ASB13 Human

What is ASB13 and what are its key structural characteristics?

ASB13 is a human protein belonging to the ankyrin repeat and SOCS box-containing (ASB) family of proteins. It contains multiple ankyrin repeat sequences and a SOCS box domain . The SOCS box domain serves to couple suppressor of cytokine signaling (SOCS) proteins and their binding partners with the elongin B and C complex, potentially targeting them for degradation . The protein is 278 amino acids in length with a molecular mass of approximately 32.4 kDa . The protein's structure includes several conserved domains that are critical for its function in protein-protein interactions and substrate recognition.

What is the primary cellular function of ASB13?

ASB13 functions as a substrate-recognition component of a SCF-like ECS (Elongin-Cullin-SOCS-box protein) E3 ubiquitin-protein ligase complex . This complex mediates the ubiquitination and subsequent proteasomal degradation of target proteins . E3 ligases are responsible for the final step in the ubiquitination cascade, where they recognize specific substrate proteins and facilitate the transfer of ubiquitin to these targets. Through this activity, ASB13 plays an important role in protein homeostasis and cellular signaling regulation.

What post-translational modifications have been identified in ASB13?

Key post-translational modifications identified in ASB13 include:

ASB13 undergoes ubiquitination at lysine residues K191 and K261 . Notably, the K261 site has been associated with a variant (N261) identified in lung cancer samples . This suggests that alterations in ASB13's own post-translational modification may play a role in disease pathology, though the functional consequences of these modifications require further investigation.

How does ASB13 regulate SNAI2 and what is its role in breast cancer metastasis?

ASB13 has been identified as an E3 ubiquitin ligase that targets SNAI2 (also known as Slug) for ubiquitination and degradation . A genome-wide E3 ligase siRNA library screen revealed ASB13 as a key regulator of SNAI2 protein levels . In breast cancer, ASB13 functions as a metastasis suppressor through this mechanism. By promoting SNAI2 degradation, ASB13 relieves SNAI2's transcriptional repression of YAP, a transcription co-activator in the Hippo pathway . The functional relationship can be summarized as:

• ASB13 targets SNAI2 for degradation

• Reduced SNAI2 levels relieve transcriptional repression of YAP

• Increased YAP expression suppresses breast cancer progression and metastasis

Experimental evidence shows that ASB13 knockout in breast cancer cells promotes cell migration and decreases F-actin polymerization, while overexpression of ASB13 suppresses lung metastasis . These findings establish ASB13 as a critical suppressor of breast cancer metastasis through a SNAI2-YAP regulatory axis.

What phenotypic changes result from ASB13 manipulation in experimental models?

Experimental manipulation of ASB13 expression reveals several important phenotypic effects:

These phenotypic changes demonstrate ASB13's role in regulating cellular behaviors relevant to cancer progression . The effect on YAP expression is particularly significant, as YAP knockout in breast cancer models increases tumorsphere formation, anchorage-independent colony formation, cell migration in vitro, and lung metastasis in vivo .

What are the optimal approaches for studying ASB13 protein interactions and activity?

Researchers studying ASB13 interactions and activity should consider these methodological approaches:

• Dual-luciferase assays: Fan et al. successfully utilized a dual-luciferase-based genome-wide E3 ligase siRNA library screen to identify ASB13 as a SNAI2-targeting E3 ligase .

• Recombinant protein studies: Use purified recombinant ASB13 protein (>95% purity, available as His-tagged protein) for in vitro binding and activity assays .

• Ubiquitination assays: In vitro and cellular ubiquitination assays to detect ASB13-mediated ubiquitination of target proteins, using antibodies specific for ubiquitin or ubiquitin chains.

• Co-immunoprecipitation: To identify ASB13 binding partners and components of the E3 ligase complex it forms.

• Cellular assays following manipulation: Knockout and overexpression studies to assess phenotypic effects on cell migration, cytoskeletal organization, and protein expression .

The choice of method should be guided by the specific research question, with combinations of approaches typically providing the most robust evidence of ASB13 function.

What expression systems and purification methods are recommended for producing recombinant ASB13 protein?

For researchers requiring recombinant ASB13 protein, several production and purification approaches have been validated:

Recombinant ASB13 produced in E. coli has been successfully used for applications including SDS-PAGE and HPLC analysis . The protein appears to require urea in the buffer, suggesting potential solubility challenges that researchers should be aware of when designing experiments. For long-term storage, it is recommended to add a carrier protein (0.1% HSA or BSA) and avoid freeze-thaw cycles . Researchers should consider these biochemical properties when planning experiments involving recombinant ASB13.

What are the most effective genetic manipulation strategies for studying ASB13 function?

Based on successful approaches in the literature, researchers should consider these genetic manipulation strategies:

• CRISPR/Cas9 knockout: Used successfully to eliminate ASB13 expression and study resulting phenotypes in breast cancer cells .

• siRNA/shRNA knockdown: For temporary reduction of ASB13 expression, useful for screening studies and when complete knockout might be lethal.

• Overexpression systems: Transfection or viral transduction of ASB13 expression constructs to study gain-of-function effects, as demonstrated in lung metastasis suppression studies .

• Domain mutation approaches: Creating point mutations or domain deletions within ASB13 to study the functional importance of specific residues or regions, particularly in the ankyrin repeats and SOCS box domains.

• Reporter systems: Fusion of ASB13 with tags such as GFP or luciferase to monitor localization, expression levels, or activity in live cells.

When designing genetic manipulation experiments, researchers should consider potential compensatory mechanisms by other ASB family members and validate knockdown/knockout efficiency using appropriate controls.

What are the unresolved questions regarding ASB13's role in normal physiology and disease?

Several important questions remain unanswered about ASB13 function:

• Complete substrate repertoire: Beyond SNAI2, what other proteins are targeted by ASB13 for ubiquitination and degradation? A comprehensive substrate identification would provide insight into the breadth of ASB13's cellular functions.

• Regulation of ASB13 itself: What factors control ASB13 expression, localization, and activity? Understanding how ASB13 is regulated could reveal additional control points in its signaling pathways.

• Role in other cancer types: While ASB13's function has been studied in breast cancer , its role in other malignancies, including the lung cancer where variants have been identified , remains to be elucidated.

• Normal physiological functions: ASB13's roles outside of pathological contexts are not well characterized. What are its functions in normal development, tissue homeostasis, and cellular processes?

• Therapeutic targeting potential: Could modulation of ASB13 activity serve as a therapeutic strategy? If so, what approaches might be most effective for enhancing its tumor-suppressive functions?

Addressing these questions will require integrated approaches combining biochemical, cellular, and in vivo studies, as well as analysis of patient samples and clinical data.