in cells with modulated FATE1 expression

• TUNEL assay for quantitative assessment of apoptotic cells

• Compare responses to different apoptotic stimuli:

  • H₂O₂ and C2-ceramide (Ca²⁺-dependent apoptosis)

  • Staurosporine (Ca²⁺-independent apoptosis)

  • Chemotherapeutic agents (mitotane, paclitaxel)

Gain/Loss-of-Function Studies:

• Doxycycline-inducible FATE1 expression systems

• siRNA-mediated knockdown for loss-of-function studies

• Rescue experiments with wild-type vs. mutant FATE1

Pro-apoptotic Protein Degradation:

• Measure levels of pro-apoptotic BH3-only protein Bik in cells with modulated FATE1 expression

• Assess protein stability and half-life through cycloheximide chase experiments

How can researchers investigate the clinical significance of FATE1 expression in cancer specimens?

Understanding the relationship between FATE1 expression and clinical outcomes requires specialized approaches:

Tissue Microarray Analysis:

• Immunohistochemical detection of FATE1 in tumor tissues

• Scoring systems for expression levels (low/medium/high)

• Correlation with clinicopathological parameters

Survival Analysis:

Response to Therapy:

• Comparison of FATE1 expression levels in treatment-responsive vs. resistant tumors

• In vitro drug sensitivity testing with modulated FATE1 expression

• FATE1 has been linked to resistance to mitotane in adrenocortical carcinoma and paclitaxel in non-small-cell lung cancer

What experimental approaches can determine structure-function relationships in FATE1?

Understanding which domains of FATE1 mediate specific functions is crucial for mechanistic studies:

Domain Mapping:

• Generate a battery of FATE1 mutants with specific domain deletions or mutations

• Key constructs to consider:

  • C-terminal domain (aa 125-183) - sufficient for mitochondrial targeting

  • N-terminal domain (aa 1-124) - shows nuclear localization

  • Basic residue mutations (RR138-139AA, RRR146-147-149AAA, RHR159-160-161AAA)

  • Coiled-coil disrupting mutations (L151D)

Structure-Function Analysis:

• Express mutant constructs as GFP fusion proteins

• Assess localization by fluorescence microscopy

• Measure functional outcomes:

  • ER-mitochondria distance

  • Calcium transfer

  • Apoptosis resistance

Protein-Protein Interaction Mapping:

• Identify which domains mediate interactions with known partners:

  • EMD (emerin)

  • Mic60/mitofilin

  • GRP75

What approaches can determine the regulation of FATE1 expression in normal and cancer cells?

Understanding FATE1 regulation provides insights into its role in cancer:

Transcriptional Regulation:

• Promoter analysis using reporter assays

• ChIP assays to confirm transcription factor binding

• Steroidogenic factor-1 (SF-1) has been identified as a key regulator of FATE1 expression in adrenocortical carcinoma cells

Expression Analysis:

• qRT-PCR for mRNA expression

• Western blot for protein levels

• Immunohistochemistry in tissue samples

• Correlate expression with cellular states (proliferation, stress response)

Epigenetic Regulation:

• DNA methylation analysis of the FATE1 promoter

• Histone modification assessment

• Effects of epigenetic modifiers (HDAC inhibitors, DNA methyltransferase inhibitors)

What are the critical quality control parameters for recombinant FATE1 protein production?

Ensuring high-quality recombinant FATE1 requires rigorous quality control:

Purity Assessment:

• SDS-PAGE with Coomassie staining (expected purity >80%)

• Western blot with anti-tag antibodies

• Analytical size exclusion chromatography (HPLC)

Functional Validation:

• Binding assays with known interaction partners

• Circular dichroism to confirm proper folding

• Activity assays based on known FATE1 functions

Storage Stability:

• Aliquot and store at -20°C

• Avoid repeated freeze/thaw cycles

• Include glycerol (typically 50%) in storage buffer

• Test stability after various storage times

What cell lines and model systems are most appropriate for FATE1 functional studies?

Selection of appropriate experimental systems is crucial:

Cell Line Models:

• H295R adrenocortical carcinoma cells (express FATE1 endogenously)

• H295R/TR cell lines with doxycycline-inducible FATE1 expression

• HeLa cells for transient transfection studies

• Non-small-cell lung cancer cell lines (show dependence on FATE1 for chemoresistance)

Knockout/Knockdown Systems:

• siRNA-mediated knockdown (validated sequences available)

• CRISPR/Cas9-mediated knockout

• Doxycycline-inducible shRNA systems

Animal Models:

• Consider tissue-specific expression pattern when designing in vivo studies

• Focus on cancer xenograft models with modulated FATE1 expression

• Assess effects on tumor growth, metastasis, and therapy response