ALKBH3 Human

What is ALKBH3 and what are its primary biological functions?

ALKBH3 (AlkB Homolog 3) belongs to the AlkB family, which in humans consists of nine highly conserved members: ALKBH1 through ALKBH8 plus FTO (fat mass and obesity-associated protein). ALKBH3 functions primarily as a demethylase that removes methyl groups from specific modified nucleobases in DNA and RNA, particularly 1-methyladenosine (m1A) and 3-methylcytosine (m3C) in single-stranded nucleic acids .

The protein demonstrates slightly higher efficiency in demethylating m1A compared to m3C in vitro. This activity maintains genome and transcriptome integrity by repairing alkylation damage and regulating RNA modifications . ALKBH3 localizes to both the nucleoplasm and cytoplasm, allowing it to function in both mRNA and tRNA m1A demethylation, enhancing ribosome assembly, and preventing apoptosis .

How does ALKBH3 differ structurally and functionally from other AlkB family members?

The AlkB family members share conserved domains but exhibit different substrate preferences and biological functions:

• ALKBH2 and ALKBH3 both catalyze m1A and m3C demethylation, but ALKBH2 preferentially acts on double-stranded DNA (dsDNA), whereas ALKBH3 primarily recognizes single-stranded substrates .

• ALKBH1, ALKBH5, and FTO catalyze RNA m6A demethylation, a distinct activity from ALKBH3 .

• Structurally, ALKBH3 contains a distinctive β-hairpin (β4-loop-β5) that prevents dsDNA binding, a key feature distinguishing it from ALKBH2 .

• Mutation experiments demonstrate that changing Glu123 and Asp124 of ALKBH3 to the corresponding ALKBH2 residues (Phe102 and Gly103) significantly increases enzymatic activity on dsDNA substrates, highlighting the importance of these structural differences .

What is the expression pattern of ALKBH3 in normal tissues versus cancer?

ALKBH3 shows distinct expression patterns between normal and cancer tissues:

• In normal tissues, ALKBH3 is expressed in various cell types, with notable expression in the prostate .

• In cancer tissues, ALKBH3 is frequently overexpressed compared to adjacent normal tissues, particularly in:

  • Lung adenocarcinoma (50% of specimens showed ≥30% of cells immunopositive for ALKBH3)

  • Squamous cell carcinoma of the lung (56.5% showed high expression)

  • Urothelial carcinoma

• High ALKBH3 expression correlates with poor prognosis, particularly in lung adenocarcinoma where the percentage of ALKBH3-positive cells statistically correlates with recurrence-free survival .

• Interestingly, ALKBH3 expression is lower in small-cell lung cancer compared to non-small-cell lung cancers, with only 25% of small-cell carcinomas showing high expression versus 75% of both adenocarcinomas and squamous cell carcinomas .

What structural features determine ALKBH3's preference for single-stranded substrates?

ALKBH3's preference for single-stranded nucleic acids is determined by specific structural elements:

• A β-hairpin (β4-loop-β5) structure physically blocks double-stranded DNA binding .

• Upon binding to single-stranded DNA (ssDNA), the outward movement of this β-hairpin prevents double-stranded DNA binding that would otherwise occur with ALKBH2 .

• Key residues Glu123 and Asp124 in the loop region of the β-hairpin move by approximately 6.6 Å and 7.2 Å respectively, which would create significant clashes with bound dsDNA strands .

• An additional β-hairpin' (β'-loop-β") originating from the α2 helix stabilizes single-stranded nucleotides and methylated base substrates inside the active site pocket .

• Superposition of the ALKBH3-ssDNA complex structure with that of human Tet2-dsDNA complex reveals completely different nucleic acid binding orientations, highlighting distinct substrate recognition mechanisms .

How does ALKBH3 achieve specificity for m1A and m3C demethylation?

ALKBH3 demonstrates remarkable specificity in its demethylation activity through several structural and biochemical mechanisms:

• A bubble region around Asp194 and a key residue Thr133 inside the active site pocket facilitate specific recognition and catalysis of m1A/1mA and m3C/3mC substrates .

• Experimental evidence shows that removal of the bubble region around Asp194 or mutagenesis of Thr133 to the corresponding residue in FTO (Arg96) or ALKBH5 (Lys132) changes the substrate selectivity of ALKBH3 from m1A to m6A .

• ALKBH3 preferentially recognizes single-stranded substrates, allowing access to modified bases that might be less accessible in double-stranded conformations .

• The protein is slightly more efficient in demethylating m1A than m3C in vitro, suggesting subtle differences in how these substrates interact with the active site .

• Unlike some other demethylases, ALKBH3 lacks a distinct sequence preference, enabling it to function broadly across different RNA molecules containing m1A modifications .

How does ALKBH3-mediated demethylation affect RNA metabolism and translation?

ALKBH3-mediated demethylation significantly impacts RNA metabolism and translation through multiple mechanisms:

• ALKBH3 demethylates m1A in mRNAs, particularly in the 5' UTR region with specific consensus sequences, promoting gene transcription and playing crucial roles in cancer development .

• In tRNA, m1A predominantly locates at positions 9, 14, 22, and 58 in the T-loop of human mitochondrial and cytoplasmic tRNAs, stabilizing tertiary structure .

• ALKBH3's demethylation activity enhances ribosome assembly, suggesting a role in promoting efficient translation .

• RNA m1A is involved in translation initiation, early elongation, ribosome release, and protein synthesis, all potentially regulated by ALKBH3 activity .

• The dynamic regulation of m1A through ALKBH3-mediated demethylation appears to be particularly important during stress conditions and environmental signaling, suggesting adaptive roles in RNA metabolism under challenging cellular conditions .

How does ALKBH3 contribute to cancer progression at the molecular level?

ALKBH3 promotes cancer progression through multiple molecular mechanisms:

• Cell Cycle Regulation: Knockdown of ALKBH3 induces expression of p21WAF1/Cip1 and p27Kip1 in lung adenocarcinoma cells, resulting in cell cycle arrest and senescence .

• Oxidative Stress Modulation: ALKBH3 knockdown induces cell cycle arrest through downregulation of NAD(P)H oxidase-2 (NOX-2)-mediated generation of reactive oxygen species (ROS) .

• Angiogenesis Promotion: ALKBH3 regulates vascular endothelial growth factor (VEGF) expression by controlling tumor necrosis factor-like weak inducer of apoptosis (Tweak) and its receptor, fibroblast growth factor-inducible 14 (Fn14) .

• Invasion and Metastasis Support: Silencing of ALKBH3 significantly suppresses invasion and angiogenesis of cancer cells both in vitro and in vivo .

• RNA Modification and Regulation: ALKBH3-mediated mRNA m1A demethylation promotes gene transcription and plays a vital role in cancer development .

• DNA Repair Functions: Initially identified as a DNA repair enzyme, ALKBH3 may contribute to cancer cell survival by repairing DNA damage caused by therapeutic agents .

What experimental evidence supports ALKBH3 as a potential therapeutic target?

Multiple lines of evidence establish ALKBH3 as a promising therapeutic target:

• Overexpression in Tumors: High ALKBH3 expression has been documented in multiple cancer types, particularly lung adenocarcinoma, squamous cell carcinoma, and urothelial carcinoma .

• Correlation with Prognosis: The percentage of cells positive for ALKBH3 statistically correlates with recurrence-free survival in lung adenocarcinoma, indicating clinical relevance .

• In Vitro Effects of Silencing: ALKBH3 knockdown significantly inhibits cancer cell proliferation, induces cell cycle arrest and senescence, and reduces migration and invasion capabilities in multiple cancer cell lines .

• In Vivo Validation:

  • Intraperitoneal injection of ALKBH3 siRNA + atelocollagen in mice inoculated with A549 cells significantly reduces both tumor number and diameter .

  • ALKBH3 knockdown significantly inhibits subcutaneous tumor growth in nude mice .

  • Silencing ALKBH3 suppresses angiogenesis in urothelial carcinoma models .

• Molecular Target Validation: Specific structural features of ALKBH3 have been identified that could facilitate the design of selective inhibitors .

What methodologies are most effective for studying ALKBH3 function in cancer models?

Research on ALKBH3 employs diverse methodological approaches:

In Vitro Approaches:

• Gene Silencing: siRNA transfection or creation of stable cell lines with ALKBH3 knockdown .

• Functional Assays:

  • CCK-8 assay for proliferation assessment

  • Wound healing assay for migration analysis

  • Transwell invasion assay for invasive capacity evaluation

  • Cell cycle analysis following ALKBH3 knockdown

  • Senescence assays after ALKBH3 inhibition

• Biochemical Approaches: Enzyme activity assays measuring demethylation of specific substrates (m1A, m3C) .

• Structural Studies: X-ray crystallography of ALKBH3-oligo complexes, often facilitated by antibody Fab fragments .

In Vivo Approaches:

• Animal Models:

  • Intraperitoneal cancer cell inoculation followed by ALKBH3 siRNA + atelocollagen treatment

  • Subcutaneous tumorigenesis models in nude mice

  • Orthotopic mouse models for tissue-specific tumor progression

• Chorioallantoic Membrane (CAM) Assay: Used to assess invasion and angiogenesis following ALKBH3 silencing .

• Clinical Sample Analysis: Immunohistochemistry, qPCR, and western blotting to correlate ALKBH3 expression with clinicopathological parameters .

How might ALKBH3 influence response to standard cancer therapies?

While direct evidence on ALKBH3's impact on treatment response is limited in the provided search results, several mechanisms suggest potential interactions:

• DNA Repair Function: As a DNA repair enzyme , ALKBH3 could potentially contribute to resistance against DNA-damaging therapies such as radiation or certain chemotherapeutics.

• Cell Survival Pathways: ALKBH3 promotes cancer cell survival through multiple pathways , suggesting that its inhibition might sensitize cells to cytotoxic therapies.

• Angiogenesis Regulation: ALKBH3's role in angiogenesis via the Tweak/Fn14 pathway could affect response to anti-angiogenic therapies.

• Prognostic Significance: The correlation between high ALKBH3 expression and poor prognosis suggests that ALKBH3 levels might identify patients requiring more aggressive treatment approaches.

• Combination Potential: Given ALKBH3's involvement in multiple cancer-promoting processes, combining ALKBH3 inhibition with standard therapies might yield synergistic effects.

What is known about ALKBH3's relationship with immune cell infiltration in tumors?

Bioinformatics analysis has revealed that "the expression of ALKBH3 is related to immune cell infiltration" , suggesting an immunomodulatory role. While detailed mechanisms aren't elaborated in the search results, this finding has several potential implications:

• ALKBH3 expression may influence the composition or extent of immune cell infiltration in tumors, potentially affecting anti-tumor immune responses.

• If ALKBH3 expression modulates the tumor immune microenvironment, this could be relevant for understanding tumor-immune interactions and potentially for immunotherapy approaches.

• The relationship between ALKBH3 and immune cell infiltration represents an emerging area for investigation, particularly given the importance of the immune microenvironment in cancer progression and treatment response.

• Understanding these relationships could potentially help identify patients who might benefit from combined ALKBH3 targeting and immunotherapy.

How can ALKBH3's crystal structure inform the development of specific inhibitors?

The elucidation of ALKBH3's crystal structure provides valuable insights for developing specific inhibitors:

• The determination of the ALKBH3-oligo crosslinked complex structure with the assistance of antibody Fab fragments has revealed atomic-level details of substrate recognition and catalytic mechanisms .

• The active site pocket contains specific features, including a bubble region around Asp194 and the key residue Thr133, that facilitate specific recognition and catalysis of m1A and m3C substrates .

• Understanding these structural features enables "future design of structure-based small molecules for additional functional studies and potential therapeutic applications" .

• The β-hairpin structures that determine substrate specificity could be targeted to develop highly selective inhibitors that don't affect other AlkB family members .

• Mutation studies have identified key residues that determine substrate selectivity, providing additional targets for inhibitor design .

What emerging delivery approaches show promise for ALKBH3-targeted therapies?

While specific advanced delivery approaches aren't detailed in the search results, the successful use of atelocollagen as a delivery system for ALKBH3 siRNA in animal models provides a foundation for future development:

• Intraperitoneal injection of ALKBH3 siRNA + atelocollagen significantly reduced tumor growth and dissemination in mice inoculated with A549 cells .

• This demonstrates that appropriate delivery methods can achieve effective ALKBH3 inhibition in tumors with meaningful biological consequences.

• Future research could explore additional delivery systems for RNA interference approaches or small molecule inhibitors targeting ALKBH3.

• The development of tumor-targeted delivery systems could enhance the specificity and efficacy of ALKBH3-directed therapies while minimizing off-target effects.

How might ALKBH3 interact with other epigenetic regulators in cancer development?

While the search results don't explicitly address ALKBH3's interactions with other epigenetic regulators, several observations suggest potential connections:

• ALKBH3 functions as a demethylase for both DNA and RNA, placing it within the broader network of epigenetic and epitranscriptomic regulation .

• Bioinformatics analysis showed that ALKBH3 interacts with ASCC family molecules , which are involved in various cellular processes including transcription regulation.

• The dynamic regulation of m1A through ALKBH3-mediated demethylation likely intersects with other RNA modification pathways and potentially with chromatin-based epigenetic mechanisms.

• Understanding how ALKBH3 cooperates with or antagonizes other epigenetic regulators could reveal new therapeutic approaches targeting multiple nodes in dysregulated epigenetic networks in cancer.