EIF5A Human

What is EIF5A and what makes it unique among translation factors?

EIF5A is a small (16.7 kDa), acidic (pI = 5.4) protein that is highly abundant among translation factors. Despite its name suggesting involvement in translation initiation, research has established that EIF5A functions primarily in translation elongation, similar to its bacterial homolog EF-P.

What makes EIF5A truly unique is that it is the only protein in eukaryotes and archaea that contains hypusine [Nε-(4-amino-2-hydroxybutyl)lysine], a post-translational modification essential for its activity . This modification is not found in any other protein in any organism studied thus far. EIF5A is present across all eukaryotic species and is highly conserved from yeast to humans, underscoring its fundamental importance in cellular function .

EIF5A is involved in diverse cellular processes including the cell cycle, apoptosis, and viral replication (particularly HIV-1) . Its primary molecular function appears to be alleviating ribosome stalling at challenging sequences, especially polyproline stretches, during protein synthesis .

What are the two human isoforms of EIF5A and how do they differ in expression and function?

Humans possess two EIF5A isoforms encoded by separate genes:

• EIF5A1 (encoded by the EIF5A1 gene):

  • Ubiquitously expressed in all tissues

  • The predominant isoform in most cellular contexts

  • Involved in general translation elongation, particularly at polyproline sequences

• EIF5A2 (encoded by the EIF5A2 gene):

  • Expression normally restricted to testis and parts of the brain

  • Often overexpressed in various cancers

  • Considered a potential oncogene

How is the hypusine modification in EIF5A formed and what enzymes are involved?

The hypusine modification in EIF5A is formed through a two-step enzymatic process:

Step 1: Deoxyhypusine formation

• Catalyzed by Deoxyhypusine Synthase (DHS; EC 2.5.1.46)

• An aminobutyl group from spermidine is transferred to the ε-amino group of a specific lysine residue (Lys50 in humans)

• This forms deoxyhypusine, creating the intermediate form EIF5A(Dhp)

Step 2: Hydroxylation

• Catalyzed by Deoxyhypusine Hydroxylase (DOHH; EC 1.14.99.29)

• The aminobutyl group is hydroxylated

• This produces the mature form EIF5A(Hyp) containing hypusine

This modification process is essential for EIF5A activity. The enzymes involved (DHS and DOHH) are potential therapeutic targets, particularly in cancer and viral infections, as inhibiting hypusine formation effectively inhibits EIF5A function .

Methodologically, researchers can track hypusine formation using radiolabeled spermidine or employ specific inhibitors of DHS (like N1-guanyl-1,7-diaminoheptane or GC7) and DOHH to block each step of the modification pathway .

How can EIF5A be detected and quantified in cellular samples?

Several methods are commonly employed to detect and quantify EIF5A in research settings:

• Western Blot Analysis:

  • Most common method for detecting EIF5A protein

  • Specific antibodies against EIF5A can detect a band at approximately 18 kDa

  • Example protocol: Use anti-EIF5A antibodies at 1 μg/mL followed by HRP-conjugated secondary antibody

  • Detects EIF5A in human, mouse, and rat samples across various cell lines

• Immunohistochemistry (IHC):

  • Detects EIF5A in tissue sections or cells

  • Example protocol for tissue sections: Use anti-EIF5A antibodies at 1.7-5 μg/mL for 1 hour at room temperature, follow with appropriate visualization systems (e.g., DAB staining)

  • Has been successfully used in human liver, mouse embryo, and other tissues

• Immunofluorescence:

  • Allows for precise subcellular localization studies

  • Can be combined with markers for different cellular compartments

• qRT-PCR:

  • For quantifying EIF5A1 and EIF5A2 mRNA levels

  • Allows discrimination between the two isoforms

• Mass Spectrometry:

  • Can definitively identify hypusinated EIF5A

  • Required for detecting post-translational modifications including hypusination and acetylation

These methods can be combined for comprehensive analysis of EIF5A expression, modification status, and localization in experimental samples.

Which amino acid residues are critical for EIF5A function based on mutational studies?

Extensive mutational analyses of human EIF5A have revealed several critical residues required for its functionality:

Critical residues for EIF5A activity include:

• Hypusination site and surrounding residues:

  • Lys50 (in human EIF5A): The site of hypusine modification; mutations (K50A, K50D, K50I, K50R) abolish activity

  • Lys47: K47D mutation abolishes activity despite allowing hypusination

  • Gly49: G49A mutation abolishes activity despite allowing hypusination, suggesting a role beyond just enabling modification

  • Gly52: G52A mutation abolishes activity

  • Lys55: K55A mutation abolishes activity

• Terminal regions:

  • Truncation of 21 amino acids from either the N-terminus or C-terminus abolishes activity

The mutational analysis methodology typically involves expressing mutant human EIF5A in Saccharomyces cerevisiae lacking its own EIF5A genes and assessing growth complementation, while also testing whether the mutants can undergo hypusination .

How does EIF5A relieve ribosome stalling at polyproline sequences and what molecular mechanisms are involved?

EIF5A plays a crucial role in resolving ribosome stalling through a precise molecular mechanism:

• Recognition of stalled ribosomes:

  • EIF5A specifically recognizes ribosomes that have stalled during the synthesis of peptides containing consecutive proline residues

  • Proline's unique cyclic structure creates steric hindrance during peptide bond formation

• Binding to the ribosome:

  • Hypusinated EIF5A binds in the E-site of the ribosome

  • The hypusine residue extends toward the peptidyl transferase center (PTC)

  • This positioning is critical for its function in alleviating stalling

• Facilitating peptide bond formation:

  • The hypusine moiety positions the P-site tRNA and nascent peptide optimally

  • This reduces the entropic cost of peptide bond formation between proline residues

  • Without EIF5A, ribosome stalling at polyproline sequences can lead to premature termination or frameshift errors

Experimental methodologies to study this mechanism include:

• Ribosome profiling to identify ribosome stalling sites genome-wide

• In vitro translation systems with defined polyproline-containing templates

• Cryo-EM to visualize EIF5A-ribosome interactions at high resolution

Numerous stalling sites have been characterized in mouse embryonic stem cell mRNAs, many of which are proline-rich sequences that depend on EIF5A for efficient translation .

What is the relationship between EIF5A and mitochondrial protein import, and how can this be experimentally investigated?

Recent research has uncovered a critical connection between EIF5A and mitochondrial protein import:

• EIF5A's role in mitochondrial function:

  • EIF5A depletion reduces translation of TCA cycle and oxidative phosphorylation proteins

  • Loss of EIF5A causes mitoprotein precursors to accumulate in the cytosol

  • Triggers a mitochondrial import stress response

• Mechanism involving Tim50:

  • Tim50, an essential mitochondrial inner membrane translocase component, contains polyproline sequences

  • EIF5A alleviates ribosome stalling during Tim50 mRNA translation at the mitochondrial surface

  • Without functional EIF5A, Tim50 translation is impaired, compromising mitochondrial protein import

• Experimental evidence and rescue:

  • Removal of polyprolines from Tim50 partially rescues the mitochondrial import stress response

  • Translation of oxidative phosphorylation genes improves when polyprolines are removed from Tim50 in EIF5A-deficient conditions

To experimentally investigate this relationship, researchers can employ:

• Mitochondrial import assays:

  • Use labeled mitochondrial precursor proteins to track import efficiency

  • Compare import rates in cells with normal vs. depleted EIF5A levels

• Ribosome profiling focused on mitochondrial proteins:

  • Assess translation efficiency of nuclear-encoded mitochondrial proteins

  • Focus particularly on proteins with polyproline sequences

• Targeted mutagenesis:

  • Express modified versions of Tim50 lacking polyproline sequences

  • Test whether this bypasses the requirement for EIF5A

This research area demonstrates how EIF5A impacts cellular physiology beyond its direct role in translation, affecting organelle function through specific proteins involved in mitochondrial import .

How is EIF5A linked to cancer development, and what therapeutic strategies target EIF5A in cancer?

EIF5A has significant connections to cancer development through multiple mechanisms:

• Expression patterns in cancer:

  • EIF5A1 is overexpressed in various cancers

  • EIF5A2 is frequently overexpressed in multiple cancer types despite being normally restricted to testis and brain

  • EIF5A2 overexpression correlates with poor prognosis in several cancers

• Oncogenic mechanisms:

  • Promotes cell proliferation and cell cycle progression

  • Enhances translation of proteins involved in epithelial-mesenchymal transition

  • Supports translation of proteins with polyproline motifs involved in cellular transformation

  • Contributes to cancer cell resistance to apoptosis

• Cancer-specific associations:

  • Linked to multiple myeloma, B-Cell lymphoma, and neuroblastoma development

  • Associated with metastasis in various solid tumors

Therapeutic strategies targeting EIF5A in cancer include:

• Inhibition of hypusination pathway:

  • Deoxyhypusine synthase (DHS) inhibitors:

    • N1-guanyl-1,7-diaminoheptane (GC7) - most widely studied

    • Other polyamine analogs that compete with spermidine

  • Deoxyhypusine hydroxylase (DOHH) inhibitors:

    • Metal chelators that inhibit the Fe(II)-dependent DOHH

    • Ciclopirox and deferiprone (clinically approved for other indications)

• Direct targeting approaches:

  • Antisense oligonucleotides to reduce EIF5A expression

  • siRNA/shRNA approaches for research applications

  • Small molecule inhibitors of EIF5A-ribosome interactions

• Combinatorial strategies:

  • Combining EIF5A targeting with conventional chemotherapy

  • Using EIF5A inhibitors to sensitize resistant cancer cells

For clinical development, researchers employ high-throughput screening for novel inhibitors, structure-based drug design, and preclinical testing in cell culture and animal models. The therapeutic potential of targeting EIF5A continues to be an active area of cancer research .

How does acetylation regulate EIF5A subcellular localization and function?

EIF5A acetylation represents an important regulatory mechanism that affects its function and localization:

• Mechanism of EIF5A acetylation:

  • Catalyzed by p300/CBP-associated factor (PCAF)

  • Occurs on specific lysine residues, including some near the hypusine modification site

  • Creates a regulatory switch between different EIF5A functions

• Effects on localization and function:

  • Promotes nuclear translocation of EIF5A from cytoplasm

  • Leads to deactivation of cytoplasmic translation functions

  • May enable alternative nuclear functions

  • Functions as a regulatory mechanism to control EIF5A activity

• Experimental approaches to study EIF5A acetylation:

  • Acetyl-lysine specific antibodies for Western blotting

  • Mass spectrometry to identify and quantify acetylated residues

  • Acetylation-mimicking mutants (lysine to glutamine substitutions)

  • Acetylation-resistant mutants (lysine to arginine substitutions)

• Methodological considerations:

  • Subcellular fractionation to separate nuclear and cytoplasmic pools

  • Immunofluorescence to visualize subcellular distribution

  • Functional assays comparing acetylated and non-acetylated EIF5A

This post-translational modification provides an additional layer of EIF5A regulation beyond hypusination, allowing cells to modulate EIF5A function in response to different cellular conditions .

What cellular compartments contain EIF5A and what functions does it serve in each location?

EIF5A has been detected in multiple cellular compartments, with distinct functions associated with each location:

• Cytoplasm:

  • Primary location of EIF5A

  • Functions in translation elongation, particularly at polyproline sequences

  • Assists in resolving ribosome stalling during protein synthesis

• Nucleus:

  • EIF5A can translocate to the nucleus when acetylated by p300/CBP-associated factor (PCAF)

  • Nuclear translocation is associated with deactivation of its cytoplasmic translation functions

  • May have roles in mRNA processing or nuclear export

• Mitochondria:

  • EIF5A has been found associated with mitochondria

  • Critical for translation of nuclear-encoded mitochondrial proteins containing polyproline sequences

  • Particularly important for Tim50 and other components of the mitochondrial import machinery

  • Regulates mitochondrial activity and apoptosis

• Endoplasmic Reticulum (ER):

  • Associated with the ER membrane

  • Contributes to maintenance of ER integrity

  • Involved in control of the unfolded protein response (UPR)

Experimental approaches to study compartment-specific functions include:

• Cell fractionation combined with Western blotting

• Immunofluorescence microscopy with organelle-specific markers

• Proximity labeling techniques to identify compartment-specific interaction partners

• Compartment-targeted EIF5A constructs to dissect location-specific functions

Understanding the compartment-specific roles of EIF5A provides insight into how this factor influences multiple cellular processes beyond its canonical role in translation .

What experimental methods are used to study EIF5A interactions with the translational machinery?

Understanding EIF5A's interactions with translational machinery requires specialized techniques:

• Structural and biochemical approaches:

  • Cryo-electron microscopy (cryo-EM) to visualize EIF5A-ribosome complexes

  • Co-immunoprecipitation to identify associated proteins

  • Pull-down assays using tagged EIF5A as bait

  • Surface plasmon resonance to measure binding kinetics and affinity constants

• Ribosome-focused techniques:

  • Ribosome profiling to identify EIF5A-dependent translation sites genome-wide

  • In vitro translation assays with purified components

  • Polysome profiling to assess how EIF5A affects ribosome distribution on mRNAs

  • Toe-printing assays to map precise locations of ribosome stalling

• Proximity-based methods:

  • BioID or TurboID proximity labeling to identify proteins near EIF5A in cells

  • Crosslinking mass spectrometry to map interaction interfaces

  • FRET-based approaches to study interactions in living cells

• Mutational analysis:

  • Testing mutant EIF5A proteins in binding and functional assays

  • Structure-function correlations using hypusination-deficient mutants

  • Competition assays between wild-type and mutant proteins

Example experimental data from studies:

• Western blot detection of EIF5A in human renal cell adenocarcinoma (786-O), mouse myoblast (C2C12), and rat alveolar macrophage (NR8383) cell lines shows a specific band at approximately 18 kDa

• Mutational studies reveal that K47D and G49A EIF5A mutants are effective substrates for deoxyhypusine synthase yet fail to support growth, indicating critical roles in EIF5A's interaction with effector molecules

These complementary approaches help build a comprehensive understanding of how EIF5A interacts with ribosomes and other components of the translational machinery.

What are the current challenges and controversies in EIF5A research?

Despite significant advances, several challenges and controversies remain in the EIF5A field:

• Functional dichotomy:

  • Despite being named an "initiation factor," EIF5A primarily functions in elongation

  • The exact extent of its roles in initiation versus elongation remains under investigation

  • Requires ribosome profiling with nucleotide resolution and reconstituted translation systems to resolve

• Differential functions of human EIF5A isoforms:

  • Understanding specific roles of EIF5A1 vs. EIF5A2 beyond their expression patterns

  • Determining whether they have distinct or redundant functions in normal physiology

  • Requires isoform-specific knockdown/knockout studies and identification of isoform-specific interaction partners

• EIF5A-dependent mRNAs beyond polyproline:

  • Identifying the complete set of mRNAs dependent on EIF5A for efficient translation

  • Determining whether there are sequence features beyond polyproline that cause EIF5A-dependent stalling

  • Requires comprehensive ribosome profiling under EIF5A depletion conditions

• Role in disease pathogenesis:

  • Understanding EIF5A's contributions to diseases beyond cancer

  • Determining which disease-associated proteins depend on EIF5A for synthesis

  • Requires analysis of EIF5A levels and modification status in patient samples

• Therapeutic targeting challenges:

  • Developing specific inhibitors of EIF5A or hypusination enzymes

  • Addressing potential side effects given EIF5A's fundamental role in translation

  • Finding ways to target EIF5A in disease contexts without disrupting essential functions

• Nuclear and mitochondrial functions:

  • Fully characterizing EIF5A's roles in the nucleus and mitochondria

  • Understanding how EIF5A regulates mitochondrial protein import at the molecular level

  • Requires compartment-specific studies and isolation of location-specific functions

Addressing these challenges requires interdisciplinary approaches combining structural biology, biochemistry, cell biology, and clinical research to fully understand this unique protein's diverse roles in cellular function and disease.