Recombinant Xenopus laevis Protein fem-1 homolog B (fem1b), partial

What is the evolutionary relationship between Xenopus laevis fem1b and its homologs in other species?

Fem1b belongs to a conserved family of proteins originally identified in C. elegans, where FEM-1 plays a central role in sex determination. In mammals, three distinct Fem1 genes (Fem1a, Fem1b, and Fem1c) have been identified, each encoding proteins with greater than 30% amino acid identity with C. elegans FEM-1 and greater than 40% identity with each other . The most conserved regions are the ankyrin repeats at the N-terminus, which mediate specific protein-protein interactions. Notably, individual Fem1 orthologs share approximately 99% amino acid identity between human and mouse .

The conservation table below illustrates the relationship between FEM1 proteins across species:

Unlike C. elegans, where fem genes are crucial for sex determination with null mutants displaying feminization of XX and XO animals , the vertebrate homologs have evolved more diverse functions while retaining certain molecular mechanisms.

What is the structure and domain organization of Xenopus laevis fem1b protein?

Xenopus laevis fem1b, like its mammalian counterparts, features a conserved domain architecture with six ankyrin repeats at the N-terminus followed by a variable C-terminal region. The ankyrin repeats are 33-amino acid motifs that mediate specific protein-protein interactions . Recent structural studies of human FEM1B have revealed its role as a substrate receptor in Cullin 2-RING ligase (CRL2) complexes, where it recognizes specific C-terminal degrons in target proteins .

A key feature of FEM1B is the presence of a critical cysteine residue (C186 in human FEM1B) that plays an essential role in substrate recognition . This cysteine has become an important target for developing covalent ligands that can modulate FEM1B function in experimental and therapeutic applications.

What is the expression pattern of fem1b during Xenopus development, and how does it compare to mammalian systems?

While specific data on Xenopus laevis fem1b expression patterns throughout development is limited in the provided search results, information from mammalian systems can provide context. In mice, Fem1b is co-expressed with Nkx3.1 in the prostate epithelium and testicular germ cells during organogenesis .

The expression pattern in mammalian systems suggests fem1b likely plays a role in sexual development and tissue differentiation in amphibians as well. In Xenopus, analyzing expression patterns would typically involve:

• Whole-mount in situ hybridization at different developmental stages

• RT-PCR analysis of tissue-specific expression

• Immunohistochemistry using antibodies against fem1b

This comparative approach allows researchers to determine whether fem1b has conserved expression patterns across vertebrates or whether it has acquired novel expression domains in amphibians.

How does fem1b function in protein degradation pathways?

FEM1B functions as a substrate receptor in Cullin 2-RING ligase (CRL2) E3 ubiquitin ligase complexes. These complexes play critical roles in targeting specific proteins for ubiquitylation and subsequent degradation by the proteasome .

Recent studies have revealed that FEM1B specifically recognizes C-degrons containing a C-terminal proline in substrate proteins . The mechanism involves:

• FEM1B associates with elongin B (EB), elongin C (EC), CUL2, and RBX1 to form the complete CRL2^FEM1B complex

• The substrate recognition domain of FEM1B, containing the critical cysteine C186, binds to target proteins bearing specific degron sequences

• This binding positions the substrate for ubiquitylation by the E2 ubiquitin-conjugating enzyme recruited by the complex

• Polyubiquitylated substrates are subsequently recognized and degraded by the 26S proteasome

In addition to recognizing C-terminal degrons, FEM1B has been shown to recognize reduced cysteines on substrates like FNIP1 under reductive stress conditions, leading to their ubiquitylation and degradation . This function helps restore redox homeostasis and maintain cellular integrity.

What are the optimal methods for expressing and purifying recombinant Xenopus laevis fem1b protein?

Based on approaches used for mammalian FEM1B, the following protocol would be suitable for Xenopus laevis fem1b:

Expression System Selection:

• E. coli for structural studies and biochemical assays (may require optimization of codon usage)

• Insect cells (Sf9 or Hi5) for studies requiring post-translational modifications

• HEK293 cells for studies of complex formation with other components

Purification Strategy:

• Clone the Xenopus laevis fem1b coding sequence into an expression vector with an appropriate tag (His6, GST, or MBP)

• For full activity, co-express with other components of the CRL2 complex (elongin B, elongin C)

• Use affinity chromatography as the initial purification step

• Further purify using ion exchange chromatography and size exclusion chromatography

Critical Considerations:

• The ankyrin repeat domain may be more soluble than the full-length protein

• Include reducing agents (such as DTT or β-mercaptoethanol) in buffers to maintain the critical cysteine residue in a reduced state

• Consider the use of protease inhibitors to prevent degradation during purification

For structural studies, researchers have successfully purified human FEM1B-elongin B-elongin C complex and combined it with CUL2Δ-RBX1 dimer at a 1:1 ratio, followed by gel filtration to obtain the CRL2^FEM1B quinary complex .

What assays can be used to study fem1b substrate recognition and ubiquitylation activity?

Several assays have been developed to study FEM1B function that could be adapted for Xenopus laevis fem1b:

1. Fluorescence Polarization Assay:

• Using TAMRA-conjugated substrate degron peptides to measure binding to fem1b

• This assay can determine binding affinities and screen for inhibitors

2. In Vitro Ubiquitylation Assay:

• Reconstitute the complete E3 ligase complex with purified components

• Include E1, E2, ubiquitin, ATP, and substrate protein

• Detect ubiquitylation by Western blot or mass spectrometry

3. Cellular Degradation Reporter System:

• Express GFP fusion proteins containing potential substrate degrons

• Co-express with fem1b and monitor GFP levels by flow cytometry or microscopy

• Compare to control reporter (e.g., mCherry) expressed from the same plasmid

4. Covalent Ligand Screening:

• Screen libraries of cysteine-reactive compounds for binding to the critical cysteine in fem1b

• Use gel-based ABPP (activity-based protein profiling) to visualize binding

• Validate hits using a competition assay with labeled substrate

These approaches have been successfully used with human FEM1B and can be adapted for the Xenopus ortholog with appropriate modifications to account for sequence differences.

How can fem1b be utilized in targeted protein degradation (TPD) applications?

Recent research has demonstrated the potential of FEM1B as a novel E3 ligase recruiter for targeted protein degradation applications . This approach could be adapted for Xenopus studies or using Xenopus fem1b for comparative analyses:

Designing FEM1B-Based PROTACs:

• Identify a covalent ligand that targets the critical cysteine in fem1b (similar to EN106 for human FEM1B)

• Link this ligand to a compound that binds the protein of interest

• Test the resulting bifunctional molecule for its ability to induce degradation of the target protein

A recent study developed the first-in-class FEM1B-recruiting histone deacetylase (HDAC) degraders, including the compound FF2049, which achieved 85% degradation of HDAC1 with a DC50 of 257 nM . Importantly, these FEM1B-based PROTACs showed different selectivity profiles compared to cereblon-recruiting degraders using the same HDAC ligand, highlighting the value of expanding the E3 ligase toolkit .

The synthetic pathway for developing such compounds typically involves:

• Creation of a covalent ligand targeting fem1b

• Addition of a linker with appropriate length and properties

• Conjugation to a ligand for the target protein

What phenotypes are associated with fem1b deficiency in model organisms, and how might these inform research in Xenopus?

In mouse models, Fem1b deficiency leads to specific developmental defects that might guide experiments in Xenopus:

Mouse Fem1b Knockout Phenotypes:

• Viable and fertile homozygous mutants with normal Mendelian ratios

• Defects in prostate ductal morphogenesis, specifically a 30% reduction in ductal tip number in the anterior prostate

• Altered expression of several secretory proteins in the dorsolateral and ventral prostate and seminal vesicles

• Defects in secretory protein production, suggesting incomplete/mis-specified epithelial differentiation

• No observable histological abnormalities in testes

• No significant differences in apoptosis rates in affected tissues

These phenotypes are relatively mild, possibly due to functional redundancy with Fem1c, which has a similar expression pattern . Researchers using Xenopus could explore:

• CRISPR/Cas9-mediated knockout of fem1b in Xenopus

• Morpholino knockdown for more transient analysis

• Tissue-specific overexpression using transgenic approaches

• Examination of potential compensatory mechanisms by other fem family members

How do post-translational modifications affect fem1b function and substrate selectivity?

While the search results don't directly address post-translational modifications (PTMs) of Xenopus fem1b, understanding potential PTMs is critical for characterizing its function:

Potential PTMs to Investigate:

• Phosphorylation: May regulate substrate binding or interaction with other CRL2 components

• Oxidation of the critical cysteine residue: Could affect substrate recognition, particularly for substrates recognized under specific redox conditions

• Ubiquitylation: Potential for auto-regulation of fem1b levels

• Neddylation of the associated cullin: Required for E3 ligase activity

Methods to investigate these PTMs include:

• Mass spectrometry to identify and map PTMs

• Site-directed mutagenesis to create phosphomimetic or phospho-deficient mutants

• Oxidative and reductive stress assays to determine how redox conditions affect fem1b function

• In vitro assays comparing native and modified fem1b proteins

What are common challenges in working with recombinant fem1b protein, and how can they be addressed?

Based on experiences with similar proteins:

Challenge 1: Low solubility and stability

• Solution: Express individual domains (e.g., ankyrin repeat domain) rather than full-length protein

• Solution: Use solubility-enhancing tags such as MBP or SUMO

• Solution: Optimize buffer conditions (pH, salt concentration, additives)

Challenge 2: Loss of the critical cysteine function through oxidation

• Solution: Include reducing agents in all buffers

• Solution: Perform experiments under anaerobic conditions when possible

• Solution: Use mutational analysis to determine the importance of the cysteine in Xenopus fem1b

Challenge 3: Difficulty in reconstituting active E3 ligase complex

• Solution: Co-express fem1b with other complex components

• Solution: Use stepwise assembly and validation of subcomplexes

• Solution: Include positive controls in activity assays

Challenge 4: Specificity of antibodies for Xenopus fem1b

• Solution: Validate commercial antibodies specifically for Xenopus fem1b

• Solution: Generate custom antibodies using unique peptide sequences

• Solution: Use epitope tags for detection when studying recombinant protein

How can researchers design experiments to study the interaction between Xenopus fem1b and potential binding partners?

To investigate protein-protein interactions involving fem1b:

Identification of Novel Interactors:

• Yeast Two-Hybrid Screening: This approach was successfully used to identify the interaction between Nkx3.1 and Fem1b in mammals

• Affinity Purification-Mass Spectrometry: Using tagged fem1b as bait to identify interacting proteins

• Proximity Labeling: BioID or APEX2 fusions to identify proximal proteins in the cellular context

Validation and Characterization of Interactions:

• GST Pull-down Assays: As used to confirm the Nkx3.1-Fem1b interaction

• Co-immunoprecipitation: To validate interactions in cell lysates

• Fluorescence Resonance Energy Transfer (FRET): To study interactions in living cells

• Surface Plasmon Resonance or Bio-Layer Interferometry: To determine binding kinetics and affinities

When designing these experiments, it's important to consider:

• The potential requirement for other components of the CRL2 complex

• The effect of redox conditions on interactions

• The tissue-specific context of the interaction

How might comparing fem1b function across species inform our understanding of E3 ligase evolution?

Comparative studies of fem1b from different species, including Xenopus laevis, could provide insights into:

• Conserved vs. Divergent Substrate Recognition:

  • Identify whether substrate degron preferences are conserved across species

  • Determine if species-specific substrates have evolved

• Evolutionary Changes in Complex Formation:

  • Compare the assembly and stability of CRL2^FEM1B complexes across species

  • Identify species-specific interacting partners

• Functional Diversification:

  • Compare phenotypes of fem1b mutants across model organisms

  • Determine whether fem1b has acquired novel functions in certain lineages

A comprehensive phylogenetic analysis combined with functional studies could map the evolutionary trajectory of fem1b from its ancestral role in sex determination in nematodes to its diverse functions in vertebrates.

What methodological advances could improve our ability to study fem1b protein dynamics in vivo?

Several cutting-edge approaches could enhance the study of fem1b:

• Live Cell Imaging of fem1b Activity:

  • FRET-based sensors for monitoring substrate ubiquitylation

  • Split fluorescent protein systems to visualize fem1b-substrate interactions

  • Optogenetic control of fem1b activity or localization

• Spatiotemporal Control of fem1b Function:

  • Photocaged inhibitors of fem1b

  • Light-activatable degrons for controlling fem1b levels

  • Temperature-sensitive fem1b variants for conditional studies

• Single-Molecule Studies:

  • Tracking individual fem1b molecules in cells

  • Analyzing the dynamics of complex assembly and substrate engagement

  • Measuring the kinetics of individual ubiquitylation events

These advanced methodologies could provide unprecedented insights into fem1b function in developmental contexts, particularly in transparent model organisms like Xenopus embryos.