Recombinant Mouse Tetratricopeptide repeat protein 16 (Ttc16)

What is Tetratricopeptide repeat protein 16 (Ttc16) and what is its basic structure?

Tetratricopeptide repeat protein 16 (Ttc16) belongs to a family of proteins characterized by tetratricopeptide repeat (TPR) motifs as supersecondary structures. Based on human TTC16 studies, the protein appears to contain approximately ten tetratricopeptide motifs, with the N-terminal portion belonging to the TPR_11 superfamily and the C-terminal region consisting of the Pumilio Superfamily . The full-length protein is estimated to be approximately 873 amino acids in human form, with mouse Ttc16 likely showing high sequence conservation due to the evolutionary conservation observed across species .

TPR motifs typically form alpha-helical structures that serve as protein-protein interaction domains, suggesting Ttc16 may function as a scaffold protein for assembling multi-protein complexes. For researchers beginning studies on mouse Ttc16, comparison of sequence homology between human and mouse variants using tools like BLAST or Clustal Omega is recommended as a first step to identify conserved functional domains.

What are the known cellular localizations and expression patterns of Ttc16?

In human cells, TTC16 has been observed primarily within the nucleus, which is consistent with the presence of four nuclear localization signals identified in the protein sequence . The protein contains post-translational modifications concentrated in its C-terminal region, with phosphorylation sites particularly abundant in the Pumilio Superfamily region .

For mouse Ttc16 localization studies, researchers should consider:

• Generating GFP-tagged Ttc16 constructs for live-cell imaging

• Performing subcellular fractionation followed by Western blotting

• Conducting immunofluorescence studies with validated anti-Ttc16 antibodies

Expression analysis suggests that transcription factors regulating TTC16 are commonly found in cells of the immune system (including various white blood cells and red blood cells), respiratory system, and endocrine system . Therefore, when designing expression studies for mouse Ttc16, these tissue types should be prioritized for investigation.

How does Ttc16 structure compare to other tetratricopeptide repeat proteins?

Ttc16 belongs to a larger family of TPR-containing proteins. While Ttc16 contains approximately ten TPR motifs, other well-characterized TPR proteins like TTC1 contain fewer motifs (TTC1 has three TPR motifs) . This structural difference likely influences their binding partners and functions.

Understanding these structural differences is essential for researchers designing experiments to investigate Ttc16-specific functions versus general TPR protein characteristics. Homology modeling approaches comparing Ttc16 with better-characterized TPR proteins can provide valuable insights for structure-function studies.

What expression systems are optimal for producing recombinant mouse Ttc16?

For optimal expression of functional recombinant mouse Ttc16, consider these methodological approaches:

• Bacterial expression: Use specialized E. coli strains like BL21(DE3) Rosetta or Arctic Express to enhance proper folding of complex proteins. Consider expressing individual TPR domains separately if full-length expression proves challenging.

• Insect cell expression: Baculovirus expression systems often yield properly folded mammalian proteins with appropriate post-translational modifications.

• Mammalian expression: For studies requiring native-like post-translational modifications, HEK293 or CHO cell expression systems are recommended, particularly when investigating protein-protein interactions.

When designing expression constructs, include an affinity tag (His-tag, GST, etc.) to facilitate purification, but ensure the tag position (N or C-terminal) minimizes interference with TPR domain folding .

What are the recommended protocols for purification and quality assessment of recombinant Ttc16?

For purification of recombinant mouse Ttc16, a multi-step approach is recommended:

• Initial capture: Using affinity chromatography (Ni-NTA for His-tagged constructs)

• Intermediate purification: Ion exchange chromatography based on Ttc16's basic isoelectric point (predicted similar to human TTC16 at pH 9.15)

• Polishing step: Size exclusion chromatography to separate properly folded protein from aggregates

Quality assessment should include:

• SDS-PAGE to confirm molecular weight and purity

• Circular dichroism to verify alpha-helical content (characteristic of TPR domains)

• Dynamic light scattering to assess monodispersity

• Limited proteolysis to evaluate domain stability

• Thermal shift assays to determine stability under various buffer conditions

For long-term storage, a buffer containing 20mM Tris-HCl (pH 8.0), 20% glycerol, 0.1M NaCl, and 1mM reducing agent (DTT or β-mercaptoethanol) at -80°C is recommended, based on successful storage conditions for similar TPR proteins .

How can recombinant Ttc16 be utilized to investigate its potential role in cancer pathways?

Recent research has identified TTC16 as one of the key driver mutations in cutaneous squamous cell carcinoma (cSCC), alongside other genes like TP53, CDKN2A, and NOTCH1 . This finding suggests Ttc16 may play important roles in pathways related to replicative senescence, cellular response to UV damage, cell-cell adhesion, and cell cycle regulation.

For researchers investigating Ttc16's role in cancer pathways, these methodological approaches are recommended:

• Ttc16 mutational analysis in mouse cancer models: Compare Ttc16 mutation frequencies and patterns in various mouse cancer models, particularly skin cancers, using whole exome sequencing.

• Protein-protein interaction studies: Utilize techniques such as co-immunoprecipitation, proximity ligation assays, or yeast two-hybrid screening to identify cancer-relevant binding partners of Ttc16.

• Functional assays: Develop Ttc16 knockout or overexpression systems in relevant cell lines to assess effects on:

  • Cell proliferation and cell cycle progression

  • Response to UV damage

  • Cell migration and invasion

  • Apoptosis resistance

• Transcriptomic analysis: Perform RNA-seq on Ttc16-modulated cells to identify downstream pathways affected by Ttc16 alteration, focusing on established cancer-related pathways.

A methodical approach combining these techniques will help establish whether mouse Ttc16 functions as a tumor suppressor or oncogene in specific contexts.

What approaches are most effective for studying Ttc16 protein-protein interactions?

Understanding Ttc16's protein-protein interactions is crucial given that TPR domains typically function as protein-protein interaction modules. Based on studies of other TPR proteins like TTC1, which interacts with G-alpha proteins and Ha-Ras , researchers should consider these methodological approaches:

• Pull-down assays: Using purified recombinant Ttc16 as bait to capture interacting proteins from cellular lysates, followed by mass spectrometry identification.

• Surface Plasmon Resonance (SPR): For quantitative measurement of binding kinetics between Ttc16 and potential partner proteins.

• Isothermal Titration Calorimetry (ITC): To determine thermodynamic parameters of Ttc16-protein interactions.

• Proximity-based labeling: BioID or APEX2 fusions with Ttc16 expressed in relevant cell types to identify proximal interacting proteins in living cells.

• Domain mapping: Creating truncated constructs of Ttc16 to identify which TPR motifs are essential for specific protein interactions.

When designing these experiments, consider potential interactions with proteins involved in cellular pathways where Ttc16 has been implicated, including cell cycle regulation and cellular response to UV damage .

What strategies are recommended for generating and validating Ttc16 knockout or knockdown models?

For researchers investigating Ttc16 function through loss-of-function approaches, several methodologies can be employed:

CRISPR-Cas9 Knockout Strategy:

• Design sgRNAs targeting early exons of mouse Ttc16, preferably exons encoding critical TPR motifs

• Validate editing efficiency using T7 endonuclease assay or Sanger sequencing

• Confirm knockout at protein level via Western blot with validated antibodies

• Assess phenotypic changes in relevant cellular processes (cell cycle, response to UV, etc.)

RNA Interference Approach:

• Design multiple siRNAs or shRNAs targeting different regions of Ttc16 mRNA

• Validate knockdown efficiency by qRT-PCR and Western blot

• Include non-targeting controls and rescue experiments with RNAi-resistant Ttc16 constructs

Validation Considerations:

• Perform off-target analysis for CRISPR-edited cell lines

• Evaluate compensatory upregulation of other TPR proteins

• Assess cell viability and basic cellular functions that might be indirectly affected

The high conservation of Ttc16 across species suggests potential developmental or physiological importance , so researchers should monitor for unexpected phenotypes that might reveal novel functions.

What are the optimal conditions for conducting functional assays with recombinant Ttc16?

When designing functional assays to investigate recombinant mouse Ttc16 activity, consider these methodological recommendations:

Buffer Conditions:

• Start with physiological-like conditions: 20mM HEPES or Tris (pH 7.4-7.6), 150mM NaCl, 2mM MgCl₂

• Include reducing agents (1-5mM DTT or β-mercaptoethanol) to maintain thiol groups

• Consider adding 5-10% glycerol to enhance protein stability

Protein-Protein Interaction Assays:

• Perform binding assays at physiologically relevant temperatures (25-37°C)

• Control for non-specific binding using unrelated TPR proteins

• Consider the potential requirement for post-translational modifications when using bacterially-expressed Ttc16

Activity Assays:

• If investigating potential enzymatic activities, include appropriate cofactors based on bioinformatic predictions

• For phosphorylation-dependent activities, include combinations of kinases predicted to modify Ttc16

• Design time-course experiments to capture both rapid and slower interactions

For challenging functional studies, consider using proximity-based approaches like FRET or BRET with Ttc16 fusion constructs to monitor interactions in real-time within living cells.

How can computational approaches enhance understanding of Ttc16 structure and function?

Computational methods offer powerful tools for predicting and understanding Ttc16 function, particularly given the limited experimental data currently available:

Structural Prediction and Analysis:

• Homology modeling based on crystal structures of related TPR proteins

• Molecular dynamics simulations to analyze flexibility of TPR domains

• Protein-protein docking studies to predict potential binding partners

Functional Annotation:

• Phylogenetic analysis across species to identify highly conserved regions

• Identification of functional motifs using pattern recognition algorithms

• Protein-protein interaction network analysis to predict biological pathways

Table: Recommended Computational Resources for Ttc16 Research

By integrating computational predictions with targeted experimental validation, researchers can develop more focused hypotheses about Ttc16 function, potentially revealing its role in various cellular processes and disease states .

What are emerging research directions for investigating Ttc16 in disease models?

Given TTC16's identification as a potential driver mutation in cutaneous squamous cell carcinoma , several promising research directions emerge:

• Cancer Research: Investigate Ttc16 mutations, expression changes, or altered localization across various cancer types in mouse models. Particular focus should be given to skin cancers given the association with cSCC.

• Immune System Function: Since TTC16 transcription factors are found in cells of the immune system , explore Ttc16's role in immune cell development, differentiation, and function using conditional knockout mouse models.

• Stress Response Pathways: The association with cellular response to UV damage pathways suggests investigating Ttc16's potential role in other cellular stress responses, including oxidative stress and heat shock.

• Drug Development: For translational applications, screen for small molecules that can modulate Ttc16 function or its protein-protein interactions, potentially developing novel therapeutic approaches for diseases where Ttc16 dysfunction is implicated.

When designing disease-related studies, researchers should consider:

• Using tissue-specific inducible knockout systems to avoid potential developmental effects

• Developing antibodies or probes that can distinguish between wildtype and mutant forms of Ttc16

• Collaborating across disciplines to integrate molecular, cellular, and physiological data

The study of Ttc16 in multiple disease contexts may reveal unexpected functions and potential therapeutic opportunities beyond what is currently known about this relatively uncharacterized protein.