RUVBL1 Human

What is the basic structure and function of human RUVBL1?

RUVBL1 is an AAA+ (ATPases Associated with diverse cellular Activities) family protein that shares sequence similarity (~30%) with bacterial RuvB helicase . The protein forms hexameric ring structures and functions primarily in complex with its paralog RUVBL2.

X-ray crystallography and cryo-EM studies have revealed that RUVBL1 contains:

• A core ATPase domain with Walker A and B motifs

• A mobile domain II that extends outward from the hexameric ring

• N-terminal and C-terminal regions involved in protein-protein interactions

Functionally, RUVBL1 participates in multiple cellular processes through:

• Integration into chromatin remodeling complexes (INO80, SWR1/SRCAP, TIP60)

• Formation of the R2TP chaperone complex

• Regulation of transcription through epigenetic mechanisms

• Participating in DNA damage repair pathways

• Facilitating proper protein complex assembly

How does RUVBL1 interact with RUVBL2 to form functional complexes?

RUVBL1 and RUVBL2 form heteromeric complexes containing equimolar amounts of each protein . These complexes typically assemble as hexameric rings where:

• The two proteins alternate within the ring structure

• Two hexameric rings can interact through the DII domains to form dodecameric structures

• ATP binding and hydrolysis regulate conformational changes within the complex

Experimental approaches to study these interactions include:

• Co-expression and co-purification of His-tagged RUVBL1 with untagged RUVBL2

• In vitro reconstitution using purified proteins

• Analytical ultracentrifugation to study oligomerization dynamics

• Cryo-EM to visualize complex formation and structural arrangements

Single-stranded DNA can promote the oligomerization of monomeric RUVBL2, suggesting nucleic acid binding may regulate complex assembly in vivo .

What macromolecular complexes incorporate RUVBL1-RUVBL2?

RUVBL1 and RUVBL2 are integral components of several multi-protein complexes with diverse cellular functions:

These proteins also associate with:

• The large RNA polymerase II holoenzyme

• TATA-binding protein (TBP)

• Certain transcription factors

• Telomerase complex

• Phosphatidylinositol 3-kinase-related protein kinases

This diversity of interactions highlights RUVBL1's role as a central mediator of protein complex assembly and function .

What role does RUVBL1 play in nonsense-mediated mRNA decay (NMD)?

RUVBL1-RUVBL2 ATPase activity is required for nonsense-mediated mRNA decay (NMD) activation, a surveillance pathway that degrades aberrant mRNAs and regulates physiological transcript expression . The mechanism involves direct interaction with DHX34, an RNA helicase regulating NMD initiation:

• Protein-Protein Interaction: DHX34 directly interacts with RUVBL1-RUVBL2 complexes, as demonstrated through:

  • In vitro pull-down assays with purified proteins

  • Immunoprecipitation from cells confirming interaction

• Mechanistic Function: Cryo-EM reveals that DHX34:

  • Induces extensive changes in the N-termini of every RUVBL2 subunit

  • Stabilizes a conformation that prevents nucleotide binding

  • Down-regulates ATP hydrolysis of the RUVBL1-RUVBL2 complex

• Subunit Specificity: Using ATPase-deficient mutants (RUVBL1-E303Q and RUVBL2-E300Q), researchers determined that DHX34 acts exclusively on the RUVBL2 subunits despite conformational changes occurring in both RUVBL1 and RUVBL2 .

These findings suggest a model where DHX34 couples RUVBL1-RUVBL2 ATPase activity to the assembly of factors required to initiate the NMD response, highlighting a specialized role in RNA quality control .

How does RUVBL1 contribute to cancer progression through chromatin remodeling?

RUVBL1 enhances malignant biological characteristics of cancer cells, particularly in uveal melanoma (UVM), through chromatin remodeling mechanisms that alter transcriptional activity :

Research Methodologies and Findings:

• Bioinformatics Analysis:

  • Expression pattern analysis shows RUVBL1 upregulation in UVM

  • RUVBL1 functions as an independent prognostic factor for UVM patients

  • Predicted association with CTNNB1 transcriptional activity through chromatin remodeling

• Functional Experiments:

  • RUVBL1 knockdown in UVM cells demonstrated:

    • Inhibited cell proliferation, invasion, and migration

    • Augmented apoptosis rates

    • Blocked cell cycle progression

• Mechanistic Investigation:

  • Co-immunoprecipitation verified interactions with predicted target genes

  • RUVBL1 increases chromatin remodeling and subsequent transcriptional activity of CTNNB1 (β-catenin)

These findings establish RUVBL1 as a potential therapeutic target in UVM and suggest a general mechanism through which RUVBL1 may contribute to malignancy in other cancers by modulating chromatin accessibility and gene expression .

What is the mechanism by which RUVBL1-RUVBL2 regulates γ-tubulin ring complex (γTuRC) assembly?

RUVBL1-RUVBL2 plays a crucial role in the assembly of the γ-tubulin ring complex (γTuRC), which is essential for microtubule organizing centers such as the centrosome :

Key Experimental Findings:

• Cellular Function:

  • RUVBL1-RUVBL2 controls both assembly and composition of γTuRC in human cells

  • Acts as an "assemblase" to build γTuRC from a minimal set of core subunits in heterologous coexpression systems

• Interaction Dynamics:

  • RUVBL1-RUVBL2 interacts with γTuRC subcomplexes during assembly

  • Importantly, it is not part of fully assembled γTuRC, suggesting a transient assembly factor role

• Functional Validation:

  • Purified, reconstituted γTuRC has nucleation activity

  • Cryo-EM structure confirms that recombinant γTuRC resembles native γTuRC

This work demonstrates RUVBL1-RUVBL2's function as a molecular chaperone/assemblase that facilitates the construction of complex macromolecular machines. Understanding this mechanism opens avenues for detailed mutational studies of both γTuRC-mediated microtubule nucleation and RUVBL assemblase function .

How does RUVBL1 contribute to protein quality control through aggresome formation?

RUVBL1 and RUVBL2 enhance aggresome formation as part of cellular protein quality control mechanisms :

Experimental Approaches and Results:

• Interaction Studies:

  • Tandem affinity purification combined with label-free quantitative mass spectrometry identified both RUVBL1 and RUVBL2 as proteins associated with the aggresome substrate synphilin-1

  • Co-immunoprecipitation confirmed interaction between RUVBL1 and the synphilin-1-derived construct ANK1-CC-ANK2

• Functional Validation:

  • Tests with yeast orthologs (Rvb1 and Rvb2) confirmed involvement in aggresome-like structure formation

  • Demonstrated that RuvbL functions as a general molecular chaperone in protein quality control

  • RuvbL proteins facilitate disaggregation of protein aggregates and amyloids

• Mechanistic Insights:

  • Despite sequence similarity to DNA helicases, experimental evidence shows chaperone-like activity

  • RUVBL1/2 appear to have dual roles in cells: DNA-related functions and protein quality control

  • This explains their presence in multiple protein complexes with diverse functions

This research establishes RUVBL1/2 as important components of cellular proteostasis mechanisms, extending their known functions beyond chromatin remodeling and transcriptional regulation .

What are the functional differences between RUVBL1 and RUVBL2 despite their structural similarity?

Although RUVBL1 and RUVBL2 are homologous proteins that often function together, research reveals significant functional specialization :

Distinguishing Characteristics:

• Enzymatic Activity:

  • RUVBL2 demonstrates several-fold higher ATPase activity than RUVBL1 when expressed separately

  • This suggests different catalytic roles within the complex

• Protein Interactions:

  • RUVBL2 binds RPAP3 and PIH1D1 (subunits of the R2TP complex), functions not shared by RUVBL1

  • Surface charge distribution differences between human RUVBL1 and RUVBL2 likely account for differential regulation and binding partners

• Nucleotide Sensitivity:

  • Crystal structure of RUVBL1-RUVBL2 bound to cordycepin (adenosine derivative) shows the compound interacts with all RUVBL2 subunits but not with RUVBL1

  • This differential sensitivity affects the circadian clock in mammals

• Opposing Functions:

  • RUVBL1 and RUVBL2 can function independently and even antagonistically in certain contexts

  • For example, during hypoxia, methylation of either protein by G9a methyltransferase leads to opposite effects on hypoxia-related gene expression

These functional differences highlight the complex interplay between these proteins despite their structural similarity and frequent co-occurrence in cellular complexes .

What experimental methods are most effective for studying RUVBL1 protein interactions and functions?

Researchers employ multiple complementary approaches to study RUVBL1 interactions and functions:

Protein-Protein Interaction Methods:

• Co-immunoprecipitation:

  • Pull-down of tagged RUVBL1 from cells followed by western blot detection of binding partners

  • Used successfully to confirm interactions with DHX34 and RUVBL2

• In vitro reconstitution:

  • Expression and purification of His-RUVBL1 and RUVBL2 as heteromeric complexes

  • Direct testing of interactions with purified potential partners

• Tandem affinity purification with mass spectrometry:

  • Identification of novel RUVBL1-associated proteins

  • Used to identify RUVBL1 interaction with aggresome substrates

Structural and Functional Analysis:

• Cryo-EM analysis:

  • Visualization of conformational changes induced by binding partners

  • Determination of complex architecture at near-atomic resolution

• Analytical ultracentrifugation:

  • Analysis of oligomerization dynamics and complex formation

  • Study of factors promoting oligomerization (e.g., ssDNA)

• Mutational analysis:

  • Generation of ATPase-dead mutants (e.g., RUVBL1-E303Q, RUVBL2-E300Q)

  • Determination of subunit-specific functions

• Cell-based functional assays:

  • RNAi knockdown to assess phenotypic consequences

  • Proliferation, migration, invasion, apoptosis, and cell cycle analysis

Recommended Methodology Combinations:
For comprehensive analysis of novel RUVBL1 functions, researchers should consider a pipeline that includes:

• Initial identification of interactions through proteomics approaches

• Validation with direct binding assays

• Structural characterization of complexes

• Functional validation in cellular systems with appropriate readouts

This multi-faceted approach has proven most effective for elucidating the diverse and context-dependent functions of RUVBL1 .

How can RUVBL1 be targeted therapeutically in cancer?

Recent research on RUVBL1's role in uveal melanoma suggests potential therapeutic applications :

• Rationale for Targeting:

  • RUVBL1 is upregulated in multiple cancer types including uveal melanoma

  • Acts as an independent prognostic factor

  • Enhances malignant characteristics through chromatin remodeling and transcriptional activation

• Potential Therapeutic Approaches:

  • Small molecule inhibitors targeting ATPase activity

  • Disruption of protein-protein interactions with key partners

  • siRNA or antisense oligonucleotides for expression knockdown

• Research Priorities:

  • Development of specific RUVBL1 inhibitors that don't affect related AAA-ATPases

  • Identification of cancer-specific interactions that could be selectively targeted

  • Understanding potential toxicity given RUVBL1's role in essential cellular processes

Researchers should focus on the pathway connecting RUVBL1 to CTNNB1 activation as a particularly promising target in uveal melanoma .

What is the significance of ATP binding and domain II motion coupling in RUVBL1/2 function?

Structural analysis suggests a mechanism for how ATP binding leads to domain II motion in RUVBL proteins :

• Structural Insights:

  • The crystallographic structure of full-length human RUVBL2 reveals the mobile domain II, responsible for protein-protein interactions and ATPase activity regulation

  • Conserved N-terminal loop histidine residues interact with ATP, potentially mediating domain II motion

• Oligomer Plasticity:

  • Analytical ultracentrifugation and cryo-EM analyses demonstrate that RUVBL2 shows oligomer plasticity

  • This plasticity may reflect different physiological conformations in the cell

  • Single-stranded DNA can promote oligomerization of monomeric RUVBL2

• Research Implications:

  • Understanding this mechanism could lead to rational design of modulators of RUVBL1/2 activity

  • The coupling between ATP binding and domain movement likely explains how these proteins translate chemical energy into mechanical force for chromatin remodeling