STUB1 Human

What is the molecular structure of human STUB1 protein?

Human STUB1/CHIP is a 303 amino acid protein with a molecular weight of approximately 35 kDa. It contains three distinct functional domains:

• N-terminal tetratricopeptide repeat (TPR) domain (amino acids 27-127) that mediates interactions with heat shock proteins

• Central charged domain that supports TPR-dependent interactions

• C-terminal U-Box domain (amino acids 226-299) that participates in ubiquitination processes

The TPR domain interacts with heat shock proteins, while the U-Box domain is essential for its E3 ubiquitin ligase activity . Human and mouse STUB1 share 97% amino acid sequence identity, indicating high evolutionary conservation .

What are the primary cellular functions of STUB1?

STUB1 functions as a quality control checkpoint in cellular protein homeostasis through several mechanisms:

• Forms complexes with molecular chaperones (Hsp70, Hsc70, and Hsp90) to modulate their activity

• Facilitates ubiquitination of chaperone substrates, targeting them for proteasomal degradation

• Regulates the stability of specific proteins including phosphorylated Tau, p53, PTEN, Synuclein-alpha, and β-APP

• Acts as an intracellular checkpoint for interferon gamma (IFNγ) sensing and response

Methodologically, these functions can be assessed through co-immunoprecipitation with chaperones, in vitro ubiquitination assays, and monitoring substrate stability in STUB1-deficient cells.

Where is STUB1 primarily expressed in human tissues?

STUB1 shows a tissue-specific expression pattern:

• Highly expressed in striated muscle and brain

• Lower expression levels in pancreas, lung, liver, and kidney

• At the cellular level, STUB1 is detected in both cytoplasm and nuclei of various cell types, as demonstrated by immunofluorescence studies in HeLa cells

To detect tissue-specific expression, researchers typically employ immunohistochemistry with anti-STUB1 antibodies or analyze tissue-specific transcriptome datasets.

What are the most reliable methods for detecting STUB1 in experimental systems?

Several complementary approaches are recommended for robust STUB1 detection:

Western Blot Analysis:

• Use specific antibodies like Goat Anti-Human CHIP/STUB1 Antigen Affinity-purified Polyclonal Antibody

• STUB1 appears as a band at approximately 39 kDa

• Recommended protocol: PVDF membrane probed with 1 μg/mL of antibody under reducing conditions

Immunofluorescence:

• Effective concentration: 25 μg/mL of Anti-Human STUB1 antibody for 3 hours at room temperature

• Look for specific staining in both cytoplasm and nuclei

• Counterstain with DAPI for nuclear visualization

Flow Cytometry:

• Particularly useful for assessing STUB1's impact on surface proteins like IFNGR1

• Use fluorochrome-conjugated secondary antibodies for detection

How can researchers generate reliable STUB1 knockout models?

Creating and validating STUB1 knockout models requires careful consideration of several factors:

CRISPR/Cas9 Knockout Strategy:

• Design guide RNAs targeting early exons to ensure complete functional disruption

• Validate knockouts through Western blotting and genomic sequencing

• Analyze phenotypic consequences, particularly related to protein quality control and IFNγ signaling

• Consider generating conditional knockouts for tissue-specific studies

Functional Validation:

• Assess accumulation of known STUB1 substrates

• Measure sensitivity to proteotoxic stress

• Evaluate IFNγ pathway activation, including STAT1 phosphorylation

• Include rescue experiments with wild-type STUB1 to confirm specificity

How does STUB1 contribute to the aging process?

STUB1 plays a significant role in age-related processes:

• STUB1-null mice exhibit deregulation of protein quality control, shortened lifespan, and accelerated age-related pathophysiological features

• It contributes to proteasomal degradation by targeting misfolded chaperone substrates and interacting with chaperone complexes, including HSPA8

• These functions are essential for maintaining protein homeostasis, which declines during aging

Methodologically, researchers should:

• Assess protein aggregation in aged tissues with and without functional STUB1

• Perform longitudinal studies in conditional knockout models

• Analyze STUB1 activity and substrate specificity changes during aging

What is STUB1's connection to neurodegenerative diseases?

Given its role in protein quality control, STUB1 has significant implications for neurodegenerative disorders:

• STUB1 facilitates the ubiquitination of neurodegeneration-associated proteins like phosphorylated Tau and Synuclein-alpha

• It plays a role in the endoplasmic reticulum unfolded protein response, which is often dysregulated in neurodegenerative diseases

• Its high expression in brain tissue suggests tissue-specific functions in neurons and glia

Research approaches should include:

• Analysis of STUB1 expression and activity in patient samples and disease models

• Investigation of STUB1 polymorphisms in neurodegenerative disease cohorts

• Assessment of STUB1's interaction with disease-specific protein aggregates

How does STUB1 regulate interferon gamma signaling in cancer cells?

Recent research has uncovered STUB1's role as a checkpoint for IFNγ signaling:

• Genetic deletion of STUB1 increases IFNGR1 abundance on the cell surface, enhancing downstream IFNγ responses

• STUB1 deletion in human prostate and breast cancer cells increases their susceptibility to cytokine-induced growth inhibition

• Loss of STUB1 leads to enhanced expression of antigen presentation machinery components, including H2-K1, B2M, PSME1, PSME2, and ERAP1

What is the relationship between STUB1 and immune checkpoint blockade therapy?

STUB1 has emerged as a potential target for enhancing immunotherapy efficacy:

• CRISPR screens have identified STUB1 as a potential target to overcome immune checkpoint blockade resistance

• STUB1 functions as a barrier to IFNγ sensing, which is critical for anti-tumor immune responses

• Despite promising in vitro findings, in vivo studies in mouse syngeneic tumor models have not demonstrated significant benefits of Stub1 inactivation, either alone or in combination with anti-PD-1 therapy

Research approaches should include:

• Single-cell analysis of STUB1 expression in tumor microenvironments

• Investigation of STUB1's role in both tumor cells and infiltrating immune cells

• Clinical correlation studies between STUB1 expression and immunotherapy response

How does STUB1 expression in testicular tissue relate to reproductive function?

STUB1 plays important roles in male reproductive biology:

• In normal human testes, STUB1 is expressed abundantly in pachytene spermatocytes and Sertoli cells, and weakly in spermatogonia and differentiating spermatids

• Sertoli-specific expression of STUB1 is significantly decreased in human testes with impaired spermatogenesis

• In mouse testis development, STUB1 is expressed exclusively in the nuclei of functionally mature Sertoli cells

• Germ cell-derived IL-1α regulates STUB1 expression through promoting ELK1-mediated transactivation

• Ablation of endogenous STUB1 causes lipid accumulation and senescence in germ cell co-incubated Sertoli cells

These findings suggest STUB1 serves as an important negative feedback signaling mechanism to modulate IL-1α levels in the testis.

What methods can identify novel STUB1 substrates and interaction partners?

Discovering novel STUB1 substrates requires multifaceted approaches:

Proteomic Approaches:

• Immunoprecipitation coupled with mass spectrometry to identify STUB1-associated proteins

• Comparative proteomics between wild-type and STUB1-knockout cells to identify proteins with altered stability

• Ubiquitinome analysis to identify differentially ubiquitinated proteins in STUB1-deficient cells

Functional Validation:

• In vitro ubiquitination assays with candidate substrates

• Protein stability assays (cycloheximide chase) in STUB1-manipulated cells

• Domain mapping to determine interaction interfaces

How can conflicting data on STUB1 function be reconciled across experimental systems?

Researchers face challenges when STUB1 studies yield contradictory results:

• The discrepancy between in vitro enhancement of IFNγ response and lack of in vivo tumor growth inhibition highlights the complexity of STUB1 function

• Different experimental systems (cell lines, primary cultures, animal models) may reveal context-dependent functions

Methodological approaches to address conflicts:

• Systematic comparison of experimental conditions (cell types, expression levels, acute vs. chronic loss)

• Integration of multiple data types (genomics, transcriptomics, proteomics)

• Investigation of compensatory mechanisms that may emerge in different model systems

• Analysis of cell type-specific effects, particularly in heterogeneous tissues

What challenges exist in developing STUB1-targeting therapeutics?

Developing STUB1-targeted therapies faces several hurdles:

• STUB1's broad substrate specificity may lead to unintended effects when inhibited

• The discrepancy between in vitro and in vivo findings needs resolution before clinical translation

• Tissue-specific functions may require targeted delivery approaches

• Optimal patient populations need identification through biomarker development

Research strategies should include:

• Structure-based drug design targeting specific STUB1 domains or interactions

• Development of proteolysis-targeting chimeras (PROTACs) for selective STUB1 degradation

• Combination therapy approaches, particularly with immune checkpoint inhibitors

• Identification of biomarkers to predict response to STUB1-targeting agents