EEF2 Human

What is the molecular function of EEF2 in human cells?

EEF2 is a critical catalyst required for the elongation of polypeptide chains during protein synthesis. It mediates the GTP-dependent translocation of peptidyl-tRNA from the A to P site and deacylated-tRNA from the P to E(xit) site, while simultaneously advancing the ribosome one codon in the 3′ direction along the mRNA . This translocation step is essential for protein synthesis across all eukaryotes, with the EEF2 protein structure being highly conserved (66% identity and 85% homology between yeast and humans) .

What is the structural organization of EEF2 and how does it relate to function?

EEF2 consists of five functionally distinct domains that collectively enable polypeptide elongation:

• Domains I and II: Form the GTP-binding pocket essential for the protein's catalytic activity

• Domains I, II, III, and V: Interact with Helices 98 (Sarcin/Ricin loop) and 43 (GTPase associated center) of the large ribosomal subunit rRNA

• Domain IV: Interacts with Helix 69 to coordinate movement of the peptidyl and deacylated tRNAs in the ribosome

These domain-specific interactions coordinate the structural changes necessary for successful translocation during protein synthesis.

What is known about EEF2 gene organization and expression patterns?

The human EEF2 gene (HAGRID: 103) is located on chromosome 19p13.3 (3,976,056-3,985,463 bp) on the minus strand . The gene is ubiquitously expressed, reflecting its fundamental role in protein synthesis across all cell types. In neural tissues, eEF2K (the kinase regulating EEF2) shows differential expression patterns, with highest expression observed in large diameter neurons . The EEF2 gene is highly constrained for both predicted loss-of-function (pLoF) variations (LOEUF = 0.172) and missense variations (misZ score = 4.878), indicating strong evolutionary pressure to maintain its precise function .

How is EEF2 activity regulated in human cells?

EEF2 activity is primarily regulated through phosphorylation/dephosphorylation cycles:

• Phosphorylation: EEF2 kinase (eEF2K) phosphorylates EEF2 at Threonine 56, inhibiting its activity and reducing translation elongation rates

• Dephosphorylation: Removal of the phosphate group reactivates EEF2, promoting protein synthesis

This kinase is calcium/calmodulin-dependent and integrates various cellular signals, including those from MAP kinase and mTOR signaling pathways . The regulation provides a mechanism for cells to modulate protein synthesis rates in response to changing cellular conditions, conserving energy under stress conditions.

What experimental approaches best assess EEF2 phosphorylation status?

To assess EEF2 phosphorylation:

• Western blot analysis: Using phospho-specific antibodies targeting Thr56 of EEF2. This approach was successfully employed to identify significantly increased levels of phosphorylated EEF2 in postmortem hippocampal tissue from Alzheimer's disease patients .

• In vivo phosphorylation studies: Comparing wild-type and eEF2K knockout/heterozygous models to correlate phosphorylation states with functional outcomes. Studies have demonstrated that genetic reduction of eEF2K alleviates eEF2 hyperphosphorylation in Alzheimer's disease model mice .

• Phosphoproteomic analysis: Mass spectrometry-based approaches can provide quantitative measurements of phosphorylation at multiple sites.

How does EEF2 phosphorylation affect de novo protein synthesis?

EEF2 phosphorylation has a direct inhibitory effect on de novo protein synthesis. This relationship can be experimentally demonstrated using SUnSET (Surface Sensing of Translation), a non-radioactive puromycin end-labeling assay . Studies in Alzheimer's disease model mice (Tg19959) revealed:

• Wild-type mice: Normal protein synthesis levels (high puromycin labeling)

• Tg19959 mice: Significantly reduced protein synthesis (low puromycin labeling) with high eEF2 phosphorylation

• Tg19959/eEF2K+/- mice: Significantly improved protein synthesis compared to Tg19959 mice, corresponding with reduced eEF2 phosphorylation

These findings demonstrate a clear inverse relationship between eEF2 phosphorylation levels and protein synthesis rates.

What are the mechanisms by which EEF2 regulates synaptic plasticity?

EEF2 serves as a critical regulator of synaptic plasticity through its control of activity-dependent protein synthesis:

• Post-training dephosphorylation: After learning events, EEF2 becomes dephosphorylated, promoting protein synthesis necessary for memory consolidation .

• Local protein synthesis: EEF2 regulation may contribute to localized translation of specific mRNAs in dendrites, essential for synapse-specific modifications.

• Interaction with BDNF signaling: Brain-derived neurotrophic factor enhances translation elongation in neurons with implications for mammalian target of rapamycin (mTOR) signaling .

The critical role of EEF2 in synaptic plasticity is evidenced by studies showing that genetic reduction of eEF2K (which lowers EEF2 phosphorylation) restores impaired long-term potentiation (LTP) in Alzheimer's disease model mice .

How does EEF2 dysfunction affect learning and memory processes?

Experimental evidence from several models indicates that EEF2 dysfunction significantly impacts learning and memory:

• Behavioral testing: Tg19959 Alzheimer's disease model mice show impaired performance in novel object recognition tasks, spending equal time with familiar and novel objects. When eEF2K expression is reduced in these mice (Tg19959/eEF2K+/-), memory performance improves to wild-type levels .

• Electrophysiological studies: Hippocampal long-term potentiation (LTP), a cellular correlate of learning and memory, is significantly impaired in Tg19959 mouse brain slices but restored in Tg19959/eEF2K+/- slices .

• Molecular mechanisms: Kinase-defective eEF2K mice demonstrate impaired associative taste learning and abnormal brain activation, further supporting the critical role of proper eEF2 regulation in learning processes .

How is EEF2 dysregulation implicated in Alzheimer's disease pathophysiology?

EEF2 dysregulation appears to play a significant role in Alzheimer's disease pathophysiology:

• Hyperphosphorylation: Western blot analysis of postmortem hippocampal tissue from AD patients reveals significantly increased levels of eEF2 phosphorylation at Thr56 compared to age-matched controls .

• Protein synthesis impairment: Hyperphosphorylation of eEF2 correlates with reduced de novo protein synthesis in AD model mice, potentially contributing to synaptic dysfunction and cognitive decline .

• Disease specificity: Interestingly, eEF2 hyperphosphorylation appears selective for Alzheimer's disease, as it was not observed in frontotemporal dementia (FTD) or Lewy body dementia (LBD) tissues compared to their respective controls .

• Therapeutic potential: Genetic reduction of eEF2K alleviates cognitive deficits, memory impairments, and synaptic failure in AD model mice, suggesting the eEF2 pathway as a potential therapeutic target .

What is the spectrum of EEF2-related genetic disorders?

EEF2 genetic variants are associated with a broader spectrum of neurological disorders than previously recognized:

• Spinocerebellar ataxia 26 (SCA26): The heterozygous p.(Pro596His) variant causes autosomal dominant late-onset spinocerebellar ataxia characterized by pure cerebellar ataxia and cerebellar atrophy .

• Neurodevelopmental disorders: De novo EEF2 missense variants have been identified in children with neurodevelopmental delays and structural brain abnormalities, including:

  • p.(Cys388Tyr): Located at a critical interface between domains I and II

  • p.(Val28Met): Located at the base of domain I where it directly contacts the SRL

  • p.(His769Tyr): Located in domain V, which interacts with the GAC

The discovery of these variants expands our understanding of EEF2-related pathologies beyond late-onset ataxia to include early-onset neurodevelopmental conditions.

What functional consequences arise from pathogenic EEF2 variants?

Functional studies in yeast models have revealed diverse effects of EEF2 variants on cellular processes:

PRF: Programmed Ribosomal Frameshifting; TCR: Termination Codon Readthrough

These functional changes demonstrate that different EEF2 variants disrupt protein synthesis in distinct ways while retaining sufficient function for cell viability.

What experimental systems are most appropriate for studying EEF2 function?

Multiple complementary experimental systems provide insights into EEF2 function:

• Yeast models: Saccharomyces cerevisiae offers a powerful system for functional analysis of EEF2 variants due to high conservation (85% homology between yeast and human EEF2 protein). Researchers can employ yeast strains where endogenous copies of EEF2 genes (EFT1 and EFT2) are disrupted and replaced with plasmids bearing wild-type or variant EEF2 genes .

• Mammalian models:

  • Transgenic mouse models: Tg19959 AD model mice crossed with eEF2K+/- mice provide insights into the role of EEF2 in disease contexts

  • Cultured neurons: Allow for detailed analysis of local translation and synaptic plasticity

• Human tissue samples: Analysis of postmortem brain tissue from patients with various neurological conditions provides clinically relevant insights into EEF2 dysfunction .

How can translational fidelity be quantitatively assessed in EEF2 variant studies?

Translational fidelity can be measured using dual-luciferase reporter assays in yeast models to assess specific aspects of translation:

• Programmed -1 ribosomal frameshifting (-1 PRF): Measures the frequency of -1 nucleotide slippage during translation, which can lead to production of alternative protein products.

• Programmed +1 ribosomal frameshifting (+1 PRF): Assesses +1 nucleotide slippage during translation.

• Termination codon readthrough (TCR): Quantifies the frequency with which ribosomes continue translation beyond stop codons .

These assays have revealed that certain EEF2 variants (e.g., P580H, C372Y, Q753Y) specifically affect termination codon readthrough, indicating altered translational fidelity .

What approaches can detect changes in EEF2-dependent protein synthesis in vivo?

Several complementary approaches can assess EEF2-dependent protein synthesis in vivo:

• SUnSET (Surface Sensing of Translation): A non-radioactive puromycin end-labeling assay that labels newly synthesized proteins. This method effectively demonstrated reduced hippocampal de novo protein synthesis in Tg19959 mice and its restoration in Tg19959/eEF2K+/- mice .

• Metabolic labeling: Using radioactive amino acids (e.g., 35S-methionine) to measure incorporation into newly synthesized proteins.

• Polysome profiling: Analyzing the distribution of ribosomes on mRNAs to assess global and transcript-specific translation efficiency.

• Ribosome profiling: High-throughput sequencing of ribosome-protected mRNA fragments to provide genome-wide information about ribosome positions and translation dynamics.

How does EEF2 regulation change under different cellular stress conditions?

EEF2 serves as a key regulator of translation during cellular stress:

• Energy stress: Under energy-limiting conditions, eEF2 becomes phosphorylated, slowing down protein synthesis to conserve energy .

• Endoplasmic reticulum stress: EEF2 responds to endoplasmic reticulum stress (GO:0034976), potentially adjusting translation rates to manage protein folding demands .

• Ischemia: EEF2 plays a role in response to ischemia (GO:0002931), likely contributing to cellular adaptation mechanisms .

• Proteostatic stress: Functional studies demonstrated that the SCA26-associated p.(Pro596His) variant resulted in greater susceptibility to proteostatic stress .

These regulatory mechanisms allow cells to modulate translation elongation rates according to cellular needs and stress conditions.

What role does EEF2 play in aging-related cellular processes?

Evidence suggests EEF2 may contribute to aging-related changes in protein synthesis:

• Age-dependent regulation: Studies in rats have demonstrated age-dependent changes to EEF2, which could account for the decline in protein synthesis observed in older animals .

• Aging processes: EEF2 has been linked to aging processes (GO:0007568), although it has not been directly related to human aging .

• Protein aggregation: EEF2 is associated with the aggresome (GO:0016235), suggesting potential roles in managing protein aggregation that may be relevant to age-related neurodegeneration .

The relationship between EEF2 and aging requires further investigation to establish direct mechanistic links in humans.

What are the most promising therapeutic strategies targeting the EEF2 pathway?

Several therapeutic strategies targeting the EEF2 pathway show promise:

• eEF2K inhibition: Genetic reduction of eEF2K alleviates cognitive deficits and synaptic failure in Alzheimer's disease model mice, suggesting pharmacological inhibitors of eEF2K could have therapeutic potential .

• Modulation of EEF2 phosphorylation: Developing compounds that specifically target the phosphorylation state of EEF2 could help restore protein synthesis in conditions where it is dysregulated.

• Transcript-specific translation restoration: Strategies to restore translation of specific transcripts affected by EEF2 dysfunction could provide targeted treatments with fewer side effects.

Research priorities should include high-throughput screening for eEF2K inhibitors and detailed characterization of downstream effects on specific mRNA translation in disease models.

How might single-cell approaches advance our understanding of EEF2 function?

Single-cell technologies offer promising new avenues for EEF2 research:

• Single-cell transcriptomics: Can reveal cell-type specific expression patterns of EEF2 and its regulatory factors across tissues and developmental stages. Current data shows that eEF2K is most highly expressed in large diameter neurons .

• Single-cell translatomics: Techniques that measure translation at the single-cell level could reveal how EEF2 regulation affects protein synthesis in specific cell populations.

• Spatial transcriptomics/translatomics: These approaches could elucidate the subcellular localization of EEF2-dependent translation, particularly important in polarized cells like neurons where local translation plays critical roles.

These approaches will help resolve cell-type specific vulnerabilities to EEF2 dysfunction in various disease contexts.

What are the challenges in developing EEF2-targeted therapeutics?

Several challenges must be addressed in developing EEF2-targeted therapeutics:

• Ubiquitous expression: EEF2's fundamental role in all cells means that systemic manipulation risks broad side effects. Strategies for tissue-specific delivery or targeting of specific EEF2 complexes will be essential.

• Context-dependent regulation: The phosphorylation state of EEF2 has different consequences depending on cellular context, developmental stage, and disease state. Therapeutic approaches must consider this complexity.

• Balancing translation rates: Too much or too little protein synthesis can be detrimental, requiring precisely calibrated interventions.

• Target validation: While genetic studies suggest EEF2 pathway as a therapeutic target, pharmacological proof-of-concept studies are needed to validate this approach for clinical development.

How conserved is EEF2 function across species and what can we learn from model organisms?

EEF2 is highly conserved across eukaryotes, making comparative studies highly informative:

• Evolutionary conservation: The eEF2 amino acid sequence is 66% identical with 85% homology between yeast and humans at the DNA and protein levels . This conservation reflects the fundamental importance of EEF2 in translation.

• Model organisms:

  • Saccharomyces cerevisiae: Provides a robust system for studying EEF2 variants and their effects on translational fidelity

  • Rodents: Mouse and rat models have revealed age-dependent changes in EEF2 regulation and its role in learning and memory

• Translational relevance: The high conservation means that findings from model organisms likely translate well to human biology, particularly for basic EEF2 functions and regulation mechanisms.