EEF1G Human

What is the structural organization of human EEF1G?

Human EEF1G is a multi-domain protein consisting of a glutathione transferase (GST)-like N-terminal domain and a C-terminal domain . Secondary structure analysis indicates that EEF1G constructs possess high α-helical structural character . The full-length protein and N-terminal domain exist as dimers, while the C-terminal domain is monomeric . This structural arrangement appears to be critical for its diverse functions in the cell, including both canonical roles in translation and various moonlighting activities .

How does EEF1G participate in the translation elongation complex?

EEF1G forms part of the eukaryotic elongation factor-1 complex, which is responsible for the enzymatic delivery of aminoacyl tRNAs to the ribosome . This complex is comprised of multiple subunits, including eEF1A (variants eEF1A1 and eEF1A2) and the eEF1B complex . The N-terminal GST-like domain of EEF1G interacts with the β subunit of the complex . This arrangement facilitates the assembly of multisubunit complexes containing elongation factors and aminoacyl-tRNA synthetases . The coordinated function of these components is essential for efficient protein synthesis in eukaryotic cells.

What are the non-canonical functions of EEF1G in human cells?

Beyond its canonical role in translation, EEF1G serves numerous moonlighting functions in cells . One notable non-canonical role is its involvement in HIV-1 reverse transcription complexes (RTCs), where it acts as a critical stability cofactor required for efficient completion of reverse transcription . Research has demonstrated that EEF1G associates with purified RTCs and colocalizes with reverse transcriptase following infection of cells . Additionally, unbalanced expression of EEF1G relative to other elongation factor components in various cancers suggests possible translation-independent functions in disease processes .

What are the established protocols for purifying and characterizing recombinant human EEF1G?

Recombinant human EEF1G can be over-expressed in the soluble Escherichia coli cell fraction and purified to homogeneity . The purification approach typically involves:

• Expression of full-length, N-terminal, and C-terminal domains as separate constructs

• Isolation from soluble bacterial fractions

• Purification to homogeneity using chromatographic techniques

• Secondary structure analysis to confirm proper folding

Characterization studies have revealed that both full-length and N-terminal domain constructs interact with 8-anilino-1-naphthalene sulfonate (ANS) with KD=70.0 (±5.7) μM and with reduced glutathione (GSH) . The N-terminal domain shows modest enzymatic activity (0.03μmol min⁻¹ mg⁻¹ protein) in the standard GSH-1-chloro-2,4-dinitrobenzene conjugation assay, whereas the full-length protein does not catalyze this reaction . These differences suggest that the C-terminal domain may regulate the activity of the N-terminal GST-like domain.

How can researchers effectively detect and quantify EEF1G in experimental samples?

Detection and quantification of EEF1G can be accomplished using several approaches:

• ELISA: Indirect sandwich assays with double antibodies (capture and detection) provide high sensitivity and specificity for EEF1G quantification in human serum, plasma, biological fluids, and cell culture supernatants .

• Immunoprecipitation: Co-immunoprecipitation studies have successfully demonstrated interactions between EEF1G and various proteins, including components of the HIV-1 reverse transcription complex .

• Western blotting: Standard immunoblotting techniques can be used to detect EEF1G in cell lysates and tissue samples, with appropriate antibodies.

• Mass spectrometry: For identification of EEF1G in complex protein mixtures, mass spectrometry following protein fractionation has proven effective .

When interpreting quantitative data, researchers should consider the potential for tissue-specific variations in EEF1G expression and interactions with other elongation factor components.

What functional assays are available to study EEF1G's role in translation and other processes?

Several functional assays have been developed to investigate EEF1G's roles:

• In vitro translation assays: Can measure the effect of EEF1G modulation on protein synthesis efficiency.

• Endogenous reverse transcription assay: Used to identify EEF1G's role in HIV-1 reverse transcription .

• siRNA knockdown: Reduction of EEF1G levels by siRNA treatment has been shown to significantly down-regulate reverse transcription efficiency in HIV-1 studies .

• GST activity assays: The standard GSH-1-chloro-2,4-dinitrobenzene conjugation assay can assess the enzymatic activity of the GST-like domain .

• Protein-protein interaction studies: Co-immunoprecipitation and colocalization studies have revealed EEF1G's associations with various cellular and viral proteins .

How is EEF1G expression altered in cancer, and what are the implications?

EEF1G is frequently overexpressed in human carcinomas . Studies examining expression patterns in cancer tissues have revealed:

Notably, concomitant cancer-related increases of both eEF1Bβ and eEF1Bγ were found in four cases of cardioesophageal cancer and five cases of lung carcinomas, suggesting functional interaction between these components . The unbalanced expression of various eEF1 subunits in cancer compared to surrounding tissues indicates potential dysregulation of translation or activation of non-canonical functions that may contribute to tumor development or progression.

What is EEF1G's significance in viral infections, particularly HIV-1?

EEF1G has been identified as a critical component of the HIV-1 reverse transcription complex (RTC) . Key findings include:

• EEF1G was identified in fractionated human T-cell lysates as a reverse transcription cofactor

• The p51 subunit of reverse transcriptase and integrase coimmunoprecipitated with EEF1G

• EEF1G associates with purified RTCs and colocalizes with reverse transcriptase following infection

• Reverse transcription is significantly down-regulated when EEF1G levels are reduced by siRNA treatment

• EEF1G appears to function as an RTC stability cofactor required for efficient completion of reverse transcription

These findings suggest that EEF1G plays a crucial role in the viral life cycle that extends beyond its canonical function in host translation. Understanding this interaction may provide insights into potential therapeutic targets for HIV-1 infection.

What is the relationship between EEF1G's GST-like domain and its potential enzymatic activities?

The N-terminal domain of EEF1G possesses a GST-like structure, raising questions about its enzymatic capabilities . Research has shown:

• The N-terminal domain exhibits modest enzyme activity (0.03μmol min⁻¹ mg⁻¹ protein) in the GSH-CDNB conjugation assay

• The full-length EEF1G does not catalyze this reaction, suggesting possible regulatory interactions between domains

• Both full-length and N-terminal constructs interact with GSH and ANS

• Glutathione sulfonate can displace ANS bound to hydrophobic binding sites in the N-terminal domain

How does EEF1G contribute to tissue-specific regulation of translation?

Research has identified tissue-specific patterns in EEF1G expression and its relationship with other elongation factor components . Notable observations include:

• Associated increase in eEF1Bβ and eEF1Bγ expression in prenatal compared to postnatal mouse brain and liver

• Evidence suggesting separate functioning of pools of individual eEF1Bα and the eEF1Bβ–eEF1Bγ subcomplex in brain tissue

• Coordinated up-regulation of eEF1Bβ and eEF1Bγ in oral squamous cell carcinoma

These patterns suggest that EEF1G may contribute to specialized translation regulation in different tissues, potentially influencing the translation of specific mRNA subsets or responding to tissue-specific demands for protein synthesis. The mechanisms controlling these tissue-specific patterns and their functional consequences remain important areas for future investigation.

What methodological approaches could advance our understanding of EEF1G structure-function relationships?

Despite significant progress, several aspects of EEF1G function remain poorly understood . Advanced methodological approaches that could address these knowledge gaps include:

• Cryo-electron microscopy to visualize EEF1G within the context of the full elongation complex

• Ribosome profiling following EEF1G modulation to identify transcript-specific effects on translation

• Proximity labeling techniques to map the dynamic interactome of EEF1G in different cellular contexts

• CRISPR-based approaches for domain-specific mutagenesis to dissect the contributions of individual EEF1G domains

• Single-molecule imaging to track EEF1G dynamics during translation and other processes

These approaches could provide insights into how EEF1G structure relates to its diverse functions and how disruptions in these relationships may contribute to disease processes.

What controls should be included when studying EEF1G expression in disease states?

When investigating EEF1G expression in disease states, researchers should consider several critical controls:

• Matched normal-diseased tissue pairs from the same patient to account for individual variation

• Examination of other eEF1 complex components to distinguish EEF1G-specific effects from general translation dysregulation

• Assessment of both mRNA and protein levels to identify post-transcriptional regulation

• Spatial analysis within tissues to detect cell type-specific alterations

• Longitudinal sampling where possible to track expression changes during disease progression

Studies have shown unbalanced changes in the expression of various eEF1 subunits in human cancers compared to tumor-surrounding tissue . This reinforces the importance of comprehensive analysis rather than focusing on EEF1G in isolation.

How can researchers effectively modulate EEF1G levels or function for mechanistic studies?

Several approaches can be employed to modulate EEF1G for functional studies:

• siRNA or shRNA: Effective for transient or stable knockdown of EEF1G, as demonstrated in HIV-1 studies where siRNA treatment significantly reduced reverse transcription efficiency .

• CRISPR-Cas9: For genomic editing to create knockout or knock-in cell lines.

• Overexpression systems: To examine the effects of increased EEF1G levels or to express mutant variants.

• Domain-specific inhibitors: Though not currently available, the development of compounds targeting the GST-like domain or other functional regions could provide valuable tools.

When designing these experiments, researchers should be mindful of potential compensatory mechanisms and the importance of appropriate controls to distinguish direct effects from secondary consequences of altered translation.

What technical challenges should be anticipated when working with EEF1G in experimental systems?

Researchers studying EEF1G should anticipate several technical challenges:

• Distinguishing canonical from non-canonical functions, as both may be affected by EEF1G modulation

• Potential embryonic lethality of complete knockout, necessitating conditional systems

• Compensatory upregulation of other translation factors following EEF1G depletion

• Distinguishing direct binding partners from components of larger complexes in interaction studies

• Tissue-specific variation in EEF1G expression and function that may limit the generalizability of findings from a single cell type

Addressing these challenges requires careful experimental design, appropriate controls, and integration of multiple complementary approaches to build a comprehensive understanding of EEF1G function.