GID8 Human

What is GID8 and what is its role in the human GID complex?

GID8 (also known as Twa1) is a central scaffold protein in the human GID (hGID) E3 ubiquitin ligase complex. It is a single, non-glycosylated polypeptide chain containing 251 amino acids (1-228 a.a. plus a 23 amino acid His-tag at N-terminus) with a molecular mass of 29.1kDa . GID8 functions as an essential structural component that bridges interactions between different modules of the complex, particularly connecting the catalytic RING-containing subunits with substrate recognition components .

How should GID8 recombinant protein be handled in laboratory settings?

For researchers working with recombinant GID8 protein, proper handling and storage are critical for maintaining functionality. The protein should be stored at 4°C if the entire vial will be used within 2-4 weeks. For longer storage periods, it should be kept frozen at -20°C .

To prevent protein degradation during long-term storage, it is recommended to add a carrier protein (0.1% HSA or BSA) . This helps maintain protein stability and activity. Multiple freeze-thaw cycles should be avoided as they can lead to protein denaturation and loss of function . When working with GID8 for biochemical assays or structural studies, proper buffer conditions should be maintained to ensure native conformation.

How does GID8 contribute to substrate recognition in the GID complex?

GID8 plays an indirect but crucial role in substrate recognition by helping to position the substrate recognition modules within the complex. The human GID complex employs two distinct modules for substrate recruitment: one dependent on GID4 and another on WDR26 .

GID8 serves as a central scaffold that helps organize these independent modules within the functional complex. Unlike direct substrate-binding components, GID8 maintains the spatial architecture that allows the complex to efficiently recognize and ubiquitinate its targets. The GID4-dependent module recognizes substrates like ZMYND19 (which interestingly lacks the Pro/N-end rule degron found in yeast GID4 substrates), while the WDR26-dependent module works with RanBP9 to ubiquitinate substrates like HBP1 .

What experimental approaches can be used to study GID8 interactions within the GID complex?

Several methodological approaches are effective for investigating GID8's interactions within the GID complex:

How are GID8 levels regulated in different cellular contexts?

While direct information on GID8 regulation is limited in the search results, inference can be made based on related findings. The GID complex is implicated in diverse biological processes including glucose metabolism and cell cycle progression , suggesting that GID8 levels may be regulated in response to metabolic cues and cell cycle phases.

The GID complex components show varying expression patterns across tissues. For instance, RanBP9 (another GID complex member) is ubiquitously expressed, with knockout mice showing severe phenotypes including post-natal lethality and impaired gametogenesis . This suggests that GID8, as part of this complex, may be subject to tissue-specific regulation.

Researchers investigating GID8 regulation should consider analyzing its expression across different tissues, cell types, and metabolic conditions. Examination of its promoter regions for transcription factor binding sites and analysis of potential post-translational modifications would provide insights into regulatory mechanisms.

How does the human GID complex differ from its yeast counterpart, and what implications does this have for GID8 function?

The human and yeast GID complexes show both conservation and divergence, with several implications for GID8 function:

These differences highlight the evolutionary adaptation of GID8 to support expanded functionality in higher organisms.

How do different ARMC8 isoforms influence GID8 incorporation and function in the GID complex?

Mammalian cells express two main ARMC8 isoforms—ARMC8α (residues 1-673) and ARMC8β (residues 1-385)—that differentially impact GID complex assembly and function, with implications for GID8 :

• Structural Differences: ARMC8β lacks the conserved C-terminal domain present in ARMC8α, which in yeast Gid5 is implicated in Gid4 binding .

• GID4 Recruitment: Immunoprecipitation assays reveal that ARMC8α readily co-purifies with GID4 complexes, while ARMC8β fails to interact with human GID4 in vivo . This suggests that ARMC8α, but not ARMC8β, can recruit GID4 to the complex.

• Complex Assembly: Both ARMC8α and ARMC8β can integrate into the GID complex and co-immunoprecipitate with WDR26, but GID4 is only present in ARMC8α-containing complexes .

• Functional Consequences: ARMC8β-containing hGID complexes show a prominent reduction in GID4-dependent ubiquitination activity compared to ARMC8α controls .

This differential interaction pattern affects how GID8 participates in complex assembly, as GID8 must interact with different ARMC8-containing modules depending on which isoform is present. Researchers studying GID8 function should consider which ARMC8 isoform predominates in their experimental system.

What methodological approaches can be used to study GID8-dependent ubiquitination in vitro?

Studying GID8-dependent ubiquitination requires specialized biochemical techniques:

• Reconstitution of the GID Complex:

  • Expression and purification of recombinant GID8 and other GID complex components

  • Assembly of complete or partial GID complexes with defined subunit composition

  • Specific inclusion or exclusion of GID8 to determine its contribution

• In Vitro Ubiquitination Assays:

  • Setting up reactions with purified E1, E2, the GID E3 complex (with or without GID8), ubiquitin, ATP, and substrate proteins

  • Time-course analysis of substrate ubiquitination

  • Western blotting or mass spectrometry to detect ubiquitinated products

• Substrate Identification and Validation:

  • Proteomic approaches such as affinity purification-mass spectrometry in the presence of proteasome inhibitors like MG132 to trap ubiquitinated substrates

  • SAINT-scoring to identify high-confidence candidate interacting proteins

  • Validation of substrate interactions through co-immunoprecipitation

• Structural Analysis:

  • Crosslinking-mass spectrometry to map interaction interfaces

  • Cryo-electron microscopy to determine structural organization

• Enzyme Kinetics:

  • Determination of kinetic parameters for GID8-containing versus GID8-depleted complexes

  • Analysis of how GID8 affects substrate binding and catalytic efficiency

The search results specifically mention reconstituting human GID complexes with different isoforms of ARMC8 to test their effects on GID4 incorporation and ubiquitination activity , providing a template for similar approaches with GID8 variants.

How does GID8 contribute to the regulation of cell cycle progression through the GID/CTLH complex?

GID8 contributes to cell cycle regulation through its role in maintaining the structural and functional integrity of the GID/CTLH E3 ubiquitin ligase complex:

• Target-Specific Regulation: The GID/CTLH complex prevents cell cycle exit in G1 phase by degrading the transcription factor Hbp1 . While GID8 doesn't directly bind Hbp1, its scaffold function is critical for the complex to recognize and ubiquitinate this substrate.

• Substrate Recognition Framework: GID8 helps position the substrate recognition modules within the complex, particularly the WDR26-dependent module responsible for targeting Hbp1 .

• Complex Assembly: As a central scaffold, GID8 ensures proper assembly of the GID/CTLH complex. Disruption of GID8 would likely impair complex formation and function, thereby affecting its ability to regulate cell cycle-related substrates.

• Pathway Integration: AP-MS experiments revealed that the GID/CTLH complex interacts with multiple proteins involved in various cellular processes , suggesting that GID8 contributes to integrating different signaling pathways.

Interestingly, unlike the autoregulated hGID subunit YPEL5 (whose levels change in MAEA-knockout cells), the gluconeogenic enzyme FBP1 levels remained unchanged in cells lacking GID activity . This suggests a preferential role for the human GID complex in cell cycle regulation rather than gluconeogenic enzyme degradation.

What structural features of GID8 are critical for its function in organizing the two independent substrate recruitment modules?

The human GID E3 ligase complex employs two distinct modules for substrate recruitment: one dependent on GID4 and another on WDR26 . GID8's structural features are crucial for organizing these modules:

Structural studies have shown that the central position of GID8 is crucial for maintaining the architecture that allows the complex to engage different types of substrates through its distinct recognition modules.

How might targeted disruption of GID8 be exploited for therapeutic applications in diseases involving GID complex dysregulation?

While the search results don't directly address therapeutic applications targeting GID8, we can make evidence-based inferences from the complex's biological functions:

• Cell Cycle Regulation: The GID/CTLH complex prevents cell cycle exit in G1 phase by degrading the transcription factor Hbp1 . In cancers characterized by uncontrolled proliferation, targeting GID8 to disrupt this activity could potentially restore normal cell cycle control.

• Substrate Specificity: GID8's role in organizing the two substrate recognition modules suggests that selective targeting of GID8 domains could disrupt specific substrate interactions while preserving others, potentially allowing for precise modulation of GID complex function.

• Complex Assembly: As a scaffold protein, GID8 is critical for proper assembly of the GID complex. Small molecules that interfere with key GID8 interaction interfaces could destabilize the complex and inhibit its E3 ligase activity.

• Tissue-Specific Functions: The GID complex components show varying expression across tissues, with knockout of the GID component RanBP9 affecting development and gametogenesis . This suggests potential applications in reproductive medicine or developmental disorders.

• Metabolic Regulation: Although human GID appears less involved in regulating gluconeogenic enzymes compared to yeast , its potential role in other metabolic pathways might be relevant for metabolic disorders.

Methodologically, researchers investigating therapeutic applications should consider:

• Structure-guided design of small molecules targeting specific GID8 interfaces

• Peptide inhibitors mimicking key interaction regions

• PROTAC approaches to selectively degrade GID8

• Cell-based screening assays measuring GID complex-dependent substrate degradation

What is the relationship between GID8 and the non-canonical substrate ZMYND19?

ZMYND19 represents an interesting non-canonical substrate of the GID complex with important implications for understanding GID8 function:

• Non-canonical Recognition: Unlike yeast GID substrates that harbor N-terminal proline residues (Pro/N-end rule degron), ZMYND19 surprisingly lacks this motif yet is still recognized by the GID4 module of the human GID complex . This suggests an evolved substrate recognition mechanism in humans.

• Stable Interaction: ZMYND19 was identified as a protein that stably interacts with the GID complex irrespective of proteasome inhibitor (MG132) treatment, distinguishing it from typical substrates whose interactions are often transient and enriched upon proteasome inhibition .

• GID4-Dependent Recognition: GID4 functions as an adaptor for ZMYND19, making this substrate dependent on the GID4 module rather than the WDR26 module . This contrasts with HBP1, which is recognized by the WDR26/RanBP9 module.

• GID8's Structural Role: While GID8 doesn't directly bind ZMYND19, its scaffold function is essential for proper positioning of GID4 within the complex, which in turn recognizes ZMYND19. The GID4-dependent mechanism requires ARMC8α for recruitment to the complex , and GID8 facilitates this interaction.

• Regulatory Implications: The stable interaction between ZMYND19 and the GID complex suggests a possible regulatory relationship beyond simple substrate-enzyme interaction, potentially involving feedback mechanisms or cofactor functions.

ZMYND19's non-canonical recognition highlights the versatility of the human GID complex and underscores GID8's importance in facilitating diverse substrate recognition mechanisms.