GLRX3 Human

What is the primary function of human GLRX3 in cellular biology?

GLRX3 functions as a [2Fe-2S] cluster chaperone in the cytosolic iron-sulfur assembly (CIA) machinery, which is essential for the maturation of cytosolic [4Fe-4S] proteins . As part of the monothiol glutaredoxin family, GLRX3 plays critical roles in maintaining iron homeostasis and facilitating efficient responses to oxidative stress . Experimental evidence demonstrates that GLRX3 can form dimeric cluster-bridged structures and transfer its [2Fe-2S] clusters to proteins like NUBP1, a P-loop NTPase essential in early CIA machinery processes .

Methodologically, researchers can study GLRX3's function through:

• Reconstitution of cluster transfer reactions using purified proteins

• Spectroscopic analyses to monitor [2Fe-2S] and [4Fe-4S] cluster formation

• Genetic complementation studies in model organisms

How does GLRX3 interact with GMP synthase and what is the significance?

GLRX3 strongly interacts with GMP synthase (GMPs) as demonstrated through multiple experimental approaches. Using two-hybrid assays, researchers identified mouse GMP synthase as a significant interaction partner (25% of identified sequences in a screening) . This interaction was subsequently validated with human GMP synthase through both two-hybrid analyses and coimmunoprecipitation approaches .

The GLRX3-GMPs interaction is functionally significant because:

• This protein complex participates in downregulating the integrated stress response (ISR) pathway

• The interaction requires iron-sulfur clusters as bridging elements

• The complex shows evolutionary conservation from yeast to humans

• This interaction connects GLRX3 to purine metabolism pathways

To study this interaction, researchers should employ two-hybrid assays, coimmunoprecipitation, and functional complementation tests under varying conditions including iron depletion .

How do iron-sulfur clusters mediate GLRX3 function and protein interactions?

Iron-sulfur clusters are central to GLRX3 function, particularly in mediating protein interactions. Experimental evidence shows that:

• GLRX3 transfers [2Fe-2S]²⁺ clusters to apo NUBP1, which are then reductively coupled to form [4Fe-4S]²⁺ clusters

• Mutations in residues involved in bridging iron/sulfur clusters severely diminish binding between glutaredoxins and GMP synthases

• Iron depletion from culture media precludes the in vivo interaction between Glrx3/Grx3/Grx4 and GMP synthases, confirming that iron bridges these complexes

• The [2Fe-2S] cluster transfer process requires glutathione as a reductant, though stronger reductants may be needed for complete efficiency

The differential binding properties of clusters are noteworthy: the [4Fe-4S]²⁺ cluster formed at the N-terminal motif of NUBP1 is tightly bound, while the cluster at the C-terminal motif is more labile .

What is the role of GLRX3 in regulating the integrated stress response (ISR) pathway?

GLRX3, in conjunction with GMP synthase, downregulates the integrated stress response (ISR) pathway, particularly under conditions of nutritional stress. Key experimental findings include:

• Coexpression of Glrx3 and human GMP synthase (hGMPs) significantly reduces GCN4 expression and eIF2α phosphorylation in wild-type, grx3 grx4, and gua1 strains under nutrient deprivation conditions

• Single overexpression of either Glrx3 or hGMPs has less impact than their coexpression

• Point mutations in GLRX3 residues critical for binding to hGMPs abrogate the negative regulatory effect on GCN4 derepression and eIF2α phosphorylation

• This regulatory mechanism appears conserved from yeast to humans, as demonstrated by complementation studies

Methodologically, researchers should analyze both GCN4 translational expression and eIF2α phosphorylation as indicators of ISR activity under various nutritional conditions.

What is the relationship between GLRX3 and cellular lifespan?

GLRX3 and its yeast homologs (Grx3/Grx4) significantly impact chronological life span. Research has demonstrated that:

• The grx3 grx4 double mutant exhibits defects in chronological life span that can be rescued by the expression of human Glrx3

• Both excess and deficiency of monothiol glutaredoxins (Glrx3/Grx3/Grx4) are detrimental to life span extension

• The addition of guanine to cultures complements growth problems in the grx3 grx4 double mutant, suggesting a functional connection between monothiol glutaredoxins and purine metabolism

This table demonstrates the impact of GLRX3 expression on cellular growth rates:

The data clearly shows that expression of Glrx3 accelerates growth in both wild-type and mutant strains, with the strongest effects seen with Glrx3 alone in wild-type cells (reducing generation time by 32%) and with Glrx3+hGMPs in the double mutant (reducing generation time by 16.7%) .

What are the optimal methods for detecting GLRX3 in experimental settings?

Multiple validated methods are available for GLRX3 detection in research applications:

• Western Blot Analysis: Using monoclonal antibodies like MAB7560, GLRX3 can be detected at approximately 40 kDa in human cell lines including HepG2 and Raji cells. Optimal results are achieved under reducing conditions using specific immunoblot buffer groups .

• Immunofluorescence: GLRX3 can be visualized in fixed cells using monoclonal antibodies (such as MAB7560 at 10 μg/mL) with appropriate secondary antibody conjugates. This has been validated in cell lines like NIH-3T3 .

• Co-localization Studies: These can determine whether GLRX3 and interaction partners (like GMP synthase) co-localize in specific cellular compartments under stress conditions. Evidence suggests GLRX3 and GMP synthase co-localize in the nucleus upon ISR pathway induction .

For optimal results, researchers should:

• Use validated antibody concentrations (0.5 μg/mL for Western blot, 10 μg/mL for immunofluorescence)

• Select appropriate detection systems (e.g., HRP-conjugated secondary antibodies for Western blot)

• Include positive control cell lines with known GLRX3 expression

How can researchers effectively study the [2Fe-2S] cluster transfer function of GLRX3?

Studying the [2Fe-2S] cluster transfer function of GLRX3 requires specialized experimental approaches:

• Protein Preparation: Express and purify dimeric cluster-bridged GLRX3 while maintaining the integrity of the [2Fe-2S]²⁺ clusters .

• Cluster Transfer Assays: Incubate GLRX3 with apo NUBP1 in the presence of glutathione to monitor transfer of [2Fe-2S]²⁺ clusters and their reductive coupling to form [4Fe-4S]²⁺ clusters .

• Spectroscopic Analysis: Use spectroscopic techniques to monitor cluster transfer and formation:

  • UV-visible spectroscopy for initial detection

  • More specialized techniques to characterize cluster oxidation states and environments

• Quaternary Structure Analysis: Monitor how cluster binding affects protein dimerization, particularly in target proteins like NUBP1 where clusters bound to the C-terminal CPXC motif promote dimerization while clusters bound to the N-terminal motif do not affect quaternary structure .

• Reductant Evaluation: Test different reductants beyond GSH to enhance transfer efficiency, as research indicates the process may not be complete with glutathione alone .

What experimental approaches best demonstrate GLRX3's role in the ISR pathway?

To study GLRX3's role in the integrated stress response (ISR) pathway, researchers should:

• Monitor Key ISR Markers: Analyze GCN4 translational expression and eIF2α phosphorylation under various conditions, particularly during nutrient deprivation or amino acid starvation .

• Gene Expression Manipulation: Design experiments with combinations of:

  • Single expression of Glrx3 or GMP synthase

  • Co-expression of both proteins

  • Point mutations in residues critical for protein-protein interactions

• Nutritional Manipulation: Conduct experiments under standard and nutrient-deprived conditions, as GLRX3-mediated effects on ISR are most pronounced during starvation .

• Cross-Species Complementation: Utilize the evolutionary conservation between human and yeast systems to conduct complementation studies:

  • Test human Glrx3 with yeast GUA1

  • Test yeast Grx3/Grx4 with human GMP synthase

  • Compare effects of various combinations on ISR pathway components

• Iron Manipulation: Given the importance of iron-sulfur clusters, manipulate iron availability to observe effects on GLRX3-mediated ISR regulation .

What is the significance of GLRX3 in human disease contexts?

GLRX3 (PICOT) is implicated in several human diseases, most notably cancer . While the search results don't provide specific disease mechanisms, the protein's fundamental roles suggest several pathways through which dysfunction might contribute to pathology:

• Iron Homeostasis Disruption: As GLRX3 is critical for iron-sulfur cluster assembly, dysfunction could lead to iron metabolism disorders .

• Stress Response Dysregulation: Given GLRX3's role in downregulating the integrated stress response pathway, alterations might impair cellular adaptation to stress conditions .

• Impaired Fe-S Protein Maturation: Since GLRX3 is essential for the maturation of cytosolic [4Fe-4S] proteins, dysfunction could broadly impact proteins requiring these clusters .

• Altered Cellular Lifespan: Experimental evidence shows that both deficiency and excess of GLRX3 affect chronological lifespan, suggesting implications for aging-related diseases .

Methodologically, researchers investigating GLRX3 in disease contexts should consider:

• Comparative expression analyses between normal and diseased tissues

• Identification of disease-associated mutations affecting GLRX3 function

• Correlation studies between GLRX3 status and clinical outcomes

How might targeting GLRX3 provide therapeutic opportunities?

Based on GLRX3's molecular functions, several therapeutic approaches might be considered:

Methodological considerations for therapeutic development include:

• Structure-based drug design targeting GLRX3 interaction interfaces

• Small molecule screening for modulators of GLRX3-GMP synthase interactions

• Evaluation of iron chelation therapies that might indirectly affect GLRX3 function

What are the key unanswered questions regarding GLRX3 biology?

Several critical questions remain unanswered about GLRX3 biology:

• Comprehensive Interactome: Beyond GMP synthase and NUBP1, what is the complete set of GLRX3 interaction partners and how do these interactions contribute to cellular homeostasis?

• Regulation Mechanisms: What factors regulate GLRX3 expression, localization, and activity under various physiological and pathological conditions?

• Human Disease Relevance: While GLRX3 is implicated in cancer, what specific mechanisms connect GLRX3 dysfunction to disease progression, and are there other diseases where GLRX3 plays a significant role?

• Therapeutic Potential: Can GLRX3 or its interactions be effectively targeted for therapeutic benefit, particularly in cancer or aging-related conditions?

• Structural Determinants: What structural features of GLRX3 determine its specificity for different interaction partners and its function in [2Fe-2S] cluster transfer?

What emerging technologies might advance GLRX3 research?

Emerging technologies that could significantly advance GLRX3 research include:

• Cryo-EM and Advanced Structural Biology: To elucidate the molecular details of GLRX3 interactions with GMP synthase and other partners, particularly the structural changes occurring during [2Fe-2S] cluster transfer .

• CRISPR-Based Technologies: For precise genomic manipulation to study GLRX3 function in various cellular contexts, including the creation of specific mutations corresponding to those potentially found in human diseases.

• Advanced Live-Cell Imaging: To monitor GLRX3 dynamics, particularly its nuclear localization upon ISR pathway induction .

• Single-Cell Analysis: To understand heterogeneity in GLRX3 expression and function across different cell populations, especially in diseased tissues.

• Systems Biology Approaches: To place GLRX3 within broader networks of iron homeostasis, stress response, and lifespan regulation, integrating transcriptomic, proteomic, and metabolomic data.

These technologies, when combined with existing methodological approaches, have the potential to address the key unanswered questions and advance our understanding of GLRX3 biology and its relevance to human health and disease.