Recombinant Mouse Deubiquitinating protein VCIP135 (Vcpip1), partial

What is VCIP135 and what is its primary cellular function?

VCIP135 (valosin-containing protein p97/p47 complex-interacting protein, p135) is a deubiquitinating enzyme that plays a crucial role in the p97/p47-mediated Golgi membrane fusion process. This protein is particularly important during cell division, as it facilitates the reassembly of Golgi membranes during the end of mitosis. The inheritance of the Golgi apparatus into daughter cells during each cycle of cell division is mediated by a precisely controlled disassembly and reassembly process that involves both phosphorylation and ubiquitination . VCIP135 functions within this system by removing ubiquitin modifications at specific stages of the cell cycle to regulate membrane dynamics.

What are the key structural domains of VCIP135?

VCIP135 contains several functional domains that contribute to its activity and interactions:

• N-terminal half (aa 1-740): Contains the enzymatic OTU (ovarian tumor) domain responsible for deubiquitinase activity

• Catalytic cysteine at position 218: Critical for enzymatic function; mutation of C218S abolishes deubiquitinase activity

• VCPID domain (aa 589-666): Forms contacts with D2 domains of VCP

• UBX domain (aa 773-852): Mediates binding to the N-domain of VCP

• Stalk region: Connects the VCPID to the OTU domain

The OTU domain provides VCIP135 with intrinsic linkage specificity for different ubiquitin chain types, while the UBX and VCPID domains mediate binding to VCP in a unique bivalent interaction mode that positions the catalytic domain near the central pore of VCP .

How is VCIP135 regulated during the cell cycle?

VCIP135 activity is tightly regulated throughout the cell cycle, primarily through phosphorylation:

• During early mitosis: VCIP135 is phosphorylated by Cdk1 at serine 130 (S130), which inactivates its deubiquitinase activity and inhibits p97/p47-mediated Golgi membrane fusion .

• At the end of mitosis: VCIP135 is dephosphorylated at S130, which restores its deubiquitinase activity and enables Golgi reassembly .

This phosphorylation-dephosphorylation cycle creates a precise temporal control mechanism that coordinates Golgi disassembly during early mitosis and reassembly at the end of mitosis. The S130 phosphorylation site is evolutionarily conserved from Xenopus to humans, suggesting this regulatory mechanism is fundamental across species .

What is the ubiquitin chain linkage specificity of VCIP135?

VCIP135 exhibits distinct preferences for different ubiquitin chain linkages. Research using endogenously expressed VCIP135 immunoprecipitated from HeLa cells revealed the following specificity profile:

This specificity pattern suggests VCIP135 may regulate diverse substrates with various ubiquitin modifications on Golgi membranes . Interestingly, the full-length VCIP135 shows different specificity compared to studies using only the OTU domain, indicating that other domains in the protein help define its linkage specificity .

How does the VCIP135-VCP interaction occur at the molecular level?

VCIP135 forms a unique bivalent interaction with VCP (valosin-containing protein) with a 2:1 VCP/VCIP135 stoichiometry:

These two interaction sites work synergistically, as demonstrated by binding studies with VCIP135 mutants:

• VCIP135WT and VCIP135ΔVCPID (with intact UBX domain) bind to VCP with comparable affinity

• VCIP135ΔUBX and VCIP135ΔVCPID ΔUBX (lacking UBX domain) show no measurable affinity for VCP

The complex is highly dynamic, with conformational changes occurring in both the N-domain of VCP and throughout VCIP135 .

How do mutations affect VCIP135 function and binding properties?

Several key mutations have been characterized that affect VCIP135 function and binding:

• Catalytic domain mutation:

  • C218S: Abolishes enzymatic activity without affecting protein structure

• Phosphorylation site mutations:

  • S130A (phosphodeficient): Maintains deubiquitinase activity even during mitosis

  • S130E (phosphomimetic): Completely lacks deubiquitinase activity in both interphase and mitosis

• VCP binding mutations:

  • L133S: Disrupts the N-terminal p97-binding region

  • F1024L/L1031P: Disrupts the C-terminal p97-binding region

  • L133S/F1024L/L1031P: Eliminates both binding sites, completely preventing p97 binding

These mutations demonstrate that:

What are the standard methods for measuring VCIP135 deubiquitinase activity?

Researchers employ several techniques to assess VCIP135 deubiquitinase activity:

• Di-ubiquitin cleavage assays:

  • Substrate: Purified di-ubiquitin chains of specific linkage types (K48, K63, etc.)

  • Detection: Western blotting or SDS-PAGE to visualize conversion of di-ubiquitin to mono-ubiquitin

  • Quantification: Densitometry analysis of mono-ubiquitin band intensity

• Fluorogenic substrate assays:

  • Substrate: Ubiquitin-rhodamine 110 (Ub-Rho)

  • Detection: Measurement of fluorescence release upon deubiquitination

  • Analysis: Determination of initial reaction velocities (v0) to assess enzyme activity

• Cell-based activity assays:

  • Immunoprecipitate VCIP135 from cells in different cell cycle stages

  • Test deubiquitinase activity against purified di-ubiquitin substrates

  • Compare activity between wild-type and mutant forms

How can researchers generate and validate VCIP135 mutants for functional studies?

The generation and validation of VCIP135 mutants typically follows this methodological workflow:

• Mutant generation:

  • Site-directed mutagenesis for specific mutations (e.g., S130A, S130E, C218S)

  • Random mutagenesis using PCR with limiting dATP to introduce random mutations

  • Construction of domain deletion mutants (e.g., ΔVCPID, ΔUBX)

• Expression systems:

  • Bacterial expression using GST-tagged or His-tagged constructs

  • Mammalian expression using GFP-tagged constructs

  • Yeast two-hybrid system for interaction screening

• Validation methods:

  • Protein purification via affinity chromatography and sucrose-gradient ultracentrifugation

  • Structural integrity assessment through circular dichroism or thermal shift assays

  • Binding assays using time-resolved fluorescence resonance energy transfer (TR-FRET)

  • Activity assays using di-ubiquitin or fluorogenic substrates

• Screening workflow:

  • Express cDNAs in yeast cells

  • Test binding affinities using yeast two-hybrid system

  • Further characterize promising candidates in mammalian cells

  • Confirm functional effects through in vitro and cell-based assays

What techniques are used to study VCIP135-VCP interactions?

Several complementary approaches are used to characterize VCIP135-VCP interactions:

• Structural techniques:

  • Cryo-electron microscopy (Cryo-EM): Reveals the 3D organization of the complex, including the 2:1 VCP/VCIP135 stoichiometry and bivalent binding mode

  • X-ray crystallography: Provides atomic-level details of specific domain interactions

• Biochemical binding assays:

  • Time-resolved fluorescence resonance energy transfer (TR-FRET): Measures binding affinities (KD values) between purified proteins

  • Pull-down assays: Identifies interacting proteins and domains

  • Yeast two-hybrid assays: Screens for protein-protein interactions

• Nucleotide dependency studies:

  • ATP/ADP/AMP-PNP comparative binding assays

  • Conformational change analysis under different nucleotide conditions

• Mutagenesis approaches:

  • Domain deletion mutants to identify binding regions

  • Point mutations to disrupt specific interactions

  • Combination mutations to eliminate multiple binding sites

How can researchers study the role of VCIP135 in Golgi dynamics?

Studying VCIP135's role in Golgi dynamics typically involves these methodological approaches:

• Cell cycle synchronization techniques:

  • Nocodazole arrest to obtain mitotic cells

  • Release from arrest to study Golgi reassembly

  • Comparison between asynchronous interphase and mitotic cells

• Imaging methods:

  • Immunofluorescence microscopy to visualize Golgi morphology

  • Live-cell imaging to track Golgi dynamics in real-time

  • Electron microscopy for ultrastructural analysis

• Functional perturbation approaches:

  • VCIP135 knockdown using siRNA or shRNA

  • Expression of phosphomutants (S130A, S130E) to manipulate activity

  • Expression of binding-deficient mutants to disrupt VCP interaction

• Biochemical fractionation:

  • Isolation of Golgi membranes at different cell cycle stages

  • Analysis of ubiquitination patterns on Golgi proteins

  • In vitro Golgi reassembly assays

How should researchers interpret contradictory findings about VCIP135 substrate specificity?

When faced with differing results regarding VCIP135 substrate specificity:

• Consider protein context: The full-length VCIP135 shows different specificity compared to the isolated OTU domain, suggesting other domains influence substrate recognition. For example, full-length VCIP135 shows highest activity toward K48- and K63-linked chains, while the OTU domain alone reportedly prefers K11- and K48-linked di-ubiquitin .

• Evaluate experimental conditions: Parameters such as pH, salt concentration, temperature, and presence of cofactors can significantly affect enzyme activity and specificity.

• Assess protein preparation methods: Recombinant protein expression systems, purification tags, and storage conditions can impact protein folding and activity.

• Consider cellular context: VCIP135 may have different specificities in vivo versus in vitro due to post-translational modifications, binding partners, or subcellular localization.

• Implement multiple substrates and techniques: Use both linkage-specific di-ubiquitin chains and fluorogenic substrates to comprehensively characterize activity.

What are common challenges in VCIP135 activity assays and how can they be addressed?

Researchers may encounter several challenges when studying VCIP135 activity:

• Protein instability issues:

  • Challenge: VCIP135 may be prone to aggregation or misfolding

  • Solution: Use sucrose-gradient ultracentrifugation to remove aggregated protein

  • Alternative: Express smaller functional domains instead of full-length protein

• Phosphorylation state heterogeneity:

  • Challenge: Mixed phosphorylation states can complicate activity measurements

  • Solution: Use phosphatase treatment to ensure homogeneous dephosphorylation

  • Alternative: Utilize phosphomimetic (S130E) or phosphodeficient (S130A) mutants

• Cell cycle-dependent activity:

  • Challenge: VCIP135 activity varies across the cell cycle

  • Solution: Carefully synchronize cells and confirm cell cycle stage

  • Alternative: Compare wild-type and phosphomutant proteins under the same conditions

• Assay sensitivity limitations:

  • Challenge: Low signal-to-noise ratio in activity assays

  • Solution: Optimize enzyme concentration and incubation time

  • Alternative: Use more sensitive fluorogenic substrates like Ub-Rho

What factors influence the effectiveness of VCIP135 knockdown experiments?

When designing and interpreting VCIP135 knockdown experiments, researchers should consider:

• Knockdown efficiency variability:

  • Research shows that initial knockdown attempts can be ineffective

  • Recommendation: Always verify knockdown efficiency by Western blot

  • Consideration: Include all data in analysis, even with partial knockdown

• Cell type considerations:

  • Different cell lines may have varying expression levels of VCIP135

  • Some cells may have compensatory mechanisms

  • Cell-type specific binding partners may influence outcomes

• Timing considerations:

  • VCIP135 has cell cycle-specific functions

  • Knockdown effects may vary depending on cell cycle stage

  • Consider synchronized versus asynchronous cell populations

• Knockdown methodology:

  • siRNA versus shRNA versus CRISPR-Cas9 approaches

  • Transient versus stable knockdown

  • Potential for off-target effects

What are the emerging questions about VCIP135's role beyond Golgi dynamics?

While VCIP135's function in Golgi dynamics is well-established, several emerging research areas warrant investigation:

• Potential roles in protein quality control pathways:

  • Given its preference for K48-linked ubiquitin chains (typically associated with proteasomal degradation), VCIP135 may participate in protein quality control mechanisms

  • Investigation of potential substrates beyond Golgi membrane proteins

  • Possible function in ER-associated degradation (ERAD) pathways

• Involvement in diverse cellular stress responses:

  • Connection between cell cycle regulation and cellular stress

  • Potential role in recovering from proteotoxic stress

  • Responses to organelle stress beyond the Golgi apparatus

• Functions related to atypical ubiquitin linkages:

  • VCIP135 shows considerable activity toward K6-, K11-, K29-, and K33-linked ubiquitin chains

  • These atypical linkages have emerging roles in DNA damage, inflammation, and mitophagy

  • Potential function in these pathways remains unexplored

• Tissue-specific functions:

  • Expression patterns and functions across different tissues

  • Role in tissue-specific development or physiological processes

  • Potential contribution to tissue-specific pathologies

How might advanced technologies improve our understanding of VCIP135?

Emerging technologies could significantly advance VCIP135 research:

• Cryo-electron tomography:

  • Visualize VCIP135 in its native cellular environment

  • Map the spatial organization of VCIP135 relative to Golgi membranes

  • Capture conformational changes during the cell cycle

• Proximity labeling proteomics:

  • Identify proximal interaction partners in different cellular contexts

  • Map the dynamic VCIP135 interactome throughout the cell cycle

  • Discover novel substrates using ubiquitination-specific proximity labeling

• Single-molecule biophysics:

  • Measure enzyme kinetics at the single-molecule level

  • Visualize conformational changes during substrate processing

  • Quantify binding/unbinding dynamics with VCP and other partners

• Artificial intelligence for structure prediction:

  • Generate comprehensive structural models of full-length VCIP135

  • Predict conformational changes upon phosphorylation

  • Model substrate recognition mechanisms