NUDT9 Human

Basic Research Questions

• What is NUDT9 and what is its primary enzymatic function?

Human NUDT9 is a member of the Nudix hydrolase superfamily that specifically catalyzes the hydrolysis of ADP-ribose (ADPR) to AMP and ribose 5'-phosphate . This enzyme belongs to a broader superfamily of Nudix hydrolases that catabolize potentially toxic compounds in the cell .

Methodological consideration: When studying NUDT9 enzymatic activity, researchers should maintain near-neutral pH conditions and include divalent metal ions, as these are required for optimal catalytic activity . Unlike some related bacterial enzymes that function as homodimers, human NUDT9 is active as a monomer, which should be considered when designing purification and activity assays .

• What is the domain structure of human NUDT9?

Human NUDT9 consists of two distinct domains with different functional roles:

• N-terminal domain: Features a novel fold structure and is proteolytically labile . This domain does not directly participate in catalysis but enhances the affinity of the C-terminal domain for ADPR .

• C-terminal domain: Contains the Nudix motif responsible for catalytic activity . This domain is proteolytically resistant and retains essentially full ADPR pyrophosphatase activity even when isolated .

The substrate ADPR binds in a cleft between these two domains . Crystal structure analysis has revealed this arrangement differs from that of the E. coli ADPRase (a close functional homolog), which functions as a homodimer .

• What are the different isoforms of NUDT9 and their subcellular localization?

Analysis of the human NUDT9 gene has identified two distinct isoforms with different subcellular targeting:

• NUDT9α (full-length): The dominant form containing an N-terminal mitochondrial targeting sequence. Using green fluorescence protein tagging, researchers have demonstrated that NUDT9α localizes specifically to mitochondria .

• NUDT9β: An alternative transcript that appears to result from a potentially aberrant splice from a cryptic donor site within the first exon. This isoform lacks the N-terminal targeting sequence and consequently exhibits no clear subcellular localization .

Methodological approach: When investigating NUDT9 localization, GFP tagging followed by fluorescence microscopy provides a reliable technique for visualizing subcellular distribution patterns. Co-localization markers for specific organelles (particularly mitochondria) should be included in experimental designs .

• How does NUDT9 relate to the TRPM2 ion channel?

NUDT9 shares 39% sequence identity with the C-terminal cytoplasmic domain of the ADPR-gated calcium channel TRPM2, a region known as the NUDT9 homology (NUDT9H) domain . This relationship has several important research implications:

• The NUDT9H domain in TRPM2 exhibits low but specific enzymatic activity compared to the standalone NUDT9 enzyme .

• In human TRPM2, gating requires the C-terminal NUDT9H domain as an ADPR-binding module .

• Species variations exist in ADPR-binding mechanisms:

  • Sea anemone TRPM2: NUDT9H is dispensable; ADPR binding occurs N-terminally .

  • Zebrafish TRPM2: Both the N-terminal ADPR-binding pocket and NUDT9H are necessary .

• The NUDT9H domains of human and zebrafish TRPM2 are interchangeable, as chimeras generate ADPR-sensitive channels .

Advanced Research Questions

• What structural features of NUDT9 are revealed by crystallography and how do they inform functional studies?

Crystal structures of human NUDT9 have been determined both with and without the reaction product ribose 5'-phosphate . These structures provide critical insights for designing experiments:

Methodological approach: When designing mutational studies, researchers should prioritize residues in the active site cleft between domains, as these are likely to be most critical for substrate recognition and catalysis, based on the crystal structure information .

• What methodologies are most effective for studying NUDT9 enzymatic activity?

Multiple complementary approaches can be employed to characterize NUDT9 enzymatic activity:

NUDT9 activity is optimal at near-neutral pH and requires divalent metal ions and an intact Nudix motif . When designing activity assays, researchers should consider these requirements for optimal results.

• How do mutations in NUDT9 affect its catalytic activity and what mechanistic insights do they provide?

Mutational studies have revealed key aspects of NUDT9's catalytic mechanism:

• Nudix motif mutations: Alterations within the conserved Nudix motif (GX₅EX₇REUXEEXGU) significantly impair catalytic activity, confirming its essential role in catalysis .

• RIF to RIL double mutation: This change mimics the Nudix signature of the TRPM2 ion channel domain and results in reduced catalytic efficiency, explaining the lower activity of the NUDT9H domain in TRPM2 .

• N-terminal domain deletions: Truncated constructs missing the N-terminal domain show reduced activity, confirming that while the C-terminal domain retains catalytic function, the N-terminal domain enhances substrate binding affinity .

• Active site mutations: Alterations of potential general bases in NUDT9 produce effects that differ from those observed in E. coli ADPRase, suggesting divergent reaction mechanisms despite functional similarity .

• What is the substrate specificity of NUDT9 and how can it be experimentally determined?

NUDT9 exhibits high specificity for ADP-ribose as its primary substrate, though recent research has identified additional substrates:

• Primary substrate: ADP-ribose (ADPR)

• Additional substrate: 2'-deoxy-ADPR has been identified as another substrate for NUDT9

Methodological approaches for determining substrate specificity include:

• Enzyme activity screening: Testing activity against a panel of structurally related compounds and measuring product formation.

• Binding studies: Using isothermal titration calorimetry (ITC) or surface plasmon resonance (SPR) to quantify binding affinities.

• Competition assays: Testing inhibition of ADPR hydrolysis by potential substrates.

• Structural studies: Co-crystallization with various ligands to identify binding modes.

• Thermal stability assays: Differential scanning fluorimetry to detect ligand-induced stabilization.

These approaches can be used in combination to comprehensively characterize substrate specificity and identify potential physiological substrates beyond ADPR.

• What techniques are used to study the conformational changes in NUDT9 upon ligand binding?

Various biophysical techniques can reveal conformational dynamics in NUDT9:

Recent studies have demonstrated that NUDT9 undergoes significant conformational changes upon ligand binding, with the structure determined in the presence of ribose 5'-phosphate revealing specific substrate-induced rearrangements . Researchers should consider using a combination of these methods for a comprehensive understanding of NUDT9 dynamics.

• How does the NUDT9H domain interact with the channel domain in TRPM2, and what methods can be used to study this interaction?

The interaction between the NUDT9H domain and the channel domain in TRPM2 is critical for channel function. Recent research has revealed that this interaction can occur both in cis (within the same polypeptide) and in trans (between separate proteins) .

Effective methodologies for studying this interaction include:

• Co-immunoprecipitation (Co-IP): This technique has successfully demonstrated specific in-trans interaction between NUDT9H domain and C-terminally truncated human TRPM2 channel .

• Proximity Ligation Assay (PLA): This method provides visualization of protein interactions in situ with single-molecule sensitivity .

• Functional reconstitution: Co-expression of separated domains (hsTRPM2ΔNUDT9H with wild-type hsNUDT9H) can partially restore channel function, as demonstrated through calcium imaging experiments .

• Electrophysiology: Patch-clamp recordings can assess ADPR-dependent current development in cells expressing wild-type or mutant constructs .

• Cryo-electron microscopy: Recent cryo-EM analyses have provided concrete structural evidence of how NUDT9H interacts with the channel domain .

Research has shown that a point mutation at a highly conserved position within NUDT9H induces loss-of-function in both human and zebrafish TRPM2 channels, highlighting critical interaction points .