Recombinant Human Protein FAM134C (FAM134C)

What is the amino acid sequence and structural characteristics of recombinant human FAM134C protein?

Recombinant human FAM134C protein (1-127 aa) has the following amino acid sequence:
MAEAEGVPTTPGPASGSTFRGRRDVSGSWERDQQVEAAQRALVEVLGPYEPLLSRVQAALVWERPARSALWCLGLNAAFWFFALTSLRLVFLLAFGLMIIVCIDQWKNKIWPEIKVPRPDALDNESW

Structurally, FAM134C contains a reticulon homology domain (RHD) that is critical for its membrane-shaping functions. The RHD structure of FAM134C was modeled using the FAM134B-RHD as a template, with alignments to map topologically equivalent positions including transmembrane segments and amphipathic helices. This modeling was performed using specialized tools like AlignME for scoring hydrophobicity patterns and Modeller for building 3D structures . The protein contains both transmembrane domains and regions that interact with autophagy machinery, particularly through its LC3-interacting region (LIR), which is essential for its biological function .

How does the structure of FAM134C compare to other FAM134 family members?

FAM134C shares structural similarities with other family members (FAM134A and FAM134B), particularly in the reticulon homology domain (RHD). Molecular dynamics simulations reveal distinct conformational dynamics among the family members. When embedded in phospholipid bilayers (POPC), FAM134C shows unique properties compared to FAM134A and FAM134B .

Analysis of the radius of gyration and root mean square fluctuations (RMSF) indicates differences in internal protein dynamics along the RHD. FAM134C shows an intermediate phenotype in terms of ER fragmentation capability compared to FAM134A and FAM134B when overexpressed. While FAM134B is the most potent inducer of ER fragmentation under basal conditions, FAM134C requires stress conditions like nutrient starvation to exhibit significant activity .

What are the optimal expression systems for recombinant human FAM134C protein?

The commercially available recombinant human FAM134C protein (amino acids 1-127) is expressed in E. coli with a His tag . While E. coli provides good yields for this truncated version, researchers should consider the following methodological approaches when designing their own expression systems:

• Expression vectors: For inducible expression in mammalian cells, doxycycline-controlled promoters have been successfully utilized for FAM134C expression. The MSCV iTAP N-FLAG-HA retroviral vector has been documented for generating stable U2OS TRex cell lines .

• Viral delivery systems: Both retroviral and lentiviral systems have been employed for FAM134C expression:

  • Retroviral systems utilize pBABE plasmids co-transfected with packaging plasmid (viral Gag-Pol) and envelope plasmid (VSV-G) in HEK293T cells .

  • Lentiviral systems involve co-transfection with packaging plasmid (pPAX2) and envelope plasmid (pMD2.G) .

• Cell culture conditions: Optimal expression occurs when cells are maintained at 37°C with 5% CO₂ in DMEM medium supplemented with 10% fetal bovine serum and 100 U/ml penicillin and streptomycin .

What purification strategy provides the highest yield and purity for recombinant FAM134C?

For His-tagged FAM134C, imidazole-based elution is the standard approach. The commercially available protein uses 300mM imidazole in the elution buffer . To achieve high purity (≥95% as verified by SDS-PAGE with Coomassie Brilliant Blue staining), researchers should consider:

• Initial capture: Immobilized metal affinity chromatography (IMAC) using Ni²⁺ or Co²⁺ resins for His-tagged proteins.

• Buffer optimization: The final preparation of FAM134C is typically lyophilized from sterile PBS (58mM Na₂HPO₄, 17mM NaH₂PO₄, 68mM NaCl, pH 7.4) with addition of 5% trehalose and 5% mannitol as protectants before lyophilization .

• Reconstitution protocol: For short-term storage, reconstitution at 0.25 μg/μl in 200 μl sterile water is recommended. For longer-term storage, reconstitution with 200 μl 50% glycerol solution is preferred to maintain protein stability .

What is the role of FAM134C in endoplasmic reticulum dynamics?

FAM134C plays a crucial role in ER shape maintenance and homeostasis through several mechanisms:

• ER remodeling: FAM134C is actively involved in ER membrane remodeling, particularly under stress conditions. When overexpressed, FAM134C leads to an intermediate phenotype of ER fragmentation, which becomes more pronounced during nutrient starvation (EBSS treatment) .

• ER-phagy regulation: FAM134C participates in ER-phagy (selective autophagy of the ER) through its LC3-interacting region (LIR). Deletion of this LIR domain (FAM134C ΔLIR) prevents the formation of LC3B- and FAM134C-positive ER fragments during starvation, indicating that the interaction with autophagy machinery is essential for its function .

• ER morphology maintenance: Knockout studies in mouse embryonic fibroblasts (MEFs) demonstrate that Fam134c is essential for maintaining normal ER morphology. Fam134c-/- MEFs exhibit swollen and dilated ER, similar to phenotypes observed in Fam134a-/- and Fam134b-/- cells. This abnormal morphology can be rescued by reconstitution with wild-type Fam134c but not with the LIR mutant (Fam134c ΔLIR) .

How does FAM134C interact with autophagy machinery to facilitate ER-phagy?

FAM134C mediates ER-phagy through a LIR-dependent mechanism:

• LC3 interaction: The LC3-interacting region (LIR) of FAM134C directly binds to LC3B, a key autophagy protein that localizes to autophagosomal membranes. This interaction is essential for targeting ER fragments to autophagosomes .

• Stress-induced activation: Unlike FAM134B, which can induce ER fragmentation under basal conditions, FAM134C requires stressors such as nutrient starvation (EBSS) to become fully activated. Under stress conditions, FAM134C-positive ER fragments become decorated with LC3B, indicating their targeting to the autophagy pathway .

• Flux measurement: ER-phagy flux mediated by FAM134C can be monitored using dual-color reporters such as ssRFP-GFP-KDEL. In this system, GFP fluorescence is quenched in the acidic environment of lysosomes while RFP fluorescence persists, allowing calculation of the RFP/GFP ratio as a measure of ER-phagy flux. Overexpression of FAM134C induces a mild increase in basal ER-phagy flux, which is further enhanced by autophagy activators like Torin1 .

What phenotypes are associated with FAM134C knockout or mutation?

Genetic ablation of FAM134C results in distinct cellular phenotypes:

• ER morphology defects: Fam134c knockout MEFs display swollen and dilated ER, visualized by both electron microscopy and immunofluorescence staining of ER markers such as Climp63 and Canx .

• ER homeostasis disruption: Loss of FAM134C leads to disturbances in ER homeostasis, likely due to impaired ER-phagy and consequent accumulation of ER components. This is evidenced by expanded areas containing ER markers in knockout cells .

• Rescue experiments: Reconstitution of Fam134c knockout MEFs with wild-type Fam134c, but not with the LIR mutant form, restores normal ER morphology. This indicates that the autophagy-dependent function of FAM134C is critical for maintaining ER shape and homeostasis .

What assays are available to measure FAM134C-mediated ER-phagy?

Researchers can employ several complementary techniques to quantify FAM134C-mediated ER-phagy:

• Dual-color reporter assay: The ssRFP-GFP-KDEL reporter system allows for real-time monitoring of ER-phagy flux. This reporter targets to the ER lumen and, upon delivery to lysosomes, exhibits differential quenching of GFP versus RFP fluorescence. The RFP/GFP ratio serves as a quantitative measure of ER-phagy flux. Experimental protocols include:

  • Infection of cells with pCW57-CMV-ssRFP-GFP-KDEL lentivirus

  • Treatment with either DMSO (control), Torin1 (autophagy activator), or Thapsigargin

  • Monitoring of fluorescence using systems like IncuCyte S3

  • Analysis of the RFP/GFP ratio from >1,000 cells across multiple wells

• Immunofluorescence analysis: Co-localization of FAM134C with LC3B and ER markers (like Climp63 and Canx) can be visualized by immunofluorescence microscopy to assess ER fragmentation and autophagy targeting. Quantification typically involves analysis of 150-200 cells per experimental condition .

• Biochemical assessment: Western blotting for ER proteins can indicate changes in their abundance due to enhanced degradation through FAM134C-mediated ER-phagy. Densitometric analysis should include at least three independent blot images .

How can researchers effectively study FAM134C interaction partners?

To investigate proteins that interact with FAM134C, researchers should consider these methodological approaches:

• Proteomics approaches: Mass spectrometry analysis of FAM134C-associated proteins can reveal novel interaction partners. In the studies reviewed, this technique identified significant changes in the proteome upon FAM134C overexpression, particularly following starvation. Pearson's correlation coefficients up to 0.9 between biological replicates indicated high reproducibility .

• Principal component analysis (PCA): This statistical method effectively visualizes global proteome changes in response to FAM134C overexpression and stress treatments. PCA revealed that FAM134C overexpression under basal conditions causes mild proteome changes, with more pronounced effects observed during starvation .

• Protein interaction network analysis: Tools like STRING can be used to generate networks of interacting proteins identified in proteomics experiments. This approach revealed that RTN4B interacts with FAM134C and promotes ER membrane dynamics .

• Functional validation: Identified interactions should be validated through complementary approaches such as co-immunoprecipitation, proximity labeling methods, or fluorescence resonance energy transfer (FRET).

How can researchers differentiate between the functions of FAM134 family members?

Distinguishing the specific roles of FAM134A, FAM134B, and FAM134C requires careful experimental design and data interpretation:

What are the key considerations when analyzing FAM134C molecular dynamics data?

Molecular dynamics (MD) simulations of FAM134C require specific analytical approaches:

• Simulation parameters optimization: For coarse-grained (CG) MD simulations of FAM134C, researchers should:

  • Use MARTINI force field for modeling protein-membrane systems

  • Employ DSSP assignments to generate backbone restraints

  • Embed models into appropriate lipid bilayers (e.g., POPC)

  • Maintain system temperature at 310 K and pressure at 1 atm

  • Use velocity rescaling thermostat and Parrinello-Rahman barostat

• Shape characterization metrics: The radius of gyration (R) computed using all protein CG-beads along the trajectory provides valuable information about FAM134C conformational dynamics. Clustering of conformations using backbone RMSD (with a cutoff of 0.8 nm) can identify long-lived, highly populated conformational states .

• Internal dynamics quantification: Root mean square fluctuations (RMSF) of atomic positions from structures sampled along the trajectory reveal regions of high flexibility, which may correspond to functionally important domains .

• Statistical analysis: For quantitative comparisons, appropriate statistical tests should be applied. The studies reviewed used Student's t-test for comparing experimental conditions, with analysis of 150-200 cells per condition to ensure statistical power .

How does FAM134C contribute to the selective regulation of ER-phagy under different stress conditions?

FAM134C exhibits stress-specific activation patterns that merit detailed investigation:

• Starvation response: FAM134C-mediated ER-phagy is strongly activated by nutrient starvation (EBSS treatment), resulting in increased formation of LC3B-positive ER fragments. This suggests a role in cellular adaptation to nutrient limitation through selective degradation of ER components .

• Downstream effects: Proteomics analysis revealed a cluster of 149 proteins that are downregulated in a FAM134-dependent manner, particularly under starvation conditions. These proteins may represent shared ER-phagy substrates of the FAM134 family and could be investigated to understand the specificity of FAM134C-mediated degradation .

• Regulatory mechanisms: The mechanisms controlling FAM134C activation under stress remain incompletely understood. Research should focus on potential post-translational modifications, conformational changes, or protein-protein interactions that might regulate FAM134C activity in response to specific stressors.

• Comparative stress responses: While starvation has been well-studied, other ER stressors (e.g., thapsigargin, tunicamycin) may differentially activate FAM134C. Systematic comparison of FAM134C activity across various stress conditions would provide valuable insights into its regulatory mechanisms.

What are the implications of FAM134C research for understanding ER-related diseases?

FAM134C's role in ER homeostasis suggests potential involvement in various pathological conditions:

• Neurodegenerative disorders: Given that FAM134B mutations have been associated with hereditary sensory and autonomic neuropathy (HSAN II), researchers should investigate whether FAM134C dysfunction contributes to similar or other neurodegenerative conditions characterized by ER stress.

• ER stress-related diseases: Conditions involving chronic ER stress, such as diabetes, obesity, and certain forms of cancer, might be influenced by alterations in FAM134C-mediated ER-phagy. Researchers could examine FAM134C expression, localization, and activity in disease models or patient samples.

• Therapeutic potential: Modulation of FAM134C activity could potentially alleviate ER stress-related pathologies. Development of small molecules or peptides that enhance or inhibit FAM134C-mediated ER-phagy might have therapeutic applications in diseases characterized by ER dysfunction.

• Compensatory mechanisms: In diseases associated with dysfunction of other FAM134 family members, FAM134C might play a compensatory role. Understanding the extent and limitations of such compensation could inform therapeutic strategies targeting the FAM134 family.