Recombinant Mouse Intraflagellar transport protein 172 homolog (Ift172), partial

What is the molecular architecture of IFT172?

IFT172 is the largest protein of the IFT complex with a complex architecture consisting of two globular domains connected by a long rod-like protrusion. This arrangement resembles the domain organization of coatomer proteins such as COPI-II or clathrin . Structural studies have revealed that IFT172 contains a C-terminal region with tetratricopeptide repeat (TPR) motifs followed by a small non-TPR C-terminal domain . Crystal structure analysis of human IFT172 has identified this C-terminal domain (residues 1683-1749) as a U-box-like domain consisting of a small 2-stranded β-sheet followed by two α-helices . This domain is connected to the IFT144 and IFT140 binding helices through a 30-residue loop region, positioning the C-terminal domain approximately 25Å away from these binding helices .

What conformational states does IFT172 adopt?

IFT172 can adopt two distinct conformational states that can be experimentally manipulated:

• An extended elongated conformation

• A globular closed architecture

These conformational changes can be induced by lipids or detergents . This conformational flexibility likely plays a functional role in IFT172's various activities, including its membrane-remodeling capabilities and interactions with other IFT proteins.

What is known about IFT172's role in ciliopathies?

Mutations in IFT172 are associated with several human diseases called ciliopathies, which result from ciliary dysfunction . Recent research has identified numerous disease-causing missense variants that map to the interface between the TPR region and the C-terminal U-box domain . One notable ciliopathy variant is C1727R, which is located in helix α2 of the U-box domain . Studies of IFT172 conditional knockout mice have demonstrated that loss of IFT172 in photoreceptors leads to rapid retinal degeneration, with severely reduced retinal thickness and electroretinogram (ERG) responses by 1 month and absent photoreceptor cells by 2 months . This mouse model recapitulates key features of IFT172-associated retinal disease in humans .

What approaches are effective for structural characterization of recombinant IFT172?

Due to IFT172's large size (approximately 172 kDa), structural characterization has been challenging and has relied on several complementary approaches:

• X-ray crystallography: Successfully applied to C-terminal fragments of human IFT172 (HsIFT172C2, residues 1470-C), yielding structures at 2.1Å resolution from native crystals and 2.8Å resolution from selenomethionine-substituted crystals . Crystallographic phase information was obtained by combining molecular replacement (using an AlphaFold model) with single-wavelength anomalous dispersion .

• Cryo-electron tomography (cryo-ET): Used to visualize IFT172 in the context of anterograde and retrograde IFT trains, revealing different arrangements of IFT172 interactions with IFT144 and IFT140 .

• Biochemical analysis of membrane interactions: Giant unilamellar vesicles (GUVs) have been employed to demonstrate IFT172's membrane-binding and remodeling activities .

For successful structural studies, researchers should consider:

• Working with defined domains rather than full-length protein

• Using selenomethionine substitution for phase determination in crystallography

• Exploring the effects of lipids or detergents on conformational states

How can the U-box domain activity of IFT172 be assessed experimentally?

Recent identification of IFT172's C-terminal domain as a U-box E3 ubiquitin ligase domain has opened new avenues for functional characterization. Methods to assess this activity include:

• Mutagenesis of key residues: Several conserved residues important for E2 binding and U-box function have been identified, including:

  • I1688: A conserved Ile/Leu residue in loop L1 involved in E2 binding

  • F1715: A conserved aromatic residue in helix α1 that contributes to E2 binding

  • P1725: A completely conserved proline in loop L2 that caps the N-terminus of helix α2

  • C1727: A residue in helix α2 associated with ciliopathy when mutated to arginine

• In vitro interaction studies: Affinity pulldowns using GST-tagged IFT172 constructs have demonstrated direct interaction between IFT172 and stable mimics of the E2∼Ub (UbcH5a∼Ub) conjugate . This interaction persisted even with UbcH5a mutants (F62A or A96D) known to disrupt binding to typical U-box domains .

• Auto-ubiquitination assays: Though not explicitly detailed in the search results, auto-ubiquitination assays are standard for characterizing E3 ligase activity and would be appropriate for evaluating IFT172's U-box domain function.

What methods can be used to study IFT172's membrane interactions?

IFT172 has been identified as a membrane-interacting protein with membrane-remodeling capabilities. Researchers can study these properties using:

• Giant unilamellar vesicles (GUVs): These model membrane systems have successfully demonstrated IFT172's ability to bind membranes and remodel large membranes into small vesicles .

• Lipid composition analysis: Since lipids can influence IFT172's conformational state, systematically varying lipid compositions can help determine specificity and requirements for membrane interaction.

• Comparison with coatomer proteins: Given IFT172's structural similarity to coatomer proteins like COPI-II and clathrin , methodologies used to study these well-characterized vesicle coat proteins could be adapted for IFT172.

• Mutational analysis: Strategic mutations could help identify regions critical for membrane binding versus protein-protein interactions, particularly given that membrane association is mutually exclusive with IFT57 binding .

How do IFT172 mutations affect ciliary protein trafficking?

Studies of IFT172 conditional knockout mice have revealed specific defects in ciliary protein trafficking:

• Mislocalization of outer segment proteins: Several key photoreceptor proteins show aberrant localization in IFT172-deficient retinas, including:

  • Rhodopsin (RHO)

  • Retinitis pigmentosa 1 (RP1)

  • IFT139

• Altered light-driven translocation: In normal photoreceptors, transducin moves from the outer segments to the inner segments in response to light. In IFT172-deficient mice, quantification of the relative fluorescence signal showed that outer segment Gαt content is 36.3% lower than in control mice (P=0.0003) .

• Transcription factor mislocalization: IFT172 deficiency also affects the localization of the GLI1 transcription factor, indicating disruption of ciliary signaling pathways .

These trafficking defects suggest that IFT172 plays a critical role in maintaining proper protein distribution within the cilium, likely through its function in IFT trains and possibly through its newly discovered ubiquitin ligase activity.

How does IFT172 contribute to IFT train assembly and function?

Recent research has revealed important details about IFT172's role in organizing IFT trains:

• Differential interactions in anterograde versus retrograde transport:

  • In anterograde IFT trains (base to tip), IFT172 interacts with IFT144

  • In retrograde IFT trains (tip to base), IFT172 interacts with IFT140

  • These mutually exclusive interactions involve the same helices of IFT172 and help establish the different architectures of anterograde and retrograde trains

• Critical binding residues: The L1615 residue is located in one of two helices of IFT172 (helix αA) that interact directly with IFT140 or IFT144. The L1615P mutation (found in the fla11 strain) disrupts this helix, impairing binding to the IFT-A complex and resulting in retrograde IFT defects and accumulation of ubiquitinated proteins .

• Membrane association versus protein binding: IFT172's association with membranes is mutually exclusive with its binding to IFT57, suggesting that IFT172 may switch between different functional roles during IFT .

This dynamic interaction pattern suggests that IFT172 serves as a critical organizational hub that helps determine the directionality and composition of IFT trains.

What is the significance of IFT172's dual roles in membrane remodeling and ubiquitination?

The discovery that IFT172 possesses both membrane-remodeling capabilities and ubiquitin ligase activity suggests intriguing possibilities for its function in ciliary biology:

• Coordinated regulation of membrane dynamics and protein turnover: IFT172 might simultaneously shape ciliary membranes while helping control protein quality or quantity through ubiquitination.

• Conformational switching between functions: The ability of IFT172 to adopt different conformations manipulated by lipids or detergents could represent a mechanism to switch between its membrane-associated and ubiquitin ligase functions.

• Potential role in ciliary vesicle formation: The structural similarity to coatomer proteins combined with membrane-remodeling activity suggests IFT172 may help form specialized vesicles during ciliogenesis.

• Links to disease mechanisms: The fla11 strain with an L1615P mutation shows both retrograde IFT defects and accumulation of ubiquitinated proteins , suggesting interconnection between these functions in disease pathogenesis.

This functional versatility makes IFT172 a particularly important target for research into ciliary biology and ciliopathy mechanisms.

What are the major difficulties in working with full-length IFT172?

Several technical challenges complicate research with full-length IFT172:

• Size constraints: As the largest protein of the IFT complex, IFT172's size has made characterization challenging . Most structural studies have focused on fragments, particularly C-terminal constructs .

• Conformational flexibility: IFT172's ability to adopt multiple conformations complicates structural analysis and may contribute to sample heterogeneity.

• Expression and purification difficulties: The search results indicate that even for some mutant constructs of the C-terminal fragment (e.g., I1688A variant), soluble protein expression could not be achieved .

• Complex interaction network: IFT172 interacts with multiple partners including IFT144, IFT140, IFT57, membranes, and potentially ubiquitination machinery, creating challenges for isolating specific functions.

What strategies can overcome challenges in IFT172 expression and purification?

Based on successful approaches described in the search results:

• Domain-based approach: Working with defined domains rather than full-length protein has proven successful, particularly:

  • C-terminal constructs containing the U-box domain (e.g., HsIFT172C2, residues 1470-C)

  • Fragments containing specific binding interfaces for interaction studies

• Expression systems and tags:

  • GST-tagged constructs have been used successfully for pulldown experiments

  • Selenomethionine substitution has been employed for phase determination in crystallography

• Purification strategy:

  • Multi-step purification including affinity chromatography and likely size exclusion chromatography

  • Careful monitoring of protein integrity during purification

• Buffer optimization:

  • Since lipids and detergents affect IFT172 conformation , careful buffer optimization is crucial

  • Consider testing various additives to improve stability and homogeneity

How can researchers study the physiological relevance of IFT172's dual functions?

To connect IFT172's biochemical activities to its physiological functions:

• Targeted mutagenesis approaches:

  • Generate mutations that specifically affect either membrane binding or U-box activity

  • Create knock-in mouse models with these selective mutations to dissect functions in vivo

• Temporal analysis in conditional knockout models:

  • The retina-specific conditional knockout model shows rapid degeneration after IFT172 depletion

  • Careful timing of analyses (e.g., examining tissue between postnatal days 25-31) can capture early defects before complete degeneration

• Combined structural and functional approaches:

  • Correlate structural states of IFT172 with specific functions

  • Use techniques like FRET to monitor conformational changes in cellular contexts

• Analysis of protein-protein interaction networks:

  • Identify binding partners specific to different conformational states

  • Map interaction changes during ciliary assembly and disassembly

What are the key unresolved questions about IFT172 function?

Despite recent advances, several important questions remain:

• Substrate specificity of the U-box domain: What are the physiological substrates of IFT172's ubiquitin ligase activity, and how does this activity contribute to ciliary function?

• Regulatory mechanisms: How is IFT172 switching between its different conformational states and functional modes regulated in vivo?

• Complete structural understanding: While fragments have been characterized, the complete structure of full-length IFT172 and its arrangement within IFT trains remains to be fully elucidated.

• Disease mechanisms: How do specific disease mutations in IFT172 selectively affect different tissues and functions, particularly in complex ciliopathy syndromes?

What experimental systems will be most valuable for future IFT172 research?

Several experimental systems show promise for advancing IFT172 research:

• Cryo-electron tomography: This technique has already provided insights into IFT172's position in IFT trains and will likely continue to reveal details about its in situ organization and conformational states.

• Human organoid models: Retinal or renal organoids derived from human stem cells with IFT172 mutations could provide physiologically relevant systems to study disease mechanisms.

• In vitro reconstitution systems: Reconstituting IFT train assembly and movement with purified components could help dissect IFT172's specific contributions.

• Live-cell imaging with tagged IFT172 variants: This approach could reveal dynamic conformational changes and interactions during ciliary transport.

How might the newly discovered functions of IFT172 inform therapeutic approaches?

The identification of IFT172's U-box domain and membrane-remodeling activities opens new possibilities for therapeutic intervention:

• Small molecule modulators: Compounds that selectively target IFT172's U-box activity or membrane-binding properties could potentially modify disease progression.

• Gene therapy approaches: For recessive IFT172-related ciliopathies, gene replacement therapies delivered to affected tissues (particularly accessible ones like the retina) could potentially restore function.

• Protein-protein interaction interventions: Molecules that stabilize critical interactions between IFT172 and its binding partners might ameliorate certain disease phenotypes.

• Pathway-based approaches: Understanding how IFT172 dysfunction affects downstream signaling pathways could reveal indirect therapeutic targets that bypass the need to directly restore IFT172 function.