CRYAB Human, His

What is the basic structure of human CRYAB protein?

Human CRYAB is a 175 amino acid protein (Met1-Lys175) with a molecular weight of approximately 21.2 kDa, though it typically appears as 22-23 kDa on SDS-PAGE gels . The protein contains an alpha-crystalline Hsp domain spanning amino acids 66-149 . CRYAB belongs to the small heat shock protein (sHSP/HSP20) family and functions primarily as a molecular chaperone . The protein's structure consists of seven regions: four homologous motifs, a connecting peptide, and N- and C-terminal extensions . The C-terminal domain (CTD) is particularly crucial for proper protein solubility and chaperone activity .

How does CRYAB function as a molecular chaperone?

CRYAB operates as a molecular chaperone by binding to improperly folded proteins to prevent protein aggregation, rather than actively renaturing and releasing proteins like traditional chaperones . This function is critical during cellular stress conditions such as heat shock, ischemia, and oxidation . CRYAB forms heterogeneous aggregates with other proteins, particularly alpha-A crystallin in the lens, where they maintain lens transparency and refractive index . The chaperone activity can be significantly impaired by post-translational modifications .

What post-translational modifications affect CRYAB function?

CRYAB undergoes several post-translational modifications that can alter its functional properties:

• Phosphorylation at Ser45 and Ser59

• Potential O-GlcNAc modification at Thr158, 162, or 170

• Acetylation at Lys92

• N-terminal modifications including cleavage of Met and sometimes the MetAspIleAlaIleHis sequence

These modifications, particularly phosphorylation and degradation during certain conditions (such as enterovirus infection), can significantly decrease chaperone activity and alter protein interactions .

How is recombinant human CRYAB protein typically produced?

Recombinant human CRYAB is commonly expressed in E. coli expression systems using the full coding sequence (Met1-Lys175) . The protein is typically tagged with a His-tag at the C-terminus to facilitate purification . The purified protein should demonstrate >95% purity as determined by reducing SDS-PAGE . Expression and purification protocols typically yield a protein with the following characteristics:

For optimal activity, the protein should be carefully reconstituted according to specific buffer conditions provided in product manuals .

What are the most effective methods for studying CRYAB expression in cell models?

Several methods have proven effective for manipulating CRYAB expression in cellular models:

• Lentiviral-mediated overexpression: CRYAB cDNA can be cloned into puromycin-resistant lentiviral vectors (e.g., pLVX-Puro) with HEK293T cells used for virus production. Target cells (e.g., THP-1, HT29, and Caco-2) can be infected with the CRYAB-expressing lentivirus and selected with puromycin to generate stable overexpression cell lines .

• CRISPR/Cas9-mediated silencing: Guide RNAs targeting CRYAB can be designed and expressed along with Cas9 using the Lenti-CRISPR V2 vector. Puromycin selection allows isolation of CRYAB-deficient cell lines. Verification should include qRT-PCR, immunoblotting, and genomic sequencing .

• Recombinant fusion proteins: Cell-permeable recombinant fusion proteins like TAT-CRYAB can be used for functional studies, particularly for assessing anti-inflammatory effects in cellular and animal models .

What analytical techniques are most informative for studying CRYAB in disease contexts?

Contemporary research employs several advanced techniques to study CRYAB in disease contexts:

• Single-cell RNA sequencing (scRNA-seq): This technique has proven valuable for identifying distinct cell populations expressing CRYAB in complex tissues. Using the Seurat R package, researchers can identify survival-related gene clusters and distinguish CRYAB expression patterns across different cell types .

• Integrated multi-omics approaches: Combining RNA-seq and microarray data from public databases (TCGA, GEO) with scRNA-seq data enables comprehensive analysis of CRYAB expression patterns and functional implications .

• Prognostic modeling: LASSO and stepwise regression algorithms applied to expression data can establish predictive models for disease outcomes, particularly in cancer contexts .

• Functional validation: In vitro experiments, including knockout studies in appropriate cell lines (e.g., U87 and LN229 for glioblastoma), can confirm the functional significance of CRYAB. Cell viability, proliferation, and invasiveness assays provide critical functional readouts .

How is CRYAB implicated in myopathies and cardiomyopathies?

Mutations in the CRYAB gene have been associated with various muscle-related disorders. Specifically:

• Myofibrillar myopathy type 2 (MFM2): CRYAB mutations can lead to progressive muscle weakness affecting both proximal and distal skeletal muscles .

• Cardiomyopathies: Several mutations in CRYAB have been linked to heart muscle disorders, with variable penetrance and expressivity .

• Combined phenotypes: Some mutations can cause a multisystem disorder characterized by a combination of myopathy, cardiomyopathy, respiratory insufficiency, and dysphagia .

The pathogenic mechanisms involve altered chaperone function, leading to protein aggregation in muscle tissues. Mutations in the C-terminal domain are particularly severe, as they can affect protein solubility and activity . The inheritance pattern can be either autosomal dominant or recessive, with variable penetrance .

What is the role of CRYAB in ocular pathologies?

CRYAB plays a crucial role in maintaining lens transparency, and mutations can lead to various eye conditions:

• Congenital cataracts: Mutations in CRYAB can cause early-onset cataracts, as seen in posterior polar cataract type 2 (CTPP2) .

• Age-related lens opacity: Altered CRYAB function has been implicated in age-related cataracts through impaired chaperone activity.

The underlying mechanism involves the formation of heterogeneous aggregates with alpha-A crystallin in the lens, which normally maintains lens transparency and refractive index . When CRYAB function is impaired, protein aggregation occurs, leading to lens opacity .

How is CRYAB involved in cancer progression, particularly in glioblastoma?

Recent research has highlighted the role of CRYAB in cancer, particularly in glioblastoma:

• Expression pattern: CRYAB is expressed in terminal-stage oligodendrocyte lineage cells in glioblastoma and serves as a marker for these cells .

• Prognostic significance: High CRYAB expression is associated with poor prognosis in glioblastoma patients .

• Functional impact: In vitro experiments have shown that knocking out CRYAB in glioblastoma cell lines (U87 and LN229) reduces cell viability, proliferation, and invasiveness .

• Solid tumor involvement: Increased expression of CRYAB has been reported in several highly invasive and metastatic solid tumors beyond glioblastoma .

These findings suggest that CRYAB may serve as both a prognostic marker and potential therapeutic target in glioblastoma and potentially other cancers.

What is the relationship between CRYAB and inflammatory conditions?

CRYAB exhibits significant anti-inflammatory properties in various contexts:

• Intestinal inflammation: CRYAB regulates inflammatory responses in intestinal mucosa by inhibiting IKKβ-mediated signaling, suggesting potential therapeutic applications in inflammatory bowel disease (IBD) .

• Neuroinflammation: CRYAB plays protective and therapeutic roles in neuroinflammation induced by injury, infection, neurodegeneration, and autoimmunity .

• Inflammatory model studies: Recombinant CRYAB fusion proteins (TAT-CRYAB) have demonstrated anti-inflammatory effects in experimental models of colitis, including dextran sulfate sodium (DSS) and 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis in mice .

The anti-inflammatory mechanism appears to involve modulation of key inflammatory pathways, suggesting CRYAB as a potential therapeutic target in inflammatory conditions.

How do mutations in different domains of CRYAB affect its function?

The functional impact of CRYAB mutations varies depending on the affected domain:

• C-terminal domain (CTD) mutations: Mutations that affect the C-terminal domain are particularly disruptive as this region is essential for protein solubility and chaperone activity. Elongation of the protein at the CTD (as seen in some dominant mutations) can lead to altered properties and severe clinical manifestations .

• Alpha-crystalline domain mutations: Alterations in the alpha-crystalline Hsp domain (aa 66-149) can impair the core chaperone function of the protein .

• N-terminal modifications: Changes in the N-terminal region can affect protein-protein interactions and oligomerization capacity .

Understanding the domain-specific effects of mutations is crucial for interpreting phenotypic variations and developing targeted therapeutic approaches.

What are the current approaches for studying CRYAB in the context of the tumor immune microenvironment?

Advanced research on CRYAB in the tumor immune microenvironment employs several sophisticated approaches:

• Immune infiltration analysis: Correlating CRYAB expression with immune cell infiltration patterns provides insights into its role in modulating the tumor immune microenvironment .

• Immunotherapy response prediction: CRYAB expression patterns may predict responses to immunotherapy, making it a potential biomarker for patient stratification .

• Single-cell analysis of immune populations: scRNA-seq enables detailed characterization of immune cell populations in relation to CRYAB expression, revealing potential immunomodulatory mechanisms .

These approaches collectively contribute to understanding how CRYAB influences the tumor immune microenvironment and potentially affects immunotherapy outcomes.

What are the challenges in developing CRYAB-targeted therapeutics?

Developing therapeutics targeting CRYAB faces several challenges:

• Multifunctional nature: CRYAB's involvement in multiple cellular processes means targeting it may have unintended consequences in unaffected tissues.

• Cell-type specific effects: CRYAB functions differently across cell types, requiring careful consideration of tissue-specific targeting strategies.

• Genetic variability: The variety of pathogenic mutations in CRYAB suggests that therapeutic approaches may need to be mutation-specific.

• Dual roles in disease: CRYAB can be both protective (in inflammation) and pathogenic (in cancer), requiring context-specific therapeutic strategies.

Despite these challenges, targeted approaches such as antisense oligonucleotides, small molecule modulators of chaperone activity, or gene therapy approaches may hold promise for CRYAB-related disorders.