Recombinant Chicken STIP1 homology and U box-containing protein 1 (STUB1)

What is STUB1 and what are its key structural domains?

STUB1 (STIP1 homology and U-box containing protein 1), also known as CHIP (Carboxyl Terminus of Hsp70-interacting Protein), is a highly conserved cytoplasmic protein of approximately 34.8 kDa that functions as a U-box ubiquitin ligase (E3). The protein consists of three key domains: an N-terminal tetratricopeptide repeat (TPR) domain (amino acids 26-127) responsible for protein-protein interactions, a central charged domain facilitating TPR-dependent interactions, and a C-terminal U-box domain (amino acids 226-300) that mediates the protein's E3 ubiquitin ligase activity . STUB1 forms dimers in its functional state, which is essential for its role in protein quality control systems .

What are the primary cellular functions of STUB1?

STUB1 participates in critical cellular processes including intracellular protein folding/refolding and degradation. The protein functions at the interface between the chaperone and ubiquitin-proteasome systems. Through its TPR domains, STUB1 complexes with several molecular chaperone proteins, including HSP70/HSPA1A, HSC70, and HSP90, modulating their activity . Simultaneously, through its U-box domain, STUB1 facilitates the ubiquitination of chaperone substrates, including nascent CFTR, phosphorylated Tau, p53, PTEN, Synuclein-alpha, and beta-APP, thereby promoting their degradation through the proteasome . This dual functionality allows STUB1 to tilt the folding-refolding machinery toward the degradative pathway, serving as a critical link between these two systems .

How is STUB1 expression distributed across tissue types?

STUB1 demonstrates a tissue-specific expression pattern that correlates with its functional roles. It is highly expressed in tissues with high metabolic activity and protein turnover rates, particularly in striated muscle and brain tissue . Lower expression levels have been observed in other organs including the pancreas, lung, liver, and kidney . This distribution pattern aligns with STUB1's role in protein quality control, as these high-metabolic tissues require robust mechanisms to maintain protein homeostasis and manage proteotoxic stress .

What are the optimal expression systems for producing recombinant STUB1 protein?

The E. coli expression system has been widely validated as an efficient platform for recombinant STUB1 production, allowing for high yields of functional protein suitable for structural and biochemical analysis . When expressing recombinant human CHIP protein in E. coli, conventional chromatography techniques are typically employed for purification, resulting in protein preparations with >90% purity as assessed by SDS-PAGE . Researchers should consider including a C-terminal 6-His tag to facilitate purification, though care must be taken to verify that the tag does not interfere with the protein's functional properties, particularly its ability to dimerize and interact with chaperone proteins . For experiments requiring mammalian post-translational modifications, alternative expression systems may be warranted, though these are less commonly reported in the literature.

What methodologies are effective for assessing STUB1 structural integrity?

Multiple complementary techniques have proven valuable for characterizing STUB1 structural properties. Limited proteolysis can be employed to evaluate protein stability and domain organization, while size-exclusion chromatography (SEC) effectively assesses STUB1's oligomeric state, particularly its dimerization capacity . Circular dichroism (CD) spectroscopy provides insights into the protein's secondary structure content, specifically the proportion of α-helical structures which is critical for STUB1 function . For more detailed structural analysis, X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy have been utilized by specialized laboratories, though these approaches require significant expertise and specialized equipment. Researchers should select methods based on the specific structural questions being addressed and available resources.

How can STUB1's ubiquitin ligase activity be assessed experimentally?

STUB1's E3 ubiquitin ligase activity can be evaluated through in vitro ubiquitination assays using purified components including E1 activating enzyme, E2 conjugating enzyme, ubiquitin, ATP, and a suitable substrate protein. The reaction products are typically analyzed by SDS-PAGE followed by Western blotting using antibodies against the substrate and/or ubiquitin . For cellular assessments, co-immunoprecipitation experiments can detect interactions between STUB1 and potential substrate proteins, followed by ubiquitination analysis. Specific substrates like GLUD1 have been established as model systems for investigating STUB1-mediated ubiquitination . When designing these experiments, researchers should include appropriate controls, including STUB1 variants with mutations in the U-box domain that abolish E3 ligase activity.

How do pathogenic STUB1 mutations impact protein structure and function?

Pathogenic STUB1 mutations associated with SCAR16 (Spinocerebellar ataxia, autosomal recessive 16) demonstrate distinct impacts on protein structure and function. Detailed characterization of six variants (E28K, N65S, K145Q, M211I, S236T, and T246M) has revealed variant-specific effects . The N65S variant shows increased CHIP dimerization, higher α-helical content, and decreased degradation rate compared to wild-type CHIP. In contrast, T246M demonstrates a strong tendency for aggregation, more flexible protein structure, decreased α-helical content, and increased degradation rate . E28K, K145Q, M211I, and S236T variants also show structural defects, though less profound than N65S and T246M. These structural alterations may impair STUB1's E3 ubiquitin ligase properties and/or interaction with chaperones, contributing to disease pathogenesis through compromised protein quality control systems .

What methodologies are most informative for characterizing novel STUB1 variants?

Comprehensive characterization of novel STUB1 variants requires a multi-faceted approach. Site-directed mutagenesis using systems such as QuikChange XL can efficiently generate mutant constructs in appropriate expression vectors . Recombinant protein expression in E. coli followed by purification enables structural and functional analyses. Limited proteolysis, size-exclusion chromatography, and circular dichroism spectroscopy provide valuable insights into protein stability, oligomerization state, and secondary structure . Functional assays examining interactions with chaperones (using co-immunoprecipitation or surface plasmon resonance) and assessment of E3 ubiquitin ligase activity are critical for establishing pathogenicity. In cellular models, examining the degradation rates of known STUB1 substrates can reveal functional consequences of variants. For novel variants of unknown significance, computational prediction tools can provide preliminary assessments, but experimental validation remains essential.

How is STUB1 involved in cancer pathways and what experimental approaches can elucidate these mechanisms?

STUB1 participates in cancer pathways through its ubiquitin-mediated regulation of oncogenic and tumor suppressor proteins. In lung adenocarcinoma, STUB1 has been identified as the key E3 ligase responsible for ubiquitin-mediated proteasomal degradation of GLUD1 (Glutamate dehydrogenase 1), a key enzyme in glutamine metabolism that supports cancer cell proliferation . The STUB1-GLUD1 interaction can be verified through co-immunoprecipitation experiments, while the specific ubiquitination site (K503) on GLUD1 was identified through detailed molecular analyses . STUB1 has also been implicated in the regulation of other cancer-related proteins, including TLR, EGFR, HER2, and other receptor proteins significantly related to cancer development . Experimental approaches to investigate these mechanisms include gene knockdown/overexpression studies, identification of ubiquitination sites through mass spectrometry, assessment of protein stability through cycloheximide chase assays, and functional studies examining cancer cell proliferation, migration, and tumor growth in both cellular and animal models.

How can protein-protein interactions between STUB1 and its partners be effectively mapped?

Mapping STUB1's protein-protein interaction network requires multiple complementary approaches. Co-immunoprecipitation (co-IP) experiments provide a foundational method to verify direct interactions between STUB1 and candidate partners, as demonstrated with its interaction with GLUD1 . For more systematic identification of interacting partners, proximity-dependent biotin identification (BioID) or affinity purification coupled with mass spectrometry offers broader screening capabilities. Yeast two-hybrid systems have also been utilized, though they may miss interactions dependent on post-translational modifications. To map specific interaction domains, truncation mutants and site-directed mutagenesis can identify critical residues mediating these interactions. Surface plasmon resonance and isothermal titration calorimetry provide quantitative binding parameters including affinity constants and thermodynamic properties. For structural characterization of complexes, X-ray crystallography, cryo-electron microscopy, or NMR spectroscopy may be employed, though these require specialized expertise and equipment.

What advanced techniques can characterize the dynamics of STUB1-mediated ubiquitination in living cells?

Modern cellular approaches have enhanced our understanding of STUB1-mediated ubiquitination dynamics. Proximity ligation assays (PLA) can visualize STUB1-substrate interactions within intact cells with high spatial resolution. For real-time monitoring of ubiquitination, fluorescence resonance energy transfer (FRET) between tagged ubiquitin and substrate proteins provides temporal resolution of modification events. Tandem ubiquitin binding entities (TUBEs) coupled with immunoprecipitation can effectively capture and preserve ubiquitinated proteins for subsequent analysis. Mass spectrometry-based proteomics using diGly remnant antibodies allows identification of specific ubiquitination sites on substrate proteins. CRISPR-Cas9 genome editing can generate endogenous tagged versions of STUB1 or its substrates, maintaining physiological expression levels. For assessing the impact of ubiquitination on substrate fate, fluorescence recovery after photobleaching (FRAP) or pulse-chase experiments using photoactivatable fluorescent proteins can track protein turnover rates with high temporal resolution.

How can STUB1's role in protein quality control be studied in disease models?

Investigating STUB1's function in disease contexts requires specialized model systems. Patient-derived induced pluripotent stem cells (iPSCs) differentiated into relevant cell types (e.g., neurons for SCAR16 studies) provide physiologically relevant models with disease-causing mutations. CRISPR-Cas9 gene editing can introduce specific STUB1 mutations into cellular or animal models to recapitulate disease phenotypes. For neurodegenerative conditions associated with STUB1 dysfunction, specialized assays examining protein aggregation, axonal transport, and synaptic function are valuable. Live-cell imaging using fluorescently tagged proteins can monitor protein aggregation dynamics and clearance mechanisms. Proteomic approaches including SILAC (stable isotope labeling with amino acids in cell culture) coupled with mass spectrometry can comprehensively assess changes in the ubiquitinated proteome upon STUB1 mutation or knockdown. For in vivo studies, conditional knockout mouse models using tissue-specific promoters allow examination of STUB1 function in specific tissues while avoiding embryonic lethality that may result from complete knockout.

How should researchers interpret contradictory findings regarding STUB1 function across different experimental systems?

When encountering contradictory results regarding STUB1 function, researchers should systematically evaluate several key factors. First, consider the experimental system—STUB1's effects may vary between in vitro biochemical assays, cell culture models, and in vivo systems due to differences in available cofactors, post-translational modifications, and cellular context. Second, evaluate the specific isoform or construct used, as truncations or tag positions may alter protein function. Third, assess expression levels, as physiological versus overexpression conditions can yield different outcomes due to stoichiometric relationships with binding partners. Fourth, consider cell-type specificity, as STUB1 may have tissue-specific functions based on the expression profile of chaperones and substrates . Finally, examine the analytical methods employed, as different assays have varying sensitivities and limitations. When publishing, researchers should explicitly report these experimental parameters and discuss how methodological differences might explain discrepancies with published literature.

What statistical approaches are most appropriate for analyzing STUB1 structural variant data?

Analyzing structural variant data for STUB1 requires tailored statistical approaches. For comparing biophysical properties (e.g., thermal stability, oligomerization state) between wild-type and variant proteins, parametric tests like Student's t-test or ANOVA with appropriate post-hoc tests are suitable for normally distributed data, while non-parametric alternatives should be employed when normality assumptions are violated. Multiple testing correction (e.g., Bonferroni or false discovery rate) is essential when analyzing multiple variants simultaneously . For structural data like circular dichroism spectra, specialized software packages that decompose spectra into secondary structure elements should be used with appropriate reference datasets. When correlating structural alterations with functional outcomes, regression analyses or machine learning approaches may identify patterns across multiple variants. For clinical data associating STUB1 variants with disease phenotypes, larger sample sizes are needed, and methods accounting for genetic background and environmental factors should be considered. Researchers should clearly report all statistical methods, including software packages, significance thresholds, and whether assumptions of tests were verified.

How can researchers integrate multi-omics data to better understand STUB1's role in cellular pathways?

Multi-omics integration provides a comprehensive understanding of STUB1's cellular functions. Researchers can combine proteomics data identifying STUB1 substrates with transcriptomics to determine whether regulation occurs at protein and/or mRNA levels, as demonstrated in studies showing STUB1 affects GLUD1 protein levels without altering mRNA expression . Interactome data from proximity labeling or co-immunoprecipitation experiments can be integrated with structural information to build detailed models of STUB1's protein interaction network. Metabolomics analyses are particularly valuable when studying STUB1's impact on metabolic enzymes like GLUD1, allowing researchers to connect ubiquitination events with downstream metabolic consequences. For data integration, computational approaches including pathway enrichment analysis, network modeling, and machine learning can identify patterns across datasets. Visualization tools that overlay multiple data types on pathway maps facilitate interpretation of complex datasets. When implementing these approaches, researchers should consider the temporal dynamics of different molecular events, as transcriptional, post-translational, and metabolic changes occur on different timescales.

What emerging technologies hold promise for advancing STUB1 research?

Several cutting-edge technologies are poised to transform STUB1 research. AlphaFold and other AI-based structural prediction tools can generate high-confidence structural models of STUB1-substrate complexes, guiding experimental design. Cryo-electron microscopy advancements now enable visualization of dynamic protein complexes in various conformational states, potentially revealing the structural basis of STUB1's interactions with chaperones and substrates. CRISPR-based technologies, including base editing and prime editing, offer precise modification of endogenous STUB1 without double-strand breaks, facilitating the study of specific variants in cellular and animal models. Single-cell multi-omics approaches can reveal cell-type-specific functions of STUB1 in complex tissues like brain, where it has been implicated in neurodegenerative conditions. Improved mass spectrometry techniques with higher sensitivity and temporal resolution will enable better characterization of the dynamics of STUB1-mediated ubiquitination events. Organoid technologies provide opportunities to study STUB1 function in physiologically relevant three-dimensional tissues, particularly valuable for neurological and cancer applications.

How might understanding STUB1 function inform therapeutic strategies for associated diseases?

Insights into STUB1 function offer multiple therapeutic opportunities. For SCAR16 and other conditions involving loss of STUB1 function, gene therapy approaches delivering functional CHIP/STUB1 to affected tissues represent a potential curative strategy. Small molecule modulators that enhance residual STUB1 activity in hypomorphic variants could provide therapeutic benefit while avoiding overexpression toxicity. For cancer contexts where STUB1 substrates like GLUD1 are overexpressed, compounds that stabilize or enhance STUB1-substrate interactions could promote degradation of oncogenic proteins . Proteolysis-targeting chimeras (PROTACs) represent another promising approach, potentially redirecting STUB1's ubiquitin ligase activity toward specific therapeutic targets. Understanding the structural basis of STUB1 dimerization and chaperone interactions could inform the development of compounds that specifically modulate these protein-protein interactions. For neurodegenerative conditions involving protein aggregation, enhancing STUB1's activity might increase clearance of aggregation-prone proteins like tau and alpha-synuclein. As therapeutic strategies advance, careful consideration of tissue-specific effects and potential off-target consequences will be essential.

What are the knowledge gaps in understanding STUB1's evolutionary conservation and species-specific functions?

Despite STUB1's clinical importance, significant knowledge gaps exist regarding its evolutionary aspects. While human STUB1 shares 97% amino acid sequence identity with mouse orthologs, it shows only 67% identity with rat orthologs, suggesting potential species-specific functional differences that remain poorly characterized . The evolutionary conservation of STUB1 across more diverse species, including avian models like chicken, requires further investigation to identify universally conserved functional domains versus species-adapted regions. Additionally, the conservation of STUB1's substrate specificity across species remains largely unexplored—whether the same proteins are targeted for ubiquitination in different organisms is unknown. The co-evolution of STUB1 with its chaperone partners (HSP70/HSC70/HSP90) represents another important research area. Comparative studies of STUB1 function across model organisms could reveal environment-specific adaptations in protein quality control systems. Understanding these evolutionary aspects would not only provide fundamental biological insights but also inform the selection of appropriate model systems for studying STUB1-related human diseases.

What are the optimal storage and handling conditions for recombinant STUB1 protein?

Proper storage and handling are critical for maintaining STUB1 activity. For short-term storage (1-2 weeks), recombinant STUB1 can be maintained at +4°C in appropriate buffer systems . For long-term preservation, aliquoting the protein and storing at -20°C or preferably -70°C is recommended to avoid repeated freeze-thaw cycles that can compromise structural integrity and function . The buffer composition significantly impacts stability—a formulation of 20 mM Tris-HCl (pH 7.5) containing 5 mM DTT and 10% glycerol has been validated for maintaining STUB1 in its native conformation . The inclusion of reducing agents like DTT is particularly important for preserving the functionality of cysteine residues within the protein. When thawing frozen samples, rapid thawing followed by gentle mixing (avoiding vortexing) helps prevent protein denaturation and aggregation. For experimental applications, researchers should verify protein activity after storage using functional assays like chaperone binding or ubiquitination activity tests to ensure the integrity of the recombinant protein preparation.

How can researchers effectively design and validate STUB1 mutations for functional studies?

Designing informative STUB1 mutations requires careful consideration of structural and functional domains. When investigating domain-specific functions, researchers should target conserved residues within the TPR domain (for chaperone interactions), the charged middle domain (for TPR-dependent interactions), or the U-box domain (for E3 ligase activity) . Site-directed mutagenesis using established protocols such as QuikChange XL represents an efficient method for generating these variants . Following mutagenesis, Sanger sequencing is essential to verify the introduction of the desired mutation without additional unintended changes. For functional validation, comparative assays between wild-type and mutant proteins should assess: (1) protein stability through thermal denaturation or limited proteolysis, (2) dimerization capacity via size-exclusion chromatography, (3) secondary structure integrity using circular dichroism, (4) chaperone interactions through co-immunoprecipitation or surface plasmon resonance, and (5) E3 ligase activity via in vitro ubiquitination assays . When possible, mutations should be designed based on naturally occurring variants with known phenotypic consequences, as these provide valuable benchmarks for interpreting experimental results.

What considerations are important when designing experiments to identify novel STUB1 substrates?

Identification of novel STUB1 substrates requires strategic experimental design. Proximity-dependent biotin identification (BioID) or APEX2-based proximity labeling using STUB1 as the bait protein can identify potential substrates based on physical proximity in cellular contexts. Quantitative proteomics comparing protein abundance or ubiquitination levels between wild-type and STUB1-deficient conditions can reveal proteins regulated by STUB1-mediated degradation . When designing these experiments, researchers should include controls with catalytically inactive STUB1 mutants to distinguish between scaffolding and enzymatic functions. Validation of candidate substrates should include: (1) verification of physical interaction with STUB1 through co-immunoprecipitation, (2) demonstration of STUB1-dependent ubiquitination using in vivo and in vitro ubiquitination assays, (3) identification of specific ubiquitination sites through mass spectrometry, and (4) assessment of protein stability dependence on STUB1 through cycloheximide chase experiments . Bioinformatic analysis of candidate substrates may reveal common structural motifs or sequence features that could predict additional STUB1 targets. When reporting new substrates, researchers should clearly delineate the evidence supporting direct versus indirect regulation by STUB1.