KAT2B Antibody

What is KAT2B and what are its primary biological functions?

KAT2B functions as a histone acetyltransferase that promotes transcriptional activation through epigenetic regulation. It binds with CBP and p300, competing with the adenoviral oncoprotein E1A for binding sites, thereby counteracting E1A's mitogenic activity . Additionally, KAT2B serves as a circadian transcriptional coactivator, enhancing the activity of circadian transcriptional activators NPAS2-ARNTL/BMAL1 and CLOCK-ARNTL/BMAL1 heterodimers . Recent research has also identified its involvement in tumor pathogenesis, particularly in epithelial ovarian cancer where it appears to function as a tumor suppressor .

How does KAT2B differ from its paralog KAT2A?

Despite their structural similarities, KAT2A and KAT2B exhibit remarkably divergent expression patterns and potentially distinct functions:

This divergent expression suggests they may have distinct roles in maintaining epidermal homeostasis .

What methods should researchers use to detect KAT2B expression when commercial antibodies yield non-specific signals?

When studying KAT2B expression in tissues where commercial antibodies produce non-specific signals (as observed in immunolabeled tissue sections), researchers should consider alternative detection methods:

How does KAT2B function as a tumor suppressor in epithelial ovarian cancer?

KAT2B exhibits characteristics of a tumor suppressor in epithelial ovarian cancer (EOC) through several mechanisms:

• Expression correlation with disease progression: KAT2B is downregulated in EOC tissues and this correlates with both FIGO stage and grade .

• Effects on cellular processes:

  • KAT2B silencing induces autophagy, enhancing cell proliferation and invasion

  • Conversely, KAT2B overexpression inhibits these processes

• In vivo effects: KAT2B silencing increases tumor volume and weight in animal models, an effect that can be mitigated by the autophagy inhibitor chloroquine .

• Molecular mechanism: KAT2B knockdown enhances autophagy via activation of the TGF-β/Smad3/7 signaling pathway, driving epithelial-mesenchymal transition (EMT), proliferation, and invasion in EOC .

• Protein interactions: Bioinformatics and co-immunoprecipitation assays have identified a KAT2B-SMAD7 interaction, suggesting direct regulation of the TGF-β pathway .

The data indicates potential therapeutic approaches targeting autophagy in EOC cases with reduced KAT2B expression.

What is the role of KAT2A/KAT2B in regulating centrosome duplication?

KAT2A/KAT2B play crucial roles in preventing aberrant centrosome amplification through the acetylation of polo-like kinase 4 (PLK4):

• Target identification: Proteomic analysis identified PLK4, a key regulator of centrosome duplication, as a substrate for KAT2A/KAT2B acetylation .

• Specific acetylation sites: KAT2A/KAT2B acetylate the PLK4 kinase domain on residues K45 and K46, with additional sites (K41 and K68) identified in vitro .

• Functional consequences:

  • Molecular dynamics modeling suggests K45/K46 acetylation impairs kinase activity by shifting PLK4 to an inactive conformation

  • PLK4 activity is reduced upon in vitro acetylation of its kinase domain

  • Overexpression of PLK4 K45R/K46R mutant (which cannot be acetylated) does not lead to centrosome overamplification, unlike wild-type PLK4

• Cellular effects: Impairing KAT2A/2B-acetyltransferase activity results in diminished phosphorylation of PLK4 and excess centrosome numbers in cells .

• Complex involvement: The ATAC complex containing KAT2A/2B plays an essential role in restricting centrosome duplication, as demonstrated by experiments showing that:

  • Overexpression of catalytically inactive KAT2A mutant leads to supernumerary centrosomes

  • shRNA-mediated depletion of KAT2A, ADA2a, or ADA3 (subunits of the AT module of ATAC complex) results in similar phenotypes

These findings establish KAT2A/2B acetylation of PLK4 as a critical regulatory mechanism preventing centrosome amplification, a hallmark of many cancers.

How can researchers resolve contradictory results when studying KAT2B function in different cancer types?

When encountering contradictory results regarding KAT2B function across different cancer types, researchers should:

• Consider context-dependent roles:

  • KAT2B functions as a tumor suppressor in EOC

  • In other contexts, KAT2B may have different roles depending on tissue type and genetic background

• Analyze signaling pathway differences:

  • Examine the status of TGF-β/Smad3/7 pathway components in different cancer types

  • Investigate potential involvement of the AKT/mTOR pathway, which has been implicated in KAT2B function

• Evaluate methodological differences:

  • Compare in vitro versus in vivo studies

  • Assess cell line differences and their genetic backgrounds

  • Consider differences in KAT2B knockdown/overexpression approaches

• Analyze substrate specificity:

  • Determine whether KAT2B targets different substrates in different cancer types

  • Characterize the acetylome in each context using proteomic approaches

• Design definitive experiments:

  • Use isogenic cell lines with controlled genetic backgrounds

  • Employ genome editing (CRISPR/Cas9) for precise manipulation of KAT2B

  • Conduct paired in vitro and in vivo studies

What are the most reliable methods for confirming KAT2B antibody specificity?

Ensuring KAT2B antibody specificity is critical for experimental validity. Recommended validation approaches include:

• Genetic knockdown/knockout controls:

  • siRNA/shRNA-mediated depletion of KAT2B

  • CRISPR/Cas9-mediated knockout of KAT2B

  • Compare antibody signals between control and KAT2B-depleted samples

• Overexpression verification:

  • Transfect cells with KAT2B expression vectors and measure increased signal

  • Include epitope-tagged versions for dual detection

• Peptide competition assays:

  • Pre-incubate antibody with immunizing peptide before application

  • Signal should be blocked by specific peptide but not by unrelated peptides

• Cross-reactivity assessment:

  • Test reactivity against recombinant KAT2A to evaluate cross-reactivity with this paralog

  • Use KAT2A-specific knockdown to distinguish signals

• Application-specific validation:

  • For immunohistochemistry/immunofluorescence: Include positive and negative control tissues

  • For Western blot: Verify expected molecular weight (approximately 93 kDa) and band pattern

  • For ChIP applications: Include IgG control and known target regions

What are the optimal experimental designs for studying KAT2B-mediated acetylation events?

To effectively study KAT2B-mediated acetylation events, researchers should consider these experimental approaches:

• Acetylome profiling:

  • Employ shotgun proteomics to identify global KAT2B substrates

  • Enrich acetylated peptides using anti-acetyllysine antibodies

  • Compare acetylomes between wild-type and KAT2B-depleted cells

• Site-specific acetylation validation:

  • Perform in vitro acetylation assays using recombinant KAT2B and substrate proteins

  • Confirm specific acetylation sites using tandem mass spectrometry

  • Develop site-specific acetylation antibodies (as demonstrated for PLK4 K45/K46)

• Functional impact assessment:

  • Generate lysine-to-arginine (KR) mutants at identified acetylation sites

  • Compare activities of wild-type and non-acetylatable mutant proteins

  • Use molecular dynamics modeling to predict structural impacts of acetylation

• Cellular consequence evaluation:

  • Compare phenotypes between cells expressing wild-type vs. non-acetylatable substrate proteins

  • Assess downstream pathway activation using phospho-specific antibodies or reporter assays

  • Evaluate subcellular localization changes of substrate proteins upon acetylation

• Acetylation dynamics analysis:

  • Use deacetylase inhibitors to trap acetylated forms

  • Perform time-course experiments after stimulus application

  • Correlate acetylation with other post-translational modifications

How can researchers effectively distinguish between KAT2A and KAT2B functions in cellular processes?

Despite their structural similarities, distinguishing between KAT2A and KAT2B functions requires specialized approaches:

• Expression pattern analysis:

  • Analyze differential expression in various tissues and cellular states

  • Track divergent expression patterns during processes like differentiation

  • KAT2A and KAT2B show opposite regulation during keratinocyte differentiation

• Selective manipulation strategies:

  • Use paralog-specific siRNAs/shRNAs with validated specificity

  • Design CRISPR/Cas9 targeting to unique regions

  • Perform rescue experiments with the other paralog to test functional redundancy

• Substrate specificity determination:

  • Compare acetylomes after selective depletion of each paralog

  • Perform in vitro acetylation assays with purified KAT2A vs. KAT2B

  • Identify unique vs. shared substrates

• Complex composition analysis:

  • Investigate differential incorporation into ATAC or SAGA complexes

  • Identify unique interaction partners for each paralog using co-immunoprecipitation followed by mass spectrometry

  • Study the effects of depleting specific complex components (e.g., ADA2a, ADA3)

• Biological outcome measurements:

  • Compare phenotypes after paralog-specific depletion

  • Assess effects on specific cellular processes (e.g., centrosome duplication, differentiation)

  • Analyze transcriptional effects using RNA-seq after selective knockdown

How can KAT2B research findings be applied to develop potential therapeutic strategies for epithelial ovarian cancer?

Based on KAT2B's tumor suppressor role in EOC, several therapeutic strategies emerge:

• Autophagy modulation:

  • Since KAT2B silencing enhances autophagy and promotes cancer progression, autophagy inhibitors like chloroquine could be particularly effective in EOC with low KAT2B expression

  • In vivo studies have already demonstrated that chloroquine can mitigate tumor growth effects of KAT2B silencing

• TGF-β pathway targeting:

  • KAT2B knockdown activates the TGF-β/Smad3/7 signaling pathway

  • TGF-β receptor inhibitors might counteract the effects of KAT2B loss

• KAT2B restoration approaches:

  • Epigenetic drugs to potentially increase KAT2B expression if silenced by methylation

  • Gene therapy approaches to restore KAT2B function

• Predictive biomarker development:

  • KAT2B expression levels could stratify patients for autophagy inhibitor treatment

  • Combined KAT2B and TGF-β pathway activation assessment may predict therapy response

• Combination therapy strategies:

  • Autophagy inhibitors plus standard chemotherapy

  • TGF-β pathway inhibitors plus agents targeting EMT

What are the implications of KAT2B's role in regulating centrosome duplication for cancer research?

The role of KAT2B in preventing centrosome amplification has significant implications for cancer research:

• Cancer diagnostic development:

  • Correlative studies of KAT2B expression/activity and centrosome abnormalities across cancer types

  • Assessment of PLK4 acetylation status as a potential biomarker

• Therapeutic targeting strategies:

  • Restoration of KAT2B activity in cancers with centrosome amplification

  • Development of mimetics that replicate the effect of KAT2B-mediated PLK4 acetylation

• Understanding cancer evolution:

  • Investigation of when KAT2B dysfunction occurs during cancer progression

  • Analysis of whether centrosome amplification drives or results from genomic instability

• Synthetic lethality approaches:

  • Identification of vulnerabilities in cancer cells with centrosome amplification due to KAT2B dysfunction

  • Testing whether such cells are particularly sensitive to mitotic checkpoint inhibitors

• Experimental systems development:

  • Creation of cellular and animal models with controlled KAT2B dysfunction to study centrosome biology

  • Development of high-throughput screening systems to identify compounds that prevent centrosome amplification

The finding that KAT2A/2B acetylate PLK4 on K45/K46 residues, thereby preventing centrosome amplification, provides a molecular mechanism that connects epigenetic regulation to centrosome biology and genomic stability .

How might KAT2B's role in circadian transcriptional regulation inform chronotherapy approaches?

KAT2B's function as a circadian transcriptional coactivator suggests potential implications for chronotherapy:

• Mechanism of action:

  • KAT2B enhances activity of circadian transcriptional activators NPAS2-ARNTL/BMAL1 and CLOCK-ARNTL/BMAL1 heterodimers

  • Likely involves histone acetylation at clock-controlled gene promoters

• Chronotherapeutic implications:

  • Drug efficacy and toxicity profiles may vary depending on time of administration

  • KAT2B activity levels might influence optimal timing for certain treatments

• Cancer chronotherapy applications:

  • Assessment of whether KAT2B dysfunction alters circadian regulation of cell cycle genes

  • Investigation of whether restoring proper circadian function might sensitize cancer cells to certain treatments

• Chronobiological research approaches:

  • Analysis of KAT2B binding to circadian gene promoters across the 24-hour cycle

  • Determination of whether KAT2B-mediated acetylation of non-histone proteins contributes to circadian regulation

  • Study of how KAT2B dysfunction affects circadian gene expression patterns

• Therapeutic timing optimization:

  • Development of strategies to time drug delivery based on KAT2B activity rhythms

  • Creation of small molecules that modulate KAT2B activity at specific times

Understanding how KAT2B contributes to circadian regulation may inform approaches to optimize treatment timing and potentially develop new strategies for treating diseases with disrupted circadian rhythms.

What are the most promising unexplored areas for KAT2B antibody applications in research?

Several promising research directions for KAT2B antibody applications include:

• Single-cell level analysis:

  • Application of KAT2B antibodies in single-cell proteomics

  • Development of proximity ligation assays to study KAT2B interactions in situ

• Cell-type specific functions:

  • Investigation of KAT2B roles in understudied cell types and tissues

  • Analysis of KAT2B contribution to tissue-specific differentiation programs

• Non-histone substrate identification:

  • Comprehensive mapping of non-histone proteins acetylated by KAT2B

  • Functional characterization of newly identified substrate acetylation events

• Therapeutic monitoring:

  • Development of assays to monitor KAT2B activity in patient samples

  • Correlation of KAT2B activity with disease progression and treatment response

• Post-translational modification interplay:

  • Investigation of how KAT2B-mediated acetylation interacts with other modifications

  • Characterization of signaling networks regulating KAT2B activity

Each of these directions would benefit from high-quality KAT2B antibodies with validated specificity and performance in diverse applications.

What technological developments might enhance KAT2B antibody research in the next five years?

Emerging technologies likely to impact KAT2B antibody research include:

• Advanced antibody engineering:

  • Development of recombinant antibodies with improved specificity

  • Creation of nanobodies targeting specific KAT2B conformations or complexes

• Spatial proteomics innovations:

  • Multiplexed imaging techniques to visualize KAT2B alongside multiple markers

  • Spatial transcriptomics combined with protein detection for correlative analysis

• Acetylation site-specific antibodies:

  • Expanded development of antibodies against specific acetylated substrates

  • Creation of conditional systems to detect acetylation events in live cells

• Integrated multi-omics approaches:

  • Combined analysis of KAT2B binding sites, acetylation targets, and transcriptional outcomes

  • Systems biology frameworks to understand KAT2B network effects

• AI-assisted antibody validation:

  • Machine learning algorithms to predict antibody specificity and optimal applications

  • Automated image analysis to quantify KAT2B localization and co-localization patterns

These technological developments will facilitate more comprehensive understanding of KAT2B biology and its therapeutic implications.