Recombinant Suncus murinus Type I iodothyronine deiodinase (DIO1)
Description
Introduction
Iodothyronine deiodinases (DIOs) are a family of selenoproteins that regulate thyroid hormone (TH) action by catalyzing the deiodination of thyroid hormones, influencing their activity and availability in a tissue-specific manner . Among these, Type I iodothyronine deiodinase (DIO1) plays a crucial role in both the activation and inactivation of thyroid hormones . Specifically, DIO1 converts thyroxine (T4) to the more active 3,3',5-triiodothyronine (T3) and can also inactivate both T4 and T3 via deiodination to reverse T3 (rT3) and 3,3'-T2, respectively .
The house musk shrew (Suncus murinus) has been noted to have low T4 to T3 converting activity . Recombinant Suncus murinus DIO1 refers to the Suncus murinus DIO1 enzyme produced through recombinant DNA technology. This approach allows for the production of large quantities of the enzyme for research purposes, overcoming the limitations of obtaining it from natural sources .
Expression and Production of Recombinant DIO1
Due to the challenges associated with purifying native DIO1, which include low expression levels and the fact that deiodinases are membrane proteins, recombinant expression systems are often employed .
Saccharomyces cerevisiae has been used to express DIO1 at high yield, but the resulting protein was not catalytically active . A more successful approach involves expressing human DIO1 recombinantly in High5 insect cells using a baculoviral transduction system . To facilitate immunodetection, a C-terminal 8xHis-tag, preceded by an enterokinase cleavage site, is often added .
3.1. Dimerization of DIO1
The possibility of DIO1 forming a homodimer has been investigated, with conserved cysteines potentially playing a role in stabilizing the dimer . Mutational analyses have indicated that Cys95 and Cys105 are involved in the formation of a stable homodimer of DIO1 .
Experiments using non-reducing SDS-PAGE followed by Western blotting revealed that a dimeric band was readily observed in DIO1 U126C (a mutant where selenocysteine (Sec) is replaced by cysteine (Cys)), which decreased in intensity upon incubation with reductants. In contrast, DIO1 U126C/C95A and DIO1 U126C/C105S mutants did not show a dimer band, suggesting the importance of these cysteine residues in dimerization .
3.2. Reduction Mechanism
The reduction of DIO1 has been compared to that of murine DIO3, with a focus on the roles of specific cysteine residues . It was found that a thioredoxin-regenerating system was unable to reduce DIO1 U126C, while a glutathione-regenerating system was effective . Mutating Cys95 or Cys105 did not reduce the activity of recombinant DIO1 U126C with DTT or the GSH-reducing system, further arguing against a functional role of potential intermolecular disulfides at these positions .
3.3. Identification of Disulfides by Mass Spectrometry
Mass spectrometry was used to identify potential inter- and intramolecular disulfides in DIO1 U126C . While no evidence for intermolecular disulfides (Cys95-Cys95, Cys95-Cys105, or Cys105-Cys105) was found, an intramolecular disulfide bond between Cys124 and Cys126 was clearly demonstrated in the 30 kDa band . This finding provides direct evidence for the existence of such an intramolecular disulfide .
3.4. Proton Relay Pathway
Conserved amino acids, previously suggested to participate in a hydrogen-bonded proton relay network in DIO3, are also conserved in DIO1 . Mutational analysis revealed that the hydroxyl function of Ser123 is involved in DIO1 activity, while the hydroxyl of Thr125 is essential .
Functional Assays and Applications
Recombinant DIO1 is utilized in various in vitro assays to assess the effects of different compounds on thyroid hormone metabolism . These assays are crucial for evaluating the potential of chemicals to disrupt the thyroid system .
One such assay, the DIO1-SK assay, has been developed to investigate DIO1 inhibition . This assay uses recombinant enzyme as the DIO1 protein source . The results of the DIO1-SK assay can be used for regulatory decision-making, with test substances classified into categories based on their inhibitory activity:
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Category 1: Substances leading to complete DIO1 inhibition (>90% inhibition)
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Category 2: Substances with inhibitory activity not resulting in complete DIO1 inhibition (between 90% and 25% inhibition)
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Category 3: Substances that do not inhibit DIO1 (<25% inhibition)
The IC50 value for DIO1 using human liver microsomes has been determined to be 7.4 μM, while the IC50 value for recombinant enzymes was 2.5-fold higher .
4.1. Data Interpretation
Data interpretation involves defining a procedure to identify substances that do not have an effect on DIO1 activity. The distribution of the IRA values of controls that can inhibit DIO1 and of controls that have no effect on DIO1 are compared.
Table 1. Distribution of IRA Values of Controls
| Control | Mean | SD | Range of mean +/−3 *SD |
|---|---|---|---|
| IRA of solvent control, % | 100.0 | 6.9 | 79.2; (120.8) |
| IRA of negative control, % | 99.9 | 9.1 | 72.6; (127.2) |
| IRA of reference item, % | 0.00 | 3.1 | (−9.4); 9.4 |
| IRA of positive control, % | 1.0 | 4.1 | (−11.2); 13.2 |
Product Specs
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Target Background
This protein is responsible for the deiodination of thyroxine (T4) to triiodothyronine (T3) and the subsequent deiodination of T3 to 3,3'-diiodothyronine (T2).
Q&A
How does Suncus murinus DIO1 compare structurally to DIO1 in other species?
The deduced amino acid sequence of Suncus murinus DIO1 shows high homology to other known mammalian DIO1 enzymes, with similar catalytic properties . Like all vertebrate DIO1 enzymes, it contains a selenocysteine residue in its catalytic center, which is essential for its enzymatic function .
Despite structural similarities, Suncus murinus DIO1 exhibits important functional differences:
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It has catalytic activity similar to other DIO1 enzymes but with unique tissue distribution
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Its expression is not upregulated by T3 administration, unlike in other mammals
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Unlike other species where DIO1 is expressed in multiple tissues, in Suncus murinus DIO1 activity is detected primarily in liver, with absence in kidney
These differences suggest that while the structural backbone of DIO1 is evolutionarily conserved, the regulatory mechanisms and physiological role of the enzyme in Suncus murinus have undergone significant adaptations.
What are the unique characteristics of thyroid hormone metabolism in Suncus murinus?
Suncus murinus displays several distinctive features in thyroid hormone metabolism that differentiate it from typical laboratory mammals:
| Parameter | Suncus murinus | Rat | Physiological Significance |
|---|---|---|---|
| Serum T4 | Higher | Lower | Different thyroid hormone set point |
| Serum rT3 | Higher | Lower | Altered peripheral metabolism |
| Serum T3 | Lower | Higher | "Low T3 state" despite normal health |
| Liver DIO1 activity | Low | High | Reduced peripheral T4-to-T3 conversion |
| Kidney DIO1 activity | Very low/absent | High | Altered organ contribution to T3 production |
| BAT DIO1 activity | High | Low | Specialized role in thermogenesis |
| DIO2 activity in brain and BAT | Very high | Moderate | Compensatory mechanism for T3 production |
| T3 regulation of DIO1 | Not upregulated by T3 | Upregulated by T3 | Different regulatory mechanisms |
Despite having lower DIO1 activity in typical T3-producing tissues (liver and kidney), Suncus murinus maintains serum T3 levels comparable to other mammals, suggesting that DIO2 contributes significantly to T3 production and maintenance . This represents a unique thyroid hormone economy that may relate to the species' metabolic needs and evolutionary history.
What experimental methods are used to measure DIO1 activity in Suncus murinus tissues?
Several complementary approaches are employed to measure DIO1 activity in Suncus murinus tissues:
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Tissue homogenate assays: Tissues (liver, kidney, BAT, etc.) are homogenized and incubated with specific substrates (T4, rT3, T3). Deiodination rates are measured to assess enzyme activity .
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Molecular characterization: The cDNA encoding DIO1 has been cloned, allowing for sequence analysis and recombinant protein expression .
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Inhibitor responses: The response to 6-n-propyl-2-thiouracil and iopanoic acid helps distinguish between DIO1 and DIO2 activity in tissue homogenates .
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Substrate preference analysis: Different substrates (T4, rT3, T3) are used to characterize the enzyme's substrate specificity .
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Standardized DIO1-SK assay: For recombinant enzyme studies, the Sandell–Kolthoff reaction-based type 1 deiodinase assay provides a standardized methodology for measuring activity and testing inhibitors .
For recombinant Suncus murinus DIO1, additional considerations include:
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Expression in appropriate cell systems with selenocysteine incorporation machinery
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Purification protocols that maintain enzyme activity
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Proper storage conditions: -20°C for standard storage, -20°C or -80°C for extended storage, avoiding repeated freeze-thaw cycles
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Buffer optimization: Tris-based buffer with 50% glycerol appears optimal
How does the distribution of DIO1 activity across tissues in Suncus murinus differ from other mammals, and what are the physiological implications?
Suncus murinus exhibits a dramatically different tissue distribution of DIO1 activity compared to typical mammals:
| Tissue | Suncus murinus DIO1 Activity | Rat DIO1 Activity | Implications |
|---|---|---|---|
| Liver | Present but <50% of rat level | High | Reduced systemic T3 production |
| Kidney | Absent/very low | High | Loss of renal contribution to T3 pool |
| BAT | Very high | Low | Enhanced local T3 production in thermogenic tissue |
| Other tissues | Intermediate/low | Variable | Tissue-specific T3 regulation |
The physiological implications of this unique distribution are significant:
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Altered thyroid hormone economy: The high BAT DIO1 activity suggests prioritization of local T3 production in thermogenic tissue, potentially serving adaptation to the species' metabolic demands .
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Compensatory mechanisms: Despite lower hepatic and renal DIO1 activity, serum T3 levels remain normal, indicating effective compensatory mechanisms primarily via DIO2 .
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Evolutionary adaptation: This pattern may represent an evolutionary adaptation in deiodinase function to meet specific metabolic needs of this insectivorous species with its simple gastrointestinal tract and unique nutritional physiology .
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Thermoregulation: The high DIO1 activity in BAT suggests specialized regulation of thermogenesis, potentially important for an animal with low body fat .
This unique distribution pattern makes Suncus murinus a valuable model for studying tissue-specific regulation of thyroid hormone metabolism and the differential roles of deiodinases in maintaining thyroid hormone homeostasis.
What is the role of DIO1 versus DIO2 in maintaining T3 levels in Suncus murinus?
In Suncus murinus, the traditional roles of DIO1 and DIO2 in thyroid hormone economy appear to be significantly reorganized:
| Parameter | Suncus murinus | Typical Mammals | Significance |
|---|---|---|---|
| Primary source of circulating T3 | DIO2 (likely) | DIO1 | Shift in enzyme contribution |
| Liver DIO1 activity | Low (<50% of rat) | High | Reduced hepatic T3 production |
| DIO1 regulation by T3 | Not upregulated | Upregulated | Different feedback mechanisms |
| DIO2 activity in BAT and brain | Very high | Moderate | Enhanced contribution to T3 pool |
| Response to physiological challenges | Not fully characterized | Well-characterized | Research opportunity |
Research explicitly concludes: "These results suggest that D2 contributes to the production and maintenance of T3 levels in the house musk shrew" . This represents a fundamental shift from the typical mammalian pattern, where DIO1 is usually considered the predominant source of circulating T3.
This adaptation may reflect:
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The specific metabolic requirements of Suncus murinus as an insectivore
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The need for precise regulation of thermogenesis given its low body fat
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Evolutionary adaptations in thyroid hormone economy
From a research perspective, this makes Suncus murinus an excellent model for understanding the plasticity of deiodinase contributions to thyroid hormone homeostasis and the redundant mechanisms that maintain T3 levels despite variations in enzyme expression patterns.
What experimental considerations should be taken into account when using recombinant Suncus murinus DIO1 in in vitro studies?
When working with recombinant Suncus murinus DIO1, researchers should consider several critical factors:
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Expression systems:
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Storage conditions:
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Buffer optimization:
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Cofactor requirements:
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Substrate considerations:
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Inhibitor responses:
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Assay validation:
These considerations are essential for generating reliable data when working with this unique selenoenzyme.
How do inhibitors like 6-n-propyl-2-thiouracil and iopanoic acid affect Suncus murinus DIO1 compared to other species?
The response of Suncus murinus DIO1 to common deiodinase inhibitors shows some distinctive features compared to other species:
| Inhibitor | Effect on Suncus murinus DIO1 | Effect on Rat/Human DIO1 | Notes |
|---|---|---|---|
| 6-n-propyl-2-thiouracil (PTU) | Sensitive, used to distinguish DIO1 from DIO2 | Highly sensitive | May show quantitative differences in IC50 |
| Iopanoic acid | Sensitive, used to identify deiodinase types | Sensitive | Used alongside substrate preference to characterize enzyme type |
While specific data on inhibition patterns are not comprehensively detailed in the available literature, research indicates that these inhibitors are useful for distinguishing between DIO1 and DIO2 activities in Suncus murinus tissues: "Liver and kidney 5'D may be type-I and that of BAT may be type-II in the shrew, judging from its response to 6-n-propyl-2-thiouracil and iopanoic acid and substrate preference" .
It's important to note that evolutionary diversity in DIO1 regarding PTU sensitivity has been observed across species. Some non-mammalian vertebrate DIO1 enzymes show significantly reduced sensitivity to PTU compared to mammalian DIO1 . While detailed comparative inhibition studies specific to Suncus murinus DIO1 are not fully characterized in the available literature, researchers should be aware that species-specific differences in inhibitor responses may exist and should empirically determine inhibition parameters when working with this enzyme.
How might the unique characteristics of Suncus murinus DIO1 contribute to research on thyroid hormone metabolic disorders?
The distinctive features of Suncus murinus DIO1 and thyroid hormone metabolism offer several valuable research opportunities:
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Model for Low T3 syndromes: Suncus murinus naturally maintains lower serum T3 levels while remaining healthy, potentially providing insights into adaptive mechanisms relevant to nonthyroidal illness syndrome and other low T3 states in humans .
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Alternative T3 production pathways: Despite reduced DIO1 activity in typical T3-producing tissues, Suncus murinus maintains normal serum T3 levels through enhanced DIO2 activity, offering a model for studying compensatory mechanisms in conditions with impaired DIO1 function .
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Tissue-specific thyroid hormone metabolism: The unique distribution of deiodinase activity (high in BAT, low in liver/kidney) provides a natural model for studying tissue-specific effects of altered thyroid hormone activation .
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Thyroid hormone metabolism in special physiological states: As an animal with minimal body fat but normal glucose metabolism, Suncus murinus may provide insights into thyroid hormone adaptation to unique metabolic demands .
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Brown adipose tissue research: The high DIO1 activity in BAT of Suncus murinus could inform research on thermogenesis and metabolic regulation, with potential relevance to obesity and metabolic disorders .
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Development of selective deiodinase modulators: Understanding the unique characteristics of Suncus murinus DIO1 could contribute to designing selective inhibitors or modulators for therapeutic purposes in thyroid disorders .
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Comparative evolutionary adaptations: Studying the differences between Suncus murinus DIO1 and other species' DIO1 can provide insights into how mammals have evolved varying strategies for thyroid hormone regulation .
The naturally occurring variations in Suncus murinus thyroid hormone economy make it a valuable comparative model for understanding the flexibility and adaptation potential of the thyroid hormone regulatory system.
