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Objective: Pro-peptide precursors are processed into biologically active peptide hormones or neurotransmitters, each playing an essential role in physiology and disease. Genetic loss of function of a pro-peptide precursor results in the simultaneous ablation of all biologically-active peptides within that precursor, often leading to a composite phenotype that can be difficult to align with the loss of specific peptide components. Due to this biological constraint and technical limitations, mice carrying the selective ablation of individual peptides encoded by pro-peptide precursor genes, while leaving the other peptides unaffected, have remained largely unaddressed. Methods: We developed and characterized a mouse model carrying the selective knockout of the TLQP-21 neuropeptide (ΔTLQP-21) encoded by the Vgf gene. To achieve this goal, we used a knowledge-based approach by mutating a codon in the Vgf sequence leading to the substitution of the C-terminal Arginine of TLQP-21, which is the pharmacophore as well as an essential cleavage site from its precursor, into Alanine (R21→A). Results: We provide several independent validations of this mouse, including a novel in-gel digestion targeted mass spectrometry identification of the unnatural mutant sequence, exclusive to the mutant mouse. ΔTLQP-21 mice do not manifest gross behavioral and metabolic abnormalities and reproduce well, yet they have a unique metabolic phenotype characterized by an environmental temperature-dependent resistance to diet-induced obesity and activation of the brown adipose tissue. Conclusions: The ΔTLQP-21 mouse line can be a valuable resource to conduct mechanistic studies on the necessary role of TLQP-21 in physiology and disease, while also serving as a platform to test the specificity of novel antibodies or immunoassays directed at TLQP-21. Our approach also has far-reaching implications by informing the development of knowledge-based genetic engineering approaches to generate selective loss of function of other peptides encoded by pro-hormones genes, leaving all other peptides within the pro-protein precursor intact and unmodified.

Summary: Structural and functional diversity of peptides and GPCR result from long evolutionary processes. Even small changes in sequence can alter receptor activation, affecting therapeutic efficacy. We conducted a structure-function relationship study on the neuropeptide TLQP-21, a promising target for obesity, and its complement 3a receptor (C3aR1). After having characterized the TLQP-21/C3aR1 lipolytic mechanism, a homology modeling and molecular dynamics simulation identified the TLQP-21 binding motif and C3aR1 binding site for the human (h) and mouse (m) molecules. mTLQP-21 showed enhanced binding affinity and potency for hC3aR1 compared with hTLQP-21. Consistently, mTLQP-21, but not hTLQP-21, potentiates lipolysis in human adipocytes. These findings led us to uncover five mutations in the C3aR1 binding pocket of the rodent Murinae subfamily that are causal for enhanced calculated affinity and measured potency of TLQP-21. Identifying functionally relevant peptide/receptor co-evolution mechanisms can facilitate the development of innovative pharmacotherapies for obesity and other diseases implicating GPCRs. : GPCRs and neuropeptide ligands are under intense evolutionary pressure and are major pharmacological targets. Sahu et al. identify a cluster of mutations within the C3aR1 receptor and the TLQP-21 peptide in the Murinae subfamily of rodents, resulting in enhanced binding affinity and potency, leading to potentiation of adrenergic-receptor-induced lipolysis. Keywords: VGF, lipolytic catecholamine resistance, granin peptides, drug discovery, obesity, innate immunity, transient receptor potential channel

3-Hydroxy-3-methylglutaryl-coenzyme A [HMG-CoA] reductase gene (Hmgcr) is a susceptibility gene for essential hypertension. Sequencing of the Hmgcr locus in genetically hypertensive BPH (blood pressure high), genetically hypotensive BPL (blood pressure low) and genetically normotensive BPN (blood pressure normal) mice yielded a number of single nucleotide polymorphisms (SNPs). BPH/BPL/BPN Hmgcr promoter-luciferase reporter constructs were generated and transfected into liver HepG2, ovarian CHO, kidney HEK-293 and neuronal N2A cells for functional characterization of the promoter SNPs. The BPH-Hmgcr promoter showed significantly less activity than the BPL-Hmgcr promoter under basal as well as nicotine/cholesterol-treated conditions. This finding was consistent with lower endogenous Hmgcr expression in liver and lower plasma cholesterol in BPH mice. Transfection experiments using 5'-promoter deletion constructs (strategically made to assess the functional significance of each promoter SNP) and computational analysis predicted lower binding affinities of transcription factors c-Fos, n-Myc and Max with the BPH-promoter as compared to the BPL-promoter. Corroboratively, the BPH promoter-luciferase reporter construct co-transfected with expression plasmids of these transcription factors displayed less pronounced augmentation of luciferase activity than the BPL construct, particularly at lower amounts of transcription factor plasmids. Electrophoretic mobility shift assays also showed diminished interactions of the BPH promoter with HepG2 nuclear proteins. Taken together, this study provides mechanistic basis for the differential Hmgcr expression in these mouse models of human essential hypertension and have implications for better understanding the role of this gene in regulation of blood pressure.