DIRAS1 Human

What is DIRAS1 and how is it structurally classified?

DIRAS1, also known as RIG (Ras-related inhibitor of cell growth), is a 22kDa GTPase that belongs to the Ras superfamily. It shares 40-50% homology with Ras and Rap proteins but differs functionally as it acts as a tumor suppressor rather than an oncogene . The DIRAS1 gene is located on chromosome band 10p13.3 and includes two exons with a 597 bp protein-coding region . Unlike typical Ras proteins which promote cell proliferation, DIRAS1 functions as a negative growth regulator, inhibiting cell growth in various contexts .

How does DIRAS1 differ from other members of the Ras superfamily?

DIRAS1 contains specific amino acid substitutions in regions critical for GTP hydrolysis, particularly in the RAF kinase and Ha-RAS domains . These substitutions result in DIRAS1 having lower GTPase activity compared to other Ras proteins, causing it to predominantly exist in the GTP-bound form . This structural difference likely explains why DIRAS1 regulates cell development differently than other Ras superfamily members, functioning as a growth inhibitor rather than a growth promoter .

What is the tissue distribution pattern of DIRAS1 in normal human tissues?

DIRAS1 is highly expressed in heart and brain tissues . The Human Protein Atlas provides comprehensive information about DIRAS1 expression across human tissues, showing distinct expression patterns that differ from other Ras family members . The tissue-specific expression suggests specialized functions in these organs and may explain why disruption of DIRAS1 expression contributes to specific cancer types, particularly those affecting tissues where it is normally expressed.

What evidence supports DIRAS1's role as a tumor suppressor in human cancers?

Studies have demonstrated that DIRAS1 expression is significantly downregulated in multiple cancer types, including ovarian, cervical, glioblastoma, colorectal, and renal cell carcinomas . In cervical cancer research, knockdown of DIRAS1 significantly promoted the proliferation, growth, migration, and invasion of C33A and SiHa cells cultured in vitro, while overexpression of DIRAS1 inhibited cell viability and motility . Furthermore, DIRAS1 expression levels in tumor tissues negatively correlate with pathological grades in cervical cancer patients, suggesting its potential as a prognostic marker .

How do DIRAS1 and DIRAS2 compare in their tumor suppressive functions?

Both DIRAS1 and DIRAS2 function as tumor suppressors in ovarian cancer. Re-expression of these genes suppresses growth of human and murine ovarian cancer cells by inducing autophagy-mediated cell death . According to TCGA analysis, higher mRNA expression of both DIRAS1 and DIRAS2 correlates with improved survival advantages for patients with high-grade serous ovarian cancer . Mechanistically, both proteins induce and regulate autophagy by inhibiting the AKT1-MTOR and RAS-MAPK signaling pathways, and by modulating nuclear localization of autophagy-related transcription factors FOXO3/FOXO3A and TFEB .

What epigenetic mechanisms regulate DIRAS1 expression in cancer cells?

DIRAS1 expression is regulated by multiple epigenetic mechanisms:

• DNA methylation and histone deacetylation: Treatment with DNA methylation inhibitor (5-Azacytidine) and histone deacetylation inhibitor (SAHA) significantly increases DIRAS1 mRNA levels in cervical cancer cells, suggesting epigenetic silencing at the transcriptional level .

• m6A RNA modification: This appears to be a critical regulatory mechanism for DIRAS1. FTO inhibitor (FB23-2) significantly down-regulates DIRAS1 mRNA levels while up-regulating DIRAS1 protein levels, indicating complex post-transcriptional regulation .

• m6A methyltransferases and demethylases: Down-regulation of METTL3 and METTL14 (m6A writers) significantly inhibits DIRAS1 protein expression, whereas down-regulation of FTO and ALKBH5 (m6A erasers) significantly increases DIRAS1 protein expression . This suggests m6A modification affects both DIRAS1 mRNA stability and translation efficiency.

What is the relationship between DIRAS family proteins and autophagy?

The DIRAS family, including DIRAS1, DIRAS2, and DIRAS3, plays essential roles in promoting autophagy:

• DIRAS3 and autophagy: DIRAS3 (ARHI) has been well-characterized for inducing autophagy in ovarian cancer cells, where re-expression promotes tumor dormancy and autophagy-mediated cell death .

• DIRAS1/2 as functional surrogates: Interestingly, the murine genome has lost DIRAS3 during evolutionary rearrangement but retained DIRAS1 and DIRAS2, which serve as functional surrogates to maintain autophagy capability . This evolutionary adaptation suggests the critical importance of DIRAS-mediated autophagy regulation.

• Mechanistic pathways: DIRAS1 and DIRAS2 promote autophagy by inhibiting AKT1-MTOR and RAS-MAPK signaling pathways, which are key negative regulators of autophagy . They also modulate the nuclear localization of autophagy-related transcription factors FOXO3/FOXO3A and TFEB .

What experimental approaches can be used to investigate DIRAS1's tumor suppressor function?

To investigate DIRAS1's tumor suppressor function, researchers can employ several complementary approaches:

• Gene expression manipulation: Knockdown of DIRAS1 using siRNA or CRISPR-Cas9 to observe changes in cancer cell proliferation, migration, and invasion as demonstrated in cervical cancer studies .

• Overexpression studies: Transfecting cancer cell lines with DIRAS1 expression vectors to assess effects on cell viability, motility, and apoptosis .

• Patient sample analysis: Comparing DIRAS1 mRNA and protein expression between cancer tissues and normal/para-cancerous tissues using qRT-PCR and immunohistochemistry, with correlation to clinical parameters and pathological grades .

• Pathway analysis: Examining the effects of DIRAS1 expression on AKT1-MTOR and RAS-MAPK signaling cascades using Western blotting and functional assays .

How can researchers study the m6A modification of DIRAS1?

Studying m6A modification of DIRAS1 requires specialized techniques:

• Pharmacological inhibitors: Using inhibitors of m6A-related enzymes, such as FTO inhibitor (FB23-2), to manipulate m6A levels and observe effects on DIRAS1 expression .

• Gene knockdown: Silencing m6A writers (METTL3, METTL14) and erasers (FTO, ALKBH5) to observe changes in DIRAS1 mRNA stability and protein translation .

• m6A-seq: Performing methylated RNA immunoprecipitation sequencing to map m6A modification sites on DIRAS1 mRNA.

• Luciferase reporter assays: Constructing wild-type and mutant DIRAS1 3'UTR reporters to examine the functional consequences of m6A modifications on translation efficiency.

What structural analysis methods have been used to characterize DIRAS1?

The crystal structure of human DIRAS1 GTPase in the inactive GDP-bound state has been determined . Key methodological approaches include:

• X-ray crystallography: Used to resolve the three-dimensional structure of DIRAS1 in complex with GDP, revealing unique structural features that differentiate it from other Ras superfamily members .

• Structural comparison: Comparing DIRAS1 with other Ras proteins to identify the critical amino acid substitutions that result in reduced GTPase activity and different functional properties .

• Protein-protein interaction studies: Investigating how DIRAS1's structure enables its interactions with downstream effectors different from those of typical Ras proteins.

How can autophagy induction by DIRAS1 be measured experimentally?

To measure DIRAS1-induced autophagy, researchers can employ:

• LC3 conversion assay: Monitoring the conversion of LC3-I to LC3-II (a marker of autophagosome formation) via Western blotting after DIRAS1 expression manipulation.

• Fluorescence microscopy: Using GFP-LC3 to visualize autophagosome formation and quantify autophagic flux in response to DIRAS1 expression.

• Autophagic flux assays: Using lysosomal inhibitors (e.g., chloroquine, bafilomycin A1) in combination with DIRAS1 expression to measure true autophagic flux rather than just autophagosome accumulation.

• Pathway intervention: Testing whether DIRAS1-induced effects can be blocked by autophagy inhibitors (3-methyladenine, wortmannin) or enhanced by autophagy inducers (rapamycin) .

How does DIRAS1 expression correlate with patient outcomes in different cancers?

DIRAS1 expression has shown significant correlations with clinical outcomes:

The consistent finding that DIRAS1 expression correlates positively with better outcomes across multiple cancer types reinforces its role as a bona fide tumor suppressor and suggests its potential utility in cancer prognosis and therapy development.

What therapeutic strategies could target or leverage DIRAS1 function in cancer?

Potential therapeutic strategies based on DIRAS1 biology include:

• Epigenetic modulators: Using DNA methylation inhibitors or histone deacetylase inhibitors to restore DIRAS1 expression in cancers where it is epigenetically silenced .

• m6A modification targeting: Developing therapies that target m6A writers or erasers to modulate DIRAS1 expression and function .

• Autophagy modulation: Since DIRAS1 functions partly through autophagy induction, combining DIRAS1-targeting approaches with autophagy modulators could enhance therapeutic efficacy .

• Gene therapy approaches: Developing vectors for DIRAS1 re-expression in tumors where it is downregulated could potentially inhibit cancer growth and enhance sensitivity to conventional therapies.

These approaches represent promising directions for translating DIRAS1 research findings into clinical applications, though each would require extensive preclinical validation before human testing.