EEF1D Human

What are the structural and functional characteristics of EEF1D in humans?

EEF1D is a subunit of the eukaryotic translation elongation 1 complex located on chromosome 8q24.3. The human EEF1D gene produces at least four protein isoforms through alternative splicing, with three shorter isoforms (NP_001951, NP_001123528, and NP_001182132) containing 281, 257, and 262 amino acids respectively, and one long isoform (NP_115754) containing 647 amino acids .

Structurally, the shorter isoforms contain a nucleotide exchange domain and six leucine zipper motif residues in the N-terminal domain, while the long isoform contains an additional 367 amino acids at the N-terminus with a nuclear localization signal (NLS) . The shorter isoforms are anchored to the endoplasmic reticulum in the cytoplasm and function as translation elongation factors, catalyzing the exchange of GDP to GTP on EEF1A to facilitate translation elongation .

The long isoform is primarily expressed in brain and testis, functioning as a transcription factor containing heat shock elements (HSE) and playing critical roles in controlling neuronal gene expression, neuron development, and synaptic strength . This tissue-specific expression pattern and functional diversity highlight EEF1D's multifaceted roles in cellular processes beyond canonical translation.

How is EEF1D expression regulated during development and in different tissues?

EEF1D exhibits distinct expression patterns across human tissues, with the three shorter isoforms expressed ubiquitously while the long isoform shows highly selective expression in brain and testis . This tissue-specific expression suggests specialized regulatory mechanisms controlling EEF1D isoform production.

During embryonic development, EEF1D expression follows a complex temporal pattern. Studies in sea urchin embryos revealed that EEF1D mRNA is present in unfertilized eggs and early embryos until 6 hours post-fertilization, after which transcript levels drop significantly during rapid cleavage and blastocyst stage . Expression then suddenly increases around 30 hours (gastrula stage) and continues to rise at 48 and 72 hours post-fertilization . This dynamic expression pattern correlates with developmental stages requiring heightened protein synthesis and cell differentiation.

In pathological conditions, EEF1D shows aberrant expression, being significantly overexpressed in numerous malignant tumors including liver cancer, esophageal cancer, small cell lung cancer, medulloblastoma, and ovarian cancer . In ovarian tissue specifically, EEF1D shows notably high expression compared to other tissues . This aberrant expression in cancer tissues suggests that EEF1D regulation is altered during malignant transformation, potentially contributing to disease progression.

What signaling pathways does EEF1D interact with beyond its role in translation?

EEF1D participates in several key signaling pathways beyond its canonical role in translation elongation. In cancer cells, EEF1D activates MAPK and PI3K/Akt signaling pathways by activating small G proteins (Ras) . This activation enhances cell repair mechanisms and anti-apoptotic capacity, contributing to drug resistance in cancer cells. Experimental evidence from ovarian cancer models shows that knockdown of EEF1D reduces phosphorylation of Akt, suggesting direct modulation of this pro-survival pathway .

Additionally, EEF1D promotes the expression of heat shock proteins through activation of heat shock promoter elements, inhibiting apoptosis induced by multiple chemotherapeutic agents . This function is particularly relevant to its role in cancer drug resistance.

EEF1D also influences apoptotic pathways by regulating the balance between pro-survival and pro-apoptotic proteins. Knockdown of EEF1D in ovarian cancer cells decreases expression of anti-apoptotic factors (p-Akt, Bcl-2) while increasing pro-apoptotic proteins (Bax, cleaved caspase-3) . Furthermore, EEF1D affects DNA damage repair pathways by influencing ERCC1 expression, a critical protein involved in nucleotide excision repair .

These diverse signaling interactions position EEF1D as a multifunctional protein that integrates translation, cell survival, and stress response pathways, explaining its importance in both normal physiology and disease states.

What mechanisms underlie EEF1D's contribution to cancer progression and drug resistance?

EEF1D contributes to cancer progression and drug resistance through several sophisticated mechanisms. In multiple cancer types, including liver, esophageal, lung, and ovarian cancers, EEF1D is significantly upregulated and associated with more aggressive disease phenotypes . This overexpression appears to promote cancer cell survival and treatment resistance.

In ovarian cancer, EEF1D mediates cisplatin (DDP) resistance through activation of the PI3K/AKT survival pathway, inhibition of apoptosis via modulation of the Bcl-2/Bax ratio, and enhancement of DNA damage repair capacity through ERCC1 upregulation . Experimental evidence from both cell lines and animal models demonstrates that reducing EEF1D expression via siRNA or CRISPR/Cas9 significantly increases cancer cell sensitivity to cisplatin, enhances drug-induced apoptosis, and inhibits tumor growth .

The molecular changes associated with EEF1D manipulation in drug-resistant cells include decreased phosphorylation of Akt, reduced expression of the anti-apoptotic protein Bcl-2, increased levels of the pro-apoptotic protein Bax, enhanced cleaved caspase-3 levels, and decreased expression of the DNA repair protein ERCC1 . These changes collectively shift the cellular balance toward apoptosis and away from survival and repair pathways that contribute to drug resistance.

Importantly, EEF1D knockdown increased cisplatin sensitivity in primary ovarian cancer cells from patients with progressive disease/stable disease (PD/SD), suggesting clinical relevance of these findings . This positions EEF1D as both a potential biomarker for predicting treatment response and a therapeutic target for overcoming drug resistance in cancer.

How does EEF1D contribute to neurological disorders, particularly autoimmune cerebellar ataxia?

EEF1D, especially its brain-specific long isoform, plays crucial roles in neurological health and disease. Mutations in the EEF1D gene have been identified as causes of several neurodevelopmental disorders, highlighting its importance in proper brain development and function . The long isoform (EEF1BδL) controls neuronal gene expression, neurodevelopment, and synaptic strength, with its selective expression in the brain suggesting specialized neurological functions .

A groundbreaking discovery is the identification of novel autoantibodies against EEF1D in patients with autoimmune cerebellar ataxia (ACA) . In a study of patients with cerebellar ataxia of unknown cause who tested positive with tissue-based indirect immunofluorescence assay (TBA) on rat cerebellum but negative for known neural autoantibodies, EEF1D was identified as the target antigen through tissue-immunoprecipitation combined with mass spectrometric analysis .

Two patients with anti-EEF1D autoantibodies presented with similar clinical manifestations of cerebellar ataxia and showed response to immunotherapy, though cerebellar atrophy that occurred before treatment appeared irreversible . Notably, symptoms worsened in one patient after tapering of pulse corticosteroid therapy, suggesting the need for sustained immunosuppression .

The specificity of anti-EEF1D as a biomarker is supported by the finding that all 30 healthy donors and 15 connective tissue disease patients without neurological disorders tested negative for these autoantibodies . This suggests that anti-EEF1D could serve as a specific biomarker for a subset of autoimmune cerebellar ataxias, potentially enabling earlier diagnosis and treatment before irreversible cerebellar damage occurs.

What functional distinctions exist between the long and short isoforms of EEF1D?

The long and short isoforms of EEF1D exhibit remarkable structural and functional divergence. The long isoform (EEF1BδL, 647 amino acids) contains an additional 367 amino acids at the N-terminus compared to the short isoforms, including a nuclear localization signal (NLS) and heat shock elements (HSE) . In contrast, the short isoforms (281, 257, and 262 amino acids) contain a nucleotide exchange domain and six leucine zipper motif residues in the N-terminal domain .

This structural differentiation correlates with distinct expression patterns: the long isoform is selectively expressed in brain and testis, while the short isoforms are ubiquitously expressed across tissues . Their subcellular localization also differs, with the long isoform found in both cytoplasm and nucleus, while short isoforms are anchored to the endoplasmic reticulum in the cytoplasm .

Functionally, the long isoform acts as a transcription factor containing heat shock elements that controls neuronal gene expression, regulates neuron development, and modulates synaptic strength . The short isoforms primarily serve as translation elongation factors, forming a complex with EEF1Bα, EEF1Bγ, and valine-tRNA synthetase to catalyze the exchange of GDP to GTP on EEF1A and promote the translation process .

These isoforms are associated with different pathologies: the long isoform is linked to neurodevelopmental disorders, with its deletion in mice causing seizures and abnormal behaviors , while the short isoforms are more commonly associated with cancer progression and drug resistance . This functional dichotomy explains how EEF1D can perform both ubiquitous housekeeping roles in translation and specialized functions in specific tissues like the brain.

What are the optimal techniques for investigating EEF1D expression and function?

Researchers investigating EEF1D should employ a comprehensive toolbox of techniques to analyze its expression and function effectively. For expression analysis, RT-PCR and qPCR are essential for quantifying EEF1D mRNA levels and distinguishing between isoforms, while western blotting using isoform-specific antibodies provides protein-level confirmation . Immunohistochemistry and immunofluorescence techniques allow visualization of tissue distribution and subcellular localization, with tissue-based indirect immunofluorescence assay (TBA) on rat cerebellum sections being particularly useful for detecting autoantibodies against EEF1D .

For functional manipulation, RNA interference using siRNA provides an effective method for transient knockdown of EEF1D gene expression, as demonstrated in SKOV3 and SKOV3/DDP ovarian cancer cells . For permanent knockout, CRISPR/Cas9 gene editing has been successfully employed with PCR primers (F: 5′-GGTTGTCCCTAGGACTGTGAG-3′, R: 5′-GCCCCAGGAAAGACAAAAACT-3′) used to amplify the edited region for validation through Sanger sequencing and western blotting .

Protein-protein interaction analysis can be performed using co-immunoprecipitation and tissue-immunoprecipitation (TIP) combined with mass spectrometric analysis, which successfully identified EEF1D as a target antigen in autoimmune cerebellar ataxia . For detecting autoantibodies against EEF1D, recombinant cell-based indirect immunofluorescence assay (CBA) using HEK293 cells overexpressing His-tagged human full-length EEF1D has proven effective .

In vivo models, including xenograft mouse models with subcutaneous inoculation of cancer cells and transgenic mouse models with EEF1D deletion, provide systems for studying EEF1D's role in tumor growth and neurological function respectively . Clinical sample analysis, including collection of patient serum and CSF for autoantibody detection and primary cell isolation for functional studies, bridges the gap between laboratory findings and clinical relevance .

What protocols are most effective for EEF1D knockdown or knockout in experimental models?

Effective manipulation of EEF1D expression requires careful methodological consideration based on research objectives. For siRNA-mediated knockdown, target sequence selection should consider all relevant isoforms, with transfection performed when cells reach 60-70% confluence using lipid-based reagents like Lipofectamine . Knockdown efficiency should be assessed at 24-72 hours post-transfection using qRT-PCR for mRNA levels and western blot for protein reduction.

CRISPR/Cas9-mediated knockout offers a more permanent approach, with guide RNA design targeting early exons common to all isoforms for complete knockout or unique exons for isoform-specific targeting . Delivery can be achieved through plasmid-based systems or lentiviral transduction for stable cell lines. Single-cell isolation and expansion followed by PCR amplification of the target region (using primers: F: 5′-GGTTGTCCCTAGGACTGTGAG-3′, R: 5′-GCCCCAGGAAAGACAAAAACT-3′) allows for validation through TA cloning and Sanger sequencing . Off-target analysis should include computational prediction of potential off-target sites followed by PCR and Sanger sequencing of these regions.

For isoform-specific targeting, researchers should design siRNAs or gRNAs targeting unique regions of short isoforms or the N-terminal region unique to the long isoform . Rescue experiments involving overexpression of wild-type or mutant EEF1D in knockout cells can help establish causality and specificity of observed phenotypes.

Functional validation assays should be tailored to the research question. For cancer studies, cell viability assays, apoptosis assays, drug sensitivity testing, and xenograft models are appropriate . For neurological studies, neuronal differentiation assays, electrophysiology for synaptic strength assessment, and behavioral testing in animal models provide relevant readouts .

Experimental design should include multiple cell lines to ensure generalizability, time course experiments to capture dynamic effects, and a combination of in vitro and in vivo approaches when possible. Consideration of compensatory mechanisms by related factors, such as other EEF1 complex components, is also important for accurate interpretation of results.

What methodologies are most reliable for detecting anti-EEF1D autoantibodies in clinical samples?

Detection of anti-EEF1D autoantibodies in clinical samples requires a multi-tiered approach combining screening and confirmation techniques. The tissue-based indirect immunofluorescence assay (TBA) on rat cerebellum sections serves as an effective initial screening tool, where patient serum or CSF is applied to the sections and binding detected with fluorescently labeled anti-human IgG antibodies . Anti-EEF1D antibodies produce characteristic neuronal soma staining patterns, though this technique lacks specificity as other neural autoantibodies may produce similar patterns.

The recombinant cell-based indirect immunofluorescence assay (CBA) provides higher specificity and has been validated for anti-EEF1D detection . This method involves transfecting HEK293 cells with human full-length EEF1D (His-tagged) and incubating them with patient samples for 2 hours at room temperature. Detection utilizes FITC-labeled anti-human IgG antibodies, with EEF1D expression visualized using Alexa Fluor 555-labeled anti-mouse IgG (targeting the His-tag) and nuclei counterstained with DAPI . This approach successfully identified two anti-EEF1D positive patients out of 33 ACA cases in published research.

Western blot serves as an important confirmation technique, where recombinant human EEF1D protein is separated by SDS-PAGE, transferred to a membrane, and incubated with patient serum . This confirms the specificity of antibody binding to EEF1D protein and has validated positive findings from patients in previous studies.

Neutralization experiments provide additional confirmation of antibody specificity, where patient samples are pre-incubated with purified EEF1D protein before application to rat cerebellum sections . If the antibodies are specific to EEF1D, the staining should be abolished, as demonstrated in published research.

For definitive antigen identification, tissue-immunoprecipitation (TIP) combined with mass spectrometric analysis can be employed, creating immunocomplexes of rat cerebellum with patient serum and analyzing precipitated complexes . In previous research, this approach identified 17 peptides in EEF1D with more than 60% protein coverage.

Clinical validation requires appropriate control groups, including healthy donors, patients with other neurological disorders, and patients with systemic autoimmune diseases without neurological involvement. In published research, all 30 healthy donors and 15 connective tissue disease patients without neurological disorders tested negative for anti-EEF1D antibodies, supporting the specificity of this biomarker .

What therapeutic strategies targeting EEF1D show promise for cancer treatment?

EEF1D presents several promising therapeutic opportunities for cancer treatment, particularly in addressing drug resistance. RNA interference-based approaches using siRNAs targeting EEF1D show significant potential, as demonstrated in ovarian cancer models where EEF1D knockdown substantially enhanced sensitivity to cisplatin both in vitro and in vivo . Delivery systems could include lipid nanoparticles, tumor-targeted vehicles, or aptamer-siRNA chimeras for cancer cell-specific delivery.

CRISPR/Cas9 gene editing strategies offer another approach, potentially delivered via viral vectors or nanoparticle-based systems for in vivo applications, or through ex vivo modification of tumor cells . These could target complete knockout for maximum sensitization to chemotherapy or isoform-specific modifications to minimize effects on normal cells.

Small molecule inhibitors targeting the nucleotide exchange domain critical for EEF1D's function or protein-protein interaction sites with other EEF1 complex components could be developed through high-throughput screening or structure-based drug design. Given EEF1D's role in activating MAPK and PI3K/Akt pathways, combination therapy approaches pairing EEF1D inhibition with conventional chemotherapy or targeted therapies like PI3K/AKT pathway inhibitors might produce synergistic effects .

EEF1D expression levels could serve as a biomarker for predicting chemotherapy response, stratifying patients for targeted therapies, and monitoring treatment efficacy. Preliminary clinical evidence supports this approach, as EEF1D knockdown increased cisplatin sensitivity in primary ovarian cancer cells from patients with progressive disease/stable disease (PD/SD) .

Considerations for clinical translation include potential toxicities affecting normal protein synthesis in healthy cells, possible neurological effects due to the long isoform's role in brain function, and resistance mechanisms such as compensatory upregulation of other EEF1 complex components. Patient selection should prioritize cancers with confirmed EEF1D overexpression and demonstrated drug resistance to maximize therapeutic benefit.

What is the clinical utility of anti-EEF1D autoantibodies as biomarkers in neurological disorders?

The discovery of anti-EEF1D autoantibodies has significant clinical implications, particularly for autoimmune cerebellar ataxia (ACA). These autoantibodies demonstrate promising diagnostic value, having been detected in patients with ACA while being absent in healthy donors and patients with connective tissue diseases without neurological disorders . This specificity suggests their potential as a diagnostic biomarker for a subset of previously unclassified ataxias.

In clinical studies, two patients with previously unknown causes of cerebellar ataxia were diagnosed with anti-EEF1D autoantibody-associated ACA, and among 33 other ACA patients without clear serological profiles, one additional patient was later identified with anti-EEF1D antibodies . These cases presented with similar clinical manifestations, including progressive cerebellar ataxia and cerebellar atrophy on neuroimaging, patterns that could help clinicians identify potential anti-EEF1D cases.

For monitoring and management, anti-EEF1D antibody titers could potentially serve as a disease activity marker, guide for immunotherapy intensity and duration, and indicator for relapse risk assessment. Long-term management considerations include the potential need for extended immunotherapy in some patients, regular monitoring for symptom recurrence, and vigilance for cerebellar atrophy progression.

Current limitations include the small sample size of confirmed cases and incomplete understanding of pathogenic mechanisms, including whether the antibodies are directly pathogenic or markers of T-cell mediated disease, and how they access intracellular EEF1D. Despite these limitations, anti-EEF1D autoantibodies represent an important advance in autoimmune neurology diagnostics.

How does EEF1D expression correlate with clinical outcomes across different diseases?

EEF1D expression patterns show emerging correlations with clinical outcomes across multiple disease categories. In cancer, particularly ovarian cancer, EEF1D overexpression correlates with resistance to cisplatin (DDP), as evidenced by experimental studies where knockdown of EEF1D significantly increased sensitivity to DDP in both cell lines and primary cancer cells from patients . This suggests EEF1D levels could serve as a predictive biomarker for treatment response in ovarian cancer patients.

Similar patterns exist in other malignancies, where EEF1D is upregulated in liver cancer, esophageal cancer, small cell lung cancer, and medulloblastoma . High expression typically associates with more aggressive disease phenotypes, positioning EEF1D as a potential prognostic biomarker across multiple cancer types.

In autoimmune cerebellar ataxia, the presence of anti-EEF1D autoantibodies identifies a specific patient subset with characteristic clinical manifestations . While these patients show response to immunotherapy, suggesting better outcomes with early detection, cerebellar atrophy that occurred before treatment appears irreversible . One patient experienced symptom worsening after tapering corticosteroid therapy, indicating the need for sustained immunosuppression and highlighting the risk of irreversible damage .

The therapeutic implications of these correlations are significant. As a predictive biomarker, EEF1D expression levels could guide treatment selection, with high expression potentially indicating the need for more aggressive therapy or combination approaches. Monitoring changes during treatment could help detect developing resistance. In autoimmune conditions, antibody status could influence selection and duration of immunosuppressive regimens.

Several clinical correlations require further investigation, including the relationship between EEF1D gene mutations and neurodevelopmental disorders, long-term outcomes of patients with these mutations, and potential roles in other autoimmune diseases beyond cerebellar ataxia . Animal studies provide additional insights, with deletion of the long EEF1D isoform in mice leading to seizures and abnormal behaviors, suggesting potential correlations between EEF1D dysfunction and epilepsy or behavioral disorders in humans .