VPS28 Human

What is the basic structure and function of VPS28 in human cells?

VPS28 (vacuolar protein sorting 28) functions as a critical subunit of the endosomal sorting complexes required for transport I (ESCRT-I). This protein plays an essential role in the biogenesis of multivesicular bodies (MVBs) and the sorting of ubiquitinated proteins into these vesicles. Structurally, human VPS28 contains domains that facilitate interactions with other ESCRT-I components, particularly VPS23/TSG101, and forms a bridge to ESCRT-II complexes. For researchers investigating VPS28's structure-function relationship, techniques such as X-ray crystallography, cryo-electron microscopy, and protein-protein interaction assays (including co-immunoprecipitation and yeast two-hybrid systems) have proven valuable for characterizing how VPS28 integrates into the larger ESCRT machinery .

How is VPS28 gene expression regulated across different human tissues?

VPS28 shows differential expression patterns across human tissues, with notably high expression in neural tissues. For researchers investigating tissue-specific expression, quantitative PCR remains the gold standard for measuring transcript levels, while immunohistochemistry provides spatial information about protein expression. Single-cell RNA sequencing data has revealed that neurons exhibit particularly high VPS28 expression compared to other central nervous system cell types, suggesting specialized functions in these cells . When designing experiments to study tissue-specific effects, researchers should consider using tissue-specific promoters for conditional expression/knockout studies to avoid global effects that might mask tissue-specific functions.

What are the key protein-protein interactions of VPS28 and how can they be studied?

VPS28 forms critical interactions with multiple proteins as part of its function:

For researchers studying these interactions, proximity-based proteomics methods (BioID, APEX) can identify novel context-specific interaction partners, while FRET and BRET approaches enable real-time monitoring of interactions in living cells . Mutational analysis of interaction interfaces can provide mechanistic insights into how these protein-protein interactions contribute to VPS28's diverse cellular functions.

How does VPS28 contribute to extracellular vesicle biogenesis and secretion?

VPS28 plays a critical role in extracellular vesicle (EV) biogenesis and secretion through its function in the ESCRT-I complex. Research has demonstrated that disruption of neuron-enriched VPS28 significantly decreases EV secretion by regulating the formation of intracellular multivesicular bodies (MVBs) . For researchers investigating this process, methodological approaches include:

• Isolation of EVs using differential ultracentrifugation or size-exclusion chromatography

• Nanoparticle tracking analysis for EV quantification and size distribution

• Western blotting for EV markers

• Electron microscopy for morphological characterization

• Comparative proteomic analysis of EVs from control and VPS28-depleted cells

Rescue experiments using purified EVs from wild-type cells to complement phenotypes in VPS28-deficient models provide compelling evidence for VPS28's specific role in EV-mediated functions .

What is the relationship between VPS28 and lipid metabolism, particularly triglyceride synthesis?

Recent research has uncovered an unexpected role for VPS28 in regulating triglyceride (TG) synthesis through ubiquitination pathways. In bovine mammary epithelial cells (MAC-T), VPS28 knockdown significantly upregulates fatty acid transporter CD36 and adipose differentiation-related protein (ADFP), leading to increased TG and fatty acid production . The effects of VPS28 manipulation on lipid metabolism markers are summarized below:

For researchers studying this relationship, methodological approaches include lentivirus-mediated gene expression manipulation, lipid droplet visualization using fluorescent dyes, and biochemical assays for triglyceride quantification .

How does VPS28 regulate neuronal VEGF trafficking and vascular development?

VPS28 plays a crucial role in neurovascular communication by controlling the trafficking of vascular endothelial growth factor A (VEGF-A) through extracellular vesicle secretion. In zebrafish models, VPS28 has been shown to be essential for the sprouting of brain central arteries (CtAs) and maintaining blood-brain barrier (BBB) integrity . Mechanistically, VPS28 regulates the formation of multivesicular bodies (MVBs) in neurons, which subsequently release VEGF-A-containing extracellular vesicles that signal to endothelial cells to promote angiogenesis.

For researchers investigating this pathway, zebrafish provide an excellent model system due to their transparent embryos that allow real-time visualization of vascular development using transgenic fluorescent reporters. Mouse models with conditional VPS28 knockout in neurons also offer valuable insights into mammalian neurovascular development . Experimental approaches include fluorescent transgenic reporter lines, confocal microscopy for vessel visualization, and functional assays for BBB integrity.

What are the most effective genetic manipulation strategies for studying VPS28 function?

Researchers have several options for manipulating VPS28 expression, each with specific advantages depending on the research question:

When designing genetic manipulation experiments, researchers should consider including appropriate controls (scrambled shRNA, empty vector) and validation of knockdown/overexpression efficiency using both RT-qPCR and western blotting .

How can researchers effectively isolate and characterize VPS28-dependent extracellular vesicles?

Isolation and characterization of VPS28-dependent extracellular vesicles (EVs) require specialized techniques to ensure purity and comprehensive analysis. A methodological workflow includes:

• Isolation: Differential ultracentrifugation (sequential centrifugation at 300g, 2000g, 10,000g, and 100,000g) or size-exclusion chromatography for purification from conditioned media

• Quantification: Nanoparticle tracking analysis (NTA) to determine concentration and size distribution

• Morphological characterization: Transmission electron microscopy (TEM) or cryo-EM for ultrastructural analysis

• Marker analysis: Western blotting for EV markers (CD63, CD9, CD81) and VPS28-dependent cargo proteins

• Cargo profiling: Mass spectrometry-based proteomics and RNA sequencing for comprehensive content analysis

Comparing EVs isolated from control and VPS28-depleted cells can reveal specific cargoes that depend on VPS28 for their sorting into EVs. Functional assays, such as EV uptake experiments and recipient cell phenotype analysis, provide insights into the biological significance of VPS28-dependent EV cargoes .

What are the optimal animal models for studying VPS28 function in vivo?

Different animal models offer complementary advantages for studying VPS28 function in vivo:

For zebrafish studies, morpholino-based knockdown or CRISPR-Cas9 gene editing in transgenic lines with fluorescently labeled vasculature (e.g., Tg(kdrl:EGFP)) enables direct visualization of vascular phenotypes . Mouse models benefit from conditional knockout approaches using tissue-specific Cre lines to circumvent potential embryonic lethality of global knockouts.

How is VPS28 dysfunction implicated in neurodegenerative disorders?

VPS28 dysfunction has been linked to several neurodegenerative conditions, most notably spastic paraplegias. According to genetic databases, diseases associated with VPS28 include Spastic Paraplegia 80 (Autosomal Dominant) and Spastic Paraplegia 53 (Autosomal Recessive) . The mechanism likely involves disrupted endosomal trafficking and protein degradation, leading to the accumulation of toxic proteins in neurons.

For researchers studying these connections, patient-derived induced pluripotent stem cells (iPSCs) differentiated into neurons provide a valuable model system. Phenotypic assays including endosomal morphology analysis, protein trafficking dynamics, and neurite outgrowth can reveal disease-specific cellular pathologies. Additionally, analysis of cerebrospinal fluid extracellular vesicles may identify biomarkers related to VPS28 dysfunction in neurodegenerative disease patients .

What is the role of VPS28 in blood-brain barrier integrity and related pathologies?

VPS28 plays a critical role in maintaining blood-brain barrier (BBB) integrity through its function in neuronal VEGF trafficking. Research in zebrafish models has demonstrated that disruption of VPS28 leads to compromised BBB integrity and intracranial hemorrhage . The mechanism involves reduced secretion of neuronal extracellular vesicles containing VEGF-A, which normally promote proper vascular development and barrier formation.

Researchers investigating this role can utilize in vivo BBB permeability assays using fluorescent tracers of various molecular weights to assess barrier function. Immunostaining for tight junction proteins (claudin-5, occludin, ZO-1) provides insights into the molecular basis of barrier defects. These findings have implications for understanding vascular contributions to neurodegenerative diseases and potential therapeutic approaches targeting the neurovascular unit .

How does VPS28 contribute to viral pathogenesis and replication?

VPS28, as part of the ESCRT-I complex, is exploited by multiple viruses during their replication cycle. According to pathway analyses, VPS28 is involved in the budding and maturation of HIV virions and the HIV life cycle . This occurs because many enveloped viruses hijack the ESCRT machinery to facilitate their budding from host cell membranes, a process that normally functions in multivesicular body formation.

For virological research, experimental approaches include:

• Viral replication assays in cells with VPS28 knockdown or overexpression

• Immunofluorescence microscopy to visualize co-localization of viral components with VPS28

• Co-immunoprecipitation to detect physical interactions between viral proteins and ESCRT components

• Electron microscopy to examine ultrastructural features of viral budding sites

Understanding these interactions has potential implications for antiviral drug development, as disrupting the virus-ESCRT interface could inhibit viral replication .

How can researchers investigate the interplay between VPS28 and the ubiquitin-proteasome system?

The relationship between VPS28 and the ubiquitin-proteasome system represents a complex regulatory network. Research has shown that VPS28 knockdown in bovine mammary epithelial cells leads to elevated ubiquitin levels while reducing proteasome activity, suggesting a compensatory relationship . For researchers investigating this interplay, several methodological approaches are valuable:

• Ubiquitin pulldown assays to identify ubiquitinated proteins that accumulate after VPS28 manipulation

• Fluorogenic substrate assays to measure proteasome activity in VPS28-modified cells

• Cycloheximide chase experiments to assess protein degradation kinetics

• Combination treatments with proteasome inhibitors (e.g., epoxomicin) and endosomal-lysosomal pathway inhibitors (e.g., chloroquine)

• Mass spectrometry-based ubiquitinomics to comprehensively profile changes in the ubiquitinated proteome

These approaches can reveal how VPS28 coordinates protein degradation between the endosomal-lysosomal and ubiquitin-proteasome systems, with implications for cellular proteostasis in both normal and disease states .

What are the emerging non-canonical functions of VPS28 beyond endosomal sorting?

Beyond its canonical role in endosomal sorting, emerging research has identified several non-canonical functions of VPS28:

For researchers exploring these emerging functions, unbiased screening approaches such as CRISPR screens for synthetic lethal interactions or proximity labeling proteomics can identify context-specific roles and interaction partners .

What cutting-edge technologies show promise for advancing VPS28 research?

Several cutting-edge technologies are poised to drive significant advances in VPS28 research:

• Spatial transcriptomics/proteomics: These approaches can reveal tissue-specific and subcellular expression patterns of VPS28 and its interaction partners with unprecedented resolution.

• Organoid models: Brain, mammary, and other tissue-specific organoids provide physiologically relevant 3D systems for studying VPS28 functions in tissue development and homeostasis.

• CRISPR base editing and prime editing: These refined gene editing technologies enable precise introduction of disease-associated VPS28 mutations without double-strand breaks.

• Lattice light-sheet microscopy: This advanced imaging technique allows long-term live imaging of VPS28-dependent trafficking events with minimal phototoxicity.

• Artificial intelligence for image analysis: Machine learning algorithms can extract subtle phenotypic features from high-content imaging data of cells with altered VPS28 function.

For researchers entering the field, combining these technologies with established biochemical and genetic approaches will provide comprehensive insights into VPS28's multifaceted roles in cellular physiology and disease .