FAM111A Antibody

What is FAM111A and what cellular functions does it perform?

FAM111A is a nuclear trypsin-like peptidase that plays multiple crucial roles in cellular function. It contains a C-terminal protease domain with a catalytic triad consisting of His385, Asp439, and Ser541, which is highly conserved among mammals . FAM111A has two primary cellular functions:

• DNA replication regulation: FAM111A localizes at replication forks and promotes DNA replication at protein obstacles through its protease activity. It interacts with proliferating cell nuclear antigen (PCNA) through its PCNA-interacting peptide (PIP) box .

• Antiviral defense: FAM111A acts as a host restriction factor against certain viruses, including simian virus 40 (SV40) and vaccinia virus lacking serine protease inhibitor 1 (VACV-ΔSPI-1) .

The protein's activity must be tightly regulated, as hyperactivation can lead to impaired DNA replication, single-strand DNA exposure, DNA damage, nuclear structure disruption, and cell death .

What are the structural characteristics of FAM111A that antibodies might target?

FAM111A has several structural domains that can serve as potential targets for antibodies:

• N-terminal domain: Contains ubiquitin-like domains (UBLs) and a single-stranded DNA-binding domain .

• PCNA-interacting peptide (PIP) box: Enables interaction with PCNA during DNA replication .

• Serine protease domain (SPD): The C-terminal region containing the catalytic triad (His385, Asp439, Ser541) .

The SPD forms a dimer that is crucial for its proteolytic activity. This dimerization occurs via the N-terminal helix within the SPD and induces an activation cascade from the dimerization sensor loop to the oxyanion hole through disorder-to-order transitions .

When selecting antibodies, researchers should consider which domain they wish to target based on their specific research questions.

What are the recommended sample preparation methods for FAM111A antibody applications?

Effective sample preparation for FAM111A antibody applications depends on the experimental technique:

For Western Blotting:

• Use RIPA buffer with protease inhibitors for cell lysis

• Include phosphatase inhibitors when studying FAM111A activation states

• Consider adding N-ethylmaleimide to preserve potential ubiquitination

For Immunofluorescence:

• 4% paraformaldehyde fixation preserves protein structure

• Permeabilization with 0.1-0.5% Triton X-100 allows antibody access to nuclear FAM111A

• BSA blocking (3-5%) reduces background signal

For Chromatin Immunoprecipitation:

• Crosslinking with formaldehyde (1%) for 10 minutes at room temperature

• Sonication to generate 200-500bp DNA fragments

• Pre-clearing with protein A/G beads to reduce non-specific binding

When working with FAM111A, it's important to note that its expression levels vary significantly between cell types. For example, FAM111A levels are much higher in U2OS cells compared to HEK293 cells , which can affect antibody detection sensitivity.

How can I validate the specificity of a FAM111A antibody for research applications?

Validating FAM111A antibody specificity requires multiple complementary approaches:

• Genetic validation:

  • Use FAM111A knockout cells as negative controls

  • Compare signal in wild-type versus FAM111A knockdown cells

  • Rescue experiments with ectopic expression of FAM111A in knockout cells

• Biochemical validation:

  • Western blot analysis using recombinant FAM111A as a positive control

  • Peptide competition assays to confirm epitope specificity

  • Immunoprecipitation followed by mass spectrometry to confirm target identity

• Cross-reactivity testing:

  • Test antibody against related family members (e.g., FAM111B)

  • Use species-specific samples to confirm cross-reactivity claims

• Application-specific validation:

  • For immunofluorescence, co-staining with a second antibody targeting a different epitope

  • For ChIP assays, testing enrichment at known FAM111A binding sites

When validating, be aware that FAM111A undergoes autocleavage, which may generate multiple bands on Western blots. Disease-associated mutations like R569H show increased autocleavage , and this can be used as a positive control for antibody validation.

What are the optimal experimental conditions for detecting FAM111A dimerization and how can antibodies facilitate this research?

Detecting FAM111A dimerization requires specialized techniques and careful experimental design:

• Biochemical approaches:

  • Size exclusion chromatography with FAM111A antibodies for detection

  • Chemical crosslinking followed by Western blot analysis

  • Blue native PAGE to preserve native protein complexes

• Biophysical methods:

  • Förster resonance energy transfer (FRET) using antibody-conjugated fluorophores

  • Bioluminescence resonance energy transfer (BRET) with luciferase-tagged FAM111A

  • Analytical ultracentrifugation with antibody detection

• Cellular assays:

  • Proximity ligation assay (PLA) using two different FAM111A antibodies targeting distinct epitopes

  • Bimolecular fluorescence complementation (BiFC)

When designing dimerization experiments, consider using the dimer-interface mutants V347D and V351D as negative controls, as these mutations disrupt dimerization and abolish proteolytic activity against substrates . Conversely, the K348A mutation affects the dimerization sensing mechanism and can serve as an additional control .

How does FAM111A interact with nuclear pore complex (NPC) components and what antibody-based methods can elucidate these interactions?

FAM111A interacts with several nuclear pore complex (NPC) components, and these interactions can be studied using the following antibody-based methods:

• Co-immunoprecipitation (Co-IP):

  • Use FAM111A antibodies to pull down protein complexes

  • Western blot with antibodies against NPC components (NUP153, POM121, NUP214, NUP50, NUP98, GANP)

  • Use protease-dead FAM111A mutants (S541A) to enhance detection of transient interactions

• Proximity-based labeling:

  • BioID approach with FAM111A fused to a biotin ligase

  • Antibody detection of biotinylated proteins after streptavidin pulldown

  • This approach has successfully identified NPC factors as FAM111A interactors

• Immunofluorescence microscopy:

  • Co-staining with FAM111A antibodies and NPC markers (e.g., Mab414)

  • Super-resolution microscopy to visualize precise co-localization patterns

  • Live-cell imaging with antibody fragments to track dynamic interactions

These interactions are particularly interesting because hyperactivation of FAM111A (as seen in disease-associated mutations) leads to compromised nuclear barrier function .

What are the differences in detecting wild-type versus mutant FAM111A proteins, and how should antibody selection account for these differences?

Detecting wild-type versus mutant FAM111A proteins presents several challenges that require careful antibody selection and experimental design:

• Expression level differences:

  • Disease-associated mutants (R569H, S342del) often show lower expression levels despite stronger effects

  • Antibodies must have sufficient sensitivity to detect lower-expressed mutants

• Autocleavage patterns:

  • Wild-type FAM111A exhibits baseline autocleavage

  • Disease-associated mutants show hyperactive autocleavage

  • Antibodies should be directed against epitopes present in both full-length and cleaved forms

• Localization differences:

  • Hyperactive mutants may show altered nuclear distribution

  • For immunofluorescence, antibodies should work under various fixation conditions

• Structural differences:

  • Mutations may alter conformation and epitope accessibility

  • Antibodies targeting conserved regions distant from mutation sites are preferable

When studying disease-associated mutants, consider using multiple antibodies targeting different regions of the protein. For example, an antibody against the N-terminal region can detect both full-length protein and N-terminal cleavage products, while a C-terminal antibody will only detect the full-length and C-terminal fragments.

How can FAM111A antibodies be utilized to study its role in antiviral defense mechanisms?

FAM111A exhibits significant antiviral activity, particularly against vaccinia virus lacking serine protease inhibitor 1 (VACV-ΔSPI-1) and simian virus 40 (SV40). Antibodies can be instrumental in studying these mechanisms:

• Infection-induced activation:

  • Use phospho-specific antibodies to detect activation state changes

  • Study FAM111A upregulation via the cGAS-STING pathway using time-course immunoblotting

  • Compare FAM111A levels before and after viral challenge

• Viral target identification:

  • Co-immunoprecipitation with FAM111A antibodies followed by mass spectrometry

  • Proximity labeling approaches to identify viral proteins in close association

  • Chromatin immunoprecipitation to identify viral DNA association

• Mechanism of restriction:

  • Immunofluorescence to track FAM111A localization during infection

  • Co-staining with viral proteins (e.g., vaccinia I3 protein)

  • Autophagic degradation assessment using LC3 co-localization

• Viral evasion strategies:

  • Study how viral proteins (e.g., SPI-1) antagonize FAM111A

  • In vitro peptidase assays with immunopurified FAM111A

FAM111A restricts VACV-ΔSPI-1 by targeting its DNA-binding protein I3 for autophagic degradation, as shown in the following experimental data:

These effects are dependent on FAM111A's functional protease domain, as the S541A mutation inactivates the enzymatic function while the R569H mutation represents a hyperactive form .

What are the key considerations for using FAM111A antibodies in chromatin immunoprecipitation (ChIP) experiments?

Chromatin immunoprecipitation with FAM111A antibodies requires special attention to several factors:

• Chromatin preparation:

  • Optimal crosslinking conditions (1% formaldehyde, 10 minutes)

  • Sonication to generate 200-500bp fragments

  • Addition of PMSF to prevent protease activity during sample processing

• Antibody selection:

  • ChIP-validated antibodies with minimal background binding

  • Epitopes that remain accessible in crosslinked chromatin

  • Concentration optimization through titration experiments

• Controls and validation:

  • IgG negative control

  • Input DNA control

  • Positive control loci (e.g., replication origins where FAM111A is known to bind)

  • FAM111A knockout cells as specificity controls

• Experimental design considerations:

  • Cell synchronization to capture cell-cycle dependent chromatin binding

  • Treatment with replication stress inducers to study FAM111A recruitment

  • Sequential ChIP to study co-occupancy with PCNA or other replication factors

FAM111A localizes to replication forks through its PIP box interaction with PCNA . When designing primers for ChIP-qPCR, focus on known replication origins or sites of replication stress.

How should researchers optimize immunofluorescence protocols for FAM111A detection in different cellular contexts?

Optimizing immunofluorescence for FAM111A detection requires addressing several technical challenges:

• Fixation method selection:

  • Paraformaldehyde (4%) preserves protein structure but may reduce epitope accessibility

  • Methanol fixation improves accessibility to some epitopes but can distort protein structure

  • Compare both methods to determine optimal detection

• Permeabilization optimization:

  • Nuclear proteins require effective permeabilization

  • Test different agents: 0.1-0.5% Triton X-100, 0.1-0.5% NP-40, or 0.1% SDS

  • Duration of permeabilization affects signal-to-noise ratio

• Antibody concentration and incubation:

  • Titrate primary antibody to determine optimal concentration

  • Extended incubation at 4°C (overnight) often improves specific signal

  • Test different blocking agents (BSA, normal serum, commercial blockers)

• Detection system selection:

  • Directly conjugated antibodies reduce background but may have lower sensitivity

  • Secondary antibody detection offers signal amplification

  • Consider tyramide signal amplification for low-abundance targets

• Context-specific considerations:

  • For cell cycle studies, co-stain with cell cycle markers

  • In virus-infected cells, include viral protein markers

  • When studying NPC interactions, use NPC-specific markers (Mab414)

FAM111A expression varies significantly between cell types, with U2OS cells showing much higher endogenous levels than HEK293 cells . Adjust antibody concentrations accordingly when working with different cell lines.

What strategies can be employed to study the post-translational modifications of FAM111A using antibodies?

Studying post-translational modifications (PTMs) of FAM111A requires specialized antibody-based approaches:

• Phosphorylation analysis:

  • Phospho-specific antibodies targeting known or predicted sites

  • Phosphatase treatment as negative control

  • Phos-tag gels to separate phosphorylated forms

  • Kinase inhibitor treatments to identify regulatory pathways

• Ubiquitination detection:

  • Co-immunoprecipitation with FAM111A antibodies followed by ubiquitin detection

  • Use of deubiquitinase inhibitors to preserve modifications

  • Expression of tagged ubiquitin for enhanced detection

• Autocleavage assessment:

  • Western blot analysis using antibodies targeting different domains

  • Comparison between wild-type and catalytic mutants (S541A)

  • Protease inhibitor treatment to distinguish between self-cleavage and degradation

• Other modifications:

  • SUMOylation analysis through SUMO-specific antibodies after immunoprecipitation

  • Acetylation detection with pan-acetyl-lysine antibodies

  • Mass spectrometry analysis of immunopurified FAM111A

FAM111A autocleavage patterns are particularly informative, as disease-associated mutations like R569H show increased autocleavage activity . When studying these patterns, use antibodies that can detect both the full-length protein and the cleaved fragments.

How can FAM111A antibodies contribute to understanding its role in genetic disorders?

FAM111A mutations cause two rare human syndromes: Kenny-Caffey Syndrome type 2 (KCS2) and the more severe Gracile Bone Dysplasia (GCLEB). Antibody-based approaches can advance our understanding of these disorders:

• Mutation-specific studies:

  • Compare wild-type and mutant protein expression in patient-derived cells

  • Assess subcellular localization changes in disease states

  • Study interaction profiles of disease-associated mutants

• Functional consequences:

  • Analyze nuclear pore complex integrity in patient cells using co-staining approaches

  • Measure DNA replication efficiency and stress markers

  • Assess cell cycle progression abnormalities

• Model systems:

  • Use CRISPR-engineered cells expressing disease mutations

  • Animal models expressing disease-associated variants

  • Patient-derived iPSCs differentiated into relevant cell types

• Potential therapeutic approaches:

  • Screen for small molecules that normalize hyperactive mutant activity

  • Test gene therapy approaches using antibodies for validation

Most disease-associated mutations in FAM111A are missense mutations clustered either within or around the peptidase domain . These mutations are thought to cause hyperactivation of the enzyme as inferred from its hyper-autocleavage activity . Ectopic expression of the disease-associated mutations causes impaired DNA replication, single-strand DNA exposure, DNA damage, nuclear structure disruption, and cell death .

What approaches can be used to study the dimerization-dependent serine protease activity of FAM111A?

The recently discovered dimerization-dependent nature of FAM111A's serine protease activity can be studied using several approaches:

• Structural analysis:

  • X-ray crystallography has revealed that FAM111A dimerizes via the N-terminal helix within the SPD

  • Use antibodies to confirm dimerization states in solution via analytical ultracentrifugation

• Functional assays:

  • In vitro peptidase assays using model peptide substrates

  • Compare wild-type FAM111A activity with dimerization-deficient mutants (V347D, V351D)

  • Assess the dimerization sensing mechanism using the K348A mutant

• Cellular studies:

  • Express dimerization mutants in FAM111A knockout cells to assess functional complementation

  • Measure TOP1cc accumulation in cells expressing dimerization mutants vs. wild-type

  • Assess chromatin localization of dimerization mutants

• Therapeutic implications:

  • Screen for small molecules that modulate dimerization

  • Design peptides that interfere with the dimerization interface

These insights provide a foundation for understanding FAM111A's enzymatic mechanism and its role in DNA replication .

How can researchers use FAM111A antibodies to study its role in preventing topoisomerase I cleavage complexes (TOP1ccs)?

FAM111A prevents the accumulation of topoisomerase I cleavage complexes (TOP1ccs), and antibody-based methods can help elucidate this function:

• TOP1cc detection methods:

  • Immunofluorescence with TOP1cc-specific antibodies

  • Slot blot assays to quantify TOP1ccs

  • RADAR (Rapid Approach to DNA Adduct Recovery) assay followed by TOP1 immunodetection

• FAM111A knockout/reconstitution studies:

  • Generate FAM111A knockout cells and measure TOP1cc accumulation

  • Reconstitute with wild-type or mutant FAM111A

  • Compare wild-type with dimerization-deficient mutants (V347D, V351D)

• Mechanistic investigations:

  • ChIP to identify FAM111A recruitment to TOP1cc sites

  • Co-immunoprecipitation to detect FAM111A-TOP1 interactions

  • In vitro protease assays with TOP1 as substrate

• Cell-based assays:

  • Treatment with camptothecin to induce TOP1ccs

  • Time-course analysis of TOP1cc resolution in wild-type vs. FAM111A knockout cells

  • Live cell imaging with FAM111A and TOP1 fluorescent fusions

Previous research has shown that FAM111A knockout causes the accumulation of TOP1ccs, and expression of wild-type FAM111A prevents this accumulation. Importantly, dimerization-deficient mutants (V347D and V351D) fail to prevent TOP1cc accumulation despite proper localization on chromatin, highlighting the importance of dimerization for FAM111A's function in DNA replication .

What are the emerging applications of FAM111A antibodies in cancer research?

FAM111A's roles in DNA replication and nuclear integrity suggest potential implications in cancer biology that can be explored using antibodies:

• Expression analysis in cancer tissues:

  • IHC studies across cancer types to assess FAM111A expression patterns

  • Correlation with clinical outcomes and treatment responses

  • Analysis of nuclear morphology abnormalities in cancer cells

• Functional studies in cancer models:

  • Knockdown/knockout effects on cancer cell proliferation and survival

  • DNA damage response assessment in cancer contexts

  • Synthetic lethality screening to identify potential therapeutic targets

• Therapeutic potential:

  • Development of inhibitors targeting FAM111A's protease activity

  • Assessment of FAM111A status as a biomarker for treatment response

  • Combination strategies with DNA-damaging agents or replication stress inducers

Given FAM111A's critical role in DNA replication and its interaction with the nuclear pore complex, further research may reveal cancer vulnerabilities that could be therapeutically exploited.

What future technologies might enhance FAM111A antibody applications in research?

Several emerging technologies promise to expand the utility of FAM111A antibodies in research:

• Advanced imaging approaches:

  • Super-resolution microscopy for detailed subcellular localization

  • Live cell nanobody-based detection for real-time dynamics

  • Correlative light and electron microscopy to link function with ultrastructure

• Single-cell technologies:

  • Single-cell proteomics to assess FAM111A levels in heterogeneous populations

  • CyTOF for multiparameter analysis of FAM111A status and cell state

  • Spatial transcriptomics combined with protein detection

• High-throughput functional genomics:

  • CRISPR screens to identify synthetic interactions with FAM111A

  • Proteomic profiling of FAM111A interactors under various conditions

  • Chemical-genetic approaches to modulate FAM111A function

• Structural biology advances:

  • Cryo-EM studies of FAM111A complexes with interaction partners

  • Hydrogen-deuterium exchange mass spectrometry to map conformational changes

  • Antibody fragments as crystallization chaperones for difficult structures