CALML5 Human

What is CALML5 and where is it expressed in humans?

CALML5 (Calmodulin-like protein 5) is a calcium binding protein related to the calmodulin family of calcium binding proteins. In humans, it is encoded by the CALML5 gene located on chromosome 10 . Functional studies with recombinant protein demonstrate that CALML5 binds calcium and undergoes conformational changes when it does so .

CALML5 expression is highly specific to the epidermis, with abundant expression detected only in reconstructed epidermis and restricted to differentiating keratinocytes . Laser capture microdissection followed by RNA sequencing has identified CALML5 as the most enriched gene in differentiating outer epidermis compared to progenitor layers .

How is CALML5 expression regulated during epidermal differentiation?

CALML5 expression is regulated through a complex transcriptional and post-transcriptional network:

• At the transcriptional level, the ZNF750 transcription factor directly upregulates CALML5 expression during epidermal differentiation . ZNF750 binding has been observed at a genomic site approximately 10 kb downstream from the CALML5 gene .

• Post-transcriptionally, the long noncoding RNA TINCR stabilizes CALML5 mRNA, enhancing its expression . TINCR RNA interactome analysis has identified a binding peak in the CALML5 3' untranslated region, indicating that CALML5 mRNA is a direct TINCR stabilization target .

• This regulation occurs downstream of p63, a master transcription factor for stratified epithelia, creating a regulatory cascade where p63 activates ZNF750, which then induces CALML5, with CALML5 mRNA subsequently stabilized by TINCR .

What happens when CALML5 function is lost in epidermal tissue?

CALML5 loss has significant consequences for epidermal differentiation and function:

• CALML5 knockout impairs differentiation, abolishes keratohyalin granules, and disrupts epidermal barrier function .

• Expression of late differentiation genes is inhibited with CALML5 loss, including filaggrin and loricrin, two proteins essential to barrier function .

• Nile Red staining reveals reduced lipid deposition, consistent with impaired lipid barrier formation .

• CALML5 depletion does not affect proliferation of basal progenitors and does not significantly impact apoptosis, suggesting that its impact on skin homeostasis is predominantly in the regulation of differentiation .

• Some differentiation genes remain unaffected by CALML5 depletion, indicating that CALML5 impacts specific aspects of differentiation rather than causing global differentiation arrest .

What molecular interactions does CALML5 participate in during epidermal differentiation?

CALML5 participates in several key molecular interactions during epidermal differentiation:

• Mass spectrometry has identified SFN (stratifin/14-3-3σ) as a CALML5-binding protein . This interaction has been validated through Far Western blotting, showing a specific interaction between CALML5 and SFN recombinant proteins .

• Proximity ligation assays (PLAs) have demonstrated that CALML5–SFN interactions occur abundantly in suprabasal strata and peak in late differentiated layers of the epidermis . At the subcellular level, their interaction occurs in the cytoplasm .

• The interaction between CALML5 and SFN occurs independently of calcium, as it is unaffected by the absence of free calcium .

• CALML5 can also associate with transglutaminase 3, a key enzyme in the terminal differentiation of keratinocytes .

• The calcium-binding EF hand motifs of CALML5, while essential for its function in differentiation, are not required for the CALML5-SFN interaction .

How does CALML5 regulate gene expression without being a transcription factor?

CALML5 regulates gene expression through several indirect mechanisms:

• CALML5 and SFN together co-control approximately 13% of late differentiation genes in the epidermis .

• CALML5 modulates the interaction of SFN with some of its binding partners . Since SFN can translocate to the nucleus and affect gene expression, CALML5 may influence transcription by regulating SFN activity .

• RNA sequencing of CALML5-depleted tissue versus normal control revealed that CALML5 depletion results in greater than twofold expression change of 547 protein-coding genes .

• Gene ontology analysis of CALML5-regulated genes shows enrichment for genes involved in epidermal development and differentiation and phenotypic terms related to palmoplantar keratoderma and congenital erythroderma .

• CALML5 functions downstream from ZNF750 to mediate up-regulation of a subset of ZNF750 differentiation gene targets but does not contribute to the repression of ZNF750-regulated progenitor genes .

What is the significance of calcium binding for CALML5 function?

Calcium binding plays a critical role in CALML5 function:

• Functional studies with recombinant CALML5 demonstrate that it binds calcium and undergoes a conformational change when it does so .

• Experiments with CALML5 calcium-binding mutants reveal that intact calcium-binding motifs are required for CALML5 effects on differentiation .

• Researchers generated alanine substitution mutants of the acidic amino acids that form the calcium-binding pocket of the N-terminal and C-terminal pair of EF hands. Only wild-type CALML5, not the EF mutants, was capable of transcriptional rescue when expressed in CALML5 knockout keratinocytes .

• Interestingly, while calcium binding is critical for CALML5's role in gene regulation, the CALML5-SFN protein interaction occurs independently of calcium binding, as demonstrated by interaction studies in the absence of free calcium .

• This suggests that calcium binding induces conformational changes in CALML5 that are essential for some functions but not for SFN binding, pointing to multiple mechanistic roles for this protein in epidermal differentiation .

What experimental approaches are most effective for studying CALML5 function in epidermal differentiation?

Several complementary experimental approaches have proven effective for studying CALML5 function:

• Gene Knockout/Knockdown:

  • CRISPR/Cas9 gene editing for complete knockout in keratinocytes

  • RNAi for transient knockdown

  • Both approaches have demonstrated CALML5's essential role in differentiation

• 3D Culture Systems:

  • Organotypic human epidermal tissue models allow assessment of stratification and differentiation

  • CALML5-depleted keratinocytes show reduced expression of late differentiation markers in these models

• In Vivo Xenografts:

  • Wild-type and CALML5 knockout keratinocytes can be xenografted onto immune-deficient mice

  • This approach allows assessment of CALML5 function in a more physiological context

• Rescue Experiments:

  • Reintroduction of wild-type or mutant CALML5 into knockout cells

  • These experiments have confirmed specificity of phenotypes and identified critical functional domains

• Proximity Ligation Assays:

  • For visualizing CALML5-protein interactions in tissue context

  • This technique has revealed the spatial and temporal pattern of CALML5-SFN interaction

• Laser Capture Microdissection:

  • For isolating specific epidermal layers for transcriptomic analysis

  • This technique identified CALML5 as the most enriched gene in differentiating outer epidermis

How can researchers identify and validate the downstream targets of CALML5 in keratinocytes?

Identifying and validating CALML5 downstream targets requires a multi-faceted approach:

What are the technical challenges in distinguishing CALML5-specific effects from those of other calmodulin family members?

Distinguishing CALML5-specific effects presents several technical challenges:

• Sequence and Structural Homology:

  • CALML5 belongs to the calmodulin family of calcium binding proteins, which share structural features including EF-hand domains

  • This homology can lead to potential cross-reactivity in antibody-based detection methods

• Functional Redundancy:

  • Other calmodulin family members may partially compensate for CALML5 loss

  • This could mask or attenuate phenotypes in knockout/knockdown models

• Expression Specificity:

  • While CALML5 is predominantly expressed in differentiating keratinocytes, other family members may be expressed in similar contexts

  • Distinguishing unique versus overlapping functions requires careful expression analysis

• Technical Approaches to Overcome These Challenges:

  • Use of multiple, highly specific antibodies validated for specificity

  • Careful analysis of related gene expression in CALML5 knockout models

  • Domain-swapping experiments between CALML5 and other family members

  • Comparative analysis of phenotypes between different family member knockouts

• Validation Standards:

  • Complete knockout verification through both DNA sequencing and protein detection

  • Use of multiple independent knockout clones to control for clonal effects

  • Rescue experiments with wild-type CALML5 to confirm phenotype specificity

How does the ZNF750–TINCR–CALML5–SFN network function in epidermal differentiation?

The ZNF750–TINCR–CALML5–SFN network represents a coordinated regulatory system for epidermal differentiation:

• Hierarchical Organization:

  • p63 activates ZNF750 expression

  • ZNF750 directly upregulates CALML5 transcription by binding to a site approximately 10 kb downstream from the CALML5 gene

  • TINCR lncRNA stabilizes CALML5 mRNA through direct interaction with the CALML5 3' UTR

  • CALML5 protein interacts with SFN in the cytoplasm of differentiating keratinocytes

• Functional Cooperation:

  • CALML5 and SFN together control approximately 13% of late differentiation genes

  • CALML5 modulates SFN's interactions with some of its binding partners

  • SFN (14-3-3σ) can affect gene expression through multiple mechanisms, including regulation of transcription factor localization

• Target Gene Specificity:

  • CALML5 mediates upregulation of a subset of ZNF750 differentiation gene targets but does not contribute to ZNF750's repression of progenitor genes

  • Depletion of SFN inhibits expression of the largest subset of CALML5 target genes in phenocopy assays

• Spatial-Temporal Dynamics:

  • CALML5–SFN interactions occur abundantly in suprabasal strata and peak in late differentiated layers

  • The interaction is predominantly cytoplasmic, suggesting regulation of SFN localization or activity

• Calcium Dependence:

  • While calcium binding is essential for CALML5's function in differentiation, the CALML5-SFN interaction itself is calcium-independent

This network illustrates the complexity of epidermal differentiation regulation, involving transcriptional, post-transcriptional, and protein-protein interaction mechanisms.

What bioinformatic approaches best reveal CALML5's position in epidermal gene regulatory networks?

Several bioinformatic approaches can effectively position CALML5 within epidermal gene regulatory networks:

• Differential Gene Expression Analysis:

  • RNA-seq comparing CALML5-depleted versus control tissue identified 547 differentially expressed genes

  • This establishes the primary gene set under CALML5 influence

• Gene Ontology and Pathway Enrichment:

  • Analysis of CALML5-regulated genes showed enrichment for epidermal development and differentiation genes

  • Phenotypic terms related to palmoplantar keratoderma and congenital erythroderma were also enriched

• Gene Set Overlap Analysis:

  • Comparison with progenitor, early, and late differentiation gene signatures showed CALML5-regulated genes are most strongly associated with late differentiation

  • Overlap analysis with 44 other epidermal regulators revealed significant connection to p63, ZNF750, and TINCR networks

• Protein-Protein Interaction Network Analysis:

  • BioID/MS identified CALML5 interaction partners in differentiated keratinocytes

  • SAINT score analysis prioritized high-confidence interactions, including SFN

  • These interactions can be mapped to understand CALML5's position in protein interaction networks

• Hierarchical Network Modeling:

  • Integration of expression data with ChIP-seq data for transcription factors like ZNF750

  • Analysis of response patterns following perturbation of different network components

  • This established the regulatory hierarchy: p63 → ZNF750 → CALML5 (with TINCR stabilization)

These approaches collectively position CALML5 as a critical downstream effector in epidermal differentiation networks, linking transcriptional regulation to functional outcomes through protein-protein interactions.

How should researchers interpret contradictory findings about CALML5 function across different experimental systems?

When interpreting contradictory findings about CALML5 function, researchers should consider:

• Experimental System Differences:

  • Cell type variations: Results from immortalized versus primary keratinocytes may differ

  • Culture conditions: 2D monolayer versus 3D organotypic models versus in vivo xenografts

  • Knockout efficiency: The referenced study observed milder phenotypes with incomplete locus deletion

• Methodological Considerations:

  • Knockdown versus knockout: The extent of protein loss affects phenotype severity

  • Timing of analysis: Acute versus chronic loss of CALML5 may yield different results

  • Clone-specific effects: The referenced study used clonal isolation and found variation in differentiation capacity

• Context-Dependent Functions:

  • Differentiation stage: CALML5 function varies based on keratinocyte differentiation status

  • CALML5 function likely requires other factors that are only produced upon differentiation

  • Forced expression of CALML5 in undifferentiated basal keratinocytes does not induce differentiation

• Resolution Strategies:

  • Rescue experiments: The referenced study confirmed phenotype specificity by restoring CALML5 expression

  • Multiple model systems: The study utilized primary keratinocytes, organotypic tissue, and in vivo xenografts

  • Functional domain analysis: Testing calcium-binding mutants revealed mechanism-specific requirements

• Example of Resolution:

  • The referenced study noted that CALML5-edited keratinocyte pools showed a milder phenotype than expected

  • Sequencing revealed incomplete locus deletion, explaining the attenuated effect

  • This was resolved by generating complete knockout clones, which displayed the full phenotype

What are promising research directions for exploring CALML5's potential roles in skin pathologies?

Several promising research directions exist for exploring CALML5's role in skin pathologies:

• Barrier Function Disorders:

  • Since CALML5 is essential for proper barrier function, investigation of its role in atopic dermatitis, ichthyosis, and other barrier-related conditions is warranted

  • Gene ontology analysis of CALML5-regulated genes showed enrichment for terms related to palmoplantar keratoderma and congenital erythroderma

• Inflammatory Skin Conditions:

  • Exploration of how dysregulation of the ZNF750–TINCR–CALML5–SFN network might contribute to inflammatory processes in conditions like psoriasis

  • Investigation of potential immune-modulating functions of CALML5 in epithelial-immune cell interactions

• Wound Healing and Regeneration:

  • Assessment of CALML5's role in re-epithelialization and barrier restoration during wound healing

  • Study of potential dysregulation in chronic wounds or hypertrophic scarring

• Skin Cancer:

  • Investigation of CALML5 expression and function in various skin cancers

  • Exploration of its potential tumor-suppressive role, given its involvement in terminal differentiation

  • Analysis of potential disruptions in the ZNF750–TINCR–CALML5–SFN network in skin malignancies

• Genetic Skin Diseases:

  • Screening for CALML5 mutations or expression abnormalities in patients with uncharacterized disorders of keratinization

  • Development of CALML5-based therapeutic approaches for genetic disorders affecting epidermal differentiation

• Aging-Related Changes:

  • Examination of age-related changes in CALML5 expression and function

  • Investigation of potential roles in age-associated barrier dysfunction

What novel methodologies could advance our understanding of CALML5 function in human skin?

Novel methodologies that could significantly advance CALML5 research include:

• Advanced Genome Editing Technologies:

  • Base editing or prime editing for precise modification of CALML5 regulatory regions

  • Inducible CRISPR systems for temporal control of CALML5 deletion

  • Domain-specific mutations to dissect functional regions beyond calcium-binding motifs

• Advanced Imaging Technologies:

  • Super-resolution microscopy to visualize CALML5-protein interactions at the nanoscale

  • Live-cell calcium imaging to correlate calcium dynamics with CALML5 function

  • Fluorescent biosensors to monitor CALML5-SFN interactions in real-time

• Single-Cell and Spatial Technologies:

  • Single-cell RNA-seq to map CALML5 expression across diverse keratinocyte states

  • Spatial transcriptomics to correlate CALML5 expression with microanatomical features

  • Spatial proteomics to map the distribution of CALML5 protein complexes in intact tissue

• Advanced Protein Analysis:

  • Hydrogen-deuterium exchange mass spectrometry to analyze calcium-induced conformational changes

  • Cryo-EM analysis of CALML5-SFN complexes

  • Proximity labeling approaches to map the complete protein interaction network in intact cells

• Human Skin Models:

  • Patient-derived organoids for personalized disease modeling

  • Skin-on-chip microfluidic systems for dynamic studies

  • Bioengineered skin with defined cellular composition for mechanistic studies

• Systems Biology Approaches:

  • Multi-omics integration (transcriptomics, proteomics, metabolomics)

  • Network modeling of the complete ZNF750–TINCR–CALML5–SFN regulatory system

  • Computational prediction of CALML5 modifiers for targeted experimental validation

How might understanding CALML5 function lead to novel therapeutic approaches for skin disorders?

Understanding CALML5 function could lead to several novel therapeutic approaches:

• Barrier Restoration Therapies:

  • Development of small molecules that enhance CALML5 activity or stability

  • Topical formulations targeting the ZNF750–TINCR–CALML5–SFN pathway to improve barrier function in conditions like atopic dermatitis

  • CALML5-based biomarkers to monitor barrier restoration during treatment

• Gene Therapy Approaches:

  • For genetic disorders involving CALML5 dysfunction, development of gene therapy vectors for epidermal delivery

  • RNA therapeutics targeting the CALML5 regulatory network

  • Ex vivo correction of patient keratinocytes followed by grafting

• Differentiation Modulators:

  • Small molecules targeting CALML5-SFN interaction as differentiation modulators

  • Peptide mimetics of functional CALML5 domains

  • Targeted stabilization of CALML5 mRNA through TINCR-inspired approaches

• Personalized Medicine Applications:

  • Genetic screening for CALML5 pathway variants to guide treatment selection

  • Patient-derived skin organoids to test treatment efficacy

  • Biomarker development to monitor therapy response

• Wound Healing Applications:

  • CALML5-pathway targeted therapies to accelerate re-epithelialization

  • Bioengineered skin grafts with optimized CALML5 expression

  • Combination approaches targeting multiple aspects of the differentiation program

• Cancer Therapeutics:

  • If CALML5 has tumor-suppressive functions, development of differentiation therapy approaches for skin cancers

  • Combination strategies targeting the ZNF750–TINCR–CALML5–SFN network alongside conventional therapies

  • Diagnostic applications based on CALML5 expression patterns in suspicious lesions

These approaches could leverage our growing understanding of CALML5 biology to address unmet needs in dermatological therapy.