Recombinant Xenopus tropicalis Leucine-rich repeat-containing protein 58 (lrrc58)

What is lrrc58 and how is it conserved across species?

Leucine-rich repeat-containing protein 58 (lrrc58) is a highly conserved protein found across various animal species. In Caenorhabditis elegans, it's known as lrr-2, while vertebrates maintain the lrrc58 nomenclature. Sequence analysis reveals significant conservation across Xenopus tropicalis, Danio rerio, Drosophila melanogaster, and mammals . The protein contains characteristic leucine-rich repeat domains that form protein-protein interaction surfaces, allowing it to function in complex molecular networks. Its high degree of conservation suggests fundamental biological importance in cellular metabolism, particularly in cysteine and sulfur homeostasis regulation.

What is the tissue expression pattern of lrrc58 in Xenopus tropicalis?

Xenopus tropicalis lrrc58 exhibits a broad expression pattern across multiple tissues and developmental stages. RNA-Seq and EST transcriptome profiles from Xenbase indicate expression in:

This widespread expression pattern suggests lrrc58 plays important roles in multiple developmental processes and adult tissue functions .

What are the known molecular interactions of lrrc58?

High-throughput bait-and-prey experiments with human cells have identified lrrc58 as interacting with:

• CDO1 (Cysteine Dioxygenase Type 1) - An enzyme that oxidizes cysteine to cysteinesulfinate

• ATG14 - A key component in autophagy regulation

• CUL5 - A cullin protein component of ubiquitin ligase complexes

These interactions suggest lrrc58 functions at the intersection of cysteine metabolism, protein degradation, and autophagy pathways. The protein likely binds to CDO1 through its leucine-rich repeat domain, potentially regulating CDO1's enzymatic activity in the conversion of cysteine to cysteinesulfinate .

What expression systems are optimal for producing recombinant Xenopus tropicalis lrrc58?

Several expression systems can be employed for producing recombinant X. tropicalis lrrc58, each with specific advantages:

For structural studies requiring high purity, bacterial expression with optimized conditions is recommended. For functional studies where proper folding is critical, mammalian or baculovirus systems may be preferable. Xenopus cell lines, such as the 91.1.F1 limb bud line, can be particularly valuable for maintaining species-specific post-translational modifications .

What are effective strategies for designing morpholinos to study lrrc58 function?

For effective morpholino-mediated knockdown of lrrc58 in Xenopus tropicalis:

• Translation-blocking morpholinos:

  • Target the 5' UTR and start codon region of lrrc58 mRNA

  • Typically 25 nucleotides in length

  • Injection concentration: 2-10 ng per embryo at 1-2 cell stage

• Splice-blocking morpholinos:

  • Target exon-intron boundaries to disrupt proper splicing

  • Verify efficacy using RT-PCR to detect altered splice products

• Validation controls:

  • Include standard control morpholino

  • Perform rescue experiments with morpholino-resistant lrrc58 mRNA

  • Confirm protein reduction via Western blot analysis

• Phenotypic analysis:

  • Monitor development at key stages where lrrc58 is expressed

  • Analyze tissue-specific effects in brain, intestine, skin, and other expressing tissues

  • Examine molecular consequences on cysteine metabolism pathways

When designing experiments, it's essential to include proper controls and validate knockdown efficiency using qPCR with housekeeping genes like odc1 (ornithine decarboxylase 1) or rpl8 (ribosomal protein L8) as reference standards .

How can CRISPR/Cas9 be implemented to generate lrrc58 mutants?

CRISPR/Cas9 genome editing in Xenopus tropicalis provides a powerful approach for generating stable lrrc58 mutant lines:

• sgRNA design:

  • Target early exons to maximize disruption probability

  • Use Xenopus-specific CRISPR design tools to minimize off-targets

  • Select target sites with high predicted efficiency and specificity

• Delivery method:

  • Microinject Cas9 protein (500-1000 pg) and sgRNA (300-500 pg) into fertilized eggs

  • Target one-cell stage for uniform distribution

• Mutation detection strategy:

  • T7 endonuclease assay for initial screening

  • Direct sequencing of PCR products from F0 embryos

  • High-resolution melt analysis for high-throughput screening

• Breeding plan:

  • Raise F0 mosaic founders to sexual maturity (4-6 months)

  • Outcross with wild-type animals

  • Screen F1 offspring for germline transmission of mutations

  • Genotype and establish lines by intercrossing F1 heterozygotes

The advantages of X. tropicalis for this approach include its diploid genome, shorter generation time (4-6 months) compared to X. laevis, and the ability to achieve homozygous mutations in F2 generation .

What is the role of lrrc58 in cysteine homeostasis?

Based on its interaction with CDO1 and studies in C. elegans, lrrc58 appears to play a critical role in cysteine homeostasis:

• Regulatory function:

  • Likely modulates CDO1 activity in oxidizing cysteine to cysteinesulfinate

  • Potentially influences downstream production of taurine and sulfate

  • May coordinate with the ubiquitin-proteasome system through CUL5 interaction

• Metabolic implications:

  • Contributes to maintaining proper cysteine levels

  • Indirectly affects H₂S production and signaling

  • Influences cellular redox state via cysteine availability for glutathione synthesis

• Physiological significance:

  • Studies in C. elegans (lrr-2) suggest involvement in fertility

  • May affect susceptibility to bacterial infections, particularly Mycobacterium

  • Potential roles in stress response pathways

The CDO pathway represents a major route for cysteine catabolism, and lrrc58's interaction with CDO1 suggests it acts as a key regulator in this pathway, potentially through stabilization, localization, or activity modulation of CDO1.

How is lrrc58 expression regulated during development?

The regulation of lrrc58 expression during X. tropicalis development likely involves multiple mechanisms:

• Temporal regulation:

  • RNA-Seq data indicates dynamic expression across developmental stages

  • Expression patterns may correlate with key developmental transitions

• Spatial regulation:

  • Tissue-specific expression suggests regulation by lineage-specific factors

  • Potential regulation by developmental signaling pathways active in expressing tissues

• Transcriptional control:

  • Promoter likely contains binding sites for developmentally regulated transcription factors

  • May share regulatory elements with other genes involved in sulfur metabolism

• Environmental responsiveness:

  • Expression may be modulated by stress conditions

  • Potential regulation by redox state and metabolic factors

Analysis of the upstream regulatory regions of lrrc58 and comparison with the expression patterns of known developmental regulators would provide deeper insights into its transcriptional control mechanisms .

What phenotypes are associated with lrrc58 disruption?

Based on its expression pattern and molecular interactions, disruption of lrrc58 in X. tropicalis might result in multiple phenotypes:

• Developmental phenotypes:

  • Potential abnormalities in tissues with high expression (brain, intestine, skin)

  • Possible developmental timing defects due to disrupted protein degradation

• Metabolic phenotypes:

  • Altered cysteine levels and metabolism

  • Disrupted redox homeostasis

  • Changes in sulfur-containing metabolites

• Cellular phenotypes:

  • Modified autophagy processes (via ATG14 interaction)

  • Altered ubiquitin-dependent protein degradation (via CUL5)

  • Potential changes in stress responses

• Physiological phenotypes:

  • Possible fertility issues (as observed in C. elegans lrr-2 mutants)

  • Potential changes in antimicrobial resistance (particularly to mycobacteria)

  • Stress response abnormalities

Comparative analysis with C. elegans data suggests that lrrc58-deficient animals might show altered responses to specific bacterial infections and changes in cysteine-dependent cellular processes.

How can protein interaction studies be designed to elucidate lrrc58 function?

To comprehensively characterize lrrc58 protein interactions:

• Affinity purification-mass spectrometry (AP-MS):

  • Express tagged lrrc58 in X. tropicalis cells or embryos

  • Perform pulldowns under different developmental stages or conditions

  • Identify interactors using mass spectrometry

  • Validate key interactions with co-immunoprecipitation

• Proximity labeling approaches:

  • Create BioID or TurboID fusions with lrrc58

  • Express in developing embryos or tissue-specific contexts

  • Identify proximal proteins through streptavidin pulldown and MS

  • Compare interactome across different tissues or conditions

• Yeast two-hybrid screening:

  • Use lrrc58 domains as bait against X. tropicalis cDNA libraries

  • Perform directed Y2H with specific candidates (CDO1, ATG14, CUL5)

  • Map interaction domains through deletion constructs

• In vitro interaction assays:

  • Produce recombinant proteins for direct binding studies

  • Measure binding kinetics using SPR or ITC

  • Determine structural basis of interactions using crystallography

These approaches would help establish the lrrc58 interactome and elucidate how it functions within protein complexes regulating cysteine metabolism and other cellular processes .

What are the implications of lrrc58 in stress response pathways?

The potential role of lrrc58 in stress response pathways presents an intriguing area for investigation:

• Oxidative stress connection:

  • Cysteine metabolism directly impacts glutathione synthesis

  • lrrc58 may influence cellular redox state through CDO1 regulation

  • Expression might be responsive to oxidative challenge

• Metabolic stress:

  • Sulfur amino acid metabolism is linked to nutritional status

  • lrrc58 may participate in adapting to nutrient availability

  • Potential crosstalk with cellular energy sensors

• Environmental stress:

  • X. tropicalis as an amphibian faces unique environmental challenges

  • Interesting connection with the wood frog (Rana sylvatica) freeze-response protein that shares similarity with retroviral envelope proteins

  • Expression may be enhanced by particular cellular stresses, similar to XTERV1 retroviral elements

• Experimental approaches:

  • Expose embryos or cells to various stressors and monitor lrrc58 expression

  • Analyze stress resistance in lrrc58-deficient models

  • Investigate transcriptional regulation under stress conditions

The potential responsiveness to stress makes lrrc58 an interesting candidate for studies on environmental adaptation and cellular resilience mechanisms.

How does lrrc58 compare between Xenopus tropicalis and other model organisms?

Comparative analysis of lrrc58 across species provides evolutionary insights:

The conservation of lrrc58 across diverse species suggests fundamental biological importance, while species-specific differences may reflect adaptation to different physiological contexts. X. tropicalis offers unique advantages for studying lrrc58 function during vertebrate development, with a simplified genome compared to X. laevis and experimental tractability for embryological studies .

What techniques are most effective for quantifying lrrc58 expression during development?

For comprehensive analysis of lrrc58 expression during X. tropicalis development:

• Quantitative RT-PCR:

  • Design primers specific to X. tropicalis lrrc58

  • Normalize to stable reference genes (odc1, rpl8)

  • Use comparative Cᴛ method for relative quantification

  • Calculate fold changes using the 2^(-ΔΔCT) formula

• RNA-Seq analysis:

  • Perform stage-specific transcriptome analysis

  • Compare expression across developmental time points

  • Analyze co-expression patterns with interacting proteins

  • Identify tissue-specific expression signatures

• Whole-mount in situ hybridization:

  • Develop specific riboprobes for X. tropicalis lrrc58

  • Analyze spatial expression patterns across developmental stages

  • Compare with expression of interacting partners (CDO1)

  • Perform double in situ hybridization for co-localization studies

• Reporter constructs:

  • Generate transgenic lines with lrrc58 promoter driving fluorescent proteins

  • Analyze temporal and spatial expression dynamics in vivo

  • Use time-lapse imaging to track expression changes

These complementary approaches provide a comprehensive view of lrrc58 expression dynamics throughout development .

How can functional genomics approaches be applied to study lrrc58?

Modern functional genomics approaches offer powerful tools to investigate lrrc58:

• ChIP-seq analysis:

  • Identify transcription factors regulating lrrc58 expression

  • Map enhancer and promoter elements

  • Analyze chromatin state at the lrrc58 locus during development

• ATAC-seq:

  • Determine chromatin accessibility at the lrrc58 locus

  • Identify potential regulatory regions

  • Compare accessibility across developmental stages and tissues

• Single-cell RNA-seq:

  • Resolve cell type-specific expression patterns

  • Identify co-expressed gene networks

  • Track expression dynamics during lineage differentiation

• Crispr screens:

  • Perform genome-wide CRISPR screens for genetic interactors

  • Identify synthetic lethal or suppressor relationships

  • Discover novel pathway connections

• Ribosome profiling:

  • Analyze translational efficiency of lrrc58 mRNA

  • Identify potential regulatory mechanisms at the translational level

These approaches would provide comprehensive insights into the regulation and function of lrrc58 within the broader genomic context .

What structural features characterize the lrrc58 protein?

The structural features of X. tropicalis lrrc58 include:

• LRR domain architecture:

  • Multiple leucine-rich repeat motifs forming a curved horseshoe structure

  • Each LRR typically containing a consensus LxxLxLxxN/CxL sequence

  • Beta-sheet forming the concave protein interaction surface

  • Alpha-helices on the convex side providing structural stability

• Predicted structural elements:

  • N-terminal and C-terminal capping domains protecting the hydrophobic core

  • Conserved residues involved in protein-protein interactions

  • Potential post-translational modification sites

• Homology modeling insights:

  • Structure likely similar to other well-characterized LRR proteins

  • Concave surface forming a binding platform for interacting proteins

  • Conservation mapping revealing functional surfaces

• Critical residues:

  • Conserved leucine residues maintaining structural integrity

  • Surface-exposed residues mediating specific interactions

  • Potential regulatory sites for post-translational modifications

Structural analysis of lrrc58 would provide important insights into its mechanism of action and interaction specificity.

What biochemical assays can assess lrrc58 function in cysteine metabolism?

To investigate lrrc58's role in cysteine metabolism:

• CDO1 activity assays:

  • Measure cysteine dioxygenase activity in presence/absence of lrrc58

  • Quantify cysteinesulfinate production using HPLC or mass spectrometry

  • Compare kinetic parameters with varying lrrc58 concentrations

• Cysteine homeostasis analysis:

  • Measure intracellular cysteine levels in lrrc58-depleted cells or tissues

  • Analyze cysteine:cystine ratio as an indicator of redox state

  • Track metabolic flux through cysteine catabolism pathways

• H₂S production assays:

  • Measure H₂S generation in presence/absence of lrrc58

  • Investigate interaction with cysteine synthase-like genes (cysl-1/2)

  • Analyze effects on bacterial growth in co-culture systems

• Protein stability assays:

  • Assess CDO1 protein half-life in the presence/absence of lrrc58

  • Investigate ubiquitination patterns of CDO1

  • Determine if lrrc58 protects CDO1 from degradation

These biochemical approaches would help elucidate the mechanistic role of lrrc58 in regulating cysteine metabolism pathways .

What is the potential role of lrrc58 in host-pathogen interactions?

The potential involvement of lrrc58 in host-pathogen interactions presents an exciting research direction:

• Mycobacterial infection models:

  • C. elegans studies suggest LRRC58 mutants might be more resistant to Mycobacterium tuberculosis

  • X. tropicalis could serve as a vertebrate model to test this hypothesis

  • Investigate whether lrrc58 knockdown affects susceptibility to mycobacterial infection

• H₂S signaling connection:

  • H₂S is beneficial to M. tuberculosis

  • lrrc58 may influence H₂S levels through regulation of cysteine metabolism

  • This could represent a novel host-pathogen interaction mechanism

• Experimental approaches:

  • Generate lrrc58-deficient X. tropicalis

  • Challenge with appropriate pathogens

  • Measure infection parameters and host survival

  • Analyze changes in sulfur metabolites during infection

• Therapeutic implications:

  • The leucine-rich repeat domain of lrrc58 could represent a drug target

  • Inhibiting lrrc58 might reduce H₂S availability to infectious bacteria

  • This could potentially reduce virulence of pathogens like M. tuberculosis

This research direction could reveal new insights into host-pathogen interactions mediated by metabolic pathways.

How might lrrc58 function during amphibian metamorphosis?

The potential role of lrrc58 during X. tropicalis metamorphosis is an intriguing area for investigation:

• Expression dynamics:

  • Analyze lrrc58 expression before, during, and after metamorphosis

  • Compare expression patterns in different tissues undergoing remodeling

  • Investigate correlation with thyroid hormone levels and signaling

• Functional significance:

  • Potential role in tissue remodeling through protein degradation pathways

  • Possible involvement in oxidative stress management during metamorphosis

  • Contribution to sulfur amino acid metabolism during this high-energy demand process

• Respiratory system connection:

  • Expression in lung tissue before and during metamorphosis

  • Potential involvement in the transition from gill to lung respiration

  • Possible role in septation of lung buds and respiratory epithelium development

• Experimental approaches:

  • Targeted knockdown or mutation of lrrc58 specifically during metamorphosis

  • Analysis of metamorphic timing and success in lrrc58-deficient animals

  • Investigation of tissue-specific phenotypes in metamorphosing structures

This research could provide insights into the unique aspects of amphibian development and the role of sulfur metabolism during major physiological transitions.

What controls are essential for interpreting lrrc58 functional studies?

For robust interpretation of lrrc58 functional studies:

• Expression analysis controls:

  • Multiple reference genes for qPCR (odc1, rpl8)

  • Stage-matched and tissue-matched controls

  • Sense probe controls for in situ hybridization

  • Antibody validation with overexpression and knockdown samples

• Genetic manipulation controls:

  • Standard control morpholinos

  • 5-base mismatch control morpholinos

  • Rescue experiments with morpholino-resistant mRNA

  • Multiple sgRNAs targeting different lrrc58 regions in CRISPR experiments

  • Off-target analysis for CRISPR/Cas9 editing

• Interaction studies controls:

  • Empty vector controls for co-immunoprecipitation

  • Unrelated protein controls of similar size/structure

  • Competition assays with purified proteins

  • Mutated binding site variants

• Phenotypic analysis controls:

  • Wild-type siblings from the same clutch

  • Careful staging to account for developmental variation

  • Blinded scoring of phenotypes

  • Appropriate statistical tests for sample sizes

Implementing these controls ensures reliable interpretation of experimental results and facilitates reproducibility across different research groups.

What are the key technical considerations for generating recombinant lrrc58?

For successful production of recombinant X. tropicalis lrrc58:

• Sequence optimization:

  • Codon optimization for the chosen expression system

  • Removal of cryptic splice sites for eukaryotic expression

  • Addition of appropriate tags for detection and purification

  • Inclusion of protease cleavage sites for tag removal

• Expression conditions:

  • For bacterial expression: low temperature (18°C) induction

  • For insect cells: optimization of MOI and harvest time

  • For mammalian cells: selection of appropriate promoters

  • For all systems: inclusion of protease inhibitors during purification

• Solubility enhancement:

  • Consider fusion partners (MBP, SUMO, Thioredoxin)

  • Test expression of individual domains if full-length is problematic

  • Optimize buffer conditions (pH, salt, additives)

  • Screen detergents if membrane association is suspected

• Functional validation:

  • Circular dichroism to confirm proper folding

  • Size exclusion chromatography to verify monodispersity

  • Binding assays with known interaction partners

  • Activity assays related to cysteine metabolism

These considerations help ensure the production of properly folded, functional protein suitable for downstream applications such as structural studies, interaction analyses, or antibody production .