Recombinant Human Protein FAM72D (FAM72D)

What is the basic molecular profile of FAM72D protein?

FAM72D (Family with Sequence Similarity 72 Member D) is a 149 amino acid protein with a molecular mass of approximately 16.6 kDa . The protein is encoded by the FAM72D gene located on chromosome 1q21, a region frequently amplified in high-risk cancers . Also known as GCUD2 (Gastric Cancer Up-regulated-2), FAM72D belongs to a family that includes several paralogs, with FAM72C being an important related member . The full amino acid sequence is: MSTNICSFKDRCVSILCCKFCKQVLSSRGMKAVLLADTEIDLFSTDIPPTNAVDFTGRCYFTKICKCKLKDIACLKCGNIVGYHVIVPCSSCLLSCNNGHFWMFHSQAVYDINRLDSTGVNVLLWGNLPEIEESTDEDVLNISAEECIRTR .

How does the expression of FAM72D differ between normal and pathological tissues?

In pathological conditions, particularly in lung adenocarcinoma (LUAD), FAM72 expression is significantly upregulated compared to normal lung tissues . This differential expression pattern makes FAM72D a potential biomarker for disease diagnosis. In multiple myeloma, FAM72D has been identified through 5-hydroxymethylcytosine (5hmC) profiling, suggesting epigenetic regulation of its expression . The amplification of the 1q21 region containing FAM72D parallels disease progression in multiple myeloma, indicating its potential role in cancer advancement .

What are the recommended experimental systems for studying FAM72D function?

For in vitro studies, HEK293 cells have been successfully used for recombinant expression of FAM72D . Cell-free protein synthesis (CFPS) systems have also been employed for producing recombinant FAM72D with various tags for detection and purification . For functional studies, both overexpression and knockdown approaches in relevant cancer cell lines (particularly multiple myeloma and lung adenocarcinoma models) are appropriate. Patient-derived xenograft models can provide more clinically relevant insights for in vivo studies, especially when investigating the role of FAM72D in tumor progression and treatment response.

What are the optimal methods for detection of FAM72D in experimental settings?

For protein detection, Western blotting using specific antibodies against FAM72D or against tags (e.g., Myc-DYKDDDDK) in recombinant versions is commonly employed . Immunohistochemistry can be used for tissue localization studies. For quantitative analysis, ELISA assays have been developed using tagged recombinant FAM72D as standards . For gene expression analysis, qRT-PCR is effective, while RNA-seq provides broader context of expression patterns alongside other genes. Detection of the native protein typically requires validation of antibody specificity given the high sequence similarity among FAM72 family members.

What expression systems yield the highest quality recombinant FAM72D protein?

HEK293 cell expression systems have demonstrated reliable production of recombinant FAM72D with protein purity typically exceeding 80% as determined by SDS-PAGE and Coomassie blue staining . For higher purity (>90%), additional purification steps may be incorporated. Cell-free protein synthesis (CFPS) systems represent an alternative approach, yielding 70-80% purity but potentially offering advantages in terms of scalability and reduced processing time . The choice between these systems depends on downstream applications, with HEK293-expressed protein being preferred for functional studies requiring post-translational modifications.

What are the critical factors in maintaining stability of recombinant FAM72D during experiments?

Recombinant FAM72D should be stored at -80°C in a buffer containing 25 mM Tris-HCl (pH 7.3), 100 mM glycine, and 10% glycerol . Repeated freeze-thaw cycles should be minimized, with the recommendation of no more than 2-3 cycles to maintain protein integrity . For working solutions, keeping the protein on ice is advisable, and for longer experiments, addition of protease inhibitors may be beneficial. The protein concentration should be maintained at approximately 50 μg/mL for optimal stability . When designing experiments, it's important to account for potential degradation over time, particularly in complex biological matrices.

How does FAM72D interact with the FOXM1 transcription factor network?

FAM72D functions as part of the FOXM1 transcription factor network, which controls cell proliferation and survival mechanisms . This interaction appears to be particularly significant in cancer biology. While the exact molecular basis of this interaction has not been fully elucidated, functional studies indicate that cells expressing high levels of both FOXM1 and FAM72D demonstrate increased sensitivity to epigenetic drugs targeting histone deacetylases and DNA methyltransferases . This suggests a potential regulatory loop involving epigenetic modifications, FOXM1-mediated transcription, and FAM72D function.

What evidence supports FAM72D's role in epigenetic regulation?

FAM72D has been identified through 5-hydroxymethylcytosine (5hmC) profiling in multiple myeloma, suggesting that its expression may be regulated by DNA demethylation processes . The 5hmC modification represents an intermediate in active DNA demethylation and is often associated with gene activation. The presence of 5hmC at the FAM72D locus correlates with its expression levels, indicating epigenetic control mechanisms . Additionally, cells with high FAM72D expression show altered sensitivity to epigenetic drugs, further supporting its connection to epigenetic processes. The protein may participate in feedback loops that influence chromatin structure and accessibility.

How does chromosome 1q21 amplification affect FAM72D expression and function in multiple myeloma?

Amplification of chromosome 1q21, where FAM72D is located, is a known driver of disease progression in multiple myeloma . This amplification typically results in increased expression of FAM72D and other genes in this region. The mechanistic connection between 1q21 amplification and disease aggression likely involves FAM72D's role in promoting cell proliferation and survival through the FOXM1 network . Patients with 1q21 amplification often show genetic instability, particularly of the pericentromeric region 1q12 and the neighboring 1q21 region, which correlates with highly proliferative plasma cells .

What molecular pathways are enriched in cells with high FAM72D expression?

Gene Ontology (GO) and Gene Set Enrichment Analysis (GSEA) have revealed that high FAM72D expression is associated with enrichment in pathways related to mitotic nuclear division and cell cycle regulation . In multiple myeloma, FAM72D appears to be part of gene networks involved in DNA damage resistance, proliferation, and survival mechanisms . The protein's association with the FOXM1 transcription factor network further implicates it in cell cycle progression, DNA repair, and anti-apoptotic functions. Additionally, immune cell infiltration correlates with FAM72D expression, suggesting potential roles in tumor-immune interactions .

What are the optimal experimental conditions for studying FAM72D's functional interactions?

For protein-protein interaction studies, co-immunoprecipitation using either endogenous protein or tagged recombinant versions can be effective. When using recombinant FAM72D, the Myc-DYKDDDDK tag can facilitate immunoprecipitation and detection . For investigating transcriptional networks, chromatin immunoprecipitation (ChIP) approaches can be used to examine FOXM1-FAM72D interactions with DNA. Cell-based functional assays should include both gain-of-function (overexpression) and loss-of-function (siRNA or CRISPR-mediated knockout) approaches to establish causality. When designing interaction studies, controls should account for potential indirect interactions through larger protein complexes.

How can researchers effectively measure the impact of FAM72D on cell proliferation and survival?

To assess FAM72D's impact on proliferation, multiple complementary approaches should be employed, including BrdU incorporation, Ki-67 immunostaining, and cell cycle analysis by flow cytometry. The plasma cell labeling index (PCLI) using BrdU incorporation has been successfully applied in multiple myeloma studies . For survival and apoptosis assessment, Annexin V/PI staining, caspase activation assays, and long-term colony formation assays provide comprehensive insights. Gene expression profiling before and after FAM72D modulation can reveal affected pathways. Correlation between FAM72D expression and proliferation markers can be determined using Spearman's test, as demonstrated in previous studies .

What methods are recommended for analyzing epigenetic modifications associated with FAM72D?

For analyzing 5-hydroxymethylcytosine (5hmC) modifications associated with FAM72D, selective chemical labeling with exonuclease digestion (SCL-exo) or without digestion (SCL-seq) has proven effective . These approaches have advantages over traditional bisulfite-based methods for distinguishing 5hmC from 5-methylcytosine. Oxidative bisulfite modification coupled with array hybridization (oxBS-450K) represents an alternative approach, though it may provide less meaningful information compared to sequencing-based methods . For data analysis, principal component analysis (PCA) of 5hmC signals and heatmap clustering of hydroxymethylated CpGs are recommended for identifying patterns across samples .

How might targeting FAM72D therapeutically affect cancer cell response to conventional treatments?

Based on the observation that cells expressing high levels of FOXM1 and FAM72D show increased sensitivity to epigenetic drugs targeting histone deacetylases and DNA methyltransferases , combination therapies targeting FAM72D alongside conventional treatments deserve exploration. Potential approaches include:

Research designs should include both in vitro drug sensitivity testing and in vivo models to assess efficacy and toxicity profiles.

What is the relationship between FAM72D expression and immune infiltration in the tumor microenvironment?

Analysis of immune infiltration has shown that FAM72D expression correlates with immune cell presence in tumor tissues . This relationship warrants deeper investigation to understand whether FAM72D directly influences immune cell recruitment or activation, or whether this correlation reflects shared regulatory mechanisms. Future studies should employ single-cell RNA sequencing to characterize the immune cell populations in FAM72D-high versus FAM72D-low tumors. Spatial transcriptomics could provide insights into the proximity relationships between FAM72D-expressing cancer cells and specific immune cell subtypes. Understanding this relationship could inform immunotherapy approaches, particularly in cancers where FAM72D serves as a poor prognostic marker.

How do the functions of FAM72D differ from other FAM72 family members, and what are the implications for targeting specificity?

While FAM72 family members (FAM72A-D) share considerable sequence homology, their functional specificity remains incompletely characterized. Comparative studies using CRISPR-based selective knockout of individual family members followed by phenotypic and transcriptomic analysis could reveal unique versus redundant functions. Structural biology approaches, including crystallography or cryo-EM of FAM72D in complex with interaction partners, could identify unique binding interfaces for specific targeting. Development of highly selective antibodies or other biologics would require careful validation of specificity across family members. The potential for compensatory upregulation of other family members following selective targeting of FAM72D represents an important consideration for therapeutic development that requires systematic investigation.