TOM1L2 Human

What are the key structural domains of TOM1L2 and how do they contribute to its function?

TOM1L2 belongs to a small gene family characterized by an N-terminal VHS (Vps27, Hrs, and STAM) domain followed by a GAT (GGA and TOM1) domain, with a C-terminal clathrin binding motif. These domains orchestrate TOM1L2's role in membrane trafficking:

• VHS domain: Involved in cargo recognition and protein-protein interactions

• GAT domain: Mediates binding to ubiquitin and sorting proteins like TOLLIP

• C-terminal domain (CTD): Contains binding sites for the BBSome and MYO6

Deletion mapping has revealed that BBSome recognition is encoded within a 30-amino acid segment (435-464) in the CTD, while MYO6 binding occurs at a separate site within the CTD . This modular organization allows TOM1L2 to function as an adaptor in multiple trafficking pathways.

How does TOM1L2 differ from other Tom1 family members (Tom1 and Tom1L1)?

The Tom1 family consists of three members with distinct functional profiles:

While all three share the conserved N-terminal VHS and GAT domains, their C-terminal regions have diverged significantly. Binding assays demonstrate that both TOM1L2 and TOM1 efficiently capture the BBSome, while TOM1L1 does not . This functional divergence explains their differential roles in membrane trafficking pathways.

What mechanisms control TOM1L2 expression and activity in human cells?

TOM1L2 activity is regulated through several mechanisms:

• Phosphorylation: ERBB2 signaling elicits indirect phosphorylation of TOM1L2 on Ser321, which promotes GAT-dependent association with the sorting protein TOLLIP .

• Protein-protein interactions: TOM1L2 associates with multiple partners including:

  • The BBSome complex for ciliary trafficking

  • MYO6 for endocytic trafficking

  • Clathrin for vesicle formation

  • TOLLIP for sorting processes

• Compartmentalization: TOM1L2 exhibits dynamic localization between cytosolic, endosomal, and ciliary compartments depending on cellular needs. In cilia, TOM1L2 accumulates more strongly when retrieval is compromised (e.g., in ARL6-depleted cells) .

• Genomic context: The TOM1L2 gene resides on chromosome 17p11.2, a region subject to deletion in Smith-Magenis syndrome, suggesting potential regulation at the genomic level .

What are the most effective methods for studying TOM1L2 protein interactions and trafficking?

Researchers employ several complementary approaches to study TOM1L2:

• Biochemical interaction assays:

  • GST pull-down assays with purified BBSome and TOM1L2 fusion proteins

  • Co-immunoprecipitation using antibodies against TOM1L2 or its interaction partners

  • Visual capture assays using fluorescent fusion proteins

• Advanced microscopy techniques:

  • Immunofluorescence microscopy to detect endogenous TOM1L2 localization

  • Live-cell imaging with fluorescently tagged TOM1L2 to track trafficking dynamics

  • Super-resolution microscopy for detailed spatial organization

• Genetic perturbation:

  • CRISPR/Cas9-mediated knockout of TOM1L2

  • Structure-function analysis using domain deletion mutants

  • Expression of phospho-mimetic or phospho-deficient mutants

• Proteomic approaches:

  • Mass spectrometry to identify TOM1L2 interactors and post-translational modifications

  • Proximity labeling to map the TOM1L2 interactome in specific compartments

For ciliary trafficking studies, these approaches are often combined with markers for cilia (such as acetylated tubulin) and cargo proteins of interest (e.g., GPCRs).

How can researchers isolate and purify TOM1L2 protein for functional studies?

For functional studies requiring purified TOM1L2, researchers can employ several strategies:

• Recombinant protein expression systems:

  • Bacterial expression: E. coli-based systems using pGEX (for GST-fusion) or pET vectors

  • Eukaryotic expression: Insect cells using baculovirus systems for properly folded mammalian proteins

  • Cell-free systems: For rapid production without cellular constraints

• Purification strategies:

  • Affinity chromatography using GST, His, or other tags

  • Ion exchange chromatography based on TOM1L2's isoelectric point

  • Size exclusion chromatography for final polishing and buffer exchange

• Domain-specific considerations:

  • The VHS-GAT domains (N-terminal region) typically express well in bacterial systems

  • Full-length protein or C-terminal regions may require eukaryotic expression for proper folding

• Quality control measures:

  • SDS-PAGE and western blotting to verify purity and identity

  • Mass spectrometry to confirm integrity and post-translational modifications

  • Functional assays (e.g., ubiquitin binding) to verify activity

Commercial recombinant TOM1L2 fragments are available as controls for antibody validation , but most research applications require custom protein production.

What genetic tools are available for studying TOM1L2 function in model organisms?

Several genetic tools and model systems have been developed for TOM1L2 research:

• Mammalian cell models:

  • CRISPR/Cas9 knockout lines: Tom1l2−/− cells show accumulation of GPCRs in cilia

  • Knockdown tools: siRNA and shRNA for transient depletion

  • Overexpression systems: Wild-type and mutant TOM1L2 variants

• Mouse models:

  • Hypomorphic mice: Show increased infections, tumors, and abnormal immune responses

  • Conditional knockouts: Allow tissue-specific TOM1L2 inactivation

• Evolutionary model systems:

  • Chlamydomonas reinhardtii: The TOM1L2 orthologue is required for clearing ubiquitinated proteins from cilia

  • Arabidopsis thaliana: TOL proteins (distantly related) function in vacuolar sorting

• Integrated genetic tools:

  • Fluorescent protein tagging: For visualization of TOM1L2 localization and dynamics

  • Ubiquitin system modifiers: UbK63-specific deubiquitinases to study ubiquitin-dependent functions

  • Inducible expression systems: For temporal control of TOM1L2 expression

When designing genetic studies, researchers should consider potential redundancy within the Tom1 family, as TOM1 and TOM1L2 share significant functional overlap in certain contexts.

What evidence connects TOM1L2 to ciliopathies and other human disorders?

TOM1L2 is implicated in several human disorders through different mechanisms:

• Ciliopathies:

  • TOM1L2 functions as an adaptor for BBSome-mediated retrieval of ubiquitinated proteins from cilia

  • Disruption of this pathway causes accumulation of signaling proteins in cilia, potentially contributing to ciliopathy phenotypes

  • TOM1L2 participates in the same pathway as proteins mutated in Bardet-Biedl Syndrome (BBS)

• Smith-Magenis Syndrome (SMS):

  • The TOM1L2 gene resides in the 3.7 Mb deletion of chromosome 17p11.2 associated with SMS

  • While RAI1 is considered the primary driver of SMS, TOM1L2 deletion may contribute to phenotypic aspects

• Neurodegenerative diseases:

  • Network analysis shows TOM1L2 exhibits significant functional coupling with Alzheimer's disease-associated genes, including sharing connections with presenilins through histone H2B proteins

  • TOM1L2's role in protein trafficking may influence pathways disrupted in neurodegeneration

• Immune disorders:

  • Tom1l2 hypomorphic mice exhibit abnormal immunologic responses and increased infection susceptibility

Further research is needed to determine whether TOM1L2 variants directly cause human disease or primarily function as modifiers of disease phenotypes.

How does TOM1L2 contribute to cancer progression, particularly in ERBB2-positive breast cancers?

Evidence suggests TOM1L2 plays important roles in cancer progression through several mechanisms:

• ERBB2 signaling and invasion:

  • ERBB2 elicits phosphorylation of TOM1L2 on Ser321

  • Phosphorylated TOM1L2 associates with TOLLIP and promotes trafficking of MT1-MMP (membrane-type 1 matrix metalloprotease)

  • This trafficking enhances invadopodia formation and extracellular matrix degradation

• Genomic alterations:

  • Members of the Tom1 family can be co-amplified with oncogenes

  • TOM1L1 is co-amplified with ERBB2 and defines a subgroup of HER2+/ER+ tumors with early metastatic relapse; similar patterns may exist for TOM1L2

• Growth factor signaling regulation:

  • TOM1L2 may regulate growth factor-induced mitogenic signaling pathways

  • These pathways often drive proliferation in cancer cells

• Immunomodulation:

  • The immunologic abnormalities in Tom1l2 hypomorphic mice suggest potential roles in tumor immunosurveillance

These findings suggest TOM1L2 could be a potential biomarker or therapeutic target in certain cancer contexts, particularly ERBB2-positive breast cancers where invasion and metastasis are enhanced through TOM1L2-dependent mechanisms.

What is the evidence linking TOM1L2 to neurodegenerative diseases?

Emerging evidence connects TOM1L2 to neurodegenerative diseases, particularly Alzheimer's disease (AD):

• Network analysis findings:

  • Analysis of the human interactome revealed TOM1L2 exhibits significant enrichment of links with AD-associated genes (APOE, APP, PSEN1, and PSEN2)

  • TOM1L2 and presenilins are functionally coupled to histone H2B proteins, suggesting shared pathways

• Trafficking machinery connections:

  • TOM1L2's role in ubiquitin-dependent trafficking may influence processing of AD-related proteins

  • Many neurodegenerative diseases involve defects in protein trafficking, sorting, and degradation

• Ciliary signaling relevance:

  • Primary cilia are important signaling hubs in neurons

  • TOM1L2's role in ciliary protein trafficking may impact neuronal function and degeneration

• Genomic context:

  • TOM1L2 resides on chromosome 17, a region with multiple genes implicated in neurodegenerative processes

While direct experimental evidence specifically linking TOM1L2 dysfunction to neurodegenerative pathology is still limited, these connections warrant further investigation into how TOM1L2 might influence disease-relevant processes like protein aggregation, sorting, and clearance.

What is the relationship between TOM1L2 phosphorylation and its trafficking functions?

Phosphorylation serves as a critical regulatory mechanism for TOM1L2 function:

• Known phosphorylation sites:

  • Ser321 phosphorylation occurs downstream of ERBB2 signaling

  • Additional phosphorylation sites likely exist but remain to be characterized

• Functional consequences:

  • Phosphorylation of TOM1L2 on Ser321 promotes GAT-dependent association with the sorting protein TOLLIP

  • This enhanced association facilitates trafficking of cargo proteins from endocytic compartments

  • In cancer cells, this mechanism directs trafficking of MT1-MMP to invasive structures

• Regulatory circuit:

  • Phosphorylation likely induces conformational changes that enhance binding capacity

  • Different phosphorylation patterns may direct TOM1L2 to distinct compartments or cargo types

  • Both kinases and phosphatases likely participate in dynamic regulation

Understanding these phosphorylation events could provide opportunities for:

• Identifying new regulatory pathways controlling TOM1L2 function

• Developing phosphorylation-state specific inhibitors for therapeutic applications

• Engineering phospho-mimetic or phospho-resistant TOM1L2 variants for research

Further research should focus on comprehensive phosphoproteomic analysis of TOM1L2 under different cellular conditions and the identification of the kinases responsible for each phosphorylation event.

What evolutionary insights can be gained from studying TOM1L2 orthologs across species?

Evolutionary analysis of TOM1L2 provides valuable insights into specialized trafficking mechanisms:

• Phylogenetic distribution:

  • TOM1L2 and related family members do not exist in yeast but appear in higher eukaryotes

  • The single-cell alga Chlamydomonas reinhardtii contains a functional TOM1L2 orthologue required for clearing ubiquitinated proteins from cilia

  • This distribution suggests TOM1L2's ciliary function emerged with the evolution of cilia themselves

• Relationship to ESCRT machinery:

  • TOM1L2 is considered an ancestral ESCRT (Endosomal Sorting Complex Required for Transport) protein

  • It shares functional similarities with ESCRT-0 components in recognizing ubiquitinated cargo

  • The ESCRT machinery shows differential retention and specialization across eukaryotic lineages

• Domain conservation:

  • The VHS and GAT domains show high conservation, reflecting fundamental trafficking roles

  • C-terminal regions display greater divergence, likely reflecting species-specific adaptations

  • Human TOM1L2 shares 96% sequence identity with mouse and rat orthologues

• Plant homologues:

  • TOL proteins in Arabidopsis function as ubiquitin receptors in early ESCRT pathways

  • They mediate vacuolar sorting of membrane proteins, paralleling TOM1L2's trafficking role

Table 1: Conservation of TOM1L2 domains across species

These patterns suggest TOM1L2 represents an example of how trafficking machinery has been adapted throughout eukaryotic evolution, with core functions in ubiquitin recognition being ancient and conserved.

How might targeting TOM1L2 functions provide therapeutic opportunities for ciliopathies or cancer?

TOM1L2's roles in key cellular processes suggest several potential therapeutic applications:

• Ciliopathy interventions:

  • Modulating TOM1L2-BBSome interactions could potentially rescue defective ciliary protein retrieval

  • Small molecules targeting the TOM1L2-BBSome interface (aa 435-464) might restore trafficking in BBSome-deficient cells

  • Enhancing TOM1L2 functions could potentially compensate for partial BBSome deficiencies in some ciliopathies

• Cancer therapeutics:

  • Inhibiting TOM1L2 phosphorylation or its interaction with TOLLIP could reduce invasiveness in ERBB2-positive cancers

  • Blocking TOM1L2-mediated trafficking of MT1-MMP might reduce matrix degradation and metastatic potential

  • TOM1L2 status could serve as a biomarker for stratifying patients most likely to benefit from specific treatments

• Neurodegenerative disease approaches:

  • Given TOM1L2's connections to AD-associated genes, enhancing its trafficking functions might improve clearance of aggregation-prone proteins

  • TOM1L2-based interventions could potentially modulate ciliary signaling in neurons, affecting neurodegenerative processes

• Delivery system opportunities:

  • Understanding TOM1L2's trafficking mechanisms could inform the design of drug delivery systems targeting specific cellular compartments

  • TOM1L2-derived peptides might direct therapeutic cargo to particular cellular destinations

Future therapeutic development will require:

• High-resolution structural studies of TOM1L2 complexes

• Development of specific chemical probes to modulate TOM1L2 functions

• Advanced in vivo models to validate TOM1L2 as a therapeutic target

• Understanding of potential compensatory mechanisms within the Tom1 family