Recombinant Human E3 ubiquitin-protein ligase HUWE1 (HUWE1), partial

What is HUWE1 and what are its key structural components?

HUWE1 (HECT, UBA, and WWE domain containing protein 1) is a large 482-kDa HECT-domain ubiquitin ligase that is evolutionarily conserved in eukaryotes. Structurally, HUWE1 contains four N-terminal armadillo repeat-like domains (ARLD1-4) and a C-terminal HECT ubiquitin ligase domain. The entire protein forms an alpha solenoid-shaped assembly with a central pore decorated with protein interaction modules. The N-terminal ~3,900 amino acids are indispensable for proper ligase function, as demonstrated by comparative activity studies between full-length HUWE1 and isolated HECT domain constructs .

How does HUWE1 function differ from other E3 ubiquitin ligases?

HUWE1 exhibits a unique ubiquitin ligase activity called Ubiquitin-Directed ubiquitin Ligase (UDL), which distinguishes it from other E3 ligases. This activity enables HUWE1 to recognize the local density of ubiquitin chains on targets and rapidly expand the ubiquitin modifications to promote both proteasomal degradation and p97-mediated unfolding of targets. Unlike many other E3 ligases, HUWE1 can mediate the ubiquitination of more than 40 diverse substrates, including key regulatory proteins involved in cell death, cellular stress response, and DNA damage repair .

What are the known substrates of HUWE1 and their biological significance?

HUWE1 targets multiple substrates with diverse biological functions, including:

The wide range of substrates highlights HUWE1's central role in coordinating various cellular processes, particularly in stress responses and cell fate decisions .

How does the three-dimensional structure of HUWE1 contribute to its ligase activity?

The three-dimensional organization of HUWE1 is critical for its ubiquitin ligase function. Cryo-EM structures reveal that HUWE1 forms a ring-shaped assembly with a central pore decorated with protein interaction modules. This architecture allows HUWE1 to engage with diverse substrates through distinct binding domains and peptide interactions with the scaffolding armadillo repeats. The full-length HUWE1 shows significantly higher activity than the isolated HECT domain for ubiquitination of substrates like Mcl1 and DDIT4, demonstrating that the N-terminal region provides critical structural elements for substrate recruitment and optimal catalytic function .

What is the mechanism of HUWE1 auto-inhibition and activation?

HUWE1 undergoes conformational regulation through an intricate mechanism involving dimerization and competing intra- and intermolecular interactions:

• Auto-inhibition: HUWE1 can form an asymmetric auto-inhibited dimer through interactions between the thumb and pointer helices.

• Activation mechanism: Disruption of the dimer interface releases inhibitory restraints on catalytic activity.

• Regulatory segment: A conserved segment (residues 3843-3895) can counteract dimer formation by associating with the dimerization region intramolecularly.

• External regulation: The tumor suppressor p14ARF binds to this segment and may shift the conformational equilibrium toward the inactive state.

This conformational control represents a sophisticated regulatory mechanism that allows HUWE1 activity to be modulated in response to physiological cues .

How do different domains of HUWE1 contribute to substrate specificity?

The substrate specificity of HUWE1 is determined by its modular architecture, with different domains contributing to substrate recognition:

• Armadillo repeat-like domains (ARLD1-4) provide scaffolding functions and are involved in protein-protein interactions.

• The catalytic HECT domain is responsible for the ubiquitin transfer activity.

• Specific regions within the N-terminal 3,900 amino acids contain determinants for substrate recognition and binding.

What are the optimal methods for purifying full-length recombinant HUWE1?

Purification of full-length HUWE1 presents challenges due to its large size (482 kDa). Based on published protocols:

• Expression system: Mammalian expression systems (e.g., Expi293 cells) have been successfully used for the production of functional full-length HUWE1.

• Purification strategy:

  • Affinity chromatography using appropriate tags (e.g., His-tag, FLAG-tag)

  • Size exclusion chromatography to separate properly folded protein from aggregates

  • Ion exchange chromatography for further purification

• Quality control: Assessment of purity using SDS-PAGE and activity using E2 discharge assays and ubiquitination assays with known substrates (e.g., Mcl1, DDIT4).

• Storage: The purified protein should be stored in buffers containing stabilizing agents to prevent aggregation and maintain activity.

Researchers have reported high purity and functional activity of HUWE1 prepared using these methods, as demonstrated by the protein's ability to ubiquitinate substrates in vitro .

What assays are available for measuring HUWE1 enzymatic activity?

Several complementary assays can be employed to assess the enzymatic activity of HUWE1:

• E2 discharge assay: A single-turnover assay that monitors the transfer of ubiquitin from a charged E2 (E2-Ub) to HUWE1. This assay measures the first step in the ubiquitination cascade mediated by HUWE1.

• Substrate ubiquitination assays: These assays use purified components (E1, E2, HUWE1, ubiquitin, and substrate) to monitor the ubiquitination of specific substrates like Mcl1 and DDIT4.

• Auto-ubiquitination assays: These can be used to assess the intrinsic catalytic activity of HUWE1.

• FRET-based assays: These assays utilize fluorescently labeled ubiquitin to monitor ubiquitin transfer in real-time.

• Cellular assays: These include monitoring substrate stability and ubiquitination in cells with modulated HUWE1 expression or activity.

These assays provide complementary information about different aspects of HUWE1 function and can be selected based on the specific research question .

How can the dimerization state of HUWE1 be experimentally determined?

Several biophysical and biochemical techniques can be used to assess the dimerization state of HUWE1:

• Size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS): This technique provides information about the molecular weight of proteins in solution, allowing determination of the oligomeric state. HUWE1 constructs containing the dimerization region show molecular weights approximately twice their calculated monomeric weights.

• Small-angle X-ray scattering (SAXS): SAXS profiles can distinguish between monomeric and dimeric states of HUWE1. Simulated scattering profiles based on crystal structures of dimeric HUWE1 show excellent fit with experimental data for dimeric samples.

• Analytical ultracentrifugation: This provides information about the sedimentation properties of proteins, which depend on their size and shape.

• Co-immunoprecipitation: In cellular contexts, this technique can be used to detect self-association of HUWE1.

These approaches have been successfully used to demonstrate that HUWE1 dimerizes in solution through the crystallographic dimer interface involving the thumb and pointer helices .

What is the evidence linking HUWE1 mutations to intellectual disability disorders?

Multiple lines of evidence connect HUWE1 mutations to intellectual disability:

• Genetic studies: HUWE1 variants have been identified in patients with X-linked intellectual disability, including Juberg-Marsidi and Brooks syndromes.

• Clinical studies: A comprehensive study of 21 patients with HUWE1 variants demonstrated dominant X-linked intellectual disability.

• Molecular mechanisms: Research indicates that HUWE1 mutations lead to increased p53 signaling, which impairs neural differentiation.

• Animal models: Studies in model organisms demonstrate the essential role of HUWE1 in nervous system development and function.

• Cellular studies: "Mini-brains" derived from patient cells have revealed mechanisms by which HUWE1 variants affect neural development.

The high evolutionary conservation of HUWE1 from C. elegans to humans (>90% identity between human and mouse) underscores its fundamental importance in development and neurological function .

How does HUWE1 contribute to tumorigenesis - oncogenic or tumor-suppressive roles?

HUWE1 exhibits context-dependent roles in cancer, functioning as either an oncogene or a tumor suppressor depending on the cellular context and specific substrates involved:

This duality highlights the complexity of HUWE1 function and suggests that therapeutic strategies targeting HUWE1 would need to be carefully tailored to specific cancer types and molecular contexts .

What is the role of HUWE1 in inflammasome activation and inflammatory responses?

Recent research has defined novel roles for HUWE1 in promoting the activation of multiple inflammasomes. Inflammasomes are multiprotein complexes that activate inflammatory caspases and trigger the release of pro-inflammatory cytokines. HUWE1-mediated inflammasome activation has significant implications for:

• Tumor immunology: Inflammasome-mediated immune responses can have multifunctional effects on tumor therapy.

• Inflammatory diseases: HUWE1 may contribute to pathological inflammation in various disease contexts.

• Autoimmune diseases: The role of HUWE1 in regulating inflammatory responses suggests potential involvement in autoimmune pathologies.

The molecular mechanisms by which HUWE1 regulates inflammasome activation likely involve ubiquitination of key inflammasome components or regulators, though detailed understanding of these pathways is still emerging .

How can CRISPR-based approaches be optimized for studying HUWE1 function?

CRISPR-based approaches offer powerful tools for studying HUWE1 function, with several optimization strategies:

• For gene knockout studies:

  • Use multiple guide RNAs targeting different exons to ensure complete loss of function

  • Consider conditional knockout strategies given potential essentiality of HUWE1 in some cell types

  • Validate knockout by Western blot and functional assays

• For gene activation studies:

  • dCas9 synergistic activation mediator (SAM) system has been successfully used to endogenously overexpress full-length HUWE1 in vitro and in glioma orthotopic xenografts

  • Design guide RNAs targeting the HUWE1 promoter region for optimal activation

• For precise gene editing:

  • Base editing or prime editing technologies can be used to introduce specific mutations found in patients with intellectual disability

  • Homology-directed repair can be employed to tag endogenous HUWE1 for localization or affinity purification studies

• For domain-specific analysis:

  • CRISPR-mediated deletion of specific domains can provide insights into domain-specific functions while maintaining expression from the endogenous locus

These approaches enable sophisticated manipulation of HUWE1 in relevant cellular and animal models .

What strategies can resolve contradictory findings about HUWE1's role in cell death and survival?

Resolving contradictions in HUWE1 research requires multifaceted approaches:

• Context-specific analysis:

  • Systematic comparison of HUWE1 function across different cell types and tissues

  • Analysis of HUWE1 activity under different stress conditions (e.g., DNA damage, hypoxia, nutrient deprivation)

  • Consideration of the expression levels of HUWE1 substrates in different contexts

• Substrate-specific effects:

  • Development of substrate-specific interaction blockers to dissect the contribution of individual HUWE1-substrate interactions

  • CRISPR-based approaches to introduce mutations that selectively disrupt specific substrate interactions

• Conformational state considerations:

  • Analysis of HUWE1 dimerization status in different cellular contexts

  • Identification of factors that modulate the conformational equilibrium of HUWE1

  • Development of conformational state-specific antibodies or biosensors

• Integration of multiple experimental systems:

  • Combination of in vitro biochemical assays, cellular studies, and animal models

  • Use of patient-derived cells to validate findings in disease-relevant contexts

These strategies can help reconcile seemingly contradictory findings by revealing how HUWE1 function is modulated by cellular context and molecular interactions .

How might the UDL activity of HUWE1 be exploited for targeting protein aggregates in neurodegenerative diseases?

The recently discovered Ubiquitin-Directed ubiquitin Ligase (UDL) activity of HUWE1, which targets the protein to both soluble factors and protein aggregates, presents intriguing therapeutic possibilities for neurodegenerative diseases:

• Mechanistic potential:

  • HUWE1's UDL activity recognizes local density of ubiquitin chains on targets

  • It rapidly expands ubiquitin modifications to promote proteasomal degradation and p97-mediated unfolding

  • This activity could potentially be directed toward disease-associated protein aggregates

• Therapeutic strategies:

  • Development of small molecules that enhance HUWE1's UDL activity

  • Engineering of HUWE1 variants with increased specificity for disease-relevant aggregates

  • Combination approaches targeting both HUWE1 and proteasome or autophagy pathways

• Experimental approaches:

  • Testing HUWE1 activity against disease-relevant protein aggregates (e.g., amyloid-β, tau, α-synuclein)

  • In vivo models expressing modulated HUWE1 in neurodegenerative disease backgrounds

  • Structure-based design of HUWE1 activators specific to the UDL mechanism

• Potential challenges to address:

  • Ensuring specificity for pathological aggregates versus normal proteins

  • Developing delivery methods for CNS-targeted therapies

  • Balancing HUWE1 activity to avoid off-target effects

This area represents a frontier in HUWE1 research with significant therapeutic potential .

What are the key considerations when designing substrate specificity studies for HUWE1?

Designing robust substrate specificity studies for HUWE1 requires careful consideration of several factors:

• Substrate selection and preparation:

  • Include both known and predicted substrates

  • Use full-length substrates as well as domain fragments

  • Consider post-translational modification status of substrates

• Experimental system:

  • In vitro reconstitution with purified components

  • Cell-based assays with endogenous or overexpressed HUWE1

  • In vivo models for physiological relevance

• Controls and validation:

  • Catalytically inactive HUWE1 (C4341S mutant)

  • HUWE1 knockdown/knockout validation

  • Competition assays with known substrates

• Analysis of ubiquitination:

  • Detection of mono- versus poly-ubiquitination

  • Determination of ubiquitin chain types (K48, K63, etc.)

  • Mapping of ubiquitination sites by mass spectrometry

• Functional consequences:

  • Assessment of substrate stability/degradation rates

  • Analysis of changes in substrate activity or localization

  • Physiological outcomes of substrate regulation

These considerations help ensure rigorous characterization of HUWE1-substrate relationships and their biological significance .

How can researchers effectively navigate the challenges of studying HUWE1 conformational regulation?

Studying HUWE1 conformational regulation presents unique challenges that can be addressed through complementary approaches:

These multidisciplinary approaches can provide comprehensive insights into the complex conformational dynamics that regulate HUWE1 activity .

What are the best approaches for integrating in vitro and cellular data when studying HUWE1 function?

Integrating in vitro biochemical data with cellular observations is essential for comprehensive understanding of HUWE1 function:

• Correlation strategies:

  • Parallel analysis of HUWE1 activity in vitro and in cells

  • Structure-function studies with the same HUWE1 variants in both contexts

  • Quantitative correlation of biochemical parameters with cellular phenotypes

• Validation approaches:

  • Confirming in vitro substrate relationships in cellular contexts

  • Testing whether mutations that affect in vitro activity show corresponding cellular phenotypes

  • Using cellular assays to validate mechanistic insights from biochemical studies

• Bridge experiments:

  • Semi-in vitro assays using cell lysates

  • Reconstitution of purified HUWE1 into permeabilized cells

  • Cellular assays with increasing levels of biochemical definition

• Computational integration:

  • Mathematical modeling to connect biochemical parameters with cellular outcomes

  • Network analysis incorporating HUWE1 and its substrates

  • Simulation of how biochemical properties translate to cellular phenotypes

• Translational relevance:

  • Testing whether disease-associated HUWE1 variants show altered biochemical properties

  • Correlating biochemical defects with cellular and organismal phenotypes

This integrated approach ensures that mechanistic insights from biochemical studies are physiologically relevant and that cellular observations have a solid mechanistic foundation .

What are the most significant recent advances in HUWE1 research methodologies?

Recent methodological advances that have significantly impacted HUWE1 research include:

These methodological advances have dramatically expanded our understanding of HUWE1 structure, function, and role in disease .

What databases and bioinformatic tools are most valuable for HUWE1 researchers?

Researchers studying HUWE1 can benefit from several specialized databases and bioinformatic tools:

• E3 ligase-specific resources:

  • Classified, annotated, and updated database of E3 ubiquitin ligase-substrate interactions

  • E3NET for E3-substrate interaction networks

  • UbiBrowser for predicted and experimentally verified ubiquitination sites

• Structural databases and tools:

  • Protein Data Bank (PDB) for HUWE1 structural data

  • SWISS-MODEL for homology modeling of HUWE1 domains

  • ConSurf Server for evolutionary conservation analysis

• Mutation and disease databases:

  • HUWE1.org for research updates and patient information

  • ClinVar for clinical variants in HUWE1

  • DECIPHER for genomic variants in developmental disorders

• Gene expression and regulation:

  • GTEx for tissue-specific expression patterns

  • ENCODE for transcriptional regulation data

  • The Cancer Genome Atlas (TCGA) for cancer-related alterations

• Analysis pipelines:

  • UbiSite for prediction of ubiquitination sites

  • PUPPCHI for prediction of protein-protein interactions

  • Molecular dynamics simulation packages for studying HUWE1 conformational dynamics

These resources help researchers integrate diverse data types and generate testable hypotheses about HUWE1 function .

How can researchers troubleshoot common problems in HUWE1 expression and purification?

Common challenges in HUWE1 expression and purification can be addressed through systematic troubleshooting:

• Low expression yields:

  • Optimize codon usage for expression system

  • Test different expression vectors and promoters

  • Consider expression of stable domains or fragments

  • Use chaperone co-expression to improve folding

• Protein aggregation:

  • Modify buffer conditions (pH, salt concentration, additives)

  • Include stabilizing agents such as glycerol or specific detergents

  • Reduce expression temperature to slow folding

  • Consider on-column refolding strategies

• Loss of activity:

  • Minimize freeze-thaw cycles

  • Include protease inhibitors throughout purification

  • Test activity immediately after purification

  • Optimize storage conditions (buffer composition, temperature)

• Proteolytic degradation:

  • Use protease-deficient expression strains

  • Include multiple protease inhibitors

  • Reduce purification time with optimized protocols

  • Consider engineering more stable variants

• Conformational heterogeneity:

  • Use size exclusion chromatography to separate conformational states

  • Include ligands or substrates that stabilize specific conformations

  • Consider mild crosslinking to capture native states

These strategies have been successfully applied to overcome challenges in working with this complex 482-kDa enzyme .