GLRX5 Human

What is the basic structure of human GLRX5 protein?

Human GLRX5 is a 156 amino acid mitochondrial protein that belongs to the monothiol glutaredoxin family with a characteristic CGFS active site motif. Crystal structure analyses reveal that holo-GLRX5 forms a tetrameric organization when bound to two [2Fe-2S] clusters and four glutathione (GSH) molecules, with the iron-sulfur clusters buried in the interior and shielded from solvent by the conserved β1-α2 loop, Phe69, and GSH molecules . Each [2Fe-2S] cluster is coordinated by the N-terminal active-site cysteine (Cys67) thiols contributed by two protomers and two cysteine thiols from two GSH molecules . The apoprotein exists as a monomer, while the holoprotein forms a tetramer, as confirmed by gel-filtration chromatography and analytical ultracentrifugation .

Where is GLRX5 predominantly localized in human cells?

GLRX5 is predominantly localized in the mitochondria, as demonstrated by immunofluorescence staining and subcellular fractionation studies. Confocal microscopic imaging has shown that GLRX5 expression colocalizes with mitochondrial markers, and Western blot analysis of subcellular fractions has confirmed its predominant presence in the mitochondrial fraction . This mitochondrial localization is consistent with its role in iron-sulfur cluster biosynthesis, which primarily occurs in the mitochondria before clusters are distributed to various cellular compartments .

What is the tissue expression pattern of GLRX5?

GLRX5 exhibits a distinctive tissue expression pattern. While it is minimally expressed in most tissues, it shows high expression in CD71+ early erythroid cells of bone marrow . This expression pattern is similar to those of human ALAS2 and FECH, which are involved in heme biosynthesis in erythroid tissues. In situ hybridization studies in adult mouse tissues have confirmed low expression levels in liver, kidney, lung, heart, testis, and skeletal muscle, with significantly higher expression in erythroid tissues . This tissue-specific expression pattern provides insight into why GLRX5 deficiency primarily affects the erythroid lineage.

How does GLRX5 participate in iron-sulfur cluster assembly?

GLRX5 functions as an essential component of the mitochondrial iron-sulfur cluster synthesis machinery. It can bind [2Fe-2S] clusters through specific coordination involving the active site cysteine residue (C67) from each monomer and two GSH molecules . In vitro reconstitution studies have demonstrated that GLRX5 can assemble a [2Fe-2S] cluster, enabling it to function as an Fe-S scaffold or sensor . Knockdown of GLRX5 affects the assembly of both [4Fe-4S] and [2Fe-2S] clusters in both mitochondria and cytosol, confirming its central role in the iron-sulfur cluster biosynthesis pathway .

Which conserved residues are critical for GLRX5's iron-sulfur cluster binding?

Mutagenesis studies have identified several conserved residues that are critical for GLRX5's ability to bind iron-sulfur clusters:

• K59 (lysine 59): Mutation to glutamine (K59Q) abolishes the characteristic [Fe-S] absorbance pattern after reconstitution

• C67 (cysteine 67): Located in the CGFS active domain, mutation to serine (C67S) prevents [Fe-S] cluster assembly

• T108 (threonine 108): Mutation to valine (T108V) eliminates [Fe-S] binding capacity

• K101 (lysine 101): Mutation to glutamine (K101Q) may prevent the binding of [Fe-S] to GLRX5 protein

• L148 (leucine 148): Mutation to serine (L148S) may interfere with [Fe-S] transfer from GLRX5 to recipient proteins

These residues are likely involved in docking of the GSH ligand to the protein or in the coordination of the iron-sulfur cluster itself .

How does GLRX5 differ from other glutaredoxins in terms of activity?

GLRX5 exhibits distinct biochemical properties compared to other glutaredoxins. Apo-GLRX5 reduces glutathione mixed disulfides at a rate approximately 100 times lower than GLRX2 . Despite this relatively low thiol-disulfide oxidoreductase activity, GLRX5 remains active as a glutathione-dependent electron donor for mammalian ribonucleotide reductase . Mass spectrometry analyses have revealed glutathionylation of cysteine residues in the absence of the [2Fe-2S] cluster, which may protect them from further oxidation and possibly facilitate cluster transfer or acceptance . These unique properties distinguish GLRX5 from other glutaredoxins and highlight its specialized role in iron-sulfur cluster metabolism.

How does GLRX5 deficiency lead to sideroblastic anemia?

GLRX5 deficiency leads to sideroblastic anemia through a complex mechanism involving disrupted iron homeostasis and impaired heme biosynthesis:

• Impaired [Fe-S] cluster biosynthesis: GLRX5 deficiency compromises the assembly of iron-sulfur clusters

• Altered IRP activity: This impairment activates iron regulatory protein 1 (IRP1) binding to iron-responsive elements (IREs) and increases IRP2 levels

• Cytosolic iron depletion: Despite mitochondrial iron overload, the cytosol experiences relative iron depletion

• Repressed ALAS2 synthesis: IRP-mediated translational repression decreases aminolevulinate δ synthase 2 (ALAS2) levels in erythroid cells

• Reduced ferrochelatase levels: The final enzyme in heme biosynthesis is diminished

• Increased ferroportin expression: Enhanced iron export further depletes cellular iron

• Failed heme synthesis: The combination of these effects impairs heme synthesis specifically in erythroid cells

The unique combination of IRP targets in erythroid cells, including IRP-repressible ALAS2 and non-IRP-repressible ferroportin 1b, accounts for the tissue-specific phenotype of human GLRX5 deficiency .

What pathogenic mutations have been identified in the GLRX5 gene?

Several pathogenic mutations in the GLRX5 gene have been identified and characterized:

• Compound heterozygous missense mutations:

  • c.301A>C (resulting in K101Q mutation)

  • c.443T>C (resulting in L148S mutation)

• Functional consequences:

  • K101Q mutation: Prevents binding of [Fe-S] to GLRX5 protein

  • L148S mutation: Interferes with [Fe-S] transfer from GLRX5 to iron regulatory protein 1 (IRP1), mitochondrial aconitase (m-aconitase), and ferrochelatase

• Other mutations:

  • K51del mutant: L148S has been shown to be functionally complementary to this mutation with respect to Fe/S-ferrochelatase, Fe/S-IRP1, Fe/S-succinate dehydrogenase, and Fe/S-m-aconitase biosynthesis and lipoylation of pyruvate dehydrogenase complex and α-ketoglutarate dehydrogenase complex

These mutations result in sideroblastic anemia and, in some cases, variant nonketotic hyperglycinemia .

How can GLRX5 function be rescued in deficient cells?

Rescue of GLRX5 function in deficient cells has been demonstrated through several approaches:

• Gene transfection: Introduction of the wild-type GLRX5 gene via transfection can reverse phenotypes in patient fibroblasts

• Viral transduction: Delivery of functional GLRX5 using viral vectors provides another effective approach

• Observed effects of successful rescue include:

  • Reversal of slow growth phenotype

  • Correction of mitochondrial iron overload

  • Increased aconitase activity

  • Restoration of normal iron homeostasis

These rescue experiments provide important proof-of-concept for potential therapeutic approaches and validate the causal relationship between GLRX5 deficiency and the observed cellular phenotypes.

What are the optimal methods for studying GLRX5's iron-sulfur cluster binding properties?

Several complementary methods can be employed to investigate GLRX5's iron-sulfur cluster binding properties:

• In vitro reconstitution: Mixing purified recombinant GLRX5 with iron, sulfide, and glutathione under anaerobic conditions to reconstitute [Fe-S] clusters

• UV-visible spectroscopy: Monitoring characteristic absorption peaks at approximately 320, 420, and 450 nm that indicate successful [Fe-S] cluster incorporation

• Site-directed mutagenesis: Systematically mutating conserved residues (like K59, C67, T108) to assess their roles in [Fe-S] binding

• Gel-filtration chromatography: Determining oligomerization state differences between apo and holo forms of GLRX5

• Analytical ultracentrifugation: Confirming tetrameric organization of holo-GLRX5

• Mass spectrometry: Identifying post-translational modifications like glutathionylation of cysteine residues

These techniques have successfully revealed the structural basis for GLRX5's interaction with [Fe-S] clusters and can be applied to investigate other aspects of GLRX5 function or the impact of mutations.

How can one develop and validate GLRX5 knockout cell models?

Development and validation of GLRX5 knockout cell models can be achieved through the following approach:

• Cell line selection: Choose appropriate cell lines, such as K562 cells, which have been successfully used for GLRX5 studies

• Knockout strategy: Implement CRISPR-Cas9 or other gene editing technologies targeting the GLRX5 gene

• Validation methods:

  • Genomic DNA sequencing to confirm targeted mutations

  • Western blotting to verify absence of GLRX5 protein

  • Phenotypic characterization:

    • Assessing growth rates

    • Measuring activities of [Fe-S]-dependent enzymes (aconitase, xanthine oxidase)

    • Evaluating mitochondrial iron levels

    • Analyzing IRP1 binding activity and IRP2 protein levels

Once validated, these knockout models provide valuable tools for studying GLRX5 function, testing the effects of mutations, and evaluating rescue strategies.

What assays can assess the functional impact of GLRX5 mutations?

To assess the functional impact of GLRX5 mutations, researchers can employ a variety of assays:

• [Fe-S] protein activity assays:

  • Aconitase activity assay (mitochondrial and cytosolic)

  • Succinate dehydrogenase activity

  • Xanthine oxidase activity (cytosolic [2Fe-2S] enzyme)

  • Ferrochelatase activity

• Iron homeostasis assessment:

  • IRP1 RNA-binding activity (electrophoretic mobility shift assay)

  • IRP2 protein levels (Western blot)

  • Mitochondrial iron content (staining or quantitative measurement)

  • Expression of iron metabolism genes

• Protein-protein interaction studies:

  • Co-immunoprecipitation to assess GLRX5 interaction with [Fe-S] cluster assembly machinery components

  • In vitro [Fe-S] transfer assays from GLRX5 to recipient proteins

• Rescue experiments:

  • Complementation of GLRX5-deficient cells with mutant variants

  • Assessment of phenotypic rescue

These assays provide comprehensive evaluation of how mutations affect GLRX5's multiple functions in [Fe-S] protein synthesis and maturation.

How do different GLRX5 mutations differentially affect downstream iron-sulfur proteins?

Different GLRX5 mutations can have distinct effects on downstream iron-sulfur proteins, revealing the multifunctional nature of GLRX5 in iron-sulfur cluster biosynthesis and transfer:

• K101Q mutation (c.301A>C):

  • Primarily affects the binding of [Fe-S] clusters to GLRX5 itself

  • Broadly impairs all downstream [Fe-S] protein maturation

• L148S mutation (c.443T>C):

  • Specifically interferes with [Fe-S] transfer from GLRX5 to recipient proteins

  • Differentially affects:

    • Iron regulatory protein 1 (IRP1)

    • Mitochondrial aconitase

    • Ferrochelatase

• Functional complementation patterns:

  • L148S is functionally complementary to the K51del mutant for:

    • Fe/S-ferrochelatase biosynthesis

    • Fe/S-IRP1 maturation

    • Fe/S-succinate dehydrogenase assembly

    • Fe/S-m-aconitase formation

    • Lipoylation of pyruvate dehydrogenase complex

    • α-ketoglutarate dehydrogenase complex formation

These differential effects indicate that GLRX5 has specific functional domains or interaction surfaces for different downstream targets, rather than a single mechanism of action for all [Fe-S] proteins.

What is the relationship between GLRX5 and mitochondrial/cytosolic iron homeostasis?

GLRX5 plays a central role in coordinating mitochondrial and cytosolic iron homeostasis through several mechanisms:

• Iron-sulfur cluster synthesis: As a component of the mitochondrial [Fe-S] biosynthesis machinery, GLRX5 is essential for the maturation of numerous [Fe-S] proteins

• IRP1 regulation:

  • Functional GLRX5 ensures proper [Fe-S] cluster loading of IRP1

  • [Fe-S]-loaded IRP1 has aconitase activity but lacks IRE-binding activity

  • GLRX5 deficiency activates the IRE-binding activity of IRP1

• Iron distribution:

  • GLRX5 deficiency leads to paradoxical iron distribution

  • Mitochondrial iron overload occurs simultaneously with cytosolic iron depletion

  • Increased IRP2 levels reflect relative cytosolic iron depletion

• Tissue-specific effects:

  • In erythroid cells, GLRX5 deficiency impacts both IRP-repressible ALAS2 and non-IRP-repressible ferroportin 1b

  • This unique combination of targets explains the tissue-specific manifestation of GLRX5 deficiency

Understanding this complex relationship is crucial for comprehending the pathophysiology of disorders like sideroblastic anemia and developing targeted interventions.

What remains unknown about GLRX5 function and research directions?

Despite significant advances, several aspects of GLRX5 function remain incompletely understood and represent promising directions for future research:

• Molecular mechanism of [Fe-S] transfer:

  • Precise structural details of how GLRX5 transfers [Fe-S] clusters to recipient proteins

  • Identification of specific protein-protein interactions that facilitate this transfer

  • Role of glutathione in the transfer process

• Regulatory mechanisms:

  • How GLRX5 expression and activity are regulated under different cellular conditions

  • Post-translational modifications that might modulate GLRX5 function

  • Signaling pathways that respond to GLRX5 deficiency

• Therapeutic approaches:

  • Development of strategies to bypass GLRX5 deficiency

  • Pharmacological approaches to correct iron distribution abnormalities

  • Gene therapy approaches for GLRX5-related disorders

• Expanded disease associations:

  • Potential role of GLRX5 in neurodegenerative disorders involving iron dysregulation

  • Contributions to aging and oxidative stress responses

  • Involvement in cancer metabolism and progression

Addressing these knowledge gaps will require interdisciplinary approaches combining structural biology, biochemistry, cell biology, and clinical research to fully elucidate GLRX5's functions and therapeutic potential.