Recombinant Eukaryotic translation initiation factor 5A-1 (iff-1)

What is eukaryotic translation initiation factor 5A (eIF5A) and what is its primary function in cells?

eIF5A is a highly conserved protein essential in all eukaryotes. Despite its name suggesting involvement in translation initiation, recent research has revealed that eIF5A primarily functions in translation elongation. Specifically, eIF5A facilitates peptide bond formation between consecutive proline residues, resolving polyproline-induced ribosomal stalling .

When studying eIF5A function, researchers should employ ribosome profiling to identify sites of ribosomal pausing, particularly at polyproline coding sequences. Additionally, in vitro translation assays using reporter constructs containing polyproline sequences can directly assess eIF5A's role in resolving translation stalling .

What is hypusination and why is it essential for eIF5A function?

Hypusination is a unique post-translational modification (PTM) that is essential for eIF5A activity. This modification occurs through a two-step enzymatic process:

• Deoxyhypusine synthase (DHS) transfers the 4-aminobutyl moiety from spermidine to a specific lysine residue in eIF5A, forming deoxyhypusine

• Deoxyhypusine hydroxylase (DOHH) hydroxylates the deoxyhypusine residue to form hypusine

This PTM is considered the most specific known to date and is absolutely required for eIF5A's biological activity in translation . Only hypusinated eIF5A can effectively stimulate translation and resolve ribosomal stalling at polyproline stretches . For experimental approaches, researchers should consider using specific inhibitors of DHS (such as GC7) or genetic approaches targeting DHS or DOHH to study the effects of blocking hypusination .

How does eIF5A interact with the ribosome during translation?

Hydroxyl radical probing experiments have localized eIF5A near the E site of the ribosome, with its hypusine residue positioned adjacent to the acceptor stem of the P-site tRNA . This strategic positioning allows eIF5A to stimulate the peptidyl-transferase activity of the ribosome, particularly when poor substrates like proline are involved .

The mechanism appears similar to that of EF-P, the bacterial ortholog of eIF5A, which also facilitates the synthesis of polyproline motifs . By interacting with components of the peptidyl transferase center, eIF5A enhances the catalytic efficiency of peptide bond formation, especially between amino acids with poor reactivity .

What evidence supports eIF5A's role in translation elongation rather than initiation?

Although eIF5A was initially classified as a translation initiation factor based on its ability to stimulate methionyl-puromycin synthesis, substantial evidence now supports its primary role in translation elongation:

• eIF5A has been shown to specifically promote translation elongation of polyproline motifs

• eIF5A associates with polysomes, indicating involvement in active translation

• Hydroxyl radical probing has localized eIF5A near the E site of the ribosome, consistent with a role in elongation

• eIF5A depletion affects polysome profiles in a manner consistent with impaired elongation

Interestingly, there has been some controversy regarding the interpretation of polysome profiles after eIF5A depletion. Some researchers have interpreted these profiles as indicating a role in initiation, while others have concluded they support an elongation function . This discrepancy may be related to experimental conditions and the specific timing of observations after eIF5A depletion.

How does eIF5A specifically facilitate the translation of polyproline motifs?

eIF5A specifically promotes peptide bond formation between consecutive proline residues, which are poor substrates for the peptidyl transferase center of the ribosome . When ribosomes encounter three or more consecutive proline codons, they tend to stall due to the unique conformational constraints of proline .

eIF5A, particularly in its hypusinated form, resolves this stalling by:

• Positioning itself near the E site of the ribosome with its hypusine residue adjacent to the P-site tRNA

• Enhancing the reactivity of the P-site peptidyl-tRNA

• Facilitating proper positioning of substrates for efficient catalysis

In vitro reconstituted translation assays have demonstrated that the addition of eIF5A relieves ribosomal stalling during translation of three consecutive proline residues, and loss of eIF5A function impairs translation of polyproline-containing proteins in vivo .

What methods are most effective for producing recombinant hypusinated eIF5A?

Producing functionally active hypusinated eIF5A presents a significant challenge due to the requirement for specific post-translational modification. Several approaches can be considered:

• Co-expression system: Express eIF5A along with DHS and DOHH enzymes in a suitable host, supplementing the culture medium with spermidine to ensure availability of the substrate for hypusination.

• In vitro hypusination: Express and purify recombinant eIF5A, then perform hypusination in vitro using purified DHS and DOHH enzymes, spermidine, and necessary cofactors.

• Expression in eukaryotic hosts: Express eIF5A in yeast or insect cells that contain endogenous hypusination machinery.

Critical considerations include confirming hypusination status using mass spectrometry and testing functionality in translation assays . Non-hypusinated variants should be prepared as important negative controls for functional studies .

What in vitro assays can effectively measure eIF5A activity?

Several in vitro assays can be used to assess eIF5A activity:

• Methionyl-puromycin synthesis assay: The classical assay measuring formation of methionyl-puromycin, which reflects peptide bond formation. This assay shows eIF5A stimulates this reaction by 2- to 3-fold .

• Reconstituted translation systems with polyproline reporters: Using purified translation components along with mRNAs encoding polyproline stretches to directly measure eIF5A's ability to resolve polyproline-induced stalling .

• [35S]methionine incorporation assays: Measuring translation in cell-free extracts with and without supplemented eIF5A. Studies show addition of hypusinated or deoxyhypusinated eIF5A to depleted lysates stimulates protein synthesis by approximately 1.8-fold .

Important methodological considerations include ensuring optimal experimental conditions (buffer composition, methionine concentration, etc.) and using appropriate controls, including non-hypusinated eIF5A .

How can researchers effectively study eIF5A depletion in vivo?

Several approaches are available for studying the consequences of eIF5A deficiency in cellular systems:

• Genetic approaches:

  • Conditional knockout systems

  • Temperature-sensitive mutants (particularly in yeast)

  • Rapid depletion systems, such as the UBHY-R strain where eIF5A expression is under GAL1 promoter control in a null background

• Analytical methods to assess translation effects:

  • Polysome profiling to examine changes in ribosome distribution

  • Metabolic labeling (e.g., with [35S]methionine) to measure global protein synthesis rates

  • Specific reporter assays to monitor translation of polyproline-containing proteins

Important considerations include the timing of observations after eIF5A depletion and growth conditions, which significantly influence the magnitude of effects observed . Immediate effects of eIF5A depletion include a 2-fold inhibition of protein synthesis and reduction in polysome size .

How does eIF5A contribute to tissue-specific functions in specialized cells?

Recent research has revealed that eIF5A plays critical roles in specialized cell types, including pancreatic β-cells. The hypusinated form of eIF5A (eIF5A-HYP) is a key translational regulator that maintains β-cell identity and function:

• eIF5A-HYP specifically promotes the translation of transcripts essential for β-cell function, including Ins1 (insulin), Slc2a2 (Glut2), Ucn3, and Chga .

• The absence of deoxyhypusine synthase (DHPS) in mouse β-cells leads to:

  • Reduced synthesis of proteins critical to β-cell identity and function

  • Impaired β-cell maturation

  • Development of diabetes

These findings suggest that eIF5A-HYP acts as a "gatekeeper" of specialized mRNA translation, allowing metabolically responsive secretory cells like β-cells to maintain protein synthesis integrity during times of increased demand .

How do the immediate and long-term effects of eIF5A depletion differ?

The temporal effects of eIF5A depletion provide important insights into its function:

• Immediate effects (within hours):

  • Approximately 2-fold reduction in global protein synthesis rates

  • Decrease in polysome size, indicating reduced ribosomal loading on mRNAs

  • These effects occur concomitantly with eIF5A depletion, suggesting a direct role in translation

• Long-term effects:

  • More pronounced inhibition of protein synthesis

  • Potential secondary effects due to impaired synthesis of critical proteins

  • In specialized cells like β-cells, loss of cellular identity and function

The rapidity with which translation is affected upon eIF5A depletion (matching the rate of factor depletion) suggests that secondary effects in the immediate timeframe are minimal, supporting a direct role for eIF5A in translation .

What is the relationship between eIF5A function and disease pathogenesis?

Given eIF5A's essential role in translation, particularly of specific protein classes, its dysregulation may contribute to various pathological conditions:

• Diabetes: Disruption of eIF5A hypusination in β-cells leads to diabetes in mouse models due to impaired translation of proteins crucial for β-cell identity and function .

• Cell proliferation disorders: As an essential protein required for cell proliferation, eIF5A may be implicated in diseases involving abnormal cell growth .

• Disorders involving proline-rich proteins: Since eIF5A is specifically required for efficient translation of polyproline sequences, diseases involving proline-rich proteins may be particularly affected by eIF5A dysfunction .

Research approaches could include analyzing eIF5A expression, mutation status, and hypusination levels in patient samples, and developing disease models with altered eIF5A function.

What are the main challenges in optimizing experimental conditions for eIF5A studies?

Several technical considerations are critical for effective eIF5A research:

• Growth media effects: The growth medium used to propagate cells significantly influences both the rate and extent of protein synthesis inhibition upon eIF5A depletion .

• Methionine concentration: For in vitro translation assays, efficient protein synthesis on endogenous mRNAs depends on raising the molar concentration of radioactive methionine by cold methionine supplementation .

• Assay optimization: Efficient quantification of eIF5A activity in translation was observed only after optimizing experimental conditions in vivo and in vitro .

• Strain-specific effects: Artifacts unique to specific experimental systems (e.g., the UBHY-R yeast strain) must be identified and controlled for .

The search results specifically note that "robust quantification of eIF5A activity in translation was observed only after optimizing experimental conditions in vivo and in vitro" , highlighting the importance of these technical considerations.

How can researchers differentiate between eIF5A's roles in initiation versus elongation?

The precise role of eIF5A in translation has been debated, with evidence supporting functions in both initiation and elongation phases:

• Polysome profile analysis: The polysome profiles observed during and after eIF5A depletion have been interpreted differently - some researchers consider them diagnostic for a role in initiation, while others interpret them as indicating an elongation function .

• Specific substrate testing: Examining eIF5A's effect on translation of polyproline sequences versus general translation initiation can help distinguish its primary function .

• Timing of effects: Immediate versus delayed effects of eIF5A depletion may provide insights into primary versus secondary functions .

As noted in the search results: "while other laboratories reported quantitatively similar effects of eIF5A depletion on global translation, some conclude that eIF5A stimulates elongation, while the polysome profiles observed during eIF5A depletion are diagnostic for a role in initiation" .

What approaches can identify mRNAs particularly dependent on eIF5A for translation?

Identifying the full complement of mRNAs whose translation depends on eIF5A is crucial for understanding its biological roles:

• Ribosome profiling in eIF5A-depleted cells to identify transcripts with altered translation efficiency, focusing particularly on sites of ribosome pausing.

• Analysis of polyproline content: Systematically identifying transcripts containing polyproline motifs, which are known to depend on eIF5A for efficient translation .

• Specialized cell type analysis: Examining translation in tissues where eIF5A plays critical roles, such as pancreatic β-cells, to identify cell-type-specific eIF5A-dependent transcripts .

• In vitro translation assays: Testing candidate mRNAs in reconstituted translation systems with and without eIF5A to directly assess dependence.

Research has already identified several eIF5A-dependent transcripts in specialized cell types, including Ins1, Slc2a2, Ucn3, and Chga in pancreatic β-cells .