Influenza A virus exploits transferrin receptor recycling to enter host cells.

Influenza A virus (IAV) enters host cells mostly through clathrin-dependent receptor-mediated endocytosis. A single bona fide entry receptor protein supporting this entry mechanism remains elusive. Here we performed proximity ligation of biotin to host cell surface proteins in the vicinity of attached trimeric hemagglutinin-HRP and characterized biotinylated targets using mass spectrometry. This approach identified transferrin receptor 1 (TfR1) as a candidate entry protein. Genetic gain-of-function and loss-of-function experiments, as well as in vitro and in vivo chemical inhibition, confirmed the functional involvement of TfR1 in IAV entry. Recycling deficient mutants of TfR1 do not support entry, indicating that TfR1 recycling is essential for this function. The binding of virions to TfR1 via sialic acids confirmed its role as a directly acting entry factor, but unexpectedly even headless TfR1 promoted IAV particle uptake in trans. TIRF microscopy localized the entering virus-like particles in the vicinity of TfR1. Our data identify TfR1 recycling as a revolving door mechanism exploited by IAV to enter host cells.

SARS-CoV-2 infection as a trigger of humoral response against apolipoprotein A-1.

BACKGROUND: Unravelling autoimmune targets triggered by SARS-CoV-2 infection may provide crucial insights into the physiopathology of the disease and foster the development of potential therapeutic candidate targets and prognostic tools. We aimed at determining (a) the association between anti-SARS-CoV-2 and anti-apoA-1 humoral response and (b) the degree of linear homology between SARS-CoV-2, apoA-1 and Toll-like receptor 2 (TLR2) epitopes. DESIGN: Bioinformatics modelling coupled with mimic peptides engineering and competition experiments were used to assess epitopes sequence homologies. Anti-SARS-CoV-2 and anti-apoA-1 IgG as well as cytokines were assessed by immunoassays on a case-control (n = 101), an intensive care unit (ICU; n = 126) and a general population cohort (n = 663) with available samples in the pre and post-pandemic period. RESULTS: Using bioinformatics modelling, linear sequence homologies between apoA-1, TLR2 and Spike epitopes were identified but without experimental evidence of cross-reactivity. Overall, anti-apoA-1 IgG levels were higher in COVID-19 patients or anti-SARS-CoV-2 seropositive individuals than in healthy donors or anti-SARS-CoV-2 seronegative individuals (P < .0001). Significant and similar associations were noted between anti-apoA-1, anti-SARS-CoV-2 IgG, cytokines and lipid profile. In ICU patients, anti-SARS-CoV-2 and anti-apoA-1 seroconversion rates displayed similar 7-day kinetics, reaching 82% for anti-apoA-1 seropositivity. In the general population, SARS-CoV-2-exposed individuals displayed higher anti-apoA-1 IgG seropositivity rates than nonexposed ones (34% vs 16.8%; P = .004). CONCLUSION: COVID-19 induces a marked humoral response against the major protein of high-density lipoproteins. As a correlate of poorer prognosis in other clinical settings, such autoimmunity signatures may relate to long-term COVID-19 prognosis assessment and warrant further scrutiny in the current COVID-19 pandemic.

Precision-engineered Peptide and Protein Analogs: Establishing a New Discovery Platform for Potent GPCR Modulators.

Numerous members of the human G protein-coupled receptor (GPCR) superfamily are receptors of therapeutic interest. GPCRs are considered to be highly tractable for drug discovery, representing the targets of approximately one-third of currently licensed drugs. These successful drug discovery outcomes cover only a relatively small subset of the superfamily, however, and many other attractive receptors have proven to present significant challenges. Among these difficult GPCRs are those whose natural ligands are peptides and proteins. In this review we explain the obstacles faced by GPCR drug discovery campaigns, with particular focus on those related to peptide and protein GPCRs. We describe a novel and promising approach for these targets based on engineering of their natural ligands and describe an integrated discovery platform that allows potent ligand analogs to be discovered rapidly and efficiently. Finally, we present a case study involving the chemokine receptor CCR5 to show that this approach can be used to generate new drugs for peptide and protein GPCR targets combining best-in-class potency with tunable signaling activity.

Arrestin Recruitment to C-C Chemokine Receptor 5: Potent C-C Chemokine Ligand 5 Analogs Reveal Differences in Dependence on Receptor Phosphorylation and Isoform-Specific Recruitment Bias.

C-C chemokine receptor 5 (CCR5) is a chemokine receptor belonging to the G protein-coupled receptor (GPCR) superfamily. An established anti-human immunodeficiency virus drug target, CCR5 is attracting significant additional interest in both cancer and neuroinflammation. Several N-terminally engineered analogs of C-C chemokine ligand 5 (CCL5), a natural ligand of CCR5, are highly potent CCR5 inhibitors. The inhibitory mechanisms of certain analogs relate to modulation of receptor desensitization, but the cellular and molecular mechanisms have not been fully elucidated. Here we made use of a collection of CCR5 phosphorylation mutants and arrestin variants to investigate how CCL5 analogs differ from CCL5 in their capacity to elicit both CCR5 phosphorylation and arrestin recruitment, with reference to the current "core" and "tail" interaction model for arrestin-GPCR interaction. We showed that CCL5 recruits both arrestin 2 and arrestin 3 to CCR5 with recruitment, particularly of arrestin 2, strongly dependent on the arrestin tail interaction. 5P12-RANTES does not elicit receptor phosphorylation or arrestin recruitment. In contrast, PSC-RANTES induces CCR5 hyperphosphorylation, driving enhanced arrestin recruitment with lower dependence on the arrestin tail interaction. 5P14-RANTES induces comparable levels of receptor phosphorylation to CCL5, but arrestin recruitment is absolutely dependent on the arrestin tail interaction, and in one of the cellular backgrounds used, recruitment showed isoform bias toward arrestin 3 versus arrestin 2. No evidence for ligand-specific differences in receptor phosphorylation patterns across the four implicated serine residues was observed. Our results improve understanding of the molecular pharmacology of CCR5 and help further elucidate the inhibitory mechanisms of a group of potent inhibitors. SIGNIFICANCE STATEMENT: C-C chemokine receptor 5 (CCR5) is a key drug target for human immunodeficiency virus, cancer, and inflammation. Highly potent chemokine analog inhibitors act via the modulation of receptor desensitization, a process initiated by the recruitment of arrestin proteins. This study shows that potent C-C chemokine ligand 5 analogs differ from each other and from the parent chemokine in the extent and quality of CCR5-arrestin association that they elicit, providing valuable insights into CCR5 pharmacology and cell biology that will facilitate the development of new medicines targeting this important receptor.

High-Affinity Binding of Chemokine Analogs that Display Ligand Bias at the HIV-1 Coreceptor CCR5.

The chemokine receptor CCR5 is a drug target to prevent transmission of HIV/AIDS. We studied four analogs of the native chemokine regulated, on activation, normal T-cell-expressed, and secreted (RANTES) (CCL5) that have anti-HIV potencies of around 25 pM, which is more than four orders of magnitude higher than that of RANTES itself. It has been hypothesized that the ultrahigh potency of the analogs is due to their ability to bind populations of receptors not accessible to native chemokines. To test this hypothesis, we developed a homogeneous dual-color fluorescence cross-correlation spectroscopy assay for saturation- and competition-binding experiments. The fluorescence cross-correlation spectroscopy assay has the advantage that it does not rely on competition with radioactively labeled native chemokines used in conventional assays. We prepared site-specifically labeled fluorescent analogs using native chemical ligation of synthetic peptides, followed by bioorthogonal fluorescent labeling. We engineered a mammalian cell expression construct to provide fluorescently labeled CCR5, which was purified using a tandem immunoaffinity and size-exclusion chromatography approach to obtain monomeric fluorescent CCR5 in detergent solution. We found subnanomolar binding affinities for the two analogs 5P12-RANTES and 5P14-RANTES and about 20-fold reduced affinities for PSC-RANTES and 6P4-RANTES. Using homologous and heterologous competition experiments with unlabeled chemokine analogs, we conclude that the analogs all bind at the same binding site, whereas the native chemokines (RANTES and MIP-1α) fail to displace bound fluorescent analogs even at tens of micromolar concentrations. Our results can be rationalized with de novo structural models of the N-terminal tails of the synthetic chemokines that adopt a different binding mode as compared to the parent compound.

Rapid and low-cost multiplex synthesis of chemokine analogs.

Peptides represent a promising source of new medicines, but improved technologies are needed to facilitate discovery and optimization campaigns. In particular, longer peptides with multiple disulfide bridges are challenging to produce, and producing large numbers of structurally related variants is dissuasively costly and time-consuming. The principal cost and time drivers are the multiple column chromatography purification steps that are used during the multistep chemical synthesis procedure, which involves both ligation and oxidative refolding steps. In this study, we developed a method for multiplex parallel synthesis of complex peptide analogs in which the structurally variant region of the molecule is produced as a small peptide on a 384-well synthesizer with subsequent ligation to the longer, structurally invariant region and oxidative refolding carried out in-well without any column purification steps. To test the method, we used a panel of 96 analogs of the chemokine RANTES (regulated on activation normal T cell expressed and secreted)/CCL5 (69 residues, two disulfide bridges), which had been synthesized using standard approaches and characterized pharmacologically in an earlier study. Although, as expected, the multiplex method generated chemokine analogs of lower purity than those produced in the original study, it was nonetheless possible to closely match the pharmacological attributes (anti-HIV potency, capacity to elicit G protein signaling, and capacity to elicit intracellular receptor sequestration) of each chemokine analog to reference data from the earlier study. This rapid, low-cost approach has the potential to support discovery and optimization campaigns based on analogs of other chemokines as well as those of other complex peptide and small protein targets of a similar size.

CCR5: Established paradigms and new frontiers for a 'celebrity' chemokine receptor.

Because of the level of attention it received due to its role as the principal HIV coreceptor, CCR5 has been described as a 'celebrity' chemokine receptor. Here we describe the development of CCR5 inhibitory strategies that have been developed for HIV therapy and which are now additionally being considered for use in HIV prevention and cure. The wealth of CCR5-related tools that have been developed during the intensive investigation of CCR5 as an HIV drug target can now be turned towards the study of CCR5 as a model chemokine receptor. We also summarize what is currently known about the cell biology and pharmacology of CCR5, providing an update on new areas of investigation that have emerged in recent research. Finally, we discuss the potential of CCR5 as a drug target for diseases other than HIV, discussing the evidence linking CCR5 and its natural chemokine ligands with inflammatory diseases, particularly neuroinflammation, and certain cancers. These pathologies may provide new uses for the strategies for CCR5 blockade originally developed to combat HIV/AIDS.

Impact of CD14 Polymorphisms on Anti-Apolipoprotein A-1 IgG-Related Coronary Artery Disease Prediction in the General Population.

OBJECTIVE: We aimed to determine whether autoantibodies against apoA-1 (apolipoprotein A-1; anti-apoA-1 IgG) predict incident coronary artery disease (CAD), defined as adjudicated incident myocardial infarction, angina, percutaneous coronary revascularization, or bypass grafting, in the general population. We further investigated whether this association is modulated by a functional CD14 receptor single nucleotide polymorphism. APPROACH AND RESULTS: In a prospectively studied, population-based cohort of 5220 subjects (mean age 52.6±10.7 years, 47.4% males), followed over a median period of 5.6 years, subjects positive versus negative for anti-apoA-1 IgG presented a total CAD rate of 3.9% versus 2.8% (P=0.077) and a nonfatal CAD rate of 3.6% versus 2.3% (P=0.018), respectively. After multivariate adjustment for established cardiovascular risk factors, the hazard ratios of anti-apoA-1 IgG for total and nonfatal CAD were: hazard ratio=1.36 (95% confidence interval, 0.94-1.97; P=0.105) and hazard ratio=1.53 (95% confidence interval, 1.03-2.26; P=0.034), respectively. In subjects with available genetic data for the C260T rs2569190 single nucleotide polymorphism in the CD14 receptor gene (n=4247), we observed a significant interaction between anti-apoA-1 IgG and rs2569190 allele status with regards to CAD risk, with anti-apoA-1 IgG conferring the highest risk for total and nonfatal CAD in non-TT carriers, whereas being associated with the lowest risk for total and nonfatal CAD in TT homozygotes (P for interaction =0.011 and P for interaction =0.033, respectively). CONCLUSIONS: Anti-apoA-1 IgG are independent predictors of nonfatal incident CAD in the general population. The strength of this association is dependent on a functional polymorphism of the CD14 receptor gene, a finding suggesting a gene-autoantibody interaction for the development of CAD.

Pharmacokinetics of the Protein Microbicide 5P12-RANTES in Sheep following Single-Dose Vaginal Gel Administration.

5P12-RANTES, a chemokine analogue that potently blocks the HIV CCR5 coreceptor, is being developed as both a vaginal and rectal microbicide for prevention of sexual transmission of HIV. Here, we report the first pharmacokinetic data for 5P12-RANTES following single-dose vaginal gel administration in sheep. Aqueous gel formulations containing low (1.24-mg/ml), intermediate (6.18-mg/ml), and high (32.0-mg/ml; suspension-type gel) concentrations of 5P12-RANTES were assessed via rheology, syringeability, and in vitro release testing. Following vaginal gel administration to sheep, 5P12-RANTES concentrations were measured in vaginal fluid, vaginal tissue, and serum over a 96-h period. All gels showed non-Newtonian pseudoplastic behavior, with the high-concentration gels exhibiting a greater viscosity and cohesive structure than the intermediate- and low-concentration gels. In in vitro release testing, >90% 5P12-RANTES was released from the low- and intermediate-concentration gels after 72 h. For the high-concentration gel, ∼50% 5P12-RANTES was detected, attributed to protein denaturation during lyophilization and/or subsequent solvation of the protein within the gel matrix. In sheep, 5P12-RANTES concentrations in vaginal fluid, vaginal tissue, and serum increased in a dose-dependent manner. The highest concentrations were measured in vaginal fluid (105 to 107 ng/ml), followed by vaginal tissue (104 to 106 ng/ml). Both of these concentration ranges are several orders of magnitude above the reported half-maximal inhibitory concentrations. The lowest concentration was measured in serum (<102 ng/ml). The 5P12-RANTES pharmacokinetic data are similar to those reported previously for other candidate microbicides. These data, coupled with 5P12-RANTES's potency at picomolar concentrations, its strong barrier to resistance, and the full protection that it was observed to provide in a rhesus macaque vaginal challenge model, support the continued development of 5P12-RANTES as a microbicide.

Enhancing Antitumor Immune Responses by Optimized Combinations of Cell-penetrating Peptide-based Vaccines and Adjuvants.

Cell penetrating peptides (CPPs) from the protein ZEBRA are promising candidates to exploit in therapeutic cancer vaccines, since they can transport antigenic cargos into dendritic cells and induce tumor-specific T cells. Employing CPPs for a given cancer indication will require engineering to include relevant tumor-associated epitopes, administration with an appropriate adjuvant, and testing for antitumor immunity. We assessed the importance of structural characteristics, efficiency of in vitro transduction of target cells, and choice of adjuvant in inducing the two key elements in antitumor immunity, CD4 and CD8 T cells, as well as control of tumor growth in vivo. Structural characteristics associated with CPP function varied according to CPP truncations and cargo epitope composition, and correlated with in vitro transduction efficiency. However, subsequent in vivo capacity to induce CD4 and CD8 T cells was not always predicted by in vitro results. We determined that the critical parameter for in vivo efficacy using aggressive mouse tumor models was the choice of adjuvant. Optimal pairing of a particular ZEBRA-CPP sequence and antigenic cargo together with adjuvant induced potent antitumor immunity. Our results highlight the irreplaceable role of in vivo testing of novel vaccine constructs together with adjuvants to select combinations for further development.

Microbicides and other topical strategies to prevent vaginal transmission of HIV.

The HIV epidemic is, by many criteria, the worst outbreak of infectious disease in history. The rate of new infections is now approximately 5 million per year, mainly in the developing world, and is increasing. Women are now substantially more at risk of infection with HIV than men. With no cure or effective vaccine in sight, a huge effort is required to develop topical agents (often called microbicides) that, applied to the vaginal mucosa, would prevent infection of these high-risk individuals. We discuss the targets for topical agents that have been identified by studies of the biology of HIV infection and provide an overview of the progress towards the development of a usable agent.

A scalable low-cost cGMP process for clinical grade production of the HIV inhibitor 5P12-RANTES in Pichia pastoris.

In the continued absence of an effective anti-HIV vaccine, approximately 2 million new HIV infections occur every year, with over 95% of these in developing countries. Calls have been made for the development of anti-HIV drugs that can be formulated for topical use to prevent HIV transmission during sexual intercourse. Because these drugs are principally destined for use in low-resource regions, achieving production costs that are as low as possible is an absolute requirement. 5P12-RANTES, an analog of the human chemokine protein RANTES/CCL5, is a highly potent HIV entry inhibitor which acts by achieving potent blockade of the principal HIV coreceptor, CCR5. Here we describe the development and optimization of a scalable low-cost production process for 5P12-RANTES based on expression in Pichia pastoris. At pilot (150 L) scale, this cGMP compliant process yielded 30 g of clinical grade 5P12-RANTES. As well as providing sufficient material for the first stage of clinical development, this process represents an important step towards achieving production of 5P12-RANTES at a cost and scale appropriate to meet needs for topical HIV prevention worldwide.

HIV-1 exploits CCR5 conformational heterogeneity to escape inhibition by chemokines.

CC chemokine receptor 5 (CCR5) is a receptor for chemokines and the coreceptor for R5 HIV-1 entry into CD4(+) T lymphocytes. Chemokines exert anti-HIV-1 activity in vitro, both by displacing the viral envelope glycoprotein gp120 from binding to CCR5 and by promoting CCR5 endocytosis, suggesting that they play a protective role in HIV infection. However, we showed here that different CCR5 conformations at the cell surface are differentially engaged by chemokines and gp120, making chemokines weaker inhibitors of HIV infection than would be expected from their binding affinity constants for CCR5. These distinct CCR5 conformations rely on CCR5 coupling to nucleotide-free G proteins ((NF)G proteins). Whereas native CCR5 chemokines bind with subnanomolar affinity to (NF)G protein-coupled CCR5, gp120/HIV-1 does not discriminate between (NF)G protein-coupled and uncoupled CCR5. Interestingly, the antiviral activity of chemokines is G protein independent, suggesting that "low-chemokine affinity" (NF)G protein-uncoupled conformations of CCR5 represent a portal for viral entry. Furthermore, chemokines are weak inducers of CCR5 endocytosis, as is revealed by EC50 values for chemokine-mediated endocytosis reflecting their low-affinity constant value for (NF)G protein-uncoupled CCR5. Abolishing CCR5 interaction with (NF)G proteins eliminates high-affinity binding of CCR5 chemokines but preserves receptor endocytosis, indicating that chemokines preferentially endocytose low-affinity receptors. Finally, we evidenced that chemokine analogs achieve highly potent HIV-1 inhibition due to high-affinity interactions with internalizing and/or gp120-binding receptors. These data are consistent with HIV-1 evading chemokine inhibition by exploiting CCR5 conformational heterogeneity, shed light into the inhibitory mechanisms of anti-HIV-1 chemokine analogs, and provide insights for the development of unique anti-HIV molecules.

Recombinant Antibodies for Academia: A Practical Approach.

After several decades of optimization, phage display technology enables the routine isolation and production of recombinant monoclonal antibodies in vitro. As such it has the potential to provide the academic community with a vast, inexpensive and renewable supply of well-characterized reagents, reducing bottlenecks in basic science, helping increase reproducibility of experiments, and phasing out the use of animals for production and discovery of antibodies. Yet the overwhelming majority of fundamental research laboratories still use incompletely characterized antibodies developed in animals. In order to promote increased use of recombinant antibodies in academia, we have recently initiated an open source recombinant antibody facility in Geneva (http://www.unige.ch/antibodies). Here we describe our experience at the Geneva Antibody Facility: the various techniques involved in isolation and production of antibodies, the strategic choices that we have made, and what we hope will be a bright future for this project as part of a growing movement in the scientific community to replace all animal-derived antibodies with recombinant antibodies.

δ-Conotoxins synthesized using an acid-cleavable solubility tag approach reveal key structural determinants for NaV subtype selectivity.

Conotoxins are venom peptides from cone snails with multiple disulfide bridges that provide a rigid structural scaffold. Typically acting on ion channels implicated in neurotransmission, conotoxins are of interest both as tools for pharmacological studies and as potential new medicines. δ-Conotoxins act by inhibiting inactivation of voltage-gated sodium channels (Nav). Their pharmacology has not been extensively studied because their highly hydrophobic character makes them difficult targets for chemical synthesis. Here we adopted an acid-cleavable solubility tag strategy that facilitated synthesis, purification, and directed disulfide bridge formation. Using this approach we readily produced three native δ-conotoxins from Conus consors plus two rationally designed hybrid peptides. We observed striking differences in Nav subtype selectivity across this group of compounds, which differ in primary structure at only three positions: 12, 23, and 25. Our results provide new insights into the structure-activity relationships underlying the Nav subtype selectivity of δ-conotoxins. Use of the acid-cleavable solubility tag strategy should facilitate synthesis of other hydrophobic peptides with complex disulfide bridge patterns.

Definition of human apolipoprotein A-I epitopes recognized by autoantibodies present in patients with cardiovascular diseases.

Autoantibodies to apolipoprotein A-I (anti-apoA-I IgG) have been shown to be both markers and mediators of cardiovascular disease, promoting atherogenesis and unstable atherosclerotic plaque. Previous studies have shown that high levels of anti-apoA-I IgGs are independently associated with major adverse cardiovascular events in patients with myocardial infarction. Autoantibody responses to apoA-I can be polyclonal and it is likely that more than one epitope may exist. To identify the specific immunoreactive peptides in apoA-I, we have developed a set of methodologies and procedures to isolate, purify, and identify novel apoA-I endogenous epitopes. First, we generated high purity apoA-I from human plasma, using thiophilic interaction chromatography followed by enzymatic digestion specifically at lysine or arginine residues. Immunoreactivity to the different peptides generated was tested by ELISA using serum obtained from patients with acute myocardial infarction and high titers of autoantibodies to native apoA-I. The immunoreactive peptides were further sequenced by mass spectrometry. Our approach successfully identified two novel immunoreactive peptides, recognized by autoantibodies from patients suffering from myocardial infarction, who contain a high titer of anti-apoA-I IgG. The discovery of these epitopes may open innovative prognostic and therapeutic opportunities potentially suitable to improve current cardiovascular risk stratification.

Targeting spare CC chemokine receptor 5 (CCR5) as a principle to inhibit HIV-1 entry.

CCR5 binds the chemokines CCL3, CCL4, and CCL5 and is the major coreceptor for HIV-1 entry into target cells. Chemokines are supposed to form a natural barrier against human immunodeficiency virus, type 1 (HIV-1) infection. However, we showed that their antiviral activity is limited by CCR5 adopting low-chemokine affinity conformations at the cell surface. Here, we investigated whether a pool of CCR5 that is not stabilized by chemokines could represent a target for inhibiting HIV infection. We exploited the characteristics of the chemokine analog PSC-RANTES (N-α-(n-nonanoyl)-des-Ser(1)-[l-thioprolyl(2), l-cyclohexylglycyl(3)]-RANTES(4-68)), which displays potent anti-HIV-1 activity. We show that native chemokines fail to prevent high-affinity binding of PSC-RANTES, analog-mediated calcium release (in desensitization assays), and analog-mediated CCR5 internalization. These results indicate that a pool of spare CCR5 may bind PSC-RANTES but not native chemokines. Improved recognition of CCR5 by PSC-RANTES may explain why the analog promotes higher amounts of ß-arrestin 2·CCR5 complexes, thereby increasing CCR5 down-regulation and HIV-1 inhibition. Together, these results highlight that spare CCR5, which might permit HIV-1 to escape from chemokines, should be targeted for efficient viral blockade.

Coordinate-based co-localization-mediated analysis of arrestin clustering upon stimulation of the C-C chemokine receptor 5 with RANTES/CCL5 analogues.

G protein-coupled receptor activation and desensitization leads to recruitment of arrestin proteins from cytosolic pools to the cell membrane where they form clusters difficult to characterize due to their small size and further mediate receptor internalization. We quantitatively investigated clustering of arrestin 3 induced by potent anti-HIV analogues of the chemokine RANTES after stimulation of the C-C chemokine receptor 5 using single-molecule localization-based super-resolution microscopy. We determined arrestin 3 cluster sizes and relative fractions of arrestin 3 molecules in each cluster through image-based analysis of the localization data by adapting a method originally developed for co-localization analysis from molecular coordinates. We found that only classical agonists in the set of tested ligands were able to efficiently recruit arrestin 3 to clusters mostly larger than 150 nm in size and compare our results with existing data on arrestin 2 clustering induced by the same chemokine analogues.

Arylsulfatases and neuraminidases modulate engagement of CCR5 by chemokines by removing key electrostatic interactions.

The chemokine receptor CCR5 is known to exist in cell surface subpopulations that differ in their capacity to engage ligands. One proposed explanation for this phenomenon is the presence of CCR5 species with different levels of post-translational modifications (PTMs). Tyrosine sulfation and O-glycan sialylation are PTMs that add negative charges to the extracellular domain of CCR5 and make strong contributions to chemokine binding but it is not known whether cellular mechanisms to control their levels exist. In this study we used a combination of sulfation-sensitive and sulfation-insensitive CCR5 ligands to show that the rate of turnover of CCR5 tyrosine sulfation is more rapid than the rate of turnover of the receptor itself. This suggests that the steady state level of CCR5 sulfation is maintained through the combination of tyrosine protein sulfotransferase (TPST), the trans-Golgi network (TGN)-resident 'source enzyme, and a 'sink' activity that removes tyrosine sulfation from CCR5. By measuring the effects on ligand binding of knockdown and overexpression experiments, we provided evidence that non-lysosomal cellular arylsulfatases, particularly ARSG, ARSI and ARSJ, are CCR5 sulfation 'sink' enzymes. We also used targeted knockdown and sialylation-sensitive and insensitive chemokines to identify the sialidase NEU3 as a candidate 'sink' enzyme for CCR5 O-glycan sialylation. This study provides the first experimental evidence of activity of sulfatase and sialidase 'sink' enzymes on CCR5, providing a potential mechanism for cells to control steady-state levels of these PTMs and thereby exert dynamic control over receptor-ligand interactions at the cell surface and during receptor desensitization.

Characterization of structure, dynamics, and detergent interactions of the anti-HIV chemokine variant 5P12-RANTES.

RANTES (CCL5) is a chemokine that recruits immune cells to inflammatory sites by interacting with the G-protein coupled receptor CCR5, which is also the primary coreceptor used together with CD4 by HIV to enter and infect target cells. Ligands of CCR5, including chemokines and chemokine analogs, are capable of blocking HIV entry, and studies of their structures and interactions with CCR5 will be key to understanding and optimizing HIV inhibition. The RANTES derivative 5P12-RANTES is a highly potent HIV entry inhibitor that is being developed as a topical HIV prevention agent (microbicide). We have characterized the structure and dynamics of 5P12-RANTES by solution NMR. With the exception of the nine flexible N-terminal residues, 5P12-RANTES has the same structure as wild-type RANTES but unlike the wild-type, does not dimerize via its N-terminus. To prepare the ground for interaction studies with detergent-solubilized CCR5, we have also investigated the interaction of RANTES and 5P12-RANTES with various commonly used detergents. Both RANTES variants are stable in Cymal-5, DHPC, Anzergent-3-12, dodecyltrimethylammonium chloride, and a DDM/CHAPS/CHS mixture. Fos-Cholines, dodecyldimethylglycine, and sodium dodecyl-sulfate denature both RANTES variants at low pH, whereas at neutral pH the stability is considerably higher. The onset of Fos-Choline-12-induced denaturation and the denatured state were characterized by circular dichroism and NMR. The detergent interaction starts below the critical micelle concentration at a well-defined mixed hydrophobic/positive surface region of the chemokine, which overlaps with the dimer interface. An increase of Fos-Choline-12 concentration above the critical micelle concentration causes a transition to a denatured state with a high α-helical content.