Recombinant Human LisH domain-containing protein ARMC9 (ARMC9), partial

What is the domain structure of human ARMC9 protein?

Human ARMC9 (Armadillo repeat-containing protein 9) contains three key functional domains:

• N-terminal lissencephaly type-1-like homology motif (LisH)

• Central coiled-coil (CC) domain

• C-terminal armadillo repeat (ARM) domain spanning residues 383-578

The ARM-type fold domain is particularly crucial for protein-protein interactions and forms helical structures arranged in tandem with interconnecting coils . This multi-domain organization allows ARMC9 to serve as a protein interaction hub within ciliary structures.

Where does ARMC9 localize within cells?

ARMC9 primarily localizes to the proximal region of cilia in cultured cells. Notably, upon Hedgehog (Hh) pathway stimulation, ARMC9 redistributes toward the ciliary tip within 6 hours before gradually returning to its original proximal location . This dynamic localization pattern can be blocked by the SMO inhibitor vismodegib, confirming that this redistribution is specifically triggered by Hh signaling . Research using ARMC9-3xFLAG constructs in both IMCD3 and NIH-3T3 cells has clearly demonstrated this localization pattern.

What is the primary function of ARMC9 in cellular physiology?

ARMC9 is required for ciliogenesis and plays a crucial role in ciliary signaling pathways, particularly the Hedgehog pathway . Specifically, ARMC9 participates in the trafficking and/or retention of GLI proteins (GLI2 and GLI3) at the ciliary tip during pathway activation . ARMC9 knockout cells exhibit reduced ciliary accumulation of these GLI proteins without affecting SMO localization, suggesting a specific role in GLI protein trafficking rather than general ciliary transport . Additionally, recent studies in zebrafish have shown that ARMC9 is essential for proper vertebral column development and axial alignment .

What protein complexes does ARMC9 form, and how have these been identified?

ARMC9 forms a distinct ciliary module comprised of:

• TOGARAM1 (TOG array regulator of axonemal microtubules 1)

• CCDC66 (Coiled-coil domain containing 66)

• CEP104

• CSPP1

This module has been identified through complementary approaches:

• Tandem affinity purification (TAP): Strep-Flag epitope-tagged ARMC9 expressed in HEK293T cells identified 106 candidate interactors including TOGARAM1 and CEP290 .

• Yeast two-hybrid (Y2H) screens: Full-length ARMC9 and four fragments were used as bait against retinal cDNA libraries, revealing direct interactions with TOGARAM1, CCDC66, CSPP1, and ARMC9 itself (suggesting self-multimerization) .

• Co-immunoprecipitation assays: Reciprocal co-IPs confirmed interactions between ARMC9 and TOGARAM1, CCDC66, CEP104, and CSPP1 .

• PalmMyr-CFP mislocalization assays: This technique provided additional validation by forcing protein mislocalization to the cell membrane through palmitoylation and myristoylation tags .

Which specific domains of ARMC9 mediate its protein interactions?

Domain-specific interaction studies have revealed:

• The N-terminal 350 amino acid region containing the LisH and coiled-coil domains (fragment 2) mediates ARMC9 self-interaction/multimerization .

• Fragment 4 (residues 150-665) containing the coiled-coil domain and the armadillo repeats domain mediates interactions with TOGARAM1 and CSPP1 .

• The ARM-type fold domain (residues 383-578) is particularly critical for protein-protein interactions, as demonstrated through molecular docking analyses .

How do molecular docking analyses inform our understanding of ARMC9 interactions?

Recent molecular docking simulations using HADDOCK and ClusPro have revealed specific details about ARMC9-TOGARAM1 interactions . Wild-type zArmc9-Tog1 interactions demonstrated a binding free energy of -124.89 kcal/mol, with 19 residues in the ARM-type fold domain contributing to the interaction . These analyses help identify the specific amino acid residues that contribute most significantly to binding energy during protein-protein interactions.

Molecular dynamics (MD) simulations over 120ns have further characterized interaction properties, examining parameters such as:

• Root-mean-square deviation (RMSD)

• Root-mean-square fluctuation (RMSF)

• Radius of gyration (Rg)

• Solvent accessible surface area (SASA)

• Hydrogen bond formation

These computational approaches provide mechanistic insights into how mutations might disrupt protein interactions.

What are the validated approaches for studying ARMC9 localization in cells?

Several complementary methods have proven effective for studying ARMC9 localization:

• Epitope tagging: ARMC9-3xFLAG and ARMC9-LAP (S-tag-HRV3C-GFP localization and affinity purification tag) constructs allow visualization via immunofluorescence microscopy .

• Immunostaining with validated antibodies: Several studies have successfully used specific antibodies against ARMC9 to visualize endogenous protein .

• Time-course experiments: To track dynamic redistribution during signaling, researchers have employed fixed timepoints after Hedgehog pathway stimulation (e.g., 6 hours post-stimulation) .

• Pharmacological manipulation: SMO inhibitors like vismodegib can be used to verify Hh-dependent localization changes .

• CRISPR-based knockout models: Comparing localization in wild-type versus mutant backgrounds helps confirm specificity of observed patterns .

What strategies are effective for measuring ARMC9 expression levels?

Researchers have successfully employed multiple approaches to quantify ARMC9 expression:

• RT-qPCR: Using validated primers specific to ARMC9 mRNA. This has been performed on both cycling cells and serum-starved cells to induce ciliogenesis, with primers targeting specific regions of the ARMC9 transcript .

• Western blotting: Using validated antibodies against ARMC9, typically with normalization to housekeeping proteins.

• RNA-seq: For transcriptome-wide analysis of ARMC9 expression across different conditions.

For optimal RT-qPCR results, researchers have used the following protocol:

• RNA extraction using commercial kits (e.g., Aurum Total RNA Mini Kit)

• cDNA generation from 2μg total RNA (iScript Reverse Transcription Supermix)

• qPCR using SYBR Green PCR Master Mix

• Thermal cycling: initial denaturation (10 min at 95°C), followed by 39 cycles of denaturation (15s at 95°C) and annealing/extension (1 min at 60°C)

What genetic screening approaches have identified ARMC9 function?

ARMC9 function has been elucidated through several genetic screening approaches:

• CRISPR-based functional screens: A Hedgehog pathway-sensitive reporter system developed in NIH-3T3 fibroblasts enabled systematic identification of ciliary components, including ARMC9 . This approach used:

  • A blasticidin-resistance reporter linked to Hh signaling

  • sgRNA lentiviral library targeting the mouse genome

  • Comparative analysis of selected versus unselected cell pools

  • Statistical analysis using maximum likelihood methods (casTLE)

• TILLING technology: Used to generate zebrafish armc9 mutants through genetic screening, revealing the protein's role in vertebral column development .

• Whole Exome Sequencing (WES): Applied to identify novel ARMC9 variants in patients with Joubert syndrome, using:

  • Agilent SureSelect Human Exon Sequence Capture Kit

  • NovaSeq6000 platform sequencing

  • Bioinformatic analysis with tools like ANNOVAR

  • Variant validation through Sanger sequencing

How do mutations in ARMC9 cause Joubert syndrome?

Biallelic variations in ARMC9 cause Joubert syndrome type thirty, a recessive neurodevelopmental ciliopathy . The pathogenic mechanisms include:

• Disruption of ciliary structure: ARMC9 mutations impair ciliogenesis, leading to shortened cilia with reduced levels of acetylated and polyglutamylated tubulin .

• Impaired Hedgehog signaling: Mutant cells show reduced ciliary accumulation of GLI2 and GLI3 transcription factors upon pathway activation, disrupting crucial developmental signaling .

• Compromise of ARM-domain function: Missense mutations (e.g., p.Arg343Cys) can alter hydrogen bond formation and protein stability, affecting interactions with other ciliary proteins .

• Altered mRNA splicing: Splice-site mutations (e.g., c.1878+1G>A) can introduce insertions into the coding sequence, resulting in altered protein structure and function .

Molecular dynamics simulations comparing wild-type and mutant ARMC9 reveal changes in protein compactness, solvent accessibility, and secondary structure stability that likely contribute to pathogenesis .

What are the phenotypic consequences of ARMC9 dysfunction in animal models?

Zebrafish armc9 mutants exhibit several phenotypic abnormalities:

• Axial skeletal defects: Significant reduction in body length along the anterior-to-posterior axis, accompanied by a pronounced dorsal hump .

• Vertebral column malformations: X-ray imaging reveals severe curvature of the vertebral column, particularly along the anterior-to-posterior axis, contrasting with the straight and orderly alignment in wild-type fish .

• Spinal cord inflammation: Histological examination shows multifocal inflammation of the spinal cord with accumulation of immune cells .

• Inflammatory marker upregulation: RT-PCR analysis shows increased levels of inflammatory markers tnfα and il1β in mutant larvae .

• Nitric oxide production: Fluorescence imaging using the NO-sensitive fluorescent sensor DAF-FM demonstrates increased accumulation of NO-producing macrophages in curved mutant larvae .

These findings support ARMC9's critical role in maintaining vertebral column structure and function, with possible inflammatory consequences when disrupted.

How does ARMC9 contribute to ciliary tip protein trafficking during Hedgehog signaling?

Current evidence suggests ARMC9 plays a specific role in GLI protein trafficking during Hedgehog signaling:

• Upon Hh pathway stimulation, ARMC9 itself redistributes from the proximal cilium to the ciliary tip, suggesting active involvement in signaling dynamics .

• ARMC9 knockout cells show normal SMO localization but impaired GLI2/GLI3 accumulation at ciliary tips during signaling, indicating a specific role in GLI protein transport or retention rather than general ciliary trafficking .

To further elucidate this mechanism, researchers should consider:

• Examining potential direct interactions between ARMC9 and GLI proteins through co-IP and proximity labeling approaches

• Investigating whether ARMC9 interacts with known intraflagellar transport (IFT) machinery

• Performing live-cell imaging with fluorescently tagged ARMC9 and GLI proteins to observe trafficking dynamics in real-time

• Mapping the specific domains required for this function through targeted mutagenesis

What is the role of ARMC9 self-interaction in its function?

Yeast two-hybrid screens have revealed that ARMC9 can interact with itself (multimerize), with this interaction mediated by the N-terminal 350 amino acid region containing the LisH and coiled-coil domains . This self-interaction ability may have significant functional implications:

• Structural roles: Self-multimerization might contribute to forming higher-order complexes or lattices within the cilium.

• Regulatory functions: The ability to multimerize might be regulated during signaling to control ARMC9 activity or localization.

• Scaffolding capacity: ARMC9 multimers could serve as scaffolds for assembling larger protein complexes.

Future research directions should include:

• Determining the stoichiometry of ARMC9 self-interactions (dimers, trimers, etc.)

• Investigating whether multimerization is regulated by post-translational modifications

• Examining whether disease-causing mutations affect self-interaction capacity

• Creating multimerization-deficient mutants to assess functional consequences

How do molecular dynamics simulations inform our understanding of ARMC9 mutations?

Molecular dynamics (MD) simulations provide detailed insights into how mutations affect ARMC9 protein structure and function:

For the p.Arg343Cys variant, 120ns MD simulations revealed several structural changes compared to wild-type:

• Hydrogen bond alterations: The mutation affects the number and stability of hydrogen bonds formed with surrounding residues, potentially destabilizing protein structure .

• Secondary structure changes: Analysis of root-mean-square deviation (RMSD) and root-mean-square fluctuation (RMSF) parameters indicates alterations in protein flexibility and secondary structure stability .

• Compactness and solvent accessibility: Changes in radius of gyration (Rg) and solvent accessible surface area (SASA) suggest alterations in protein compactness and exposure to solvent environment .

These computational approaches can predict the structural consequences of novel ARMC9 variants identified in patients, helping to establish pathogenicity and understand molecular mechanisms of disease. Future research should expand these analyses to additional variants and attempt to correlate computational predictions with experimental findings.