Recombinant Drosophila melanogaster Probable insulin-like peptide 5 (Ilp5)

What is the structural characterization of DILP5?

DILP5 has been analyzed through X-ray crystallography at a high resolution of 1.85 Å. The protein maintains the basic fold of the insulin peptide family (T conformation), but displays a disordered B-chain C terminus. The N-terminus of the B-chain exhibits folding surprisingly comparable to the T-state of human insulin, with the first residues B1-5 (NSLRA) extended similarly to the N-terminal B1-6 residues of insulin (FVNQHL) . The major conformational differences between DILP5 and human insulin are found in the C-terminal region of the B-chain, which is more flexible and consequently more disordered in DILP5 .

When superimposing 37 corresponding C-α atoms, DILP5 and the T-form of insulin display a root mean square deviation of 1.57 Å (with remaining residues rejected using a 3.67 Å cut-off) . This indicates significant structural similarity despite moderate sequence identity (27.8%) between DILP5 and human insulin . For comparison, DILP5 shows lower sequence identity to human relaxin (17.2%) and bombyxin-II (20.8%) .

How does DILP5 dimerize and how is this different from vertebrate insulin?

DILP5 demonstrates a unique dimerization mechanism that fundamentally differs from that observed in vertebrate insulins. In DILP5, dimerization occurs through an anti-parallel β-sheet involving the N-termini of the B-chains (residues B1-5 NSLRA) . This contrasts sharply with the dimerization mechanism of vertebrate insulin, which forms through an anti-parallel β-sheet involving the B-chain C termini .

Analysis of protein packing using the PDBe PISA-server revealed the dimerization interface of DILP5 has a total buried accessible surface area of 1129.6 Ų (representing 17.65% of total solvent-accessible area), which supports DILP5's capacity to dimerize in solution . Comparatively, human insulin has a buried interface area of 1252.8 Ų (17.2% of total solvent-accessible area) . This evolutionary divergence in dimerization mechanism while maintaining functionality represents a fascinating example of molecular evolution.

What are the functional properties of DILP5?

DILP5 exhibits remarkable cross-species functionality. It binds to and activates the human insulin receptor and shows physiological effects by lowering blood glucose levels in rats . In its native context, DILP5 lowers trehalose levels (the primary circulating sugar) in Drosophila . This functional conservation is reciprocal, as human insulin can bind to the Drosophila insulin receptor and induce negative cooperativity similar to its effect on the human receptor .

DILP5 also binds to insect insulin-binding proteins, including the insulin-related peptide-binding protein from Spodoptera frugiperda (sf-IBP), and the imaginal morphogenesis protein-Late 2 (IMP-L2) from D. melanogaster . This multi-receptor binding capacity makes DILP5 an excellent model for studying insulin receptor signaling mechanisms across species.

What expression systems are effective for recombinant DILP5 production?

For effective recombinant DILP5 production, researchers have successfully employed expression from cloned cDNAs rather than total peptide synthesis methods used previously for other invertebrate insulin-like peptides . Two variants of DILP5 have been successfully expressed: DB and C4, which differ by the absence or presence of an Asp-Phe-Arg sequence extension at the N terminus of the A-chain .

Bacterial expression systems can be utilized, but require careful optimization of culture conditions to prevent formation of inclusion bodies. For functional studies, mammalian or insect cell expression systems may yield better results due to their capacity for proper disulfide bond formation and folding. When designing expression constructs, researchers should consider that DILP5 has multiple Cys residues that form disulfide bonds critical to proper folding.

What purification strategies yield the highest purity and bioactivity of DILP5?

Effective purification of recombinant DILP5 requires a multi-step approach to ensure high purity while preserving bioactivity. Initial capture typically employs immobilized metal affinity chromatography (IMAC) when using His-tagged constructs. This is followed by size exclusion chromatography to separate monomeric and dimeric forms, which is particularly important given DILP5's tendency to dimerize in solution .

For applications requiring verification of proper folding, reverse-phase HPLC can be employed as a final polishing step. Throughout the purification process, it's critical to maintain reducing conditions that prevent inappropriate disulfide bond formation while allowing native bonds to form. Buffer conditions should be optimized to prevent aggregation, with typical buffers containing stabilizing agents such as glycerol or low concentrations of non-ionic detergents.

How can researchers validate the correct folding of recombinant DILP5?

Validating correct folding of recombinant DILP5 requires multiple complementary approaches. Structural analysis through circular dichroism spectroscopy provides information about secondary structure content, while thermal shift assays assess protein stability. For definitive structural characterization, X-ray crystallography has been successfully applied to DILP5, yielding a high-resolution (1.85 Å) structure .

Functional validation is equally important and can be performed through receptor binding assays using either the Drosophila insulin receptor or the human insulin receptor, as DILP5 activates both . Bioactivity can be confirmed through in vivo assays measuring the ability of DILP5 to lower trehalose levels in Drosophila or glucose levels in mammalian models . The ability of DILP5 to bind to insect insulin-binding proteins can also serve as a functional validation method .

How can DILP5 be used to study cross-species insulin receptor interactions?

DILP5 provides a valuable tool for investigating cross-species insulin receptor interactions due to its ability to activate both insect and mammalian insulin receptors. Experiments can be designed using purified DILP5 in binding assays with different species' insulin receptors to determine binding affinities, activation kinetics, and signaling outcomes . The unique property of DILP5 to induce negative cooperativity in both Drosophila and human insulin receptors allows for comparative studies of receptor allostery .

For mechanistic studies, researchers can create chimeric receptors containing domains from both Drosophila and mammalian insulin receptors to identify the structural elements responsible for cross-species recognition. Site-directed mutagenesis of specific residues in DILP5 can help identify the binding interface critical for receptor activation. These approaches can provide insights into the evolutionary conservation of insulin signaling mechanisms despite divergent sequence identity.

What are the protocols for using DILP5 in metabolic studies?

When using DILP5 in metabolic studies, researchers have established several validated protocols. For in vivo studies in Drosophila, DILP5 can be injected into the intersegmental integument of the abdomen at a concentration of 0.01 mg/ml diluted in PBS . Trehalose levels can then be measured by collecting flash-frozen samples (typically five flies pooled together) and determining glucose content before and after digestion with trehalase enzyme .

For mammalian studies, DILP5's effect on blood glucose can be measured using protocols similar to those established for insulin. In rats, DILP5 can be injected intravenously in the tail, followed by blood sample collection at various time points . Each experimental group should include at least four animals, with experiments repeated at least three times for statistical robustness .

For in vitro metabolic studies, cell culture systems expressing either Drosophila or human insulin receptors can be treated with DILP5 to measure downstream signaling events such as receptor phosphorylation, Akt activation, and glucose uptake. These studies help elucidate the conserved and divergent aspects of insulin signaling pathways.

How can DILP5 be utilized in structural biology research?

DILP5 offers unique opportunities for structural biology research due to its distinct dimerization mechanism and cross-species receptor interactions. Crystallographic studies of DILP5 have already yielded high-resolution structures (1.85 Å) , enabling detailed comparative analyses with other insulin family peptides. Future structural studies could focus on co-crystallizing DILP5 with receptor fragments to elucidate binding interfaces.

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) can be employed to study DILP5's conformational dynamics and how these change upon receptor binding. Cryo-electron microscopy (cryo-EM) could be used to visualize the full-length insulin receptor in complex with DILP5, potentially revealing conformational changes induced by ligand binding.

The unusual dimerization of DILP5 through N-terminal rather than C-terminal B-chain interactions makes it valuable for studying alternative protein-protein interaction mechanisms within the insulin family . This property can be exploited to engineer novel protein interfaces or to design insulin variants with modified oligomerization properties.

How does DILP5 structurally compare to other DILPs and mammalian insulin?

DILP5 shares the basic fold of the insulin peptide family but exhibits notable structural differences compared to both mammalian insulin and other DILPs. Among the seven DILPs in Drosophila (DILP1-7), DILP2 is most related to human insulin with 35% sequence identity, while DILP5 has 27.8% identity . This moderate sequence conservation translates into structural similarities and differences.

The following table summarizes structural comparisons between DILP5 and other insulin family peptides:

What functional differences exist between DILP5 and other insulin-family peptides?

Despite structural similarities, DILP5 and other insulin family peptides exhibit functional differences that reflect their evolutionary adaptations. Unlike mammalian insulin, which primarily regulates glucose metabolism, DILPs in Drosophila collectively regulate a broader range of physiological processes including growth, development, metabolism, reproduction, and lifespan .

The seven DILPs in Drosophila are expressed in highly tissue- and stage-specific patterns . Functional studies using genetic deletion approaches have shown that while DILP2 can rescue growth defects in flies lacking DILPs1-5, suggesting functional redundancy, specific DILPs may have specialized roles . For instance, DILP6 does not affect global metabolic rate in Drosophila larvae, unlike DILPs1-5 .

How can researchers use comparative studies to understand insulin evolution?

Comparative studies of DILP5 with other insulin-family peptides provide crucial insights into the evolution of insulin signaling. The finding that DILP5 dimerizes through an anti-parallel β-sheet involving the B-chain N termini, rather than the C termini as in vertebrate insulin, suggests independent evolutionary paths to achieve similar functions . This represents a case of functional conservation despite structural divergence.

Researchers can employ several approaches for comparative evolutionary studies:

• Phylogenetic analysis comparing DILP sequences across insect species can reveal conserved motifs and lineage-specific adaptations.

• Receptor-ligand co-evolution studies can identify correlated mutations between DILPs and their receptors.

• Functional substitution experiments, where DILP5 is tested for its ability to activate insulin receptors from different species, can reveal the molecular basis of signaling conservation .

• Structural analysis of insulin peptides across species, focusing on differences in oligomerization interfaces and receptor binding regions, can illuminate divergent evolutionary paths.

These comparative approaches can reveal how insulin signaling has evolved over hundreds of millions of years while maintaining core functionality in regulating metabolism and growth.

What genetic approaches are used to study DILP5 function in vivo?

Several genetic approaches have been successfully employed to study DILP5 function in vivo. Since DILPs1-5 are clustered on chromosome III in Drosophila, researchers have generated small deficiencies that simultaneously delete multiple genes by taking advantage of FRT sites in the Drosophila genome . These deficiencies can be verified using PCR to confirm the absence of coding regions for specific DILPs .

For more targeted studies of individual DILPs, researchers can use:

• RNA interference (RNAi) to knockdown specific DILPs in different tissues using the GAL4-UAS system.

• CRISPR-Cas9 gene editing to create precise mutations or deletions in individual DILP genes.

• Tissue-specific or inducible expression systems to overexpress specific DILPs in different contexts.

• "Add-back" experiments where individual DILPs are expressed in a deficiency background to test for functional rescue .

The versatility of Drosophila genetic tools makes it possible to conduct sophisticated genetic analyses that can dissect the specific roles of individual DILPs versus redundant functions within the family.

What are the phenotypic consequences of DILP5 deletion?

The phenotypic consequences of DILP deletion have been primarily studied using deficiencies that remove multiple DILPs simultaneously. Animals lacking DILPs1-5 (Df[dilp1-5] homozygotes) are homozygous viable and can reproduce, but display several striking phenotypes :

These phenotypes resemble those seen in "diabetic" flies with impaired insulin signaling, suggesting DILPs1-5 function collectively in regulating growth, development, and metabolism.

How can researchers differentiate between DILP5-specific effects and redundant DILP functions?

Differentiating between DILP5-specific effects and redundant functions shared with other DILPs requires sophisticated experimental designs:

• Comparative analysis of single-gene deletions: Generate and compare phenotypes of flies with individual deletions of each DILP to identify unique versus overlapping functions.

• Rescue experiments: In Df[dilp1-5] homozygotes, express individual DILPs to determine which can rescue specific phenotypes. For example, expression of DILP2 alone was sufficient to rescue growth defects in Df[dilp1-5] homozygotes, suggesting functional redundancy for this particular phenotype .

• Spatiotemporal expression analysis: Determine where and when each DILP is expressed using reporter constructs, fluorescent tagging, or RNA in situ hybridization. The seven DILPs in Drosophila are expressed in highly tissue- and stage-specific patterns , which may correlate with specific functions.

• Receptor and binding protein interaction studies: Compare binding affinities and signaling outcomes of different DILPs with the insulin receptor and insulin-binding proteins to identify specific versus shared interaction patterns.

• Transcriptomic and proteomic analyses: Profile gene expression and protein changes in tissues from flies lacking specific DILPs to identify unique downstream targets.

Through these complementary approaches, researchers can build a comprehensive understanding of specific versus redundant DILP functions.

What is the role of DILP5 in Drosophila metabolism?

DILP5, along with other DILPs, plays crucial roles in Drosophila metabolism. Deletion of DILPs1-5 leads to metabolic defects reminiscent of diabetes in mammals, including elevated circulating sugar levels during both feeding and fasting states . This suggests that DILP5 contributes to maintaining proper sugar homeostasis in flies, similar to insulin's role in mammals.

In vivo experiments have shown that DILP5 efficiently lowers trehalose levels (the primary circulating sugar in insects) when injected into Drosophila, confirming its direct role in sugar metabolism . The conserved ability of DILP5 to lower blood glucose in rats further demonstrates its fundamental role in metabolic regulation across species .

How does DILP5 influence growth and development?

DILP5 and other DILPs have profound effects on growth and development in Drosophila. Flies lacking DILPs1-5 show significant developmental delay, taking more than 9 days to reach the wandering third-instar stage compared to 5 days for controls . The time to reach pupation is also extended, although the duration from pupation to eclosion is less affected .

DILP5 also plays a role in regulating allometry, the relative proportioning of body parts. In Df[dilp1-5] homozygotes, male genital arch posterior lobes fail to scale proportionately with the smaller body size, being reduced only approximately 15% compared to the 29% reduction in wing size . This differential scaling resembles the allometric effects seen in insulin receptor (dinr) and insulin receptor substrate (chico) mutants, suggesting that DILPs function upstream in this pathway .

What is known about DILP5's role in tissue-specific responses?

DILP5, like other DILPs, contributes to tissue-specific responses in Drosophila, though our understanding of its exact tissue-specific functions is still developing. The allometric scaling differences observed in Df[dilp1-5] homozygotes provide evidence for tissue-specific responses to DILP signaling . While most body organs scale down proportionately with smaller body size, male genitals do not scale proportionately, suggesting differential sensitivity to DILP signaling across tissues .

Fat body cells show clear responses to DILP signaling, with Df[dilp1-5] homozygotes displaying reduced fat body cell size that can be rescued by DILP2 expression . The fat body also exhibits metabolic responses to DILP deficiency, including premature activation of autophagy and decreased triglyceride levels . These responses indicate that the fat body is a key target tissue for DILP action in regulating both growth and metabolism.

The expression patterns of different DILPs are highly tissue- and stage-specific , suggesting specialized roles in different developmental contexts. Future research using tissue-specific knockdown or overexpression of DILP5 will be needed to fully characterize its tissue-specific functions and to differentiate these from the functions of other DILPs.