ZNF514 Human

What is the basic structure of human ZNF514 protein?

Human ZNF514 is a zinc finger protein characterized by multiple C2H2-type zinc finger domains. The full-length protein consists of approximately 400 amino acids. The recombinant form includes multiple zinc finger motifs that are typically involved in DNA binding. The protein sequence contains several conserved KRAB domains and multiple zinc finger domains with the characteristic C2H2 pattern (Cys-X2-4-Cys-X12-His-X3-5-His) that fold into a finger-like structure stabilized by zinc ions . This structural arrangement is critical for the protein's ability to recognize and bind specific DNA sequences.

What is the presumed biological function of ZNF514?

ZNF514 is believed to be involved in transcriptional regulation, like most members of the krueppel C2H2-type zinc-finger protein family . These proteins typically function as transcription factors that regulate gene expression by binding to specific DNA sequences through their zinc finger domains. The presence of multiple zinc finger domains in ZNF514 suggests it may have sequence-specific DNA-binding capabilities and potentially regulate the expression of target genes, though specific targets have not yet been definitively identified in the provided research.

What post-translational modifications have been identified in ZNF514?

Multiple post-translational modifications have been identified in ZNF514 according to iPTMnet data, including:

These modifications likely play roles in regulating protein function, stability, localization, or interaction with other molecules .

How might phosphorylation affect ZNF514 function?

The extensive phosphorylation pattern observed in ZNF514 (with at least 11 phosphorylation sites identified) suggests complex regulation of this protein's function. Phosphorylation of transcription factors typically modulates their DNA-binding affinity, nuclear localization, protein-protein interactions, or stability. For ZNF514, the clustering of phosphorylation sites at specific regions (such as S78/S80 and S354/S357/S358) may indicate regulatory hotspots that control its activity in response to various cellular signals . Experimental approaches to study these effects would include phosphomimetic mutations (substituting serine/threonine with aspartic acid or glutamic acid) and phospho-null mutations (substituting with alanine) to assess functional consequences.

What disease-associated variants have been identified in ZNF514?

Several disease-associated variants affecting PTM sites in ZNF514 have been identified:

These variants occur at sites that normally undergo post-translational modifications, suggesting that disruption of these modifications might contribute to disease development .

How do we interpret the functional significance of cancer-associated mutations at PTM sites?

The co-occurrence of cancer-associated mutations at post-translational modification sites in ZNF514 suggests these modifications may have functional significance. When mutations replace modifiable amino acids (e.g., serine to leucine), they permanently eliminate the potential for regulation at that site. For example, the S99L, S111L, and S357L variants eliminate phosphorylation sites, potentially leading to constitutive activity or inactivity of the protein. Similarly, the K180T variant eliminates a ubiquitination site, which might affect protein stability or turnover . This pattern of mutations at PTM sites suggests that proper regulation of ZNF514 may be important for preventing oncogenic processes, though confirmatory functional studies would be needed to establish causality.

What are the optimal methods for producing recombinant ZNF514 protein for in vitro studies?

For in vitro studies of ZNF514, recombinant protein production has been successfully accomplished using Escherichia coli expression systems. The available recombinant Human ZNF514 protein covers the full-length protein (1-400 amino acids) and has been produced with a histidine tag (His-tag) for purification purposes. According to commercial sources, this approach yields >85% purity, making it suitable for SDS-PAGE analysis and various biochemical assays . For functional studies, researchers should consider that the denatured form may be sufficient for antibody production and immunological assays, but native folding would be critical for DNA-binding or protein-interaction studies.

What techniques are recommended for detecting ZNF514 protein in experimental samples?

Based on available research, SDS-PAGE analysis has been successfully used to detect recombinant ZNF514 protein . For detecting endogenous ZNF514 in biological samples, Western blotting with specific antibodies would be the method of choice. Given the multiple post-translational modifications identified, researchers investigating specific modified forms should consider using modification-specific antibodies or enrichment techniques like phospho-peptide enrichment followed by mass spectrometry. For localization studies, immunofluorescence microscopy using validated antibodies would be appropriate to determine the subcellular distribution of ZNF514, which is expected to be predominantly nuclear given its presumed function in transcriptional regulation.

How do copy number variations (CNVs) impact the study of ZNF514?

While the provided search results don't specifically mention CNVs affecting the ZNF514 gene itself, understanding CNVs is important for genomic studies involving zinc finger proteins. Research on clinical-grade human embryonic stem cell lines reveals that approximately 72% of cell lines harbor CNVs greater than 100kb, comparable to the general population . When studying ZNF514, researchers should be aware that CNVs affecting this gene or its regulatory regions could influence expression levels and potentially function. Methods such as whole-genome SNP genotyping arrays (like the Illumina HumanCytoSNP-12 array) can be used to detect such variations, though targeted approaches like quantitative PCR or digital droplet PCR might provide more focused analysis for ZNF514 specifically.

What considerations should be made when analyzing ZNF514 genetic data from different cell sources?

When analyzing ZNF514 genetic data, researchers should consider several factors that might influence interpretation:

• Natural genetic variation: Like other genes, ZNF514 may exhibit natural polymorphisms in the population that don't necessarily indicate pathology

• Copy-neutral loss of heterozygosity (CN-LOH): These regions can affect gene expression through altered allelic representation

• Cell line artifacts: Extended culture of cell lines can introduce genomic alterations

• Tissue-specific expression patterns: ZNF514 expression may vary across tissue types

The molecular karyotypic evaluation of cell lines used for research is crucial, especially before their application in clinical settings . For ZNF514 specifically, researchers should verify that observed variations are not artifacts of cell culture or sample preparation before attributing functional significance to them.

How might ZNF514 function in transcriptional networks?

As a member of the krueppel C2H2-type zinc-finger protein family, ZNF514 likely functions within complex transcriptional networks. These proteins typically recognize specific DNA sequences through their zinc finger domains and can act as transcriptional activators or repressors. Advanced research approaches might include:

• ChIP-seq (Chromatin Immunoprecipitation followed by sequencing) to identify genomic binding sites

• RNA-seq after ZNF514 knockout/knockdown to identify regulated genes

• Protein-protein interaction studies (co-IP, BioID, or yeast two-hybrid) to identify cofactors

• ATAC-seq to examine chromatin accessibility changes upon ZNF514 manipulation

These approaches could help place ZNF514 within the broader context of transcriptional regulation networks and potentially identify its role in specific biological processes or disease states.

What is the significance of the multiple zinc finger domains in ZNF514?

The presence of multiple zinc finger domains in ZNF514 suggests a complex DNA-binding specificity. Each C2H2 zinc finger typically recognizes approximately 3-4 base pairs, and the arrangement of multiple fingers allows for recognition of extended DNA sequences with high specificity. Advanced research questions might address:

• Which zinc fingers are essential for DNA binding versus protein-protein interactions

• Whether different combinations of fingers recognize distinct DNA sequences

• If post-translational modifications differentially affect the function of specific fingers

• Whether the protein can adopt different conformations to recognize diverse targets

Methodological approaches might include mutational analysis of individual zinc fingers, structural studies using X-ray crystallography or cryo-EM, and high-throughput binding assays like SELEX (Systematic Evolution of Ligands by Exponential Enrichment) to define binding preferences.

What are the critical unanswered questions about ZNF514 function?

Despite our understanding of ZNF514's basic characteristics, several critical questions remain unanswered:

• What are the specific target genes regulated by ZNF514?

• What signaling pathways regulate ZNF514 activity through its various PTM sites?

• Does ZNF514 have different functions in different cell types or developmental stages?

• What is the three-dimensional structure of ZNF514, particularly its DNA-binding domains?

• How do cancer-associated mutations functionally impact ZNF514 activity?

Addressing these questions would significantly advance our understanding of this protein's biological roles and potential contributions to disease processes.

What emerging technologies might advance ZNF514 research?

Several emerging technologies could significantly advance ZNF514 research:

• CRISPR-Cas9 gene editing for precise modification of endogenous ZNF514

• Single-cell technologies to examine ZNF514 expression and function at cellular resolution

• Proteomics approaches like BioID or APEX to map the protein's interaction network

• Cryo-EM for structural determination of the full-length protein

• Organoid models to study ZNF514 function in more physiologically relevant contexts

These approaches would provide deeper insights into ZNF514's function and potentially reveal new therapeutic strategies for diseases involving dysregulation of this protein.