MRFAP1L1 Human

What is MRFAP1L1 and what is its relation to the MORF4 family?

MRFAP1L1 is a 127 amino acid protein that belongs to the MORF4 family-associated protein family . It is closely related to MRFAP1 (MORF4 family-associated protein 1) and interacts with mortality factor 4-like protein 1 (MORF4L1, also known as MRG15), which functions as a chromatin regulatory protein . The mortality factor family includes mortality factor 4 (MORF4), MORF4L1 (MRG15), and MORF4-related gene X (MRGX) . MRFAP1L1's association with MORF4L1 suggests a potential role in chromatin modification and transcriptional regulation processes .

What are the optimal expression systems for producing recombinant MRFAP1L1?

Several expression systems have been validated for MRFAP1L1 production with varying yields and purity levels:

The choice of expression system should be determined based on downstream applications, required purity, and whether post-translational modifications are essential for the research question .

How can researchers effectively detect and quantify MRFAP1L1 in experimental samples?

For effective detection and quantification of MRFAP1L1, researchers should consider these methodological approaches:

• Western Blotting (WB): The most common method using MRFAP1L1-specific antibodies, with optimal working dilutions determined empirically for each investigative context .

• ELISA: Useful for quantitative detection in complex samples, particularly when using recombinant MRFAP1L1 as a standard .

• Mass Spectrometry (MS): Provides detailed analysis of protein modifications and can be combined with SILAC (Stable Isotope Labeling with Amino acids in Cell culture) for turnover studies .

• SDS-PAGE with Coomassie staining: For purified recombinant protein quality assessment, typically showing >95% purity for E. coli-expressed protein .

• Immunohistochemistry: For tissue expression analysis, particularly useful for examining cellular localization patterns in specific tissues .

Researchers should note that detection sensitivity varies between methods, with MS offering the highest sensitivity for low-abundance samples and western blotting providing good specificity when optimized with appropriate controls .

What purification strategies yield the highest purity MRFAP1L1 protein preparations?

To achieve high-purity MRFAP1L1 preparations, researchers should implement the following strategies:

• Affinity Chromatography: Using His-tag affinity with Ni-NTA resins typically yields >95% purity as determined by SDS-PAGE . This approach represents the primary purification step for recombinant MRFAP1L1.

• Filtration: Implementation of 0.2 μm filtration for sterility without compromising protein integrity is recommended for preparations intended for cellular applications .

• Expression System Selection: E. coli expression systems consistently yield the highest purity (>95%) for MRFAP1L1 when combined with appropriate affinity purification techniques .

• Tag Selection: His-tagged MRFAP1L1 appears to consistently yield higher purity than other tags in published preparations, though GST and Strep tags also produce acceptable results for specific applications .

Researchers should validate purification success through SDS-PAGE analysis, with high-quality preparations showing a predominant band at approximately 14-15 kDa (for the untagged protein) or slightly higher depending on the fusion tag used .

What are the known protein-protein interactions of MRFAP1/MRFAP1L1?

The MRFAP1/MRFAP1L1 interaction network demonstrates its importance in chromatin regulation and cell cycle control. Key interaction partners include:

• MORF4L1 (MRG15): Both MRFAP1 and likely MRFAP1L1 interact with MORF4L1, a chromatin regulatory protein containing a chromo domain with affinity for di- or tri-methylated histone H3 on K36 . This interaction occurs via MORF4L1's C-terminal MRG domain.

• E3 Ubiquitin Ligases: MRFAP1 binds to several E3 ubiquitin ligases, including CUL4B, which may regulate its turnover and stability . This suggests a mechanism for the rapid degradation observed for these proteins.

• Retinoblastoma protein (Rb1): MRFAP1 associates with Rb1, suggesting a potential role in cell cycle regulation and transcriptional control processes .

• Competitive Interaction with MRGBP: MRFAP1 appears to compete with MRG-binding protein (MRGBP) for binding to MORF4L1, potentially regulating MORF4L1's ability to interact with chromatin-modifying enzymes like those in the NuA4 histone acetyltransferase complex .

These interactions position MRFAP1/MRFAP1L1 as potential regulators of chromatin modification and transcriptional control networks .

How does the NEDD8-Cullin E3 ligase pathway regulate MRFAP1 turnover?

The NEDD8-Cullin E3 ligase pathway plays a critical role in regulating MRFAP1 turnover, which may have implications for MRFAP1L1 as well:

• Rapid Protein Turnover: MRFAP1 exhibits a fast turnover rate under normal conditions and is degraded via the ubiquitin-proteasome system .

• NEDD8 Pathway Inhibition Effects: Treatment with MLN4924, a small molecule inhibitor of the NEDD8 conjugation pathway, leads to significant upregulation of MRFAP1 in multiple human cell lines . This increased abundance results from decreased degradation rates rather than increased synthesis.

• E3 Ligase Binding: MRFAP1 binds to several E3 ubiquitin ligases, including CUL4B, which likely mediates its ubiquitination and subsequent proteasomal degradation .

• Regulatory Implications: The rapid turnover suggests that MRFAP1 levels need to be tightly controlled, implying its importance in regulating dynamic cellular processes including chromatin modification .

This regulatory mechanism positions MRFAP1/MRFAP1L1 as downstream targets of the NEDD8-Cullin pathway, potentially connecting protein degradation systems with chromatin regulation mechanisms .

How might MRFAP1L1 function in chromatin modification pathways?

MRFAP1L1 likely plays a regulatory role in chromatin modification pathways through mechanisms similar to MRFAP1:

• Competitive Binding Model: Research suggests MRFAP1 competes with MRGBP for binding to MORF4L1 . Since MRGBP interaction with MORF4L1 recruits the NuA4 histone acetyltransferase complex to chromatin, MRFAP1 binding may inhibit this recruitment, thus regulating histone H4 acetylation.

• Tissue-Specific Chromatin Regulation: The restricted expression pattern of MRFAP1, particularly in testis and brain, suggests tissue-specific functions in chromatin regulation . Similar patterns might exist for MRFAP1L1.

• Dynamic Regulation through Protein Turnover: The rapid turnover of MRFAP1 provides a mechanism for dynamic regulation of chromatin modification activities . By adjusting MRFAP1 levels through protein degradation, cells could rapidly alter chromatin modification states in response to stimuli.

• Developmental Regulation: The expression pattern of MRFAP1 during spermatogenesis (high in spermatogonia, low in spermatocytes and spermatids) inversely correlates with MRGBP expression, suggesting regulated exchange of MORF4L1 interaction partners during meiosis .

These mechanisms position MRFAP1L1 as a potential regulatory factor in chromatin modification pathways with implications for cell differentiation and development .

What experimental approaches can resolve the competing interactions between MRFAP1L1, MORF4L1, and chromatin modification complexes?

To investigate the competing interactions model involving MRFAP1L1, researchers should consider these advanced experimental approaches:

• Quantitative Protein-Protein Interaction Analysis:

  • Isothermal titration calorimetry (ITC) to determine binding affinities between MRFAP1L1, MORF4L1, and MRGBP

  • Surface plasmon resonance (SPR) to measure association/dissociation kinetics

  • Competition binding assays with purified proteins to directly test the mutual exclusivity hypothesis

• Structural Biology Approaches:

  • Cryo-electron microscopy of MORF4L1 complexes with either MRFAP1L1 or MRGBP

  • X-ray crystallography of binding domains to characterize interaction interfaces

  • NMR spectroscopy to map binding regions in solution

• Chromatin Modification Assays:

  • In vitro histone acetyltransferase assays with reconstituted NuA4 complex in the presence/absence of MRFAP1L1

  • ChIP-seq analysis to identify genomic binding sites affected by MRFAP1L1 levels

  • ATAC-seq to assess chromatin accessibility changes when manipulating MRFAP1L1 expression

• Advanced Cell Biology Techniques:

  • FRET or BRET assays to measure protein interactions in living cells

  • Proximity ligation assays (PLA) to visualize protein complexes in situ

  • Optogenetic approaches to temporally control MRFAP1L1 interactions

These methodologies would provide complementary evidence to elucidate the precise molecular mechanisms by which MRFAP1L1 may regulate chromatin modification through competitive protein interactions .

How can researchers investigate the tissue-specific functions of MRFAP1L1 in development and disease?

To investigate tissue-specific functions of MRFAP1L1, researchers should employ these comprehensive approaches:

• Tissue Expression Profiling:

  • Single-cell RNA-seq analysis in tissues of interest, particularly testis and brain where MRFAP1 shows highest expression

  • Spatial transcriptomics to map expression patterns with anatomical context

  • Immunohistochemistry with validated antibodies across developmental timepoints and disease states

• Functional Genomics:

  • CRISPR/Cas9-mediated knockout or knockdown in relevant cell types or organoid models

  • Conditional knockout mouse models to assess tissue-specific roles while avoiding potential embryonic lethality

  • Rescue experiments with wild-type vs. mutant MRFAP1L1 to identify critical functional domains

• Disease Association Studies:

  • Analysis of MRFAP1L1 expression in tissue samples from various pathological conditions

  • Examination of genetic variations in MRFAP1L1 associated with specific diseases

  • Investigation of epigenetic changes at MRFAP1L1 target genes in disease states

• Developmental Biology Approaches:

  • Time-course analysis of MRFAP1L1 expression during spermatogenesis and neural development

  • Co-expression analysis with MORF4L1 and MRGBP during cellular differentiation

  • Experimental manipulation of MRFAP1L1 levels during critical developmental windows

These multidisciplinary approaches would provide insights into the biological significance of MRFAP1L1 in specific tissues and potentially reveal its relevance to developmental processes and disease mechanisms .