Recombinant Mus caroli Nucleoside diphosphate-linked moiety X motif 19, mitochondrial (Nudt19)

What is the subcellular localization of Nudt19?

Despite previous reports suggesting mitochondrial localization, current evidence strongly supports that Nudt19 is predominantly a peroxisomal enzyme. Fluorescence microscopy studies have confirmed this localization through colocalization experiments. When expressed with an N-terminal GFP tag that leaves the PTS1 (peroxisomal targeting signal 1) exposed, Nudt19 colocalizes with the peroxisomal marker PMP70, visualized as yellow pixels resulting from the overlap of green (GFP-Nudt19) and red (PMP70) signals . Importantly, masking the PTS1 of Nudt19 with a C-terminal GFP tag prevents peroxisomal localization but does not redirect the protein to mitochondria, resulting instead in diffuse cytoplasmic localization . This confirms that Nudt19 is specifically targeted to peroxisomes via its PTS1 signal.

How does Nudt19 differ structurally from other Nudix hydrolases?

Nudt19 exhibits several unique structural features compared to other Nudix hydrolases. It displays modest sequence similarity (~30%) to the better-characterized Nudt7 enzyme . Notably, Nudt19 is larger than typical Nudix hydrolases and contains a distinctive 45-49 amino acid insertion within its Nudix box, which is the catalytic domain characteristic of this enzyme family . This structural difference may contribute to Nudt19's substrate specificity and regulatory properties. When designing experiments to study Nudt19, researchers should account for these structural differences when applying methods optimized for other Nudix hydrolases.

What is the tissue distribution of Nudt19?

Nudt19 exhibits a highly tissue-specific expression pattern. Both mRNA and protein analyses consistently demonstrate that Nudt19 is most abundantly expressed in the kidneys . Lower but detectable levels can be found in skeletal muscle and brain . This distinctive tissue distribution suggests a specialized role for Nudt19 in kidney metabolism. When designing tissue-specific studies, researchers should consider this distribution pattern and prioritize kidney samples for most Nudt19-focused investigations.

How is Nudt19 activity regulated in vivo?

Nudt19 activity appears to be regulated by metabolites rather than changes in expression levels. While Nudt19 mRNA and protein expression remain stable during feeding and fasting conditions (even during prolonged fasting of up to 48 hours), its activity appears to be higher in the fed state compared to the fasted state, as evidenced by increased kidney CoA levels in Nudt19-/- mice specifically in the fed state .

Regulatory metabolite screening has identified a select group of bile acids as potent inhibitors of Nudt19 activity. Specifically, chenodeoxycholic acid (CDCA) and its conjugated derivatives (taurochenodeoxycholic acid and glycochenodeoxycholic acid) decrease Nudt19 activity by 60-75% compared to controls . Other hydrophobic bile acids including lithocholic acid and ursocholanic acid also potently inhibit Nudt19, while α-muricholic acid (derived from CDCA) retains significant inhibitory potency . This inhibition appears highly specific, as other steroid compounds (progesterone, pregnenolone, and corticosterone) show no significant effect on enzyme activity .

What phenotypic changes occur in Nudt19-/- mice fed different diets?

Nudt19-/- mice exhibit diet-dependent phenotypic changes that provide insight into the functional role of this enzyme:

The exacerbated albuminuria in Nudt19-/- mice fed HFD suggests that Nudt19 may play a protective role against diet-induced kidney damage. The absence of significant changes in total kidney CoA levels in HFD-fed mice despite the enzyme's known CoA diphosphohydrolase activity suggests that Nudt19 may only access and regulate a specific subcellular pool of CoA, likely the peroxisomal pool .

How does Nudt19 deletion affect the kidney metabolome and proteome?

Untargeted metabolomics analysis of kidneys from HFD-fed mice identified 126 significantly changed metabolites out of 839 detected compounds when comparing Nudt19-/- to wild-type mice . Among these, 55 were increased and 71 were decreased in Nudt19-/- mice . Pathway enrichment analysis revealed significant alterations in lipid metabolism, particularly affecting non-esterified fatty acids (NEFAs) .

Long-chain fatty acids showed a global decrease across all classes (including essential, monounsaturated, polyunsaturated, and branched-chain fatty acids) . This correlated with reduced albumin reabsorption and may explain the decreased concentrations of short branched-chain acyl-CoA species such as isovaleryl/methylbutyryl-CoA, which are generated in peroxisomes through oxidation and shortening of branched-chain fatty acids .

Proteomics analysis identified several significantly altered proteins, including increased expression of enzymes involved in peroxisomal and mitochondrial β-oxidation (ECH1, THIKB, ECHD2), suggesting compensatory metabolic adaptations in response to Nudt19 deletion .

What are optimal conditions for measuring Nudt19 enzymatic activity in vitro?

When assessing Nudt19 enzymatic activity, several key experimental parameters should be optimized:

How can researchers generate and validate Nudt19 knockout models?

The generation and validation of Nudt19 knockout (Nudt19-/-) models involve several critical steps:

• Acquisition of heterozygous mice: Nudt19+/- mice can be obtained from repositories such as the KOMP (Knockout Mouse Project) repository .

• Breeding strategy: Heterozygous mice can be bred to generate Nudt19-/- mice and wildtype littermate controls for experimental comparisons .

• Genotype verification: PCR analysis of tail biopsies can be used to verify the genotype of the mice .

• Protein expression confirmation: Western blotting using a specific anti-Nudt19 antibody (such as a polyclonal antibody against full-length mouse Nudt19) can confirm the absence of Nudt19 protein expression in knockout mice .

• Enzymatic activity validation: Measuring CoA diphosphohydrolase activity in tissue homogenates, particularly kidney samples where Nudt19 is highly expressed, can confirm the functional consequence of gene deletion. Specifically, the absence of 3',5'-ADP formation in kidney homogenates confirms successful functional deletion of Nudt19 .

How should researchers interpret changes in CoA levels in Nudt19-/- mice?

The interpretation of CoA level changes in Nudt19-/- mice requires consideration of several factors:

What are the recommended approaches for studying Nudt19 in kidney disease models?

When investigating Nudt19 in kidney disease models, researchers should consider the following approaches:

• Diet intervention: High-fat diet (HFD) feeding provides a valuable model as it reveals Nudt19-dependent phenotypes not observed with standard chow. HFD feeding for 15 weeks has been shown to decrease NUDT19 and NUDT7 protein levels in wild-type mouse kidneys .

• Histological analysis: Examination of renal cortex areas can reveal vacuolated tubular epithelial cells, which increase in frequency with HFD feeding in both wild-type and Nudt19-/- mice .

• Kidney function assessment:

  • 24-hour urine collection for sodium excretion analysis

  • Measurement of TBARS (thiobarbituric acid reactive substances) as a marker of lipid peroxidation

  • Determination of albumin-to-creatinine ratio (ACR), which is significantly elevated in Nudt19-/- mice fed HFD

• Metabolic profiling: Untargeted metabolomics can identify broad metabolic changes, with particular attention to lipid metabolism pathways that are significantly altered in Nudt19-/- mice .

• Proteomic analysis: Mass spectrometry-based proteomic approaches can identify changes in protein expression, such as the increased levels of enzymes involved in peroxisomal and mitochondrial β-oxidation observed in Nudt19-/- mice fed HFD .

• Blood pressure measurement: Assessment of systolic and diastolic blood pressure may reveal genotype-dependent effects, as Nudt19-/- mice fed HFD tend to have lower blood pressure compared to wild-type males .

How does Nudt19 function compare to other CoA-degrading Nudix hydrolases?

Nudt19 shares functional similarity with other CoA-degrading Nudix hydrolases, particularly Nudt7 and Nudt8, but exhibits distinct characteristics:

• Substrate specificity: While both Nudt19 and Nudt7 hydrolyze various acyl-CoAs, they show different substrate preferences. Nudt19 readily hydrolyzes short and medium chain acyl-CoAs, malonyl-CoA, succinyl-CoA, and free CoA, but has poor activity against acetyl-CoA and mBB-CoA, which are excellent substrates for Nudt7 .

• Enzymatic efficiency: Under comparable conditions (4 mM MgCl₂), the specific activity per pmol of Nudt19 is lower than that of Nudt7 . This suggests differences in catalytic efficiency between these enzymes.

• Tissue distribution: Nudt19 is predominantly expressed in the kidneys, with lower levels in skeletal muscle and brain . In contrast, Nudt7 is primarily expressed in the liver. This distinct tissue distribution suggests specialized roles for these enzymes in different organs.

• Regulatory mechanisms: Nudt19, but not Nudt7, is competitively inhibited by specific bile acids, particularly chenodeoxycholic acid and its derivatives . This unique regulatory mechanism could allow for tissue-specific modulation of CoA metabolism.

• Response to nutritional status: While Nudt19 activity appears higher in the fed state (based on CoA accumulation in knockout mice), the expression levels of the enzyme remain stable during feeding and fasting . This suggests post-translational regulation that differs from other Nudix hydrolases.

What are the key unanswered questions about Nudt19?

Despite significant advances in understanding Nudt19 function, several important questions remain: