Recombinant Salmo salar F-box/LRR-repeat protein 5 (fbxl5), partial

What is the basic structure of FBXL5 protein and how does it compare between mammals and Salmo salar?

FBXL5 is a 691 amino acid protein (in humans) with a predicted molecular weight of 78.6 kD. The protein contains an N-terminal hemerythrin-like domain, followed by an F-box domain and seven leucine-rich repeats . The Salmo salar FBXL5 shares conserved domains with its mammalian counterparts, particularly in the hemerythrin-like domain which is critical for iron and oxygen sensing. Unlike mammalian FBXL5, which has been extensively characterized, the salmon variant may exhibit species-specific adaptations related to the aquatic environment and evolutionary divergence, though structural conservation of the functional domains is expected.

Researchers should note that when working with the recombinant partial FBXL5 from Salmo salar, domain-specific structural analyses are recommended to confirm conservation of key functional regions before extrapolating findings from mammalian studies.

How does the hemerythrin-like domain in FBXL5 function as a cellular sensor for iron and oxygen?

The hemerythrin-like domain (FBXL5-Hr) employs distinct mechanisms to signal changes in iron and oxygen availability. For iron sensing, FBXL5-Hr undergoes substantial conformational changes when iron becomes limiting, exhibiting switch-like behavior between two very distinct conformations depending on cellular iron status . This conformational change affects the protein's stability and subsequent ubiquitination.

In contrast, oxygen depletion does not induce the same gross structural rearrangements used to communicate cellular iron status . This suggests that FBXL5 uses different molecular mechanisms to respond to iron versus oxygen availability. Importantly, FBXL5-Hr can only incorporate iron during or shortly after its synthesis rather than continuously sampling the cellular environment . This restricted sensing window has significant implications for understanding how cells monitor and respond to changes in these critical metabolites.

What is the cellular distribution and expression pattern of FBXL5?

FBXL5 is predominantly localized in the cytosol and nucleus, with ubiquitous expression across tissues . In experimental settings, understanding this distribution is critical when designing immunocytochemistry or subcellular fractionation experiments with Salmo salar FBXL5. When working with the recombinant protein, researchers should consider that the partial protein may lack sequences that determine natural subcellular localization.

For studies investigating tissue-specific expression patterns in salmon, it's advisable to develop species-specific antibodies or nucleic acid probes that can reliably detect endogenous FBXL5 across different tissue types, as expression patterns may differ from those observed in mammalian systems.

What are the optimal storage and handling conditions for recombinant Salmo salar FBXL5?

Recombinant Salmo salar FBXL5 should be stored at -20°C for regular use or -80°C for long-term storage. The protein is typically supplied in a liquid formulation containing glycerol . For working applications, store aliquots at 4°C for up to one week to avoid repeated freeze-thaw cycles which can compromise protein integrity .

For experimental work, consider the following evidence-based handling protocol:

• Prepare single-use aliquots upon receipt to minimize freeze-thaw cycles

• Maintain a consistent reducing environment if studying iron-binding properties

• When investigating iron-dependent conformational changes, ensure iron supplementation occurs during or immediately after protein synthesis

• For structural studies, note that the protein may exist in distinct conformational states depending on iron availability during preparation

This approach maintains protein stability and functional integrity while accommodating FBXL5's unique properties as a metalloproteiin.

What methodologies are effective for analyzing iron-dependent conformational changes in FBXL5?

Several complementary approaches have proven effective for analyzing iron-dependent conformational changes in FBXL5-Hr:

• Limited proteolysis assays: Trypsin digestion patterns differ significantly between iron-bound and iron-free FBXL5, reflecting their distinct conformational states . Protocol: Add 3 μg trypsin to 100 μg total protein and incubate at 37°C for 1 hour. Quench the reaction with 0.2 mM PMSF and assess digestion patterns via immunoblotting .

• Circular dichroism (CD) spectroscopy: While secondary structure remains relatively unchanged between apoHr and holoHr forms (as shown in Fig. 3A from source material), subtle differences can be detected that reflect tertiary structural changes .

• Half-life determination: Under iron-replete conditions, FBXL5-Hr exhibits a substantially longer half-life (~7.3 hours) compared to iron-deplete conditions (~1.9 hours) . This provides a functional readout of conformational state.

• Ubiquitination assays: Polyubiquitination increases when FBXL5-Hr undergoes conformational changes due to iron depletion, serving as an indirect measure of structural state .

When adapting these methods for Salmo salar FBXL5, researchers should validate each assay with appropriate controls, as species-specific differences may affect proteolytic patterns or spectroscopic properties.

How can researchers effectively express and purify recombinant Salmo salar FBXL5 for experimental studies?

For optimal expression and purification of recombinant Salmo salar FBXL5, consider the following validated methodology:

Expression System Selection:
While commercial recombinant FBXL5 is available from expression in E. coli, yeast, baculovirus, or mammalian cells , each system offers distinct advantages:

• E. coli expression: Highest yield but may lack post-translational modifications

• Insect cell/baculovirus system: Better for preserving structural integrity of the hemerythrin domain

• Mammalian expression: Optimal for functional studies requiring authentic folding and modification

Purification Protocol:

• Use affinity chromatography with appropriate tags (His-tag or FLAG-tag)

• Consider the iron status during purification - supplement with ferrous iron during lysis if studying the holo-form

• Employ size exclusion chromatography as a final purification step

• Validate protein purity (>90%) using SDS-PAGE

Critical Considerations:

• The timing of iron supplementation is crucial as FBXL5-Hr incorporates iron primarily during synthesis

• If studying the apo-form, include chelating agents in buffers to ensure iron-free status

• For functional assays, verify that the recombinant protein retains iron-binding capacity using spectroscopic methods

How does FBXL5 participate in the SCF ubiquitin ligase complex and what are the implications for iron homeostasis research?

FBXL5 functions as the substrate recognition component of the SCF (SKP1-Cullin-1-F-box) E3 ubiquitin ligase complex. This complex contains SKP1, Cullin-1, RBX1, and FBXL5 . Under iron-replete conditions, FBXL5 recruits Iron Regulatory Protein 2 (IRP2) to the SCF complex, promoting its ubiquitination and subsequent proteasomal degradation .

The detailed molecular mechanism involves:

• Iron binding to the hemerythrin-like domain of FBXL5, stabilizing the protein

• Stabilized FBXL5 incorporates into the SCF complex via its F-box domain

• The leucine-rich repeat region recognizes and binds IRP2

• The complex facilitates ubiquitin transfer to IRP2, targeting it for degradation

For research with Salmo salar FBXL5, investigators should determine whether the partial recombinant protein contains the domains necessary for both iron sensing and SCF complex formation. If studying the complete iron-regulatory pathway, complementary experiments with SKP1 and Cullin-1 from Salmo salar may be necessary to reconstitute the entire complex.

The evolutionary conservation of this pathway in fish provides valuable comparative insights into iron homeostasis mechanisms across vertebrates, potentially revealing adaptations specific to aquatic environments.

What experimental approaches are recommended for investigating iron-dependent protein-protein interactions of FBXL5?

To investigate iron-dependent protein-protein interactions of FBXL5, several complementary methodologies are recommended:

Co-immunoprecipitation under varying iron conditions:

• Treat cells with either iron supplementation (ferric ammonium citrate) or iron chelation (deferoxamine)

• Lyse cells under non-denaturing conditions

• Immunoprecipitate FBXL5 using specific antibodies

• Analyze co-precipitating proteins by immunoblotting or mass spectrometry

Proximity-based labeling:
BioID or APEX2 fusion proteins can identify transient or weak interactors in living cells under different iron conditions, capturing the dynamic interactome of FBXL5.

Quantitative Analysis of Interaction Partners:
The table below summarizes key FBXL5 interaction partners and their iron-dependency:

When working with Salmo salar FBXL5, researchers should validate these interactions in fish cell lines or tissues, as evolutionary differences may affect interaction specificities or regulatory mechanisms.

How does the oxygen-sensing mechanism of FBXL5 differ from its iron-sensing properties, and what methodologies can distinguish between these functions?

The oxygen and iron-sensing mechanisms of FBXL5 employ distinct molecular approaches, despite both being mediated through the hemerythrin-like domain:

Key Differences:

• Iron limitation induces substantial conformational changes in FBXL5-Hr, while oxygen depletion does not cause the same gross structural rearrangements

• Under hypoxia but with sufficient iron, FBXL5-Hr half-life decreases to ~3 hours (compared to ~7.3 hours under normal oxygen with iron and ~1.9 hours without iron)

• The molecular basis for oxygen sensing likely involves the oxidation state of iron within the hemerythrin domain rather than major conformational changes

Methodologies to Distinguish Between These Functions:

• Differential proteolysis patterns:

  • Compare trypsin digestion patterns of FBXL5-Hr under four conditions: +Fe/+O₂, +Fe/-O₂, -Fe/+O₂, and -Fe/-O₂

  • Perform experiments under controlled atmospheric conditions (e.g., anoxic chamber with <1 ppm O₂)

• Spectroscopic analysis:

  • UV-visible spectroscopy to monitor the characteristic absorption of the diferric center

  • Circular dichroism to detect subtle structural differences between oxygen-depleted and oxygen-replete states

• Mutational analysis:

  • Generate point mutations in the iron-coordinating residues versus mutations that would affect oxygen binding

  • Assess differential effects on protein stability and function

• Half-life determination under varying conditions:

  • Use cycloheximide chase experiments under combinations of iron and oxygen availability

  • Quantify degradation rates to distinguish between iron and oxygen effects

For studies with Salmo salar FBXL5, researchers should consider potential evolutionary adaptations that might alter the oxygen-sensing properties, particularly given the aquatic environment where oxygen availability may fluctuate significantly.

What are the recommended approaches for analyzing FBXL5 function in fish models compared to mammalian systems?

When analyzing FBXL5 function in fish models compared to mammalian systems, researchers should consider methodological adaptations to account for biological differences:

Experimental System Selection:

• Cell culture models: Establish salmon-derived cell lines for in vitro studies, as mammalian cell lines may not recapitulate fish-specific regulatory mechanisms

• Primary tissue explants: Employ liver, gill, or intestinal explants to study FBXL5 in tissues central to iron homeostasis in fish

• Whole organism studies: Utilize embryonic development stages for gene knockdown/knockout experiments using CRISPR-Cas9 or morpholinos

Physiological Parameter Considerations:

• Temperature: Fish FBXL5 may function optimally at lower temperatures than mammalian counterparts

• Oxygen levels: Adjust experimental conditions to reflect the dissolved oxygen levels in aquatic environments

• Iron bioavailability: Consider that fish acquire iron primarily from water and diet in different forms than terrestrial animals

Assessment Methods:

• Develop fish-specific antibodies for immunodetection

• Design qPCR primers optimized for salmon FBXL5 and related genes

• Establish appropriate internal controls and reference genes for fish systems

Comparative Table of Experimental Approaches:

These methodological adaptations will help researchers obtain physiologically relevant results when studying FBXL5 function in fish models.

How might the FBXL5-mediated iron-sensing mechanism in Salmo salar adapt to fluctuating environmental conditions?

Fish encounter highly variable environmental conditions that may necessitate specialized adaptations in iron-sensing mechanisms. For Salmo salar FBXL5, researchers might investigate:

• Temperature-dependent iron sensing:

  • Examine FBXL5 stability across temperature ranges encountered during salmon migration

  • Investigate potential temperature-sensitive structural elements in the hemerythrin domain

• Adaptations to environmental iron fluctuations:

  • Compare FBXL5 responsiveness to iron between freshwater and seawater phases of the salmon lifecycle

  • Analyze potential adjustments in iron affinity or sensing threshold

• Integration with other environmental sensing pathways:

  • Explore crosstalk between FBXL5 and pathways sensing pH, salinity, or pollutants

  • Investigate potential secondary modifications that modulate FBXL5 function in response to environmental stressors

Recommended experimental approach: Develop an in vitro system using salmon hepatocytes maintained under controlled conditions where temperature, pH, salinity, and iron availability can be independently varied. Monitor FBXL5 protein levels, stability, and activity under these different conditions to identify environment-specific regulatory mechanisms.

What are the methodological considerations for investigating the role of FBXL5 in regulating systemic versus cellular iron homeostasis in fish?

Investigating FBXL5's role in systemic versus cellular iron homeostasis in fish requires specialized methodological approaches:

For Systemic Iron Regulation:

• Tissue-specific expression analysis:

  • Quantify FBXL5 expression across tissues involved in iron metabolism (liver, intestine, gills, kidney)

  • Compare expression patterns during different life stages and environmental conditions

• Whole-organism manipulation:

  • Generate FBXL5 knockdown/knockout models in zebrafish as a proxy for salmonid studies

  • Monitor systemic iron parameters including serum iron, transferrin saturation, and tissue iron distribution

• Physiological challenges:

  • Subject fish to iron overload or deficiency conditions

  • Measure compensatory responses in FBXL5 levels across relevant tissues

For Cellular Iron Regulation:

• Primary cell isolation:

  • Establish protocols for isolating hepatocytes, enterocytes, and macrophages from Salmo salar

  • Characterize FBXL5-dependent responses to iron challenges in these cell types

• Subcellular iron distribution:

  • Employ cellular fractionation combined with iron quantification methods

  • Use fluorescent iron sensors to track iron redistribution in response to FBXL5 manipulation

Bridging Cellular and Systemic Approaches:
Develop ex vivo perfused organ systems (particularly liver) that maintain physiological iron handling while allowing experimental manipulation of FBXL5 levels or activity.

Critical Control Considerations:
When working with fish models, researchers must account for the significant influence of environmental variables on experimental outcomes, necessitating rigorous standardization of water chemistry, temperature, and feeding regimens.

What are the emerging technologies and methodologies that could advance our understanding of FBXL5 function across species?

Several cutting-edge technologies offer promising approaches for advancing our understanding of FBXL5 function across species:

• Cryo-electron microscopy:

  • Resolve high-resolution structures of FBXL5 in different conformational states

  • Compare structures between mammalian and fish FBXL5 to identify species-specific adaptations

  • Visualize complexes with interaction partners under varying iron conditions

• Single-cell multi-omics:

  • Combine single-cell RNA-seq, proteomics, and metabolomics to map FBXL5-dependent pathways

  • Identify cell type-specific responses to iron fluctuations

  • Reveal heterogeneity in iron sensing within tissues

• Genome editing with temporal control:

  • Deploy inducible CRISPR-Cas9 systems to manipulate FBXL5 with precise timing

  • Generate conditional knockouts in specific tissues to dissect systemic versus local functions

  • Create precise point mutations to separate iron-sensing from oxygen-sensing functions

• Live iron sensing:

  • Develop fluorescent biosensors based on the FBXL5 hemerythrin domain

  • Create fusion proteins that report on FBXL5 conformational states in living cells

  • Employ these tools across species to identify differences in sensing dynamics

• Computational approaches:

  • Apply molecular dynamics simulations to predict species-specific differences in FBXL5 dynamics

  • Use machine learning to identify patterns in iron-responsive gene expression across vertebrates

  • Develop systems biology models of iron homeostasis incorporating FBXL5-dependent regulation

Implementation strategy for Salmo salar studies:
Begin with comparative genomics to identify conserved and divergent features in salmon FBXL5, then develop species-specific tools (antibodies, expression constructs) for experimental validation. Apply emerging technologies in a stepwise manner, starting with structural studies and progressing to functional genomics approaches.

How does FBXL5 research in diverse species, including Salmo salar, contribute to our broader understanding of iron homeostasis mechanisms?

Studying FBXL5 across diverse species provides unique insights into the evolution and adaptation of iron homeostasis mechanisms:

• Evolutionary conservation: The presence of FBXL5-mediated iron sensing across vertebrates highlights the fundamental importance of this regulatory mechanism. Comparative studies between mammals and fish reveal which aspects have remained invariant over hundreds of millions of years of evolution.

• Environmental adaptations: Salmo salar experiences dramatic changes in iron availability during its lifecycle, transitioning between freshwater and marine environments. The adaptations in FBXL5 function to accommodate these changes may reveal novel regulatory mechanisms not apparent in terrestrial mammals.

• Specialized physiological requirements: Fish obtain oxygen from water through gills rather than lungs, potentially necessitating specialized coupling between oxygen and iron sensing. The dual-sensing capacity of FBXL5 may have evolved different sensitivities or response characteristics in aquatic vertebrates.

• Therapeutic implications: Understanding the conserved cores and variable regions of FBXL5 function across species helps identify potential therapeutic targets for iron disorders. Features conserved from fish to mammals likely represent essential mechanisms that cannot be altered without severe consequences.

For researchers exploring this area, we recommend a collaborative approach that integrates evolutionary biology, structural biochemistry, and physiological studies across model organisms. Such comprehensive investigation will not only advance our understanding of iron homeostasis but also provide insights into how fundamental cellular sensing mechanisms adapt to diverse environmental challenges.