Recombinant Human Williams-Beuren syndrome chromosomal region 28 protein (WBSCR28)

What is WBSCR28 and what are its fundamental characteristics?

WBSCR28 (Williams-Beuren syndrome chromosomal region 28), also known by its synonym TMEM270, is one of approximately 25-28 genes deleted in individuals with Williams-Beuren syndrome . It is identified with NCBI Gene ID 135886 and its protein product is cataloged as WBS28_HUMAN . WBSCR28 is part of the critical region on chromosome 7q11.23 that, when hemizygously deleted, results in the multisystemic disorder characterized by distinctive facial features, cardiovascular abnormalities, and a unique cognitive profile with relative strengths in language and facial processing but weaknesses in visuospatial construction . The specific function of WBSCR28 in normal physiology and its precise contribution to Williams-Beuren syndrome pathology remain areas of active investigation.

What functional associations has WBSCR28 been demonstrated to have?

According to the Harmonizome database, WBSCR28 has 904 functional associations with biological entities spanning 8 distinct categories: molecular profiles, organisms, functional terms, phrases or references, chemicals, diseases, phenotypes or traits, structural features, cell lines, cell types or tissues, genes, proteins or microRNAs . These associations have been extracted from 42 different datasets providing a comprehensive network of potential interactions and functions. The breadth of these associations suggests WBSCR28 may have pleiotropic effects across multiple biological systems, consistent with the multisystemic nature of Williams-Beuren syndrome.

How is WBSCR28 expressed across different tissues and developmental stages?

Expression data for WBSCR28 is available from several tissue-specific databases. The Allen Brain Atlas Developing Human Brain database contains microarray data showing relative expression levels of WBSCR28 in various brain tissues throughout development . Additionally, BioGPS Human Cell Type and Tissue Gene Expression Profiles database documents WBSCR28 expression patterns across diverse human tissues . The CCLE database provides information on WBSCR28 expression in different cell lines, as well as copy number variation profiles . These resources collectively indicate that WBSCR28 has tissue-specific expression patterns that may vary throughout development, potentially contributing to the developmental aspects of Williams-Beuren syndrome phenotypes.

What experimental approaches are most effective for studying WBSCR28 function?

Based on methodologies employed for similar genes in the Williams-Beuren syndrome region, effective approaches for studying WBSCR28 function may include:

• Gene knockout or knockdown studies in cellular or animal models to observe resulting phenotypic changes

• Protein-protein interaction studies to identify binding partners

• Subcellular localization experiments using tagged versions of the protein

• Comparative expression analyses between typical and Williams-Beuren syndrome-affected tissues

• Structural biology techniques to elucidate protein conformation and potential function

Similar to the approach taken with WBSCR27, researchers could generate cell lines with WBSCR28 gene knockout using CRISPR-Cas9 technology and employ molecular biology and mass spectrometry techniques to study the resulting cellular consequences . Such knockout models would allow investigation of phenotypic changes and potential pathway disruptions resulting from WBSCR28 deficiency.

How might recombinant WBSCR28 protein be effectively produced and purified for structural studies?

Drawing from protocols established for the related protein WBSCR27, researchers could employ the following methodology for WBSCR28:

• Clone the WBSCR28 coding sequence into a bacterial expression vector with an affinity tag (such as a His-tag)

• Express the protein in E. coli grown in appropriate media, potentially using isotope enrichment for NMR studies (15N and/or 13C)

• Purify using affinity chromatography on a Ni-NTA column

• Perform tag cleavage if necessary

• Include a refolding protocol if the protein forms inclusion bodies, similar to the procedure described for WBSCR27 (denaturation in 6M urea followed by controlled refolding)

• Perform additional purification steps such as size exclusion chromatography to ensure sample homogeneity

This approach would yield purified protein suitable for structural and functional characterization. For NMR studies specifically, isotope labeling strategies as detailed for WBSCR27 could be adapted, including selective labeling of specific amino acid residues to facilitate structural determination .

What techniques are appropriate for characterizing WBSCR28's potential binding partners and cellular localization?

To identify binding partners and determine cellular localization of WBSCR28, researchers could implement:

• Proximity-based labeling techniques like BioID (as used for WBSCR27), which involves fusion of a promiscuous biotin ligase to WBSCR28 to biotinylate neighboring proteins in living cells, followed by streptavidin pulldown and mass spectrometry identification

• Co-immunoprecipitation experiments using antibodies against tagged versions of WBSCR28

• Fluorescence microscopy using fusion proteins (such as WBSCR28-mKate2 or WBSCR28-GFP) to visualize subcellular localization

• Immunofluorescence with antibodies against epitope-tagged WBSCR28 (e.g., HA-WBSCR28) combined with DAPI nuclear staining

• Cell fractionation followed by Western blotting to biochemically determine the subcellular distribution

These approaches would provide complementary data about WBSCR28's cellular context and potential interaction network.

What expression systems are optimal for different experimental applications of recombinant WBSCR28?

The choice of expression system depends on the specific research application:

For structural studies of WBSCR28, bacterial expression similar to that used for WBSCR27 would be appropriate, particularly if isotope labeling is required for NMR studies . For functional studies where post-translational modifications may be critical, mammalian expression systems would be preferable.

What purification challenges might be anticipated with recombinant WBSCR28?

Based on experience with related proteins:

• Solubility issues - WBSCR28 may form inclusion bodies in bacterial expression systems, necessitating refolding procedures similar to those described for WBSCR27

• Protein stability - The protein may require specific buffer conditions or additives to maintain stability

• Co-purifying contaminants - WBSCR28 might co-purify with endogenous molecules (similar to how WBSCR27 co-purifies with SAH)

• Aggregation tendencies - Careful optimization of concentration and storage conditions may be required

• Maintaining native conformation - Verification of proper folding through functional or structural assays would be crucial

A multi-step purification strategy would likely be necessary, potentially including refolding steps if the protein is expressed in inclusion bodies, followed by multiple chromatography steps to achieve high purity.

What analytical methods are most appropriate for confirming the identity and purity of recombinant WBSCR28?

Researchers should employ multiple complementary techniques:

• SDS-PAGE and Western blotting to confirm molecular weight and immunoreactivity

• Mass spectrometry for accurate mass determination and sequence verification

• Size exclusion chromatography to assess homogeneity and aggregation state

• Circular dichroism spectroscopy to evaluate secondary structure content

• Dynamic light scattering to analyze size distribution and potential aggregation

• Limited proteolysis followed by mass spectrometry to probe structural integrity

• Thermal shift assays to assess stability under various conditions

For tagged versions of WBSCR28, verification of tag accessibility using appropriate antibodies or affinity reagents would be important to ensure the tag doesn't interfere with protein structure or function .

How can knockout or knockdown models be effectively designed to study WBSCR28 function?

Following methodologies similar to those used for WBSCR27 , researchers could:

• Design CRISPR-Cas9 targeting strategies focusing on critical exons of WBSCR28

• Incorporate reporter systems to facilitate identification of successful editing events

• Validate knockout efficiency at both DNA level (sequencing), RNA level (RT-PCR), and protein level (Western blot with tagged versions if antibodies are unavailable)

• Create rescue lines expressing wild-type WBSCR28 to confirm phenotype specificity

• Consider conditional knockout systems for developmental studies

• Establish appropriate control lines, including those with non-targeting gRNAs

For cell line models, both NIH3T3 fibroblasts and neural-derived cell lines would be appropriate given the neurological aspects of Williams-Beuren syndrome . For animal models, mouse knockouts would be valuable given the conservation of the Williams-Beuren syndrome critical region.

What assays would be most informative for studying WBSCR28's potential role in Williams-Beuren syndrome pathology?

Given the neurological and developmental aspects of Williams-Beuren syndrome:

• Neurite outgrowth and branching assays in neural cell models with WBSCR28 deletion or overexpression

• Electrophysiological studies to assess potential impacts on neuronal function

• RNA-Seq and proteomics comparisons between wild-type and WBSCR28-deficient cells to identify affected pathways

• Cell migration and adhesion assays to investigate potential roles in neural crest development

• Developmental timing studies if using embryonic models

• Behavioral assessments in animal models focusing on cognitive domains affected in Williams-Beuren syndrome

Additionally, ERP (Event-Related Potential) markers might be valuable for assessing brain function in animal models with WBSCR28 modifications, similar to approaches used in human Williams-Beuren syndrome research .

How might structural studies inform understanding of WBSCR28 function?

Structural characterization of WBSCR28 could provide significant functional insights:

• NMR spectroscopy or X-ray crystallography to determine three-dimensional structure

• Identification of structural motifs or domains that suggest biochemical function

• Characterization of potential binding pockets for ligands or interaction partners

• Conformational dynamics studies to identify flexible regions that might mediate protein-protein interactions

• Comparative structural analysis with other proteins in the Williams-Beuren syndrome region

• Molecular dynamics simulations to predict functional movements and potential binding sites

The approach used for WBSCR27, which revealed a canonical Rossman fold typical of Class I methyltransferases , demonstrates how structural studies can provide functional clues for poorly characterized proteins in the Williams-Beuren syndrome region.

What are the current limitations in WBSCR28 research?

Several challenges currently hamper WBSCR28 research:

• Limited specific literature focusing directly on WBSCR28 function

• Potential lack of specific antibodies for detecting endogenous WBSCR28

• Incomplete understanding of its expression pattern across developmental stages

• Unknown binding partners or substrates that would clarify its biochemical function

• Uncertainty regarding its specific contribution to Williams-Beuren syndrome phenotypes

• Technical challenges in producing recombinant protein if it has solubility issues

• Difficulty in distinguishing its individual effects from the broader consequences of the Williams-Beuren syndrome deletion

These limitations necessitate careful experimental design and interpretation of results within the broader context of Williams-Beuren syndrome research.

How can integrative approaches advance understanding of WBSCR28's role in Williams-Beuren syndrome?

Progress will likely require multidisciplinary strategies:

• Combining genetic, biochemical, and cellular approaches to build a comprehensive functional profile

• Integrating data from individuals with partial deletions in the Williams-Beuren syndrome region to isolate WBSCR28-specific effects

• Leveraging large-scale omics data (genomics, transcriptomics, proteomics) to place WBSCR28 in biological pathways

• Using developmental models to track WBSCR28's role across critical timepoints

• Implementing brain imaging and electrophysiological methods in conjunction with genetic models

• Establishing collaborations between clinical researchers and basic scientists to correlate molecular findings with clinical observations

As demonstrated by research on WBSCR27 and other Williams-Beuren syndrome genes, an integrated approach combining structural biology, biochemistry, cell biology, and developmental neuroscience will be necessary to fully elucidate WBSCR28's function .