Recombinant Gillichthys mirabilis Urotensin-2

What is Gillichthys mirabilis Urotensin-2 and what is its significance in research?

Urotensin-II (U-II) is a cyclic peptide first isolated from the urophysis (neurosecretory system) of the teleost fish Gillichthys mirabilis by Karl Lederis and Howard Bern in the 1960s . This peptide was originally characterized based on its potent smooth muscle contracting and hypertensive effects . The structure of the goby fish U-II is H-Ala-Gly-Thr-Ala-Asp-c[Cys-Phe-Trp-Lys-Tyr-Cys]-Val-OH .

Initially, U-II was thought to be exclusively produced by fish and involved in osmoregulation. Subsequent research revealed that U-II exists across vertebrate species from frogs to humans, making it a significant research target for understanding evolutionarily conserved signaling systems . The human isoform identification in 1998 and the subsequent discovery of its receptor (UT receptor, previously known as GPR14) in 1999 catalyzed intensive research into its role in human physiology and pathophysiology, particularly in cardiovascular regulation .

How does the structure of Urotensin-II vary across species and what regions are crucial for its activity?

The length of urotensin-II peptides varies across species, ranging from 17 amino acid residues in mice to 11 in humans, depending on the proteolytic cleavages that occur in precursors . The N-terminus region demonstrates high variability among different animal species, reflecting potential species-specific adaptations in function .

In contrast, the C-terminal amino acids organized in a disulphide-linked cyclic array, c[Cys-Phe-Trp-Lys-Tyr-Cys], are continuously conserved from species to species, suggesting their critical role in biological activity . Structure-function studies involving alanine scanning of truncated goby U-II demonstrated that the replacement of Trp, Lys, and Tyr is crucial for maintaining biological activity, indicating that the sequence Trp-Lys-Tyr within U-II is essential for binding and activation of the receptor .

How is Urotensin-II synthesized in biological systems?

Like other peptide ligands, U-II is synthesized from a larger precursor molecule known as Prepro-urotensin-II . In humans, two isoforms of this precursor have been identified, with lengths of 124 and 139 residues . Cleavage of either of these precursors produces identical mature U-II peptides consisting of eleven residues .

The gene encoding prepro-U-II is expressed not only in the caudal portion of the spinal cord but also in brain neurons, from frogs to humans . This widespread expression pattern indicates that U-II serves functions beyond its originally identified role in fish osmoregulation.

What is known about the Urotensin-II receptor and its signaling pathway?

The human U-II receptor (UT, formerly known as GPR14) was identified in 1999 and is a G-protein coupled receptor (GPCR) that primarily signals through the Gq pathway . Human UT is encoded on chromosome 17q25.3, is intronless, and comprises 389 amino acids . Several single nucleotide polymorphisms of both U-II and UT have been identified, although their association with various cardiovascular disease phenotypes remains largely undefined .

When U-II binds to UT, it activates phospholipase C, leading to the liberation of inositol (1,4,5) trisphosphate [Ins(1,4,5)P3] . This second messenger interacts with the Ins(1,4,5)P3 receptor located on the endoplasmic/sarcoplasmic reticulum to release Ca2+ from intracellular stores, triggering tissue-dependent responses . In cardiomyocytes, this typically leads to increased contractility, while in the vasculature, both constrictor and dilator responses have been observed depending on whether the receptor is located on vascular smooth muscle cells or endothelium, respectively .

How does the pharmacological profile of Urotensin-II vary across different experimental models?

The pharmacological responses to U-II show remarkable variability across experimental models and tissue types . While U-II is currently described as the most potent vasoconstrictor known, the responses it produces are extremely variable and often of low efficacy . This variability presents significant challenges for researchers attempting to characterize its function.

For example, SB-710411, a UT receptor ligand, displays antagonistic effects at the rat UT receptor but acts as a full agonist at the recombinant monkey UT receptor, inducing the maximal response although with approximately 100-fold less potency than U-II . These findings suggest that the functional response of UT receptor modulators at rodent UT receptors does not necessarily predict the functional response at non-rodent UT receptors. This inconsistency could result from alterations in receptor number, coupling efficiency, or other factors .

What techniques are most effective for producing recombinant Gillichthys mirabilis Urotensin-2?

Recombinant production of Gillichthys mirabilis Urotensin-2 typically involves expression systems utilizing bacterial (E. coli), yeast, insect, or mammalian cells. For research applications, E. coli systems are often preferred due to their cost-effectiveness and high yield, though proper folding of the disulfide bond in the cyclic region requires careful optimization .

The production process generally involves:

• Gene synthesis or cloning of the U-II sequence

• Insertion into an appropriate expression vector with a purification tag (His-tag, GST, etc.)

• Transformation into the expression host

• Induction of protein expression

• Cell lysis and initial purification

• Tag cleavage (if applicable)

• Secondary purification and folding optimization to ensure proper disulfide bond formation

• Quality control testing for purity and biological activity

Commercial recombinant Urotensin-2 preparations are available for research purposes, including specific preparations of mouse and human variants .

What analytical methods are recommended for characterizing Urotensin-II structure and function?

Multiple analytical approaches are employed to characterize both the structure and function of U-II:

Structural Analysis:

• Nuclear magnetic resonance (NMR) spectroscopy has revealed that U-II adopts a preferential conformation in both DMSO and water solution, with amino acids in the core region identified in a highly compact conformation forming a hydrophobic pocket .

• Mass spectrometry for sequence verification and purity assessment

• Circular dichroism for secondary structure analysis

• X-ray crystallography (when co-crystallized with receptor fragments)

Functional Analysis:

• Binding assays to determine receptor affinity (K_i values)

• Functional assays measuring calcium mobilization or inositol phosphate production

• Tissue bath experiments for contractile responses

• In vivo hemodynamic measurements for cardiovascular effects

Structure-activity relationship studies have provided valuable insights, including the determination that replacement of the Tyr residue with the bulkier 2-Nal [(2-naphthyl)-L-alanine] in the goby U-II sequence showed similar potency in agonist activity while improving binding affinity approximately 6-fold .

How does Urotensin-II influence cardiovascular function in experimental models?

Urotensin-II has significant effects on cardiovascular function, although these effects show considerable variability across species and experimental conditions . U-II is widely expressed in several peripheral tissues including the cardiovascular system .

In rats, chronic high-dose U-II infusion over 4 weeks significantly impairs diastolic function . Research suggests that U-II likely plays a key causal role in cardiac remodeling that ultimately leads to heart failure . The peptide appears to enhance collagen production, contributing to structural changes in cardiac tissue .

The cardiovascular effects of U-II are complex and context-dependent:

• In cardiomyocytes, U-II binding to its receptor typically increases contractility through calcium mobilization

• In the vasculature, U-II can cause constriction when acting on vascular smooth muscle cells or dilation when acting on endothelial cells (through nitric oxide production)

• Chronic exposure to elevated U-II levels can lead to pathological cardiac remodeling

What role does the Rho kinase (ROCK) signaling pathway play in Urotensin-II-induced effects?

Cell culture studies have identified the Rho kinase (ROCK) signaling pathway as an important downstream mediator of Urotensin-II effects, particularly in relation to cardiovascular pathology . While the specific mechanisms are still being elucidated, research indicates that ROCK signaling contributes to the pathophysiological changes observed following chronic U-II exposure, including diastolic dysfunction and enhanced collagen production .

Additionally, U-II has been linked to NOX4 activation, which mediates activation of FoxO3a and matrix metalloproteinase-2 expression, further contributing to tissue remodeling processes .

What is the current state of development for Urotensin-II-based therapeutics?

The modulation of the U-II system offers significant potential for therapeutic strategies related to the treatment of several diseases, particularly cardiovascular conditions . Research into selective and potent ligands targeting the UT receptor remains an active area of investigation .

Both peptide and non-peptide structures have been developed to target the U-II system . The design and development of novel U-II analogues has improved understanding of structure-activity relationships (SAR) while also generating potential therapeutic candidates .

Pharmacophore models have been developed through docking studies, such as the model proposed by Kinney et al., which identified key interaction points including the essential role of the Lys₈ amino group for interaction with Asp₁₃₀ on transmembrane helix TM3 of UT receptor . Such models facilitate rational design of new molecules with high affinity for the UT receptor.

What challenges exist in translating Urotensin-II research to clinical applications?

Several challenges complicate the translation of U-II research to clinical applications:

• Species-specific differences: The functional response of UT receptor modulators varies significantly between species, complicating preclinical testing and translation to humans .

• Complex physiological effects: U-II can produce both vasoconstriction and vasodilation depending on the vascular bed and experimental conditions, making it difficult to predict the net effect of U-II modulation in patients .

• Receptor distribution heterogeneity: The distribution of UT receptors varies across tissues, including brain, respiratory system, heart, vasculature, and kidney, creating the potential for wide-ranging effects beyond the targeted therapeutic benefit .

• Polymorphisms: Several single nucleotide polymorphisms of both U-II and UT exist, and their association with various cardiovascular disease phenotypes remains largely undefined, potentially affecting individual responses to therapeutic interventions .

Structure Comparison of Urotensin-II Across Species

Key Pharmacological Properties of Urotensin-II and Analogues

What are the most promising areas for future Urotensin-II research?

Based on current knowledge and gaps identified in the literature, several promising research directions emerge:

• Development of selective UT receptor modulators: Continued refinement of both peptide and non-peptide structures targeting the UT receptor could yield therapeutically valuable compounds with improved pharmacological profiles .

• Clarification of species-specific differences: Further investigation of the molecular basis for species differences in U-II responses could improve translation of preclinical findings to human applications .

• Elucidation of tissue-specific signaling pathways: Deeper understanding of how U-II activates different signaling cascades in various tissues could help explain its diverse and sometimes contradictory physiological effects .

• Role in non-cardiovascular systems: While most research has focused on cardiovascular effects, investigating U-II's function in other systems where it and its receptor are expressed could reveal new therapeutic applications.

• Clinical relevance of genetic polymorphisms: Determining how polymorphisms in U-II and UT affect disease susceptibility and progression could enable personalized therapeutic approaches .

Through continued investigation of these areas, researchers will gain a more comprehensive understanding of the Urotensin-II system and its potential therapeutic applications.