VISTA drives macrophages towards a pro-tumoral phenotype that promotes cancer cell phagocytosis yet down-regulates T cell responses.

BACKGROUND: VISTA is a well-known immune checkpoint in T cell biology, but its role in innate immunity is less established. Here, we investigated the role of VISTA on anticancer macrophage immunity, with a focus on phagocytosis, macrophage polarization and concomitant T cell activation. METHODS: Macrophages, differentiated from VISTA overexpressed THP-1 cells and cord blood CD34+ cell-derived monocytes, were used in phagocytosis assay using B lymphoma target cells opsonized with Rituximab. PBMC-derived macrophages were used to assess the correlation between phagocytosis and VISTA expression. qRT-PCR, flow cytometry, and enzyme-linked immunosorbent assay were performed to analyze the impact of VISTA on other checkpoints and M1/M2-like macrophage biology. Additionally, flow cytometry was used to assess the frequency of CD14+ monocytes expressing VISTA in PBMCs from 65 lymphoma patients and 37 healthy donors. RESULTS: Ectopic expression of VISTA in the monocytic model cell line THP-1 or in primary monocytes triggered differentiation towards the macrophage lineage, with a marked increase in M2-like macrophage-related gene expression and decrease in M1-like macrophage-related gene expression. VISTA expression in THP-1 and monocyte-derived macrophages strongly downregulated expression of SIRPα, a prominent 'don't eat me' signal, and augmented phagocytic activity of macrophages against cancer cells. Intriguingly, expression of VISTA's extracellular domain alone sufficed to trigger phagocytosis in ∼ 50% of cell lines, with those cell lines also directly binding to recombinant human VISTA, indicating ligand-dependent and -independent mechanisms. Endogenous VISTA expression was predominantly higher in M2-like macrophages compared to M0- or M1-like macrophages, with a positive correlation observed between VISTA expression in M2c macrophages and their phagocytic activity. VISTA-expressing macrophages demonstrated a unique cytokine profile, characterized by reduced IL-1ß and elevated IL-10 secretion. Furthermore, VISTA interacted with MHC-I and downregulated its surface expression, leading to diminished T cell activation. Notably, VISTA surface expression was identified in monocytes from all lymphoma patients but was less prevalent in healthy donors. CONCLUSIONS: Collectively, VISTA expression associates with and drives M2-like activation of macrophages with a high phagocytic capacity yet a decrease in antigen presentation capability to T cells. Therefore, VISTA is a negative immune checkpoint regulator in macrophage-mediated immune suppression.

Dimerization of European Robin Cryptochrome 4a.

Homo-dimer formation is important for the function of many proteins. Although dimeric forms of cryptochromes (Cry) have been found by crystallography and were recently observed in vitro for European robin Cry4a, little is known about the dimerization of avian Crys and the role it could play in the mechanism of magnetic sensing in migratory birds. Here, we present a combined experimental and computational investigation of the dimerization of robin Cry4a resulting from covalent and non-covalent interactions. Experimental studies using native mass spectrometry, mass spectrometric analysis of disulfide bonds, chemical cross-linking, and photometric measurements show that disulfide-linked dimers are routinely formed, that their formation is promoted by exposure to blue light, and that the most likely cysteines are C317 and C412. Computational modeling and molecular dynamics simulations were used to generate and assess a number of possible dimer structures. The relevance of these findings to the proposed role of Cry4a in avian magnetoreception is discussed.

Molecular tuning of calcium dependent processes by neuronal calcium sensor proteins in the retina.

Vertebrate photoreceptor cells are exquisite light detectors operating under very dim and bright illumination mediated by phototransduction, which is under control of the two secondary messengers cGMP and Ca2+. Feedback mechanisms enable photoreceptor cells to regain their responsiveness after light stimulation and involve neuronal Ca2+-sensor proteins, named GCAPs (guanylate cyclase-activating proteins) and recoverins. This review compares the diversity in Ca2+-related signaling mediated by GCAP and recoverin variants that exhibit differences in Ca2+-sensing, protein conformational changes, myristoyl switch mechanisms, diversity in divalent cation binding and dimer formation. In summary, both subclasses of neuronal Ca2+-sensor proteins contribute to a complex signaling network in rod and cone cells, which is perfectly suited to match the requirements for sensitive cell responses and maintaining this responsiveness in the presence of different background light intensities.

Kinetics of cone specific G-protein signaling in avian photoreceptor cells.

Cone photoreceptor cells of night-migratory songbirds seem to process the primary steps of two different senses, vision and magnetoreception. The molecular basis of phototransduction is a prototypical G protein-coupled receptor pathway starting with the photoexcitation of rhodopsin or cone opsin thereby activating a heterotrimeric G protein named transducin. This interaction is well understood in vertebrate rod cells, but parameter describing protein-protein interactions of cone specific proteins are rare and not available for migratory birds. European robin is a model organism for studying the orientation of birds in the earth magnetic field. Recent findings showed a link between the putative magnetoreceptor cryptochrome 4a and the cone specific G-protein of European robin. In the present work, we investigated the interaction of European robin cone specific G protein and cytoplasmic regions of long wavelength opsin. We identified the second loop in opsin connecting transmembrane regions three and four as a critical binding interface. Surface plasmon resonance studies using a synthetic peptide representing the second cytoplasmic loop and purified G protein α-subunit showed a high affinity interaction with a K D value of 21 nM. Truncation of the G protein α-subunit at the C-terminus by six amino acids slightly decreased the affinity. Our results suggest that binding of the G protein to cryptochrome can compete with the interaction of G protein and non-photoexcited long wavelength opsin. Thus, the parallel presence of two different sensory pathways in bird cone photoreceptors is reasonable under dark-adapted conditions or during illumination with short wavelengths.

Direct Interaction of Avian Cryptochrome 4 with a Cone Specific G-Protein.

BACKGROUND: Night-migratory birds sense the Earth's magnetic field by an unknown molecular mechanism. Theoretical and experimental evidence support the hypothesis that the light-induced formation of a radical-pair in European robin cryptochrome 4a (ErCry4a) is the primary signaling step in the retina of the bird. In the present work, we investigated a possible route of cryptochrome signaling involving the α-subunit of the cone-secific heterotrimeric G protein from European robin. METHODS: Protein-protein interaction studies include surface plasmon resonance, pulldown affinity binding and Förster resonance energy transfer. RESULTS: Surface plasmon resonance studies showed direct interaction, revealing high to moderate affinity for binding of non-myristoylated and myristoylated G protein to ErCry4a, respectively. Pulldown affinity experiments confirmed this complex formation in solution. We validated these in vitro data by monitoring the interaction between ErCry4a and G protein in a transiently transfected neuroretinal cell line using Förster resonance energy transfer. CONCLUSIONS: Our results suggest that ErCry4a and the G protein also interact in living cells and might constitute the first biochemical signaling step in radical-pair-based magnetoreception.

The Transition of Photoreceptor Guanylate Cyclase Type 1 to the Active State.

Membrane-bound guanylate cyclases (GCs), which synthesize the second messenger guanosine-3', 5'-cyclic monophosphate, differ in their activation modes to reach the active state. Hormone peptides bind to the extracellular domain in hormone-receptor-type GCs and trigger a conformational change in the intracellular, cytoplasmic part of the enzyme. Sensory GCs that are present in rod and cone photoreceptor cells have intracellular binding sites for regulatory Ca2+-sensor proteins, named guanylate-cyclase-activating proteins. A rotation model of activation involving an α-helix rotation was described as a common activation motif among hormone-receptor GCs. We tested whether the photoreceptor GC-E underwent an α-helix rotation when reaching the active state. We experimentally simulated such a transitory switch by integrating alanine residues close to the transmembrane region, and compared the effects of alanine integration with the point mutation V902L in GC-E. The V902L mutation is found in patients suffering from retinal cone-rod dystrophies, and leads to a constitutively active state of GC-E. We analyzed the enzymatic catalytic parameters of wild-type and mutant GC-E. Our data showed no involvement of an α-helix rotation when reaching the active state, indicating a difference in hormone receptor GCs. To characterize the protein conformations that represent the transition to the active state, we investigated the protein dynamics by using a computational approach based on all-atom molecular dynamics simulations. We detected a swinging movement of the dimerization domain in the V902L mutant as the critical conformational switch in the cyclase going from the low to high activity state.

Molecular Properties of Human Guanylate Cyclase-Activating Protein 3 (GCAP3) and Its Possible Association with Retinitis Pigmentosa.

The cone-specific guanylate cyclase-activating protein 3 (GCAP3), encoded by the GUCA1C gene, has been shown to regulate the enzymatic activity of membrane-bound guanylate cyclases (GCs) in bovine and teleost fish photoreceptors, to an extent comparable to that of the paralog protein GCAP1. To date, the molecular mechanisms underlying GCAP3 function remain largely unexplored. In this work, we report a thorough characterization of the biochemical and biophysical properties of human GCAP3, moreover, we identified an isolated case of retinitis pigmentosa, in which a patient carried the c.301G>C mutation in GUCA1C, resulting in the substitution of a highly conserved aspartate residue by a histidine (p.(D101H)). We found that myristoylated GCAP3 can activate GC1 with a similar Ca2+-dependent profile, but significantly less efficiently than GCAP1. The non-myristoylated form did not induce appreciable regulation of GC1, nor did the p.D101H variant. GCAP3 forms dimers under physiological conditions, but at odds with its paralogs, it tends to form temperature-dependent aggregates driven by hydrophobic interactions. The peculiar properties of GCAP3 were confirmed by 2 ms molecular dynamics simulations, which for the p.D101H variant highlighted a very high structural flexibility and a clear tendency to lose the binding of a Ca2+ ion to EF3. Overall, our data show that GCAP3 has unusual biochemical properties, which make the protein significantly different from GCAP1 and GCAP2. Moreover, the newly identified point mutation resulting in a substantially unfunctional protein could trigger retinitis pigmentosa through a currently unknown mechanism.

Retinal degeneration protein 3 controls membrane guanylate cyclase activities in brain tissue.

The retinal degeneration protein RD3 is involved in regulatory processes of photoreceptor cells. Among its main functions is the inhibition of photoreceptor specific membrane guanylate cyclases during trafficking from the inner segment to their final destination in the outer segment. However, any physiological role of RD3 in non-retinal tissue is unsolved at present and specific protein targets outside of retinal tissue have not been identified so far. The family of membrane bound guanylate cyclases share a high homology of their amino acid sequences in their cytoplasmic domains. Therefore, we reasoned that membrane guanylate cyclases that are activated by natriuretic peptides are also regulated by RD3. We analyzed transcript levels of the rd3 gene and natriuretic peptide receptor genes Npr1 and Npr2 in the mouse retina, cerebellum, hippocampus, neocortex, and the olfactory bulb during development from the embryonic to the postnatal stage at P60. The rd3 gene showed a lower expression level than Npr1 and Npr2 (encoding for GC-A and GC-B, respectively) in all tested brain tissues, but was at least one order of magnitude higher in the retina. RD3 and natriuretic peptide receptor GCs co-express in the retina and brain tissue leading to functional tests. We expressed GC-A and GC-B in HEK293T cells and measured the inhibition of GCs by RD3 after activation by natriuretic peptides yielding inhibitory constants around 25 nM. Furthermore, endogenous GCs in astrocytes were inhibited by RD3 to a similar extent. We here show for the first time that RD3 can inhibit two hormone-stimulated GCs, namely GC-A and GC-B indicating a new regulatory feature of these hormone receptors.

A hybrid stochastic/deterministic model of single photon response and light adaptation in mouse rods.

The phototransduction cascade is paradigmatic for signaling pathways initiated by G protein-coupled receptors and is characterized by a fine regulation of photoreceptor sensitivity and electrical response to a broad range of light stimuli. Here, we present a biochemically comprehensive model of phototransduction in mouse rods based on a hybrid stochastic and deterministic mathematical framework, and a quantitatively accurate description of the rod impedance in the dark. The latter, combined with novel patch clamp recordings from rod outer segments, enables the interconversion of dim flash responses between photovoltage and photocurrent and thus direct comparison with the simulations. The model reproduces the salient features of the experimental photoresponses at very dim and bright stimuli, for both normal photoreceptors and those with genetically modified cascade components. Our modelling approach recapitulates a number of recent findings in vertebrate phototransduction. First, our results are in line with the recently established requirement of dimeric activation of PDE6 by transducin and further show that such conditions can be fulfilled at the expense of a significant excess of G protein activated by rhodopsin. Secondly, simulations suggest a crucial role of the recoverin-mediated Ca2+-feedback on rhodopsin kinase in accelerating the shutoff, when light flashes are delivered in the presence of a light background. Finally, stochastic simulations suggest that transient complexes between dark rhodopsin and transducin formed prior to light stimulation increase the reproducibility of single photon responses. Current limitations of the model are likely associated with the yet unknown mechanisms governing the shutoff of the cascade.

Magnetic sensitivity of cryptochrome 4 from a migratory songbird.

Night-migratory songbirds are remarkably proficient navigators1. Flying alone and often over great distances, they use various directional cues including, crucially, a light-dependent magnetic compass2,3. The mechanism of this compass has been suggested to rely on the quantum spin dynamics of photoinduced radical pairs in cryptochrome flavoproteins located in the retinas of the birds4-7. Here we show that the photochemistry of cryptochrome 4 (CRY4) from the night-migratory European robin (Erithacus rubecula) is magnetically sensitive in vitro, and more so than CRY4 from two non-migratory bird species, chicken (Gallus gallus) and pigeon (Columba livia). Site-specific mutations of ErCRY4 reveal the roles of four successive flavin-tryptophan radical pairs in generating magnetic field effects and in stabilizing potential signalling states in a way that could enable sensing and signalling functions to be independently optimized in night-migratory birds.

Farnesylation of Zebrafish G-Protein-Coupled Receptor Kinase Using Bio-orthogonal Labeling.

G-protein-coupled receptors are deactivated or desensitized by phosphorylation by respective G-protein-coupled receptor kinases (GRKs). In zebrafish rod and cone photoreceptor cells, four orthologous GRKs are expressed participating in the deactivation of rod and cone opsins. An important feature of GRKs in general is the consensus sites for lipid modification, which would allow the posttranslational attachment of isoprenoids facilitating membrane association and enzymatic performance. Because direct proof is missing for isoprenoid modification of zebrafish GRKs, we used a semichemical approach to study the incorporation of a farnesyl moiety into a GRK and its cellular consequences. The approach involves organic synthesis of a functionalized farnesyl derivative that is suitable for a subsequent alkyne-azide cycloaddition (click reaction). For this purpose, zebrafish GRK was expressed in HEK293 cells and modified in situ with the synthetic farnesyl moiety. Successful farnesylation by an endogenous farnesyltransferase was detected by immunoblotting and immunocytochemistry using a biotin-streptavidin-coupled assay and ligation with a fluorescence dye, respectively. Immunocytochemical detection of farnesylated GRK in different cell compartments indicates the applicability of the approach for studying the transport of cellular components.

An Assessment of GUCA1C Variants in Primary Congenital Glaucoma.

In the special issue "Molecular Genetics of Retinal Dystrophies", Morales-Cámara and colleagues reported the association of a new candidate gene with primary congenital glaucoma (PCG) [...].

Molecular properties of human guanylate cyclase-activating protein 2 (GCAP2) and its retinal dystrophy-associated variant G157R.

In murine and bovine photoreceptors, guanylate cyclase-activating protein 2 (GCAP2) activates retinal guanylate cyclases (GCs) at low Ca2+ levels, thus contributing to the Ca2+/cGMP negative feedback on the cyclase together with its paralog guanylate cyclase-activating protein 1, which has the same function but different Ca2+ sensitivity. In humans, a GCAP2 missense mutation (G157R) has been associated with inherited retinal degeneration (IRD) via an unknown molecular mechanism. Here, we characterized the biochemical properties of human GCAP2 and the G157R variant, focusing on its dimerization and the Ca2+/Mg2+-binding processes in the presence or absence of N-terminal myristoylation. We found that human GCAP2 and its bovine/murine orthologs significantly differ in terms of oligomeric properties, cation binding, and GC regulation. Myristoylated GCAP2 endothermically binds up to 3 Mg2+ with high affinity and forms a compact dimer that may reversibly dissociate in the presence of Ca2+. Conversely, nonmyristoylated GCAP2 does not bind Mg2+ over the physiological range and remains as a monomer in the absence of Ca2+. Both myristoylated and nonmyristoylated GCAP2 bind Ca2+ with high affinity. At odds with guanylate cyclase-activating protein 1 and independently of myristoylation, human GCAP2 does not significantly activate retinal GC1 in a Ca2+-dependent fashion. The IRD-associated G157R variant is characterized by a partly misfolded, molten globule-like conformation with reduced affinity for cations and prone to form aggregates, likely mediated by hydrophobic interactions. Our findings suggest that GCAP2 might be mostly implicated in processes other than phototransduction in human photoreceptors and suggest a possible molecular mechanism for G157R-associated IRD.

First 3D-Structural Data of Full-Length Guanylyl Cyclase 1 in Rod-Outer-Segment Preparations of Bovine Retina by Cross-Linking/Mass Spectrometry.

The rod-outer-segment guanylyl cyclase 1 (ROS-GC1) is a key transmembrane protein for retinal phototransduction. Mutations of ROS-GC1 correlate with different retinal diseases that often lead to blindness. No structural data are available for ROS-GC1 so far. We performed a 3D-structural analysis of native ROS-GC1 from bovine retina by cross-linking/mass spectrometry (XL-MS) and computational modeling. Absolute quantification and activity measurements of native ROS-GC1 were performed by MS-based assays directly in bovine retina samples. Our data present the first 3D-structural analysis of active, full-length ROS-GC1 derived from bovine retina. We propose a novel domain organization for the intracellular domain ROS-GC1. Our XL-MS data of native ROS-GC1 from rod-outer-segment preparations of bovine retina agree with a dimeric architecture. Our integrated approach can serve as a blueprint for conducting 3D-structural studies of membrane proteins in their native environment.

Interaction of G protein-coupled receptor kinases and recoverin isoforms is determined by localization in zebrafish photoreceptors.

The zebrafish retina expresses four recoverin genes (rcv1a, rcv1b, rcv2a and rcv2b) and four opsin kinase genes (grk1a, grk1b, grk7a and grk7b) coding for recoverin and G protein-coupled receptor kinase (opsin kinase) paralogs, respectively. Both protein groups are suggested to form regulatory complexes in rod and cone outer segments, but at present, we lack information about co-localization of recoverin and opsin kinases in zebrafish retinae and which protein-protein interacting pairs form. We analyzed the distribution and co-localization of recoverin and opsin kinase expression in the zebrafish retina. For this purpose, we used custom-tailored monospecific antibodies revealing that the amount of recoverin paralogs in a zebrafish retina can differ by more than one order of magnitude with the highest amount for recoverin 1a and 2b. Further, immunohistochemical labelling showed presence of recoverin 1a in all rod cell compartments, but it only co-localized with opsin kinase 1a in rod outer segments. In contrast, recoverin 2b was only detected in double cones and co-localized with opsin kinases 1b, 7a and 7b. Further, we investigated the interaction between recoverin and opsin kinase variants by surface plasmon resonance spectroscopy indicating interaction of recoverin 1a and recoverin 2b with all opsin kinases. However, binding kinetics for recoverin 1a differed from those observed with recoverin 2b that showed slower association and dissociation processes. Our results indicate diverse recoverin and opsin kinase properties due to differential expression and interaction profiles.

Bringing the Ca2+ sensitivity of myristoylated recoverin into the physiological range.

The prototypical Ca2+-sensor protein recoverin (Rec) is thought to regulate the activity of rhodopsin kinase (GRK1) in photoreceptors by switching from a relaxed (R) disc membrane-bound conformation in the dark to a more compact, cytosol-diffusing tense (T) conformation upon cell illumination. However, the apparent affinity for Ca2+ of its physiologically relevant form (myristoylated recoverin) is almost two orders of magnitude too low to support this mechanism in vivo. In this work, we compared the individual and synergistic roles of the myristic moiety, the GRK1 target and the disc membrane in modulating the calcium sensitivity of Rec. We show that the sole presence of the target or the disc membrane alone are not sufficient to achieve a physiological response to changes in intracellular [Ca2+]. Instead, the simultaneous presence of GRK1 and membrane allows the T to R transition to occur in a physiological range of [Ca2+] with high cooperativity via a conformational selection mechanism that drives the structural transitions of Rec in the presence of multiple ligands. Our conclusions may apply to other sensory transduction systems involving protein complexes and biological membranes.

Label-free Quantification of Direct Protein-protein Interactions with Backscattering Interferometry.

The functional performance of a cell depends on how macromolecules, in particular proteins, come together in a precise orientation, how they assemble into protein complexes and interact with each other. In order to study protein-protein interactions at a molecular level, a variety of methods to investigate these binding processes yield affinity constants and/or the identification of binding regions. There are several well-established biophysical techniques for biomolecular interaction studies, such as fluorescence spectroscopy and surface plasmon resonance. Although these techniques have been proven to be efficient, they either need labeling or immobilization of one interaction partner. Backscattering interferometry (BSI) is a label-free detection method, which allows label- and immobilization-free interaction analysis under physiologically relevant conditions with high sensitivity and in small volumes. We used BSI to measure the interaction of the neuronal calcium sensor recoverin with its target G protein-coupled receptor kinase 1 (GRK1) as a model system. Increasing concentrations of purified recoverin were mixed with a specific concentration of a GRK1 fusion protein. In this protocol, we provide a full description of the instrumental setup, data acquisition, and evaluation. Equilibrium dissociation constants of recoverin-GRK1 interaction determined by the BSI instrumental setup are in full agreement with affinity constants obtained by different methods as described in the literature.

Protein-protein interaction of the putative magnetoreceptor cryptochrome 4 expressed in the avian retina.

Migratory birds can sense the Earth's magnetic field and use it for orientation over thousands of kilometres. A light-dependent radical-pair mechanism associated with the visual system is currently discussed as the underlying mechanism of the magnetic compass sense. The blue light receptor cryptochrome 4 (Cry4) is considered as the most likely primary sensory protein that detects the geomagnetic field. Since the protein interaction partners of Cry4 are completely unknown at present, here, we aim to identify potential candidate interaction partners of Cry4 in the avian retina. We used the yeast-two-hybrid system to screen avian cDNA libraries for possible interaction partners of Cry4 in the European robin. The UAS-GAL yeast two hybrid system was applied to confirm a group of candidate Cry4 interaction partners. Six proteins were found to be particularly promising candidates for interacting with European robin Cry4. The identified genes code for guanine nucleotide-binding protein G(t) subunit alpha-2 (GNAT2), long-wavelength-sensitive opsin (LWS, also called iodopsin), guanine nucleotide-binding protein subunit gamma 10 (GNG10), potassium voltage-gated channel subfamily V member 2 (KCNV2), retinol binding protein 1 (RBP1) and retinal G protein-coupled receptor (RGR). All genes are known to be expressed in vertebrate retinae of different species. We conclude by discussing putative signalling pathways that could connect cryptochrome 4 to one or more of these 6 candidates.

Neuronal Calcium Sensor GCAP1 Encoded by GUCA1A Exhibits Heterogeneous Functional Properties in Two Cases of Retinitis Pigmentosa.

Genetic heterogeneity leading to retinal disorders impairs biological processes by causing, for example, severe disorder of signal transduction in photoreceptor outer segments. A normal balance of the second messenger homeostasis in photoreceptor cells seems to be a crucial factor for healthy and normal photoreceptor function. Genes like GUCY2D coding for guanylate cyclase GC-E and GUCA1A coding for the Ca2+-sensor guanylate cyclase-activating protein GCAP1 are critical for a precisely controlled synthesis of the second messenger cGMP. Mutations in GUCA1A frequently correlate in patients with cone dystrophy and cone-rod dystrophy. Here, we report two mutations in the GUCA1A gene that were found in patients diagnosed with retinitis pigmentosa, a phenotype that was rarely detected among previous cases of GUCA1A related retinopathies. One patient was heterozygous for the missense variant c.55C > T (p.H19Y), while the other patient was heterozygous for the missense variant c.479T > G (p.V160G). Using heterologous expression and cell culture systems, we examined the functional and molecular consequences of these point mutations. Both variants showed a dysregulation of guanylate cyclase activity, either a profound shift in Ca2+-sensitivity (H19Y) or a nearly complete loss of activating potency (V160G). Functional heterogeneity became also apparent in Ca2+/Mg2+-binding properties and protein conformational dynamics. A faster progression of retinal dystrophy in the patient carrying the V160G mutation seems to correlate with the more severe impairment of this variant.