Targeted modulation of gene expression through receptor-specific delivery of small interfering RNA peptide conjugates.

Small interfering RNA (siRNA) has emerged as a valuable tool to address RNA interference (RNAi) to modulate gene expression also in therapy. However, challenges such as inefficient cell targeting and rapid degradation in biological systems have limited its success. To address these issues, the development of a receptor-specific shuttle system represents a promising solution. [F7,P34]-NPY analogues were modified by solid-phase peptide synthesis, enabling non-covalent conjugation with siRNA. This modification yielded an efficient siRNA vehicle capable of binding and transporting its cargo into target cells without adversely affecting receptor activation or cell viability. Mass spectrometry and gel shift assays confirmed successful and stable siRNA binding under various conditions. Microscopy experiments further demonstrated the co-internalization of labeled peptides and siRNA in Hepa1c1 cells, highlighting the stability of the complex. In vitro quantitative RT-PCR experiments, targeting the TSC22D4 gene to normalize systemic glucose homeostasis and insulin resistance, revealed a functional peptide-based siRNA shuttle system with the ability to decrease mRNA expression to approximately 40%. These findings strengthen the potential of receptor-specific siRNA shuttle systems as efficient tools for gene therapy that offer a possibility for reducing side effects.

Stable Binding of Full-Length Chemerin Is Driven by Negative Charges in the CMKLR1 N Terminus.

The adipokine chemerin is the endogenous ligand of the chemokine-like receptor 1 (CMKLR1), a member of the family of G protein-coupled receptors (GPCRs). This protein ligand plays an important role in obesity and inflammatory processes. Stable receptor-ligand interactions are highly relevant for its different physiological effects such as the migration of immune cells towards sites of inflammation. Here, we demonstrate that negative charges in the CMKLR1 N terminus are involved in the formation of strong contacts with a specific positively charged patch at the surface of full-length chemerin, which is absent in the short nonapeptide agonist chemerin-9, thus explaining its reduced affinity. Using receptor chimera of G protein-coupled receptor 1 (GPR1) and CMKLR1, we were able to identify the residues of this interaction and its relevance for stable full-length chemerin binding. This could help to develop more potent ligands for the treatment of inflammation-related diseases.

Receptor-specific recognition of NPY peptides revealed by structures of NPY receptors.

In response to three highly conserved neuropeptides, neuropeptide Y (NPY), peptide YY, and pancreatic polypeptide (PP), four G protein-coupled receptors mediate multiple essential physiological processes, such as food intake, vasoconstriction, sedation, and memory retention. Here, we report the structures of the human Y1, Y2, and Y4 receptors in complex with NPY or PP, and the Gi1 protein. These structures reveal distinct binding poses of the peptide upon coupling to different receptors, reflecting the importance of the conformational plasticity of the peptide in recognizing the NPY receptors. The N terminus of the peptide forms extensive interactions with the Y1 receptor, but not with the Y2 and Y4 receptors. Supported by mutagenesis and functional studies, subtype-specific interactions between the receptors and peptides were further observed. These findings provide insight into key factors that govern NPY signal recognition and transduction, and would enable development of selective drugs.

Orthogonal Peptide-Templated Labeling Elucidates Lateral ETA R/ETB R Proximity and Reveals Altered Downstream Signaling.

Fine-tuning of G protein-coupled receptor (GPCR) signaling is important to maintain cellular homeostasis. Recent studies demonstrated that lateral GPCR interactions in the cell membrane can impact signaling profiles. Here, we report on a one-step labeling method of multiple membrane-embedded GPCRs. Based on short peptide tags, complementary probes transfer the cargo (e. g. a fluorescent dye) by an acyl transfer reaction with high spatial and temporal resolution within 5 min. We applied this approach to four receptors of the cardiovascular system: the endothelin receptor A and B (ETA R and ETB R), angiotensin II receptor type 1, and apelin. Wild type-like G protein activation after N-terminal modification was demonstrated for all receptor species. Using FRET-competent dyes, a constitutive proximity between hetero-receptors was limited to ETA R/ETB R. Further, we demonstrate, that ETA R expression regulates the signaling of co-expressed ETB R. Our orthogonal peptide-templated labeling of different GPCRs provides novel insight into the regulation of GPCR signaling.

Shuttling of Peptide-Drug Conjugates by G Protein-Coupled Receptors Is Significantly Improved by Pulsed Application.

G protein-coupled receptors (GPCRs) can be used to shuttle peptide-drug conjugates into cells. But, for efficient therapy, a high concentration of cargo needs to be delivered. To explore this, we studied the pharmacologically interesting neuropeptide Y1 receptor (Y1 R) in one recombinant and three oncogenic cell systems that endogenously express the receptor. We demonstrate that recycled receptors behave identically to newly synthesized receptors with respect to ligand binding and internalization pathways. Depending on the cell system, biosynthesis, recycling efficiency, and peptide uptake differ partially, but shuttling was efficient in all systems. However, by comparing continuous application of the ligand for four hours to four cycles of internalization and recycling in between, a significantly higher amount of peptide uptake was achieved in the pulsed application (150-250 % to 300-400 %). Accordingly, in this well-suited drug shuttle system pulsed application is superior under all investigated conditions and should be considered for innovative, targeted drug delivery in general.

A Venomics Approach Coupled to High-Throughput Toxin Production Strategies Identifies the First Venom-Derived Melanocortin Receptor Agonists.

Animal venoms are rich in hundreds of toxins with extraordinary biological activities. Their exploitation is difficult due to their complexity and the small quantities of venom available from most venomous species. We developed a Venomics approach combining transcriptomic and proteomic characterization of 191 species and identified 20,206 venom toxin sequences. Two complementary production strategies based on solid-phase synthesis and recombinant expression in Escherichia coli generated a physical bank of 3597 toxins. Screened on hMC4R, this bank gave an incredible hit rate of 8%. Here, we focus on two novel toxins: N-TRTX-Preg1a, exhibiting an inhibitory cystine knot (ICK) motif, and N-BUTX-Ptr1a, a short scorpion-CSαß structure. Neither N-TRTX-Preg1a nor N-BUTX-Ptr1a affects ion channels, the known targets of their toxin scaffolds, but binds to four melanocortin receptors with low micromolar affinities and activates the hMC1R/Gs pathway. Phylogenetically, these two toxins form new groups within their respective families and represent novel hMC1R agonists, structurally unrelated to the natural agonists.

Unusually persistent Gαi-signaling of the neuropeptide Y2 receptor depletes cellular Gi/o pools and leads to a Gi-refractory state.

BACKGROUND: A sensitive balance between receptor activation and desensitization is crucial for cellular homeostasis. Like many other GPCR, the human neuropeptide Y2 receptor (hY2R) undergoes ligand dependent activation and internalization into intracellular compartments, followed by recycling to the plasma membrane. This receptor is involved in the pathophysiology of distinct diseases e.g. epilepsy and cancer progression and conveys anorexigenic signals which makes it an interesting and promising anti-obesity target. However, Y2R desensitization was observed after daily treatment with a selective PYY13-36 analog in vivo by a yet unknown mechanism. MATERIALS: We studied the desensitization and activatability of recycled Y2R in transiently transfected HEK293 cells as well as in endogenously Y2R expressing SH-SY5Y and SMS-KAN cells. Results were evaluated by one-way ANOVA and Tukey post test. RESULTS: We observed strong desensitization of the Y2R in a second round of stimulation despite its reappearance at the membrane. Already the first activation of the Y2R leads to depletion of the functional cellular Gαi/o protein pool and consequently desensitizes the linked signal transduction pathways, independent of receptor internalization. This desensitization also extends to other Gαi/o-coupled GPCR and can be detected in transfected HEK293 as well as in SH-SY5Y and SMS-KAN cell lines, both expressing the Y2R endogenously. By overexpression of chimeric Gαqi proteins in a model system, activation has been rescued, which identifies a critical role of the G protein status for cellular signaling. Furthermore, Y2R displays strong allosteric coupling to inhibitory G proteins in radioligand binding assays, and loses 10-fold affinity in the G protein-depleted state observed after activation, which can be largely abrogated by overexpression of the Gαi-subunit. CONCLUSION: The unusually persistent Gαi-signaling of the Y2R leads to a state of cellular desensitization of the inhibitory Gαi-pathway. The strong allosteric effects of the Y2R-Gαi-interaction might be a mechanism that contributes to the burst of Gαi-signaling, but also serves as a mechanism to limit the Y2-mediated signaling after recycling. Thus, the cell is left in a refractory state, preventing further Gαi-signaling of the Y2R itself but also other Gαi/o-coupled receptors by simply controlling the repertoire of downstream effectors. Video abstract.

NPY1R-targeted peptide-mediated delivery of a dual PPARα/γ agonist to adipocytes enhances adipogenesis and prevents diabetes progression.

OBJECTIVE: PPARα/γ dual agonists have been in clinical development for the treatment of metabolic diseases including type 2 diabetes and dyslipidemia. However, severe adverse side effects led to complications in clinical trials. As most of the beneficial effects rely on the compound activity in adipocytes, the selective targeting of this cell type is a cutting-edge strategy to develop safe anti-diabetic drugs. The goal of this study was to strengthen the adipocyte-specific uptake of the PPARα/γ agonist tesaglitazar via NPY1R-mediated internalization. METHODS: NPY1R-preferring peptide tesaglitazar-[F7, P34]-NPY (tesa-NPY) was synthesized by a combination of automated SPPS and manual couplings. Following molecular and functional analyses for proof of concept, cell culture experiments were conducted to monitor the effects on adipogenesis. Mice treated with peptide drug conjugates or vehicle either by gavage or intraperitoneal injection were characterized phenotypically and metabolically. Histological analysis and transcriptional profiling of the adipose tissue were performed. RESULTS: In vitro studies revealed that the tesaglitazar-[F7, P34]-NPY conjugate selectively activates PPARγ in NPY1R-expressing cells and enhances adipocyte differentiation and adiponectin expression in adipocyte precursor cells. In vivo studies using db/db mice demonstrated that the anti-diabetic activity of the peptide conjugate is as efficient as that of systemically administered tesaglitazar. Additionally, tesa-NPY induces adipocyte differentiation in vivo. CONCLUSIONS: The use of the tesaglitazar-[F7, P34]-NPY conjugate is a promising strategy to apply the beneficial PPARα/γ effects in adipocytes while potentially omitting adverse effects in other tissues.

Different mode of arrestin-3 binding at the human Y1 and Y2 receptor.

GPCR internalization, which is induced by arrestin recruitment, is an important mechanism for the regulation of signaling and receptor quantity at the cell surface. In this study, differences in the mechanism of arrestin-3 (arr-3) recruitment to the neuropeptide Y1 and Y2 receptor were identified. These receptors play an essential role in the regulation of feeding, energy homeostasis and cancer. The Y1R displays high affinity to arr-3, which induces rapid internalization of the arrestin/receptor complex. In contrast, the Y2R has a lower affinity for arr-3. Internalization is induced by arrestin binding, but arr-3 is released from the receptor and remains at the membrane while the receptor internalizes. Moreover, the deletion of the finger loop region of arr-3 reduces its agonist-dependent recruitment to the Y2R significantly, but not to the Y1R suggesting different binding conformations. For the first time, the formation of a supercomplex consisting of Y receptor, Gα0 protein and arrestin was studied by BRET-assay. We demonstrated that the Y1R is able to bind Gα0 protein as well as arr-3 simultaneously and internalizes as a supercomplex. For the Y2R no supercomplex formation was observed. By substituting the C-terminus or specific residues within the intracellular loop 1 and 2 of the receptors, the arr-3 recruitment of the Y1R and Y2R can be switched. Thus, we shed light on the specific spatio-temporal distribution of Gα0 protein and arrestin in response to Y1 versus Y2 receptor activation and identified the molecular determinants.

Time-Resolved Tracking of Separately Internalized Neuropeptide Y2 Receptors by Two-Color Pulse-Chase.

Internalization and intracellular trafficking of G protein-coupled receptors (GPCR) plays an important role in the signal transduction. These processes are often highly dynamic and take place rapidly. In the past 10 years, it became obvious that internalized GPCRs are also capable of signaling via arrestin or heterotrimeric G proteins within the endosomal compartment. Real-time imaging of receptors in living cells can help to evaluate the temporal and spatial localization. We achieved a two-color pulse-chase labeling approach, which allowed the tracking of the human neuropeptide Y2 receptor (hY2R) in the same cell at different times. The ability to visualize the internalization pathway of two separately labeled and separately stimulated subsets of hY2R in a time-resolved manner revealed a rapid trafficking. Fusion of the two hY2R subsets was already observed 10 min after stimulation in the early endosomal compartment without subsequent separation of the fused receptor populations. The results demonstrate that the cells do not discriminate between receptors that were stimulated and internalized at different time points.

C-terminal motif of human neuropeptide Y4 receptor determines internalization and arrestin recruitment.

The human neuropeptide Y4 receptor is a rhodopsin-like G protein-coupled receptor (GPCR), which contributes to anorexigenic signals. Thus, this receptor is a highly interesting target for metabolic diseases. As GPCR internalization and trafficking affect receptor signaling and vice versa, we aimed to investigate the molecular mechanism of hY4R desensitization and endocytosis. The role of distinct segments of the hY4R carboxyl terminus was investigated by fluorescence microscopy, binding assays, inositol turnover experiments and bioluminescence resonance energy transfer assays to examine the internalization behavior of hY4R and its interaction with arrestin-3. Based on results of C-terminal deletion mutants and substitution of single amino acids, the motif 7.78EESEHLPLSTVHTEVSKGS7.96 was identified, with glutamate, threonine and serine residues playing key roles, based on site-directed mutagenesis. Thus, we identified the internalization motif for the human neuropeptide Y4 receptor, which regulates arrestin-3 recruitment and receptor endocytosis.

Rapid Covalent Fluorescence Labeling of Membrane Proteins on Live Cells via Coiled-Coil Templated Acyl Transfer.

Fluorescently labeled proteins enable the microscopic imaging of protein localization and function in live cells. In labeling reactions targeted against specific tag sequences, the size of the fluorophore-tag is of major concern. The tag should be small to prevent interference with protein function. Furthermore, rapid and covalent labeling methods are desired to enable the analysis of fast biological processes. Herein, we describe the development of a method in which the formation of a parallel coiled coil triggers the transfer of a fluorescence dye from a thioester-linked coil peptide conjugate onto a cysteine-modified coil peptide. This labeling method requires only small tag sequences (max 23 aa) and occurs with high tag specificity. We show that size matching of the coil peptides and a suitable thioester reactivity allow the acyl transfer reaction to proceed within minutes (rather than hours). We demonstrate the versatility of this method by applying it to the labeling of different G-protein coupled membrane receptors including the human neuropeptide Y receptors 1, 2, 4, 5, the neuropeptide FF receptors 1 and 2, and the dopamine receptor 1. The labeled receptors are fully functional and able to bind the respective ligand with high affinity. Activity is not impaired as demonstrated by activation, internalization, and recycling experiments.

Intracellular Trafficking of Neuropeptide Y Receptors.

The multireceptor multiligand system of neuropeptide Y receptors and their ligands is involved in the regulation of a multitude of physiological and pathophysiological processes. Specific expression patterns, ligand-binding modes, and signaling properties contribute to the complex network regulating distinct cellular responses. Intracellular trafficking processes are important key steps that are regulated in context with accessory proteins. These proteins exert their influence by interacting directly or indirectly with the receptors, causing modification of the receptors, or operating as scaffolds for the assembly of larger signaling complexes. On the intracellular receptor faces, sequence-specific motifs have been identified that play an important role in this process. Interestingly, it is also possible to influence the receptor internalization by modification of the peptide ligand.

Peptide-templated acyl transfer: a chemical method for the labeling of membrane proteins on live cells.

The development of a method is described for the chemical labeling of proteins which occurs with high target specificity, proceeds within seconds to minutes, and offers a free choice of the reporter group. The method relies upon the use of peptide templates, which align a thioester and an N-terminal cysteinyl residue such that an acyl transfer reaction is facilitated at nanomolar concentrations. The protein of interest is N-terminally tagged with a 22 aa long Cys-E3 peptide (acceptor), which is capable of forming a coiled-coil with a reporter-armed K3 peptide (donor). This triggers the transfer of the reporter to the acceptor on the target protein. Because ligation of the two interacting peptides is avoided, the mass increase at the protein of interest is minimal. The method is exemplified by the rapid fluorescent labeling and fluorescence microscopic imaging of the human Y2 receptor on living cells.

Peptide modifications differentially alter G protein-coupled receptor internalization and signaling bias.

Although G protein-coupled receptors (GPCRs) are targeted by more clinically used drugs than any other type of protein, their ligand development is particularly challenging. Humans have four neuropeptide Y receptors: hY1R and hY5R are orexigenic, while hY2R and hY4R are anorexigenic, and represent important anti-obesity drug targets. We show for the first time that PEGylation and lipidation, chemical modifications that prolong the plasma half-lives of peptides, confer additional benefits. Both modifications enhance pancreatic polypeptide preference for hY2R/hY4R over hY1R/hY5R. Lipidation biases the ligand towards arrestin recruitment and internalization, whereas PEGylation confers the opposite bias. These effects were independent of the cell system and modified residue. We thus provide novel insights into the mode of action of peptide modifications and open innovative venues for generating peptide agonists with extended therapeutic potential.

Towards improved receptor targeting: anterograde transport, internalization and postendocytic trafficking of neuropeptide Y receptors.

The neuropeptide Y system is known to be involved in the regulation of many central physiological and pathophysiological processes, such as energy homeostasis, obesity, cancer, mood disorders and epilepsy. Four Y receptor subtypes have been cloned from human tissue (hY1, hY2, hY4 and hY5) that form a multiligand/multireceptor system together with their three peptidic agonists (NPY, PYY and PP). Addressing this system for medical application requires on the one hand detailed information about the receptor-ligand interaction to design subtype-selective compounds. On the other hand comprehensive knowledge about alternative receptor signaling, as well as desensitization, localization and downregulation is crucial to circumvent the development of undesired side-effects and drug resistance. By bringing such knowledge together, highly potent and long-lasting drugs with minimized side-effects can be engineered. Here, current knowledge about Y receptor export, internalization, recycling, and degradation is summarized, with a focus on the human Y receptor subtypes, and is discussed in terms of its impact on therapeutic application.

The anterograde transport of the human neuropeptide Y2 receptor is regulated by a subtype specific mechanism mediated by the C-terminus.

The export of newly synthesized proteins, including G protein-coupled receptors (GPCR), from the endoplasmic reticulum (ER) and further transport to the plasma membrane is a tightly regulated process. ER export and subsequent cell surface targeting of GPCR is initially mediated through COPII-coated vesicles. It is governed by specific amino acid sequences located in extracellular as well as intracellular receptor domains, for example in the C-terminus (CT) of the receptor. Herein, we determined the role of the CT in the anterograde transport of the human neuropeptide Y receptor (hYR) type 2. We identified a short sequence motif in the membrane proximal CT: Y(x)(3)F(x)(3)F in the region of the putative 8th helix has a critical functional relevance for the anterograde transport of hY(2)R, since its deletion leads to accumulation of the receptor in the ER. It is sequence and position specific. Furthermore we identified a distinct role of C-terminal sequences in hY(1)R, hY(2)R, hY(4)R and hY(5)R. Regulation of hY(5)R export is regulated by a different mechanism as compared to hY(2)R. Different sequence elements with respect to function and localization are involved as demonstrated by the construction of a hY(2)/hY(5) receptor chimera and a noneffective deletion in the region of helix eight in the hY(5)R. In contrast to hY(2)R, deletion of the corresponding helical segment F(x)(3)L(x)(3)F has no influence on anterograde transport of hY(1)R, whereas deletion of F(x)(3)I(x)(3)V in hY(4)R restrains the receptor to the Golgi apparatus. Interestingly this pattern is not mirrored by repression of COPII vesicle transport by Sar1[H79G] overexpression. Whereas the 8th helix is involved before or at the level of Sar1 dependent export pathways in the ER for the hY(2)R, in hY(4)R helix eight is involved at later stages of anterograde transport.

Adiponectin receptor 1 interacts with both subunits of protein kinase CK2.

Adiponectin is an adipose tissue-derived hormone that is involved in the inhibition of metabolic syndrome, protection of hypertension, and suppression of atherosclerosis. Since these effects are not understood in detail, adiponectin signaling has to be clarified for therapeutic applications. Adiponectin activities are mediated by its two receptors adiponectin receptor 1 and adiponectin receptor 2, which consist of seven transmembrane helices. Previous studies revealed the beta subunit of protein kinase CK2 as an interaction partner of the adiponectin receptor 1 N-terminus using a yeast-two-hybrid screen, co-immunoprecipitation, ELISA experiments, and co-localization studies. Inhibition of CK2 activity by 2-dimethylamino-4,5,6,7-tetrabromo-1H-benz-imidazole led to a decrease of ACC phosphorylation and indicates an important role of CK2 in adiponectin signaling. CK2 is characterized as a heterotetramer that consists of two regulatory beta and two catalytic alpha subunits, but a holoenzyme-independent role for both subunits is described as well. Therefore, we analyzed the role of the catalytic subunit in this interaction by co-immunoprecipitation and bimolecular fluorescence complementation studies and found CK2 alpha as an interaction partner of the receptor. Treatment with full-length adiponectin resulted in no dissociation of the catalytic alpha subunit. Consequently, our data suggest an interaction of the adiponectin receptor 1 with the tetrameric complex and identified protein kinase CK2 as a key player in adiponectin signaling.

Neuropeptide Y receptors: ligand binding and trafficking suggest novel approaches in drug development.

NPY, PYY and PP constitute the so-called NPY hormone family, which exert its biological functions in humans through YRs (Y1, Y2, Y4 and Y5). Systematic modulation of YR function became important as this multireceptor/multiligand system is known to mediate various essential physiological key functions and is involved in a variety of major human diseases such as epilepsy, obesity and cancer. As several YRs have been found to be overexpressed on different types of malignant tumors they emerge as promising target in modern drug development. Here, we summarize the current understanding of YRs function and the molecular mechanisms of ligand binding and trafficking. We further address recent advances in YR-based drug design, the development of promising future drug candidates and novel approaches in YR-targeted tumor diagnostics and therapy opportunities.

Ligand-induced internalization and recycling of the human neuropeptide Y2 receptor is regulated by its carboxyl-terminal tail.

Agonist-induced internalization of G protein-coupled receptors plays an important role in signal regulation. The underlying mechanisms of the internalization of the human neuropeptide Y(2) receptor (hY(2)R), as well as its desensitization, endocytosis, and resensitization are mainly unknown. In the present study we have investigated the role of carboxyl-terminal (C-terminal) Ser/Thr residues and acidic amino acids in regulating receptor internalization, arrestin interaction, and recycling by fluorescence microscopy, cell surface enzyme-linked immunosorbent assay, and bioluminescence resonance energy transfer in several cell lines. Strikingly, C-terminal truncation mutants revealed two different internalization motifs. Whereas a distal motif (373)DSXTEXT(379) was found to be the primary regulatory internalization sequence acting in concert with arrestin-3, the proximal motif (347)DXXXSEXSXT(356) promoted ligand-induced internalization in an arrestin-3-independent manner. Moreover, we identified a regulatory sequence located between these internalization motifs ((357)FKAKKNLEVRKN(368)), which serves as an inhibitory element. We found that hY(2)R recycling is also governed by structural determinants within the proximal internalization motif. In conclusion, these results indicate that the hY(2)R C terminus is involved in multiple molecular events that regulate internalization, interaction with arrestin-3, and receptor resensitization. Our findings provide novel insights into complex mechanisms of controlled internalization of hY(2)R, which is likely applicable to other GPCRs.