Functional studies on twenty novel naturally occurring melanocortin-4 receptor mutations

The melanocortin-4 receptor (MC4R) is a G protein-coupled receptor critically involved in regulating energy balance. MC4R activation results in decreased food intake and increased energy expenditure. Genetic and pharmacological studies demonstrated that the MC4R regulation of energy balance is conserved from fish to mammals. In humans, more than 150 naturally occurring mutations in the MC4R gene have been identified. Functional study of mutant MC4Rs is an important component in proving the causal link between MC4R mutation and obesity as well as the basis of personalized medicine. In this article, we studied 20 MC4R mutations that were either not characterized or not fully characterized. We showed that 11 mutants had decreased or absent cell surface expression. D126Y was defective in ligand binding. Three mutants were constitutively active but had decreased cell surface expression. Eleven mutants had decreased basal signaling, with two mutants defective only in this parameter, suggesting that impaired basal signaling might also be a cause of obesity. Five mutants had normal functions. In summary, we provided detailed functional data for further studies on identifying therapeutic approaches for personalized medicine to treat patients harboring these mutations.     

The Asp298Asn polymorphism of melanocortin-4 receptor (MC4R) in pigs: evidence for its potential effects on MC4R constitutive activity and cell surface expression

In humans and mice, melanocortin receptor 4 (MC4R) and melanocortin receptor accessory protein 2 (MRAP2) can form a complex and control energy balance, thus regulating body weight and obesity. In pigs, a missense variant (p.Asp298Asn) of MC4R has been suggested to be associated with growth and fatness; however, the effect of Asp298Asn substitution on MC4R function is controversial, limiting its application in animal breeding. Here we examined the effect of this polymorphism on MC4R constitutive activity, cell surface expression and signaling, and its interaction with MRAP2 in pigs. We found that: (i) both pig MC4R^Asp and MC4R^Asn can be activated by its ligands (α-MSH and ACTH) and stimulate cAMP/PKA signaling pathway, as detected by pGL3–CRE–luciferase reporter assay, indicating that, like pMC4R^Asp, pMC4R^Asn is coupled to the cAMP/PKA signaling pathway; (ii) compared with pMC4R^Asp, pMC4R^Asn loses the basal constitutive activity and shows a decreased surface expression, as detected by dual-luciferase reporter assay and Nano-HiBiT system; (iii) as in other vertebrates, both pMC4R^Asp and pMC4R^Asn can interact with pMRAP2, thus decreasing receptor surface expression and enhancing ligand sensitivity, although, in contrast to pMC4R^Asp, the basal constitutive activity of pMC4R^Asn cannot be affected by pMRAP2; and (iv) RNA-seq data analysis revealed a co-expression of MC4R and MRAP2 in pig hypothalamus. Taken together, our data provide convincing evidence that Asp298Asn substitution decreases the constitutive activity and cell surface expression of MC4R or MC4R–MRAP2 complex, which may affect energy balance and be a valuable selection marker for breeding programs in pigs.     

A new G protein-coupled receptor from a primitive metazoan shows homology with vertebrate aminergic receptors and displays constitutive activity in mammalian cells

Biogenic amine receptors mediate wide-ranging hormonal and modulatory functions in vertebrates, but are largely unknown in primitive invertebrates. In a representative of the most basal multicellular animals possessing a nervous system, the cnidarian Renilla koellikeri, aminergic-like receptors were previously characterized pharmacologically and found to engender control of the animal's bioluminescent and peristaltic reactions. Using degenerate oligonucleotides in a RT-PCR strategy, we obtained a full-length cDNA encoding a polypeptide with typical G protein-coupled receptor (GPCR) characteristics and which displayed a significant degree of sequence similarity (up to 45%) to biogenic amine receptors, particularly dopamine and adrenergic receptors. The new receptor, named Ren1, did not resemble any one specific type of amine GPCR and thus could not be identified on the basis of sequence. Ren1 was expressed transiently and stably in cultured mammalian cells, as demonstrated by immunocytochemistry and western blotting. Functional analysis of transfected HEK293, LTK- and COS-7 cells, based on both cAMP and Ca2+ signalling assays, revealed that Ren1 was not activated by any of the known biogenic amines tested and several related metabolites. The results indicated, however, that cells stably expressing Ren1 contained, on average, an 11-fold higher level of cAMP than the controls, in the absence of agonist stimulation. The high basal cAMP levels were shown to be specific for Ren1 and to vary proportionally with the level of Ren1 expressed in the transfected cells. Taken together, the data suggested that Ren1 was expressed as a constitutively active receptor. Its identification provides a basis for examination of the early evolutionary emergence of GPCRs and their functional properties.     

Agonist-activated glucagon receptors are deubiquitinated at early endosomes by two distinct deubiquitinases to facilitate Rab4a-dependent recycling

The glucagon receptor (GCGR) activated by the peptide hormone glucagon is a seven-transmembrane G protein–coupled receptor (GPCR) that regulates blood glucose levels. Ubiquitination influences trafficking and signaling of many GPCRs, but its characterization for the GCGR is lacking. Using endocytic colocalization and ubiquitination assays, we have identified a correlation between the ubiquitination profile and recycling of the GCGR. Our experiments revealed that GCGRs are constitutively ubiquitinated at the cell surface. Glucagon stimulation not only promoted GCGR endocytic trafficking through Rab5a early endosomes and Rab4a recycling endosomes, but also induced rapid deubiquitination of GCGRs. Inhibiting GCGR internalization or disrupting endocytic trafficking prevented agonist-induced deubiquitination of the GCGR. Furthermore, a Rab4a dominant negative (DN) that blocks trafficking at recycling endosomes enabled GCGR deubiquitination, whereas a Rab5a DN that blocks trafficking at early endosomes eliminated agonist-induced GCGR deubiquitination. By down-regulating candidate deubiquitinases that are either linked with GPCR trafficking or localized on endosomes, we identified signal-transducing adaptor molecule–binding protein (STAMBP) and ubiquitin-specific protease 33 (USP33) as cognate deubiquitinases for the GCGR. Our data suggest that USP33 constitutively deubiquitinates the GCGR, whereas both STAMBP and USP33 deubiquitinate agonist-activated GCGRs at early endosomes. A mutant GCGR with all five intracellular lysines altered to arginines remains deubiquitinated and shows augmented trafficking to Rab4a recycling endosomes compared with the WT, thus affirming the role of deubiquitination in GCGR recycling. We conclude that the GCGRs are rapidly deubiquitinated after agonist-activation to facilitate Rab4a-dependent recycling and that USP33 and STAMBP activities are critical for the endocytic recycling of the GCGR.     

Neutrophil Elastase Activates Protease-activated Receptor-2 (PAR2) and Transient Receptor Potential Vanilloid 4 (TRPV4) to Cause Inflammation and Pain

Proteases that cleave protease-activated receptor-2 (PAR₂) at Arg³⁶↓Ser³⁷ reveal a tethered ligand that binds to the cleaved receptor. PAR₂ activates transient receptor potential (TRP) channels of nociceptive neurons to induce neurogenic inflammation and pain. Although proteases that cleave PAR₂ at non-canonical sites can trigger distinct signaling cascades, the functional importance of the PAR₂-biased agonism is uncertain. We investigated whether neutrophil elastase, a biased agonist of PAR₂, causes inflammation and pain by activating PAR₂ and TRP vanilloid 4 (TRPV4). Elastase cleaved human PAR₂ at Ala⁶⁶↓Ser⁶⁷ and Ser⁶⁷↓Val⁶⁸_. Elastase stimulated PAR₂-dependent cAMP accumulation and ERK1/2 activation, but not Ca²⁺ mobilization, in KNRK cells. Elastase induced PAR₂ coupling to Gα_s but not Gα_q in HEK293 cells. Although elastase did not promote recruitment of G protein-coupled receptor kinase-2 (GRK2) or β-arrestin to PAR₂, consistent with its inability to promote receptor endocytosis, elastase did stimulate GRK6 recruitment. Elastase caused PAR₂-dependent sensitization of TRPV4 currents in Xenopus laevis oocytes by adenylyl cyclase- and protein kinase A (PKA)-dependent mechanisms. Elastase stimulated PAR₂-dependent cAMP formation and ERK1/2 phosphorylation, and a PAR₂- and TRPV4-mediated influx of extracellular Ca²⁺ in mouse nociceptors. Adenylyl cyclase and PKA-mediated elastase-induced activation of TRPV4 and hyperexcitability of nociceptors. Intraplantar injection of elastase to mice caused edema and mechanical hyperalgesia by PAR₂- and TRPV4-mediated mechanisms. Thus, the elastase-biased agonism of PAR₂ causes Gα_s-dependent activation of adenylyl cyclase and PKA, which activates TRPV4 and sensitizes nociceptors to cause inflammation and pain. Our results identify a novel mechanism of elastase-induced activation of TRPV4 and expand the role of PAR₂ as a mediator of protease-driven inflammation and pain.     

Metal ions in macrophage antimicrobial pathways: emerging roles for zinc and copper

The immunomodulatory and antimicrobial properties of zinc and copper have long been appreciated. In addition, these metal ions are also essential for microbial growth and survival. This presents opportunities for the host to either harness their antimicrobial properties or limit their availability as defence strategies. Recent studies have shed some light on mechanisms by which copper and zinc regulation contribute to host defence, but there remain many unanswered questions at the cellular and molecular levels. Here we review the roles of these two metal ions in providing protection against infectious diseases in vivo, and in regulating innate immune responses. In particular, we focus on studies implicating zinc and copper in macrophage antimicrobial pathways, as well as the specific host genes encoding zinc transporters (SLC30A, SLC39A family members) and CTRs (copper transporters, ATP7 family members) that may contribute to pathogen control by these cells.     

敲减MC4R表达对牛胎儿成纤维细胞CMS系统关键因子的影响

为获得敲减黑素皮质素4受体（melanocortin 4 receptor,MC4R）基因的牛胎儿成纤维细胞,并探讨其在能量平衡神经调节系统中的作用,将构建成功并已鉴定为有效序列的短发夹状RNA (short hairpin RNA, shRNA)真核表达载体pGSH1 GFP MC4R,利用阳离子脂质体转染牛胎儿成纤维细胞并使用G418筛选稳定转染细胞株.利用实时荧光定量和Western印迹检测MC4R及中枢黑素皮质素系统(central melanocortin system, CMS)关键因子的表达水平变化.结果表明,在稳定转染的牛胎儿成纤维细胞系中, MC4R表达显著抑制,瘦蛋白(leptin)和阿黑色素原(POMC)表达下调,黑素皮质素拮抗物agouti相关蛋白(AGRP)和MC3R表达上调,而神经肽Y (NPY)表达无明显改变.综上所述,本研究成功获得了敲减MC4R基因表达的牛胎儿成纤维细胞.相关基因表达水平检测结果提示, MC4R的表达水平对CMS系统中的各关键基因的表达有不同的抑制或促进影响.     

The Activation Pathway of Human Rhodopsin in Comparison to Bovine Rhodopsin

Rhodopsin, the photoreceptor of rod cells, absorbs light to mediate the first step of vision by activating the G protein transducin (G_t). Several human diseases, such as retinitis pigmentosa or congenital night blindness, are linked to rhodopsin malfunctions. Most of the corresponding in vivo studies and structure-function analyses (e.g. based on protein x-ray crystallography or spectroscopy) have been carried out on murine or bovine rhodopsin. Because these rhodopsins differ at several amino acid positions from human rhodopsin, we conducted a comprehensive spectroscopic characterization of human rhodopsin in combination with molecular dynamics simulations. We show by FTIR and UV-visible difference spectroscopy that the light-induced transformations of the early photointermediates are very similar. Significant differences between the pigments appear with formation of the still inactive Meta I state and the transition to active Meta II. However, the conformation of Meta II and its activity toward the G protein are essentially the same, presumably reflecting the evolutionary pressure under which the active state has developed. Altogether, our results show that although the basic activation pathways of human and bovine rhodopsin are similar, structural deviations exist in the inactive conformation and during receptor activation, even between closely related rhodopsins. These differences between the well studied bovine or murine rhodopsins and human rhodopsin have to be taken into account when the influence of point mutations on the activation pathway of human rhodopsin are investigated using the bovine or murine rhodopsin template sequences.     

Association of melanocortin 4 receptor gene variation with satiation and gastric emptying in overweight and obese adults

Melanocortin 4 receptor (MC4R) has a major role in energy homeostasis. The rs17782313 polymorphism, mapped 188 kb downstream from MC4R, has been associated with satiety, higher body mass index (BMI) and total calorie intake in adults. To assess the association of rs17782313 with gastric functions, satiation, or satiety, we studied 178 predominantly Caucasian overweight and obese people: 120 females, 58 males; mean BMI 33.4 ± 5.3 kg/m² (SD); age 37.7 ± 11.2 years. Quantitative traits assessed were gastric emptying (GE) of solids and liquids; fasting and postprandial gastric volume; satiation by maximum tolerated volume and 4 symptoms by 100-mm visual analog scales (VAS); and satiety by ad libitum buffet meal. Associations of genotype and quantitative traits were assessed by analysis of covariance (using gender and BMI as covariates), based on a dominant [TC (n = 72) − CC (n = 12) vs. TT (n = 94)] genetic model. rs17782313(C) was associated with postprandial satiation symptoms (median Δ total VAS 26.5 mm, p = 0.036), reduced proportion of solid GE at 2 h (median Δ 6.7 %, p = 0.008) and 4 h (median Δ 3.2 %, p = 0.006), and longer t_½ (median Δ 6 min, p = 0.034). Associations of rs17782313 with obesity may be explained by reduced satiation and GE. The role of MC4R mechanisms in satiation and gastric function deserves further study.     

应用进化踪迹及分子动力学模拟研究beta2肾上腺素受体突变活性

β2肾上腺素受体(β2adrenergic receptor,β2AR)是G蛋白耦联受体(G protein coupled receptors,GPCRs)超家族中的一员,也是研究治疗哮喘的关键药物受体靶标.采用进化踪迹(evolutionary trace,ET)方法分析肾上腺素受体家族跨膜区片段序列,识别出了44个保守的残基,然后将β2肾上腺素受体以及受体D130N活性突变体、D79N失活突变体进行分子动力学模拟,试图找出与受体不同功能状态相关的结构动力学特征.发现受体DRY motif中的D130远离R131而转向K149残基这一结构特征与受体活性高度关联,此外,从残基相互作用的变化推断出了受体helix 2,4 and 6伴随着受体活化而发生的运动.这些研究结果对进一步探索β2肾上腺素受体突变体的激活机制以及所诱发疾病的分子机理提供了依据.     

P2Y1 Receptor Activation of the TRPV4 Ion Channel Enhances Purinergic Signaling in Satellite Glial Cells

Transient receptor potential (TRP) ion channels of peripheral sensory pathways are important mediators of pain, itch, and neurogenic inflammation. They are expressed by primary sensory neurons and by glial cells in the central nervous system, but their expression and function in satellite glial cells (SGCs) of sensory ganglia have not been explored. SGCs tightly ensheath neurons of sensory ganglia and can regulate neuronal excitability in pain and inflammatory states. Using a modified dissociation protocol, we isolated neurons with attached SGCs from dorsal root ganglia of mice. SGCs, which were identified by expression of immunoreactive Kir4.1 and glutamine synthetase, were closely associated with neurons, identified using the pan-neuronal marker NeuN. A subpopulation of SGCs expressed immunoreactive TRP vanilloid 4 (TRPV4) and responded to the TRPV4-selective agonist GSK1016790A by an influx of Ca²⁺ ions. SGCs did not express functional TRPV1, TRPV3, or TRP ankyrin 1 channels. Responses to GSK1016790A were abolished by the TRPV4 antagonist HC067047 and were absent in SGCs from Trpv4^−/− mice. The P2Y₁-selective agonist 2-methylthio-ADP increased [Ca²⁺]_i in SGCs, and responses were prevented by the P2Y1-selective antagonist MRS2500. P2Y₁ receptor-mediated responses were enhanced in TRPV4-expressing SGCs and HEK293 cells, suggesting that P2Y₁ couples to and activates TRPV4. PKC inhibitors prevented P2Y₁ receptor activation of TRPV4. Our results provide the first evidence for expression of TRPV4 in SGCs and demonstrate that TRPV4 is a purinergic receptor-operated channel in SGCs of sensory ganglia.     

Enhancement of arachidonic acid signaling pathway by nicotinic acid receptor HM74A

HM74A is a G protein-coupled receptor for nicotinic acid (niacin), which has been used clinically to treat dyslipidemia for decades. The molecular mechanisms whereby niacin exerts its pleiotropic effects on lipid metabolism remain largely unknown. In addition, the most common side effect in niacin therapy is skin flushing that is caused by prostaglandin release, suggesting that the phospholipase A(2) (PLA(2))/arachidonic acid (AA) pathway is involved. Various eicosanoids have been shown to activate peroxisome-proliferator activated receptors (PPAR) that play a diverse array of roles in lipid metabolism. To further elucidate the potential roles of HM74A in mediating the therapeutic effects and/or side effects of niacin, we sought to explore the signaling events upon HM74A activation. Here we demonstrated that HM74A synergistically enhanced UTP- and bradykinin-mediated AA release in a pertussis toxin-sensitive manner in A431 cells. Activation of HM74A also led to Ca(2+)-mobilization and enhanced bradykinin-promoted Ca(2+)-mobilization through Gi protein. While HM74A increased ERK1/2 activation by the bradykinin receptor, it had no effects on UTP-promoted ERK1/2 activation.Furthermore, UTP- and bradykinin-mediated AA release was significantly decreased in the presence of both MAPK kinase inhibitor PD 098059 and PKC inhibitor GF 109203X. However, the synergistic effects of HM74A were not dramatically affected by co-treatment with both inhibitors, indicating the cross-talk occurred at the receptor level. Finally, stimulation of A431 cells transiently transfected with PPRE-luciferase with AA significantly induced luciferase activity, mimicking the effects of PPARgamma agonist rosiglitazone, suggesting that alteration of AA signaling pathway can regulate gene expression via endogenous PPARs.     

Role of third intracellular loop of the melanocortin 4 receptor in the regulation of constitutive activity

The melanocortin 4 receptor (MC4R) has been reported to display constitutive activity, which is probably relevant to the maintenance of a normal energy balance. Among the clinically reported mutants of MC4R in human obesity patients, we investigated the functional characteristics of seven mutants characterized by mutations in the third intracellular (i3) loop of MC4R. Via a CRE (cAMP responsive element)-mediated luciferase reporter gene assay, we show that most of these mutants displayed significantly reduced basal activity with reduced reporter gene activity, whereas the P230L mutant manifested significantly increased basal activity. When the dominant negative G_s mutant was co-expressed, the majority of the mutants, including the P230L mutant, showed reduced basal activity. These results suggest that the i3 loop of MC4R is essential not only for the functional activity but also for the regulation and maintenance of an optimal constitutive activity of MC4R in association with G protein coupling, in the control of energy homeostasis.     

Intramolecular fluorescence resonance energy transfer (FRET) sensors of the orexin OX1 and OX2 receptors identify slow kinetics of agonist activation

Intramolecular fluorescence resonance energy transfer (FRET) sensors able to detect changes in distance or orientation between the 3rd intracellular loop and C-terminal tail of the human orexin OX(1) and OX(2) G protein-coupled receptors following binding of agonist ligands were produced and expressed stably. These were directed to the plasma membrane and, despite the substantial sequence alterations introduced, in each case were able to elevate [Ca(2+)](i), promote phosphorylation of the ERK1/2 MAP kinases and become internalized effectively upon addition of the native orexin peptides. Detailed characterization of the OX(1) sensor demonstrated that it was activated with rank order of potency orexin A > orexin B > orexin A 16-33, that it bound antagonist ligands with affinity similar to the wild-type receptor, and that mutation of a single residue, D203A, greatly reduced the binding and function of orexin A but not antagonist ligands. Addition of orexin A to individual cells expressing an OX(1) sensor resulted in a time- and concentration-dependent reduction in FRET signal consistent with mass-action and potency/affinity estimates for the peptide. Compared with the response kinetics of a muscarinic M(3) acetylcholine receptor sensor upon addition of agonist, response of the OX(1) and OX(2) sensors to orexin A was slow, consistent with a multistep binding and activation process. Such sensors provide means to assess the kinetics of receptor activation and how this may be altered by mutation and sequence variation of the receptors.     

The Activation Mechanism of Glycoprotein Hormone Receptors with Implications in the Cause and Therapy of Endocrine Diseases

Glycoprotein hormones (GPHs) are the main regulators of the pituitary-thyroid and pituitary-gonadal axes. Selective interaction between GPHs and their cognate G protein-coupled receptors ensure specificity in GPH signaling. The mechanisms of how these hormones activate glycoprotein hormone receptors (GPHRs) or how mutations and autoantibodies can alter receptor function were unclear. Based on the hypothesis that GPHRs contain an internal agonist, we systematically screened peptide libraries derived from the ectodomain for agonistic activity on the receptors. We show that a peptide (p10) derived from a conserved sequence in the C-terminal part of the extracellular N terminus can activate all GPHRs in vitro and in GPHR-expressing tissues. Inactivating mutations in this conserved region or in p10 can inhibit activation of the thyroid-stimulating hormone receptor by autoantibodies. Our data suggest an activation mechanism where, upon extracellular ligand binding, this intramolecular agonist isomerizes and induces structural changes in the 7-transmembrane helix domain, triggering G protein activation. This mechanism can explain the pathophysiology of activating autoantibodies and several mutations causing endocrine dysfunctions such as Graves disease and hypo- and hyperthyroidism. Our findings highlight an evolutionarily conserved activation mechanism of GPHRs and will further promote the development of specific ligands useful to treat Graves disease and other dysfunctions of GPHRs.     

Pharmacological and signalling properties of a D2-like dopamine receptor (Dop3) in Tribolium castaneum

Dopamine is an important neurotransmitter in the central nervous system of vertebrates and invertebrates. Despite their evolutionary distance, striking parallels exist between deuterostomian and protostomian dopaminergic systems. In both, signalling is achieved via a complement of functionally distinct dopamine receptors. In this study, we investigated the sequence, pharmacology and tissue distribution of a D2-like dopamine receptor from the red flour beetle Tribolium castaneum (TricaDop3) and compared it with related G protein-coupled receptors in other invertebrate species.The TricaDop3 receptor-encoding cDNA shows considerable sequence similarity with members of the Dop3 receptor class. Real time qRT-PCR showed high expression in both the central brain and the optic lobes, consistent with the role of dopamine as neurotransmitter. Activation of TricaDop3 expressed in mammalian cells increased intracellular Ca²⁺ signalling and decreased NKH-477 (a forskolin analogue)-stimulated cyclic AMP levels in a dose-dependent manner. We studied the pharmacological profile of the TricaDop3 receptor and demonstrated that the synthetic vertebrate dopamine receptor agonists, 2 – amino- 6,7 – dihydroxy – 1,2,3,4 – tetrahydronaphthalene hydrobromide (6,7-ADTN) and bromocriptine acted as agonists. Methysergide was the most potent of the antagonists tested and showed competitive inhibition in the presence of dopamine. This study offers important information on the Dop3 receptor from Tribolium castaneum that will facilitate functional analyses of dopamine receptors in insects and other invertebrates.     

Agouti signalling protein is an inverse agonist to the wildtype and agonist to the melanic variant of the melanocortin-1 receptor in the grey squirrel (Sciurus carolinensis)

The melanocortin-1 receptor (MC1R) is a key regulator of mammalian pigmentation. Melanism in the grey squirrel is associated with an eight amino acid deletion in the mutant melanocortin-1 receptor with 24 base pair deletion (MC1RΔ24) variant. We demonstrate that the MC1RΔ24 exhibits a higher basal activity than the wildtype MC1R (MC1R-wt). We demonstrate that agouti signalling protein (ASIP) is an inverse agonist to the MC1R-wt but is an agonist to the MC1RΔ24. We conclude that the deletion in the MC1RΔ24 leads to a receptor with a high basal activity which is further activated by ASIP. This is the first report of ASIP acting as an agonist to MC1R.     

Trypsin activity in the small intestine of rats after application of copper and zinc

In vivo experiments with Sprague-Dawley rats were conducted in order to explore the influence of Cu²⁺, Zn²⁺ as well as of the combinations of both on the activity of trypsin. The solutions of the trace elements were given per os, the animals were killed 30 min after the applications, and the activity of trypsin was determined in the juice of the small intestine by usingN ^α-benzoyl-L-arginine-p-nitroanilide (L-BAPA) as the substrate. The activity of trypsin depends on the concentration of the trace elements. When Cu²⁺ ions are applied, there is a minimum activity at 10⁻⁵ mol Cu²⁺/L and a maximum at 10⁻⁴ mol Cu²⁺/L. When giving Zn²⁺ ions, a minimum of trypsin activity is found at 10⁻⁵ mol Zn²⁺/L and a maximum at 5×10⁻⁶ mol Zn²⁺/L. On the whole, the trypsin activity is lower when the Cu²⁺/Zn²⁺ combinations are applied compared to the addition of the single trace elements. On principle, a good conformity of the in vivo results was found with in vitro results.     

Dual Targeting of the Chemokine Receptors CXCR4 and ACKR3 with Novel Engineered Chemokines

The chemokine CXCL12 and its G protein-coupled receptors CXCR4 and ACKR3 are implicated in cancer and inflammatory and autoimmune disorders and are targets of numerous antagonist discovery efforts. Here, we describe a series of novel, high affinity CXCL12-based modulators of CXCR4 and ACKR3 generated by selection of N-terminal CXCL12 phage libraries on live cells expressing the receptors. Twelve of 13 characterized CXCL12 variants are full CXCR4 antagonists, and four have K_d values <5 nm. The new variants also showed high affinity for ACKR3. The variant with the highest affinity for CXCR4, LGGG-CXCL12, showed efficacy in a murine model for multiple sclerosis, demonstrating translational potential. Molecular modeling was used to elucidate the structural basis of binding and antagonism of selected variants and to guide future designs. Together, this work represents an important step toward the development of therapeutics targeting CXCR4 and ACKR3.