VPS24 Human

What is the primary function of human VPS24 protein?

Human VPS24 functions as a core component of the ESCRT-III complex, which is essential for multivesicular body (MVB) formation and sorting of endosomal cargo proteins into MVBs. The protein is involved in membrane invagination and scission processes that generate intraluminal vesicles (ILVs) within MVBs. These processes are crucial for degradation of membrane proteins such as stimulated growth factor receptors, lysosomal enzyme trafficking, and lipid metabolism . VPS24 also participates in topologically equivalent membrane fission events, including cytokinesis and viral budding (particularly HIV-1 and other lentiviruses) . The protein selectively binds to specific phosphoinositides, particularly phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2) and PtdIns(3,4)P2, which helps target it to appropriate membrane domains .

How is VPS24 structurally organized?

VPS24 is a 222-amino acid protein with several functional domains. The full-length protein (referred to as VPS24alpha) contains an N-terminal lipid binding domain essential for membrane association, followed by a core region with multiple alpha-helical segments that mediate protein-protein interactions within the ESCRT-III complex . The C-terminal region contains a MIM (MIT-Interacting Motif) that binds to the AAA-ATPase VPS4, which provides the mechanical force for ESCRT-III disassembly . VPS24 adopts a "semi-open" conformation in polymers, distinct from the fully extended conformations of other ESCRT-III proteins like Snf7 and Did2 . This structural property is likely critical for its specific function within the ESCRT-III assembly sequence.

What are the different isoforms of human VPS24 and how do they function?

The human VPS24 gene produces at least two functionally distinct protein isoforms through alternative splicing:

• VPS24alpha (222 amino acids): The canonical full-length protein that functions within the ESCRT pathway for endosomal sorting and multivesicular body formation. It contains the N-terminal lipid binding domain necessary for membrane association .

• VPS24beta (156 amino acids): A shorter variant that lacks the N-terminal lipid binding domain. Unlike VPS24alpha, this isoform does not participate in the ESCRT pathway but instead serves an anti-apoptotic role. Research has demonstrated that VPS24beta can suppress the lethal effects of the pro-apoptotic protein BAX . Additionally, VPS24beta prevents stress-mediated cell death and accumulation of reactive oxygen species when expressed in yeast cells .

Experimental evidence clearly demonstrates their functional divergence: VPS24alpha, but not VPS24beta, can rescue the temperature and salt sensitivity of yeast lacking a functional VPS24 gene. Conversely, VPS24beta, but not VPS24alpha, suppresses BAX-mediated growth inhibition in yeast .

What expression systems are optimal for producing recombinant VPS24 protein?

For obtaining functional recombinant VPS24 protein, Escherichia coli expression systems have been successfully employed, as demonstrated in commercial preparations with >90% purity suitable for SDS-PAGE and functional studies . When expressing VPS24 in E. coli, using an N-terminal His-tag (typically 6× histidine) facilitates purification through nickel affinity chromatography. The specific tag sequence "MGSSHHHHHH SSGLVPRGSH" has been validated for VPS24 expression .

For purification, a multi-step approach is recommended:

• Initial capture using nickel affinity chromatography

• Secondary purification using ion-exchange chromatography

• Final polishing with size-exclusion chromatography to ensure monodisperse protein preparation

Critical considerations include buffer optimization to maintain VPS24 in its monomeric state, as ESCRT-III proteins tend to polymerize spontaneously. For functional validation, the recombinant protein should be tested for its ability to bind physiological lipid partners (PtdIns(3,5)P2 and PtdIns(3,4)P2) .

How can researchers effectively study VPS24 function through depletion approaches?

For investigating VPS24 function through depletion strategies, several complementary approaches are effective:

• RNA interference (RNAi):

  • siRNA transfection provides transient knockdown (typically 72-96 hours)

  • shRNA expression enables stable long-term knockdown

  • Multiple siRNA/shRNA sequences targeting different regions of VPS24 mRNA should be employed to confirm phenotypes

  • Knockdown efficiency should be validated by qRT-PCR and Western blotting

• CRISPR/Cas9 gene editing:

  • Complete knockout using guide RNAs targeting early exons

  • For studying essential functions, inducible or conditional knockout systems may be necessary

  • Edited clones must be validated by sequencing and protein expression analysis

• Dominant-negative approaches:

  • Overexpression of truncated VPS24 forms that incorporate into ESCRT-III but disrupt function

  • Expression of VPS24 lacking its MIM domain disrupts VPS4 recruitment and function

• Yeast complementation studies:

  • As demonstrated in search result , deletion of the VPS24 homolog in yeast (vps24Δ) followed by complementation with human VPS24 variants provides valuable insights into structure-function relationships

When conducting VPS24 depletion experiments, researchers should consider potential compensatory mechanisms by related ESCRT-III proteins. For instance, search result demonstrates that overexpression of VPS2 can partially compensate for VPS24 deletion in yeast, restoring approximately 40% of cargo sorting function .

What assays are most informative for studying VPS24's role in membrane remodeling?

For studying VPS24's role in membrane remodeling, several complementary assays provide mechanistic insights:

• Cargo sorting assays:

  • In yeast systems, tracking fluorescently tagged cargo proteins (e.g., Mup1-pHluorin) provides a quantitative readout of MVB pathway function

  • Canavanine sensitivity assays in yeast provide a phenotypic assessment of MVB function

  • In mammalian cells, EGFR degradation kinetics following EGF stimulation offers a physiologically relevant cargo sorting assay

• Microscopy-based approaches:

  • Immunofluorescence using antibodies against endogenous VPS24 combined with markers for endosomal compartments

  • Electron microscopy to directly visualize MVB morphology and intraluminal vesicle formation

  • Live-cell imaging with fluorescently tagged VPS24 to monitor dynamics of recruitment

• Reconstitution assays with purified components:

  • Giant unilamellar vesicles (GUVs) with defined lipid compositions including PtdIns(3,5)P2

  • Sequential addition of ESCRT-III components (starting with Snf7/CHMP4, followed by VPS24/CHMP3, VPS2/CHMP2)

  • Direct visualization of membrane deformation and scission events

• Functional complementation studies:

  • Testing VPS24 variants and chimeras (such as those described in search result ) in VPS24-depleted systems

  • Assessing the ability of specific domains to rescue function

These assays should be selected based on the specific aspect of VPS24 function being investigated, from initial membrane recruitment to ESCRT-III assembly and membrane scission.

How does VPS24 cooperate with VPS2 to form a functional module within ESCRT-III?

VPS24 and VPS2 form a critical functional module within the ESCRT-III complex with several distinctive properties:

• Cooperative recruitment:

  • VPS24 and VPS2 require each other for efficient recruitment to membranes

  • Neither protein efficiently localizes to endosomal membranes in the absence of the other

• Sequential assembly in the ESCRT-III pathway:

  • The VPS24-VPS2 module functions downstream of Snf7 (CHMP4) polymerization

  • Together, they induce lateral association and bundling of ESCRT-III filaments, as well as helicity in ESCRT-III spirals

• Distinct structural roles:

  • VPS24 adopts a "semi-open" conformation in polymers, distinct from the fully extended conformations of other ESCRT-III proteins

  • This structural property may be critical for the transition from initial membrane deformation to neck constriction

• VPS4 recruitment:

  • The VPS24-VPS2 module is crucial for recruiting the AAA ATPase VPS4

  • VPS2 possesses a higher affinity VPS4 binding site compared to VPS24

  • This recruitment provides the mechanical force needed for membrane scission and ESCRT-III disassembly

• Partial functional redundancy:

  • Overexpression of VPS2 can partially compensate for VPS24 deletion in yeast, restoring approximately 40% of cargo sorting function

  • An "activated" VPS24 mutant (E114K) carrying the VPS4 binding motif of VPS2 can function in place of VPS2

These findings highlight how the VPS24-VPS2 module represents a specialized functional unit within the ESCRT-III complex, with both proteins contributing distinct properties to membrane remodeling and scission.

What structural transitions does VPS24 undergo during ESCRT-III assembly?

VPS24 undergoes specific structural transitions during ESCRT-III assembly that distinguish it from other complex members:

• Conformational states:

  • Cytosolic VPS24 exists in an auto-inhibited "closed" conformation

  • Upon membrane recruitment, VPS24 transitions to a "semi-open" conformation, distinct from the fully extended conformations adopted by Snf7 (CHMP4) and Did2 (CHMP1)

  • This semi-open state likely represents a specific functional adaptation for VPS24's role in the ESCRT-III assembly sequence

• Critical structural elements:

  • Interactions between helix 2 and helix 3 are particularly important for conformational control

  • The E114K mutation weakens these interactions, creating an "activated" VPS24 that can partially function in place of VPS2 when it also carries VPS4 binding motifs

  • This suggests that regulated conformational opening is essential for proper function

• Domain-specific roles:

  • The N-terminal region (residues ~1-152) contains the core structural elements necessary for ESCRT-III assembly

  • The C-terminal region (beyond residue ~152) can be replaced with that of VPS2 while maintaining function

  • This indicates that the N-terminal domain mediates the critical structural transitions

• Activation mechanisms:

  • Unlike Snf7, which readily forms extended polymers, VPS24 appears to have more tightly regulated conformational activation

  • This likely prevents premature or inappropriate assembly and ensures proper sequential assembly of the ESCRT-III complex

These structural transitions are essential for VPS24's proper function within the ESCRT-III complex and highlight its specialized role in membrane remodeling events.

How does VPS24 contribute to neurodegenerative disease mechanisms?

VPS24's contribution to neurodegenerative disease mechanisms centers on its essential role in protein aggregate clearance:

• Autophagy pathway involvement:

  • Functional multivesicular bodies (MVBs) are required for autophagic clearance of protein aggregates associated with neurodegenerative diseases

  • Depletion of ESCRT subunits, including VPS24, inhibits autophagic degradation, leading to accumulation of ubiquitinated proteins, p62, and Alfy (autophagy-linked FYVE protein)

• Disease-specific protein clearance:

  • MVB function is specifically required for clearance of TDP-43 (TAR DNA-binding protein 43), the major ubiquitinated protein in ALS and frontotemporal lobar degeneration with ubiquitin deposits (FTLD-U)

  • VPS24 is also required for efficient clearance of huntingtin (Htt) polyQ aggregates associated with Huntington's disease

• Relationship to known disease mutations:

  • Mutations in the related ESCRT-III subunit CHMP2B (charged multivesicular body protein 2B) are associated with frontotemporal dementia and amyotrophic lateral sclerosis

  • Given that VPS24 (CHMP3) and CHMP2B function in the same complex, VPS24 dysfunction likely contributes to similar pathogenic mechanisms

• Mechanistic pathway:

  • Dysfunctional ESCRT-III → Impaired MVB formation → Defective autophagy → Accumulation of toxic protein aggregates → Neurodegeneration

  • This represents a fundamental cellular quality control mechanism that, when disrupted, leads to the characteristic protein aggregation seen in multiple neurodegenerative conditions

This research highlights how VPS24, as an essential component of the ESCRT-III complex, plays a critical role in maintaining neuronal health through efficient clearance of protein aggregates via the autophagy-lysosomal pathway.

What are the unexplored roles of VPS24 beyond the canonical ESCRT pathway?

Several unexpected roles of VPS24 beyond the canonical ESCRT pathway warrant further investigation:

• Anti-apoptotic functions:

  • The VPS24beta isoform (156 amino acids) functions independently of the ESCRT pathway as an anti-apoptotic factor

  • It suppresses the effects of pro-apoptotic BAX in a manner that does not require other VPS proteins

  • The molecular mechanisms through which VPS24beta interfaces with apoptotic machinery remain largely unexplored

• Oxidative stress protection:

  • VPS24beta prevents stress-mediated cell death and accumulation of reactive oxygen species when expressed in yeast cells

  • The mechanisms underlying this protective effect and its relevance to mammalian cells require further investigation

• Growth factor signaling modulation:

  • Beyond its mechanical role in EGFR degradation, potential direct effects of VPS24 on signal transduction pathways remain unexplored

  • The selective binding of VPS24 to specific phosphoinositides may influence signaling platforms beyond endosomal sorting

• Interaction with insulin-like growth factor system:

  • VPS24 has been shown to interact with IGFBP7 (insulin-like growth factor binding protein 7)

  • The functional significance of this interaction and how it relates to VPS24's canonical or non-canonical functions represents an important knowledge gap

• Potential chromatin-associated functions:

  • The alternative name "Chromatin-modifying protein 3" suggests possible nuclear roles

  • Whether VPS24 has direct or indirect effects on chromatin structure or gene expression requires investigation

These unexplored aspects of VPS24 biology highlight the potential complexity of its cellular functions beyond the well-established role in ESCRT-III-mediated membrane remodeling.

How might targeting VPS24 provide therapeutic approaches for neurodegenerative diseases?

Based on current understanding of VPS24 function, several therapeutic approaches could be developed:

• Enhancing autophagic clearance of protein aggregates:

  • Since functional MVBs requiring VPS24 are essential for clearance of disease-associated proteins like TDP-43 and polyQ-expanded huntingtin , enhancing VPS24 function could promote more efficient clearance

  • Potential approaches include small molecules that stabilize or enhance VPS24 assembly into functional ESCRT-III complexes

  • Cell-penetrating peptides derived from VPS24 activation domains could potentially boost function

• Leveraging VPS24beta's anti-apoptotic properties:

  • The anti-apoptotic role of VPS24beta could be exploited to protect neurons from degeneration

  • Approaches might include promoting alternative splicing to favor VPS24beta production or delivering VPS24beta-derived peptides to affected neurons

• Compensatory strategies for ESCRT dysfunction:

  • Research shows that overexpression of VPS2 can partially compensate for VPS24 deletion

  • Similarly, "activated" versions of VPS24 (like the E114K mutant) could potentially bypass requirements for other ESCRT components

  • These insights suggest possible approaches for restoring function when the ESCRT machinery is compromised

• Targeted modulation of VPS24-lipid interactions:

  • VPS24 selectively binds phosphatidylinositol 3,5-bisphosphate and PtdIns(3,4)P2

  • Small molecules that enhance these specific lipid interactions could potentially boost VPS24 recruitment to endosomal membranes

  • This approach might enhance MVB function and subsequent clearance of neurotoxic proteins

When developing these therapeutic approaches, researchers must consider the fundamental nature of ESCRT pathway functions and aim for targeted interventions that enhance function without disrupting essential cellular processes.

What is the significance of VPS24's selective phosphoinositide binding?

VPS24's selective binding to specific phosphoinositides, particularly PtdIns(3,5)P2 and PtdIns(3,4)P2 , has several important implications:

• Spatial regulation of ESCRT-III assembly:

  • The specific phosphoinositide composition of different endosomal membranes helps target VPS24 to appropriate cellular compartments

  • This ensures that ESCRT-III assembly occurs at the correct location for MVB biogenesis

  • The preference for PtdIns(3,5)P2, which is enriched in late endosomal membranes, likely helps coordinate the timing of VPS24 recruitment

• Conformational activation:

  • Interaction with specific phosphoinositides may promote the structural transition of VPS24 from its closed cytosolic conformation to the membrane-bound "semi-open" state

  • This lipid-induced conformational change could be a key regulatory step in ESCRT-III assembly

• Pathological implications:

  • Disorders of phosphoinositide metabolism could indirectly affect VPS24 function through altered membrane recruitment

  • Such disruptions could contribute to neurodegenerative conditions by impairing protein aggregate clearance

• Potential for targeted intervention:

  • The specific phosphoinositide binding properties of VPS24 could be exploited for therapeutic targeting

  • Small molecules that mimic these lipid interactions might enhance VPS24 recruitment and function

• Evolutionary significance:

  • The selective lipid binding likely represents an evolved mechanism to ensure proper spatiotemporal control of ESCRT-III assembly

  • This selectivity distinguishes VPS24 from other ESCRT-III components and contributes to the ordered assembly process

Further research into the structural basis and functional consequences of VPS24's phosphoinositide selectivity will provide important insights into ESCRT-III regulation and potentially reveal new therapeutic approaches.

What strategies can overcome the challenges of studying VPS24 isoforms?

Research on VPS24 isoforms presents several technical challenges that can be addressed through strategic approaches:

• Isoform-specific detection:

  • Develop antibodies targeting the unique N-terminal regions of each isoform

  • For VPS24alpha, target epitopes within the N-terminal lipid binding domain that is absent in VPS24beta

  • For VPS24beta, target the unique junction created by alternative splicing

  • Validate specificity through Western blotting against recombinant proteins of both isoforms

• Expression system considerations:

  • Use isoform-specific expression constructs with appropriate tags that don't interfere with function

  • Consider inducible expression systems to control expression levels and avoid toxicity

  • For studying isoform-specific functions, create cell lines with CRISPR/Cas9 knockout of endogenous VPS24 complemented with individual isoforms

• Functional differentiation approaches:

  • Leverage the yeast complementation system described in search result , where VPS24alpha, but not VPS24beta, rescues VPS24 deletion phenotypes

  • Similarly, use the BAX suppression assay where VPS24beta, but not VPS24alpha, shows activity

  • Develop mammalian cell assays that can distinguish isoform-specific functions

• RNA-level analysis:

  • Design isoform-specific primers for qRT-PCR to quantify relative expression levels

  • Use RNA-seq analysis to identify cell types or conditions where isoform ratios vary

  • Consider RNA hybridization techniques to visualize isoform localization patterns

• Structural studies:

  • Express and purify individual isoforms for comparative structural analysis

  • Use techniques like hydrogen-deuterium exchange mass spectrometry (HDX-MS) to identify structural differences

  • Perform comparative interaction studies to map isoform-specific protein binding partners

These strategic approaches can help overcome the inherent challenges in studying highly similar protein isoforms while revealing their distinct functional roles.

What are the most reliable animal models for studying VPS24 in neurodegenerative disease contexts?

For studying VPS24's role in neurodegenerative diseases, several animal model approaches offer complementary advantages:

• Conditional knockout mouse models:

  • Complete VPS24 knockout may cause embryonic lethality due to its essential cellular functions

  • Neuron-specific conditional knockout using Cre-lox systems (e.g., CaMKII-Cre for forebrain neurons)

  • Inducible knockout systems to trigger VPS24 deletion in adult animals, avoiding developmental effects

  • These approaches permit investigation of VPS24 loss in specific neural populations or at defined timepoints

• Transgenic models expressing disease-related proteins:

  • Cross VPS24 conditional knockout mice with models expressing neurodegenerative disease proteins (mutant huntingtin, TDP-43)

  • This approach directly tests the hypothesis from search result that "functional multivesicular bodies are required for autophagic clearance of protein aggregates associated with neurodegenerative disease"

  • Allows assessment of how VPS24 dysfunction exacerbates protein aggregation phenotypes

• Drosophila models:

  • The Drosophila ESCRT system is highly conserved with simpler genetic redundancy

  • CRISPR/Cas9 or RNAi approaches to manipulate the VPS24 ortholog

  • Combined with neurodegeneration models (polyQ, TDP-43) to assess effects on protein aggregation

  • Permits high-throughput screening of genetic modifiers or potential therapeutic compounds

• C. elegans models:

  • Transparent nematode allows real-time visualization of protein aggregates

  • CRISPR/Cas9 modification of the VPS24 ortholog

  • Combination with fluorescently tagged disease proteins provides a system for direct observation of aggregate dynamics

  • Short lifespan enables rapid assessment of age-dependent phenotypes

• iPSC-derived neuronal models:

  • While not traditional animal models, human induced pluripotent stem cell-derived neurons provide a human-specific context

  • CRISPR/Cas9 engineering to create isogenic lines with VPS24 mutations

  • Can be derived from patients with neurodegenerative diseases to study interactions with disease-specific backgrounds

For all these models, key readouts should include:

• Protein aggregate formation and clearance

• Autophagic flux measurements

• Endosomal-lysosomal morphology

• Neuronal survival and function

• Behavioral phenotypes (where applicable)

Using multiple model systems provides the most comprehensive understanding of VPS24's role in neurodegenerative disease pathways.

How can researchers effectively distinguish between the functions of VPS24 and other ESCRT-III components?

Distinguishing between the functions of VPS24 and other ESCRT-III components requires sophisticated experimental approaches:

• Selective depletion strategies:

  • Use siRNA/shRNA targeting specific ESCRT-III components

  • Employ CRISPR/Cas9 to create single and combinatorial knockouts

  • Utilize auxin-inducible degron (AID) systems for rapid and reversible protein depletion

  • These approaches allow assessment of phenotypes resulting from loss of individual components

• Structure-guided mutational analysis:

  • Based on search result , specific mutations like E114K can create "activated" forms of VPS24

  • Similar targeted mutations in other ESCRT-III components create a panel of proteins with altered function

  • This approach helps identify the unique structural features that determine component-specific functions

• Chimeric protein approaches:

  • Create fusion proteins exchanging domains between VPS24 and other ESCRT-III components

  • Search result demonstrates the utility of this approach, showing that replacing regions beyond residue ~152 of VPS24 with the homologous region of VPS2 maintains function

  • This identifies which domains confer component-specific functions

• Temporal recruitment analysis:

  • Use live-cell imaging with differently colored fluorescent tags to monitor the recruitment sequence of ESCRT-III components

  • Super-resolution microscopy to determine the spatial organization of different components within ESCRT-III assemblies

  • This approach reveals the dynamic assembly process and component-specific localization

• Component-specific interactome mapping:

  • Immunoprecipitation followed by mass spectrometry for each ESCRT-III component

  • BioID or APEX proximity labeling to identify neighboring proteins in living cells

  • These approaches identify component-specific protein interactions that may reveal unique functions

• In vitro reconstitution with purified components:

  • Systematic addition and omission of individual components in membrane remodeling assays

  • Direct visualization of how each component contributes to membrane deformation

  • This reductionist approach helps define the minimal requirements for specific functions

• Assessing specific cargo dependencies:

  • Determine whether particular cargo proteins require specific ESCRT-III components for sorting

  • This may reveal specialized functions for different components in handling distinct cargo types

These approaches collectively enable researchers to disentangle the specific contributions of VPS24 from other ESCRT-III components, revealing both shared and unique functions within this complex system.