Recombinant Dictyostelium discoideum Transmembrane protein 208 homolog (tmem208)

What are the known cellular locations and functions of TMEM208?

TMEM208 is primarily localized to the endoplasmic reticulum (ER) membrane. Studies using fluorescent tagging of TMEM208 homologs have confirmed this localization .

Key cellular functions include:

Additionally, STRING database analysis indicates TMEM208 in D. discoideum has predicted functional partnerships with:

• ATG9 (autophagy-related protein 9) - score: 0.814

• ARSA (ATPase ASNA1 homolog) - score: 0.603

• SEC61a (protein transport protein) - score: 0.540

These interactions suggest broader roles in autophagy and protein trafficking pathways.

How is TMEM208 structurally and functionally conserved across species?

TMEM208 exhibits significant evolutionary conservation:

The functional conservation is most clearly demonstrated by complementation experiments where the expression of reference human TMEM208 in flies fully rescues the loss of D. discoideum TMEM208 , indicating that despite evolutionary distance, the protein's core functions remain preserved. This conservation makes D. discoideum a valuable model organism for studying TMEM208-related human conditions.

What methodologies are most effective for studying TMEM208 function in Dictyostelium discoideum?

Several complementary approaches have proven effective for investigating TMEM208 function:

Gene Manipulation Techniques:

• CRISPR-Cas9 Gene Editing: Researchers have generated CRISPR-induced null alleles by replacing the gene with reporter constructs (e.g., Kozak-GAL4 sequence)

• RNAi-Mediated Knockdown: Useful for studying partial loss-of-function effects

• Creation of GFP-Tagged Alleles: Insertion of GFP sequence (with linkers) between specific amino acids (e.g., between R99 and E100) using CRISPR-mediated homologous recombination

Functional Analysis Methods:

• Rescue Experiments: Testing wild-type and mutant constructs for ability to rescue mutant phenotypes

• Protein-Protein Interaction Assays: Identifying binding partners like Frizzled

• ER Stress Assessment:

  • Western blot analyses for ER stress markers (Bip, p-Eif2α)

  • Immunostaining with anti-Bip antibody

  • Xbp1-GFP reporter assays to monitor ER stress-induced Xbp1 mRNA splicing

Phenotypic Characterization:

• Developmental Assays: Tracking multicellular development

• Lifespan Analysis: Comparing longevity between wild-type and mutant strains

• Cell Polarity Assessments: Examining wing and eye development defects

A comprehensive approach combining these methods has proven most informative, with genetic manipulation followed by functional and phenotypic analyses providing the most complete picture of TMEM208 biology.

How does TMEM208 contribute to cell polarity mechanisms and development?

TMEM208 plays a critical role in planar cell polarity (PCP) through several mechanisms:

Direct Interaction with PCP Components:

• TMEM208 physically interacts with Frizzled (Fz), a key PCP receptor

• This interaction helps maintain proper levels of Fz at the cell membrane

• Loss of TMEM208 results in altered distribution and reduced levels of Fz

Developmental Consequences of TMEM208 Loss:

TMEM208 mutant escapers (rare surviving mutants) exhibit clear PCP defects:

Human Disease Connection:

A human patient with biallelic TMEM208 variants presented with:

• Developmental delay

• Skeletal abnormalities

• Multiple hair whorls (a hallmark of PCP defects)

• Cardiac and neurological issues

These symptoms are consistent with PCP defects observed in mouse models and human patients with other PCP pathway mutations, reinforcing TMEM208's critical role in this developmental process .

What is the relationship between TMEM208 and ER stress, and how can it be experimentally assessed?

TMEM208 plays a role in maintaining ER homeostasis, with its loss leading to mild ER stress. This relationship has been established through several experimental approaches:

Evidence of ER Stress in TMEM208 Mutants:

• Increased Bip Levels: Approximately 1.5-fold increase in Bip (a key ER stress marker) protein levels observed in both wing discs and whole mutants

• Elevated p-Eif2α: Phosphorylated Eif2α levels were higher in TMEM208 mutants compared to controls, indicating activation of the ER stress response

• Enhanced Xbp1 Splicing: Using an Xbp1-GFP reporter system, increased GFP expression was observed in the pouch region of larval wing discs upon TMEM208 knockdown

• Human Patient Fibroblasts: Fibroblasts from a proband with TMEM208 variants also displayed mild ER stress

Experimental Methods to Assess TMEM208-Related ER Stress:

• Western Blot Analysis:

  • Quantification of ER stress markers (Bip, p-Eif2α) in wild-type vs. mutant samples

  • This approach allows for quantitative comparison of protein levels

• Immunostaining:

  • Using anti-Bip antibodies on fixed tissues (e.g., wing discs)

  • Enables visualization of spatial distribution of ER stress

• Reporter Systems:

  • Xbp1-GFP reporter transgene: Allows visualization of Xbp1 splicing, which occurs during ER stress

  • When Xbp1 mRNA is spliced due to ER stress, the GFP becomes in-frame and is expressed

• Co-localization Studies:

  • Immunostaining for GFP-tagged TMEM208 together with ER markers like Calnexin

  • Confirms localization of TMEM208 to the ER

The mechanistic link appears to involve TMEM208's role in protein translocation into the ER, as its absence could lead to accumulation of untranslocated proteins, triggering an ER stress response.

How can TMEM208 dysfunction contribute to developmental disorders, and what experimental models best elucidate these mechanisms?

TMEM208 dysfunction has been directly linked to developmental disorders through both clinical observations and experimental models:

Human Clinical Evidence:

A child with compound heterozygous variants in TMEM208 presented with:

• Global developmental delay

• Skeletal abnormalities

• Multiple hair whorls

• Cardiac issues

• Neurological problems including seizures

These symptoms align with those seen in other PCP-related disorders, suggesting a mechanistic connection.

Experimental Models and Approaches:

Molecular Mechanisms:

• Disrupted Protein Trafficking: TMEM208 is part of the signal-independent pathway for protein translocation into the ER

• PCP Pathway Disruption: TMEM208 physically interacts with Frizzled (Fz) and helps maintain proper Fz levels

• ER Stress: Loss of TMEM208 induces mild ER stress, which can disrupt developmental processes

The most comprehensive approach combines:

• Creation of model organism mutants (especially Drosophila)

• Testing human variants in these models through rescue experiments

• Analysis of patient-derived cells

• Detailed phenotypic characterization across development

This multi-faceted strategy has successfully confirmed TMEM208 mutations as causative for a new developmental disorder by establishing genotype-phenotype correlations across species .

What is the role of TMEM208 in cancer progression and immune evasion?

Research indicates TMEM208 may play important roles in cancer, particularly in head and neck squamous cell carcinoma (HNSCC):

Immune Evasion Mechanisms:

TMEM208 appears to influence tumor immune microenvironment through:

• Negative Correlation with Immune Cell Infiltration:

  • Reduced infiltration of B cells, CD8+ T cells, CD4+ T cells, neutrophils, dendritic cells, T follicular helper cells, NK cells, NKT cells, and mast cells in high TMEM208-expressing tumors

  • Specifically, CIBERSORTx analysis showed significantly reduced infiltration of B cell naive, T cell CD4 memory resting, NK cells resting, NK cells activated, and neutrophils in high expression group

• Correlation with Immune Checkpoint Molecules:

  • Positive correlation with immune checkpoint inhibitors:

    • CD24 (correlation coefficient: positive)

    • CD276 (correlation coefficient: positive)

    • LAG3 (correlation coefficient: positive)

    • HVEM (correlation coefficient: positive)

• Association with Cellular Functions:

  • Positive correlation with ribosomal and mitochondrial functions, ATP biosynthesis

  • Negative correlation with immune system activation and differentiation of immune cells

Experimental Approaches for Studying TMEM208 in Cancer:

• Expression Analysis:

  • TCGA database mining for expression differences between tumor and normal tissues

  • Paired analysis of tumor vs. adjacent normal tissues

  • Immunohistochemical staining to confirm protein-level expression

• Survival Analysis:

  • Kaplan-Meier analysis for OS, PFI, and DSS

  • Cox regression modeling to identify independent prognostic factors

• Immune Correlation Studies:

  • CIBERSORTx analysis of 22 immune cell types

  • TIMER and TISIDB database analyses to examine correlation with immune cell infiltration

  • Correlation analysis with immune checkpoints

These findings suggest TMEM208 could serve as both a prognostic biomarker and a potential therapeutic target in HNSCC, particularly in the context of immunotherapy approaches .

How can Dictyostelium discoideum serve as a model for studying TMEM208-related human diseases?

Dictyostelium discoideum offers several advantages as a model system for studying TMEM208-related human diseases:

Key Advantages of D. discoideum as a Disease Model:

Methodological Approaches Using D. discoideum:

• Humanized D. discoideum Models:

  • Expression of human TMEM208 variants in D. discoideum TMEM208-null background

  • Assessment of functional rescue to determine pathogenicity of human variants

• Cellular Phenotype Analysis:

  • Study of ER stress responses

  • Autophagy pathway analysis (TMEM208 has predicted functional partnership with ATG9)

  • Protein trafficking and localization studies

• Protein Interaction Mapping:

  • Identification of TMEM208 interactors in a simplified cellular context

  • Validation of conserved interactions between D. discoideum and humans

• Drug Screening Platforms:

  • Testing compounds that may alleviate TMEM208-related cellular defects

  • Simplified system allows for more direct interpretation of drug effects

D. discoideum has already proven valuable for studying various human disease-related proteins, including those involved in Alzheimer's disease (such as presenilin proteins), where both proteolytic and non-proteolytic functions were investigated . This precedent suggests D. discoideum could similarly provide insights into TMEM208-related developmental disorders and potentially cancer through investigation of basic cellular mechanisms that are disrupted when TMEM208 function is compromised.

What protein interactions are known for TMEM208 and how can they be experimentally validated?

TMEM208 interacts with several proteins that contribute to its cellular functions:

Key Protein Interactions:

Experimental Methods for Validating Interactions:

• Co-Immunoprecipitation:

  • Precipitate TMEM208 using specific antibodies and identify interacting partners

  • Can be performed with endogenous proteins or tagged versions

  • Western blot analysis for known interactors or mass spectrometry for unbiased discovery

• Proximity Labeling Approaches:

  • BioID or APEX2 fusion proteins to identify proximal proteins in living cells

  • Particularly useful for membrane proteins like TMEM208

• Yeast Two-Hybrid Screening:

  • Can identify direct protein-protein interactions

  • May require optimization for membrane proteins like TMEM208

• Fluorescence Resonance Energy Transfer (FRET):

  • Tag TMEM208 and potential interactors with appropriate fluorophores

  • Detect energy transfer indicating close proximity (<10 nm)

• Bimolecular Fluorescence Complementation (BiFC):

  • Split fluorescent protein approach where each half is fused to potential interacting partners

  • Fluorescence is reconstituted when proteins interact

• Quantitative Assessment of Target Protein Levels:

  • For interactions that stabilize proteins (like TMEM208-Fz)

  • Western blot analysis of Fz levels in wild-type vs. TMEM208 mutant backgrounds

• Recombinant Antibody Approaches:

  • Development of specific recombinant antibodies against D. discoideum proteins

  • Provides reliable tools for immunoprecipitation and detection

The research community has successfully applied several of these approaches to validate TMEM208's interaction with Frizzled, demonstrating both physical interaction and functional consequences of this interaction for Frizzled protein levels . For newly predicted interactions, combining multiple orthogonal approaches would provide the most convincing validation.