HIV-1 hypermethylated guanosine cap licenses specialized translation unaffected by mTOR.

Appended to the 5' end of nascent RNA polymerase II transcripts is 7-methyl guanosine (m7G-cap) that engages nuclear cap-binding complex (CBC) to facilitate messenger RNA (mRNA) maturation. Mature mRNAs exchange CBC for eIF4E, the rate-limiting translation factor that is controlled through mTOR. Experiments in immune cells have now documented HIV-1 incompletely processed transcripts exhibited hypermethylated m7G-cap and that the down-regulation of the trimethylguanosine synthetase-1-reduced HIV-1 infectivity and virion protein synthesis by several orders of magnitude. HIV-1 cap hypermethylation required nuclear RNA helicase A (RHA)/DHX9 interaction with the shape of the 5' untranslated region (UTR) primer binding site (PBS) segment. Down-regulation of RHA or the anomalous shape of the PBS segment abrogated hypermethylated caps and derepressed eIF4E binding for virion protein translation during global down-regulation of host translation. mTOR inhibition was detrimental to HIV-1 proliferation and attenuated Tat, Rev, and Nef synthesis. This study identified mutually exclusive translation pathways and the calibration of virion structural/accessory protein synthesis with de novo synthesis of the viral regulatory proteins. The hypermethylation of select, viral mRNA resulted in CBC exchange to heterodimeric CBP80/NCBP3 that expanded the functional capacity of HIV-1 in immune cells.

Activated Clec4nhi Neutrophils Aggravate Lung Injury in an Endothelial IGFBP7-Dependent Manner.

The role of endothelial cells in acute lung injury (ALI) has been widely elaborated, but little is known about the role of different subtypes of endothelial cells in ALI. ALI models were established by lipopolysaccharide. Single-cell RNA sequencing was used to identify differential molecules in endothelial subtypes and the heterogeneity of lung immune cells. Specific antibodies were used to block insulin-like growth factor binding protein 7 (IGFBP7), and AAVshIGP7 was used to specifically knock down IGFBP7. Here, we found that IGFBP7 was the most differentially expressed molecule in diverse subsets of endothelial cells and that IGFBP7 was strongly associated with inflammatory responses. Elevated IGFBP7 significantly exacerbated barrier dysfunction in ALI, whereas blockade of IGFBP7 partially reversed barrier damage. General capillary cells are the primary source of elevated serum IGFBP7 after ALI. Using single-cell RNA sequencing, we identified significantly increased Clec4nhi neutrophils in mice with ALI, whereas IGFBP7 knockdown significantly reduced infiltration of Clec4nhi cells and mitigated barrier dysfunction in ALI. In addition, we found that IGFBP7 activated the NF-κB signaling axis by promoting phosphorylation and ubiquitination degradation of F-box/WD repeat-containing protein 2 (FBXW2), thereby exacerbating barrier dysfunction in ALI. Taken together, our data indicate that blockade of serum IGFBP7 or IGFBP7 depletion in general capillary cells reversed barrier damage in ALI. Therefore, targeting IGFBP7 depletion could be a novel strategy for treating ALI.

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.

Retinal degeneration-3 protein attenuates photoreceptor degeneration in transgenic mice expressing dominant mutation of human retinal guanylyl cyclase.

Different forms of photoreceptor degeneration cause blindness. Retinal degeneration-3 protein (RD3) deficiency in photoreceptors leads to recessive congenital blindness. We proposed that aberrant activation of the retinal membrane guanylyl cyclase (RetGC) by its calcium-sensor proteins (guanylyl cyclase-activating protein [GCAP]) causes this retinal degeneration and that RD3 protects photoreceptors by preventing such activation. We here present in vivo evidence that RD3 protects photoreceptors by suppressing activation of both RetGC1 and RetGC2 isozymes. We further suggested that insufficient inhibition of RetGC by RD3 could contribute to some dominant forms of retinal degeneration. The R838S substitution in RetGC1 that causes autosomal-dominant cone-rod dystrophy 6, not only impedes deceleration of RetGC1 activity by Ca2+GCAPs but also elevates this isozyme's resistance to inhibition by RD3. We found that RD3 prolongs the survival of photoreceptors in transgenic mice harboring human R838S RetGC1 (R838S+). Overexpression of GFP-tagged human RD3 did not improve the calcium sensitivity of cGMP production in R838S+ retinas but slowed the progression of retinal blindness and photoreceptor degeneration. Fluorescence of the GFP-tagged RD3 in the retina only partially overlapped with immunofluorescence of RetGC1 or GCAP1, indicating that RD3 separates from the enzyme before the RetGC1:GCAP1 complex is formed in the photoreceptor outer segment. Most importantly, our in vivo results indicate that, in addition to the abnormal Ca2+ sensitivity of R838S RetGC1 in the outer segment, the mutated RetGC1 becomes resistant to inhibition by RD3 in a different cellular compartment(s) and suggest that RD3 overexpression could be utilized to reduce the severity of cone-rod dystrophy 6 pathology.

Retinal degeneration-3 protein promotes photoreceptor survival by suppressing activation of guanylyl cyclase rather than accelerating GMP recycling.

Retinal degeneration-3 protein (RD3) deficiency causes photoreceptor dysfunction and rapid degeneration in the rd3 mouse strain and in human Leber's congenital amaurosis, a congenital retinal dystrophy that results in early vision loss. However, the mechanisms responsible for photoreceptor death remain unclear. Here, we tested two hypothesized biochemical events that may underlie photoreceptor death: (i) the failure to prevent aberrant activation of retinal guanylyl cyclase (RetGC) by calcium-sensor proteins (GCAPs) versus (ii) the reduction of GMP phosphorylation rate, preventing its recycling to GDP/GTP. We found that GMP converts to GDP/GTP in the photoreceptor fraction of the retina ∼24-fold faster in WT mice and ∼400-fold faster in rd3 mice than GTP conversion to cGMP by RetGC. Adding purified RD3 to the retinal extracts inhibited RetGC 4-fold but did not affect GMP phosphorylation in wildtype or rd3 retinas. RD3-deficient photoreceptors rapidly degenerated in rd3 mice that were reared in constant darkness to prevent light-activated GTP consumption via RetGC and phosphodiesterase 6. In contrast, rd3 degeneration was alleviated by deletion of GCAPs. After 2.5 months, only ∼40% of photoreceptors remained in rd3/rd3 retinas. Deletion of GCAP1 or GCAP2 alone preserved 68% and 57% of photoreceptors, respectively, whereas deletion of GCAP1 and GCAP2 together preserved 86%. Taken together, our in vitro and in vivo results support the hypothesis that RD3 prevents photoreceptor death primarily by suppressing activation of RetGC by both GCAP1 and GCAP2 but do not support the hypothesis that RD3 plays a significant role in GMP recycling.

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.

Two clusters of surface-exposed amino acid residues enable high-affinity binding of retinal degeneration-3 (RD3) protein to retinal guanylyl cyclase.

Retinal degeneration-3 (RD3) protein protects photoreceptors from degeneration by preventing retinal guanylyl cyclase (RetGC) activation via calcium-sensing guanylyl cyclase-activating proteins (GCAP), and RD3 truncation causes severe congenital blindness in humans and other animals. The three-dimensional structure of RD3 has recently been established, but the molecular mechanisms of its inhibitory binding to RetGC remain unclear. Here, we report the results of probing 133 surface-exposed residues in RD3 by single substitutions and deletions to identify side chains that are critical for the inhibitory binding of RD3 to RetGC. We tested the effects of these substitutions and deletions in vitro by reconstituting purified RD3 variants with GCAP1-activated human RetGC1. Although the vast majority of the surface-exposed residues tolerated substitutions without loss of RD3's inhibitory activity, substitutions in two distinct narrow clusters located on the opposite sides of the molecule effectively suppressed RD3 binding to the cyclase. The first surface-exposed cluster included residues adjacent to Leu63 in the loop connecting helices 1 and 2. The second cluster surrounded Arg101 on a surface of helix 3. Single substitutions in those two clusters drastically, i.e. up to 245-fold, reduced the IC50 for the cyclase inhibition. Inactivation of the two binding sites completely disabled binding of RD3 to RetGC1 in living HEK293 cells. In contrast, deletion of 49 C-terminal residues did not affect the apparent affinity of RD3 for RetGC. Our findings identify the functional interface on RD3 required for its inhibitory binding to RetGC, a process essential for protecting photoreceptors from degeneration.

GUCY2D mutations in retinal guanylyl cyclase 1 provide biochemical reasons for dominant cone-rod dystrophy but not for stationary night blindness.

Mutations in the GUCY2D gene coding for the dimeric human retinal membrane guanylyl cyclase (RetGC) isozyme RetGC1 cause various forms of blindness, ranging from rod dysfunction to rod and cone degeneration. We tested how the mutations causing recessive congenital stationary night blindness (CSNB), recessive Leber's congenital amaurosis (LCA1), and dominant cone-rod dystrophy-6 (CORD6) affected RetGC1 activity and regulation by RetGC-activating proteins (GCAPs) and retinal degeneration-3 protein (RD3). CSNB mutations R666W, R761W, and L911F, as well as LCA1 mutations R768W and G982VfsX39, disabled RetGC1 activation by human GCAP1, -2, and -3. The R666W and R761W substitutions compromised binding of GCAP1 with RetGC1 in HEK293 cells. In contrast, G982VfsX39 and L911F RetGC1 retained the ability to bind GCAP1 in cyto but failed to effectively bind RD3. R768W RetGC1 did not bind either GCAP1 or RD3. The co-expression of GUCY2D allelic combinations linked to CSNB did not restore RetGC1 activity in vitro The CORD6 mutation R838S in the RetGC1 dimerization domain strongly dominated the Ca2+ sensitivity of cyclase regulation by GCAP1 in RetGC1 heterodimer produced by co-expression of WT and the R838S subunits. It required higher Ca2+ concentrations to decelerate GCAP-activated RetGC1 heterodimer-6-fold higher than WT and 2-fold higher than the Ser838-harboring homodimer. The heterodimer was also more resistant than homodimers to inhibition by RD3. The observed biochemical changes can explain the dominant CORD6 blindness and recessive LCA1 blindness, both of which affect rods and cones, but they cannot explain the selective loss of rod function in recessive CSNB.

Regulation of retinal membrane guanylyl cyclase (RetGC) by negative calcium feedback and RD3 protein.

This article presents a brief overview of the main biochemical and cellular processes involved in regulation of cyclic GMP production in photoreceptors. The main focus is on how the fluctuations of free calcium concentrations in photoreceptors between light and dark regulate the activity of retinal membrane guanylyl cyclase (RetGC) via calcium sensor proteins. The emphasis of the review is on the structure of RetGC and guanylyl cyclase activating proteins (GCAPs) in relation to their functional role in photoreceptors and congenital diseases of photoreceptors. In addition to that, the structure and function of retinal degeneration-3 protein (RD3), which regulates RetGC in a calcium-independent manner, is discussed in detail in connections with its role in photoreceptor biology and inherited retinal blindness.

Structural Insights into Retinal Guanylate Cyclase Activator Proteins (GCAPs).

Retinal guanylate cyclases (RetGCs) promote the Ca2+-dependent synthesis of cGMP that coordinates the recovery phase of visual phototransduction in retinal rods and cones. The Ca2+-sensitive activation of RetGCs is controlled by a family of photoreceptor Ca2+ binding proteins known as guanylate cyclase activator proteins (GCAPs). The Mg2+-bound/Ca2+-free GCAPs bind to RetGCs and activate cGMP synthesis (cyclase activity) at low cytosolic Ca2+ levels in light-activated photoreceptors. By contrast, Ca2+-bound GCAPs bind to RetGCs and inactivate cyclase activity at high cytosolic Ca2+ levels found in dark-adapted photoreceptors. Mutations in both RetGCs and GCAPs that disrupt the Ca2+-dependent cyclase activity are genetically linked to various retinal diseases known as cone-rod dystrophies. In this review, I will provide an overview of the known atomic-level structures of various GCAP proteins to understand how protein dimerization and Ca2+-dependent conformational changes in GCAPs control the cyclase activity of RetGCs. This review will also summarize recent structural studies on a GCAP homolog from zebrafish (GCAP5) that binds to Fe2+ and may serve as a Fe2+ sensor in photoreceptors. The GCAP structures reveal an exposed hydrophobic surface that controls both GCAP1 dimerization and RetGC binding. This exposed site could be targeted by therapeutics designed to inhibit the GCAP1 disease mutants, which may serve to mitigate the onset of retinal cone-rod dystrophies.

Apo-Opsin Exists in Equilibrium Between a Predominant Inactive and a Rare Highly Active State.

Bleaching adaptation in rod photoreceptors is mediated by apo-opsin, which activates phototransduction with effective activity 105- to 106-fold lower than that of photoactivated rhodopsin (meta II). However, the mechanism that produces such low opsin activity is unknown. To address this question, we sought to record single opsin responses in mouse rods. We used mutant mice lacking efficient calcium feedback to boosts rod responses and generated a small fraction of opsin by photobleaching ∼1% of rhodopsin. The bleach produced a dramatic increase in the frequency of discrete photoresponse-like events. This activity persisted for hours, was quenched by 11-cis-retinal, and was blocked by uncoupling opsin from phototransduction, all indicating opsin as its source. Opsin-driven discrete activity was also observed in rods containing non-activatable rhodopsin, ruling out transactivation of rhodopsin by opsin. We conclude that bleaching adaptation is mediated by opsin that exists in equilibrium between a predominant inactive and a rare meta II-like state.SIGNIFICANCE STATEMENT Electrophysiological analysis is used to show that the G-protein-coupled receptor opsin exists in equilibrium between a predominant inactive and a rare highly active state that mediates bleaching adaptation in photoreceptors.

Retinal guanylyl cyclase activation by calcium sensor proteins mediates photoreceptor degeneration in an rd3 mouse model of congenital human blindness.

Deficiency of RD3 (retinal degeneration 3) protein causes recessive blindness and photoreceptor degeneration in humans and in the rd3 mouse strain, but the disease mechanism is unclear. Here, we present evidence that RD3 protects photoreceptors from degeneration by competing with guanylyl cyclase-activating proteins (GCAPs), which are calcium sensor proteins for retinal membrane guanylyl cyclase (RetGC). RetGC activity in rd3/rd3 retinas was drastically reduced but stimulated by the endogenous GCAPs at low Ca2+ concentrations. RetGC activity completely failed to accelerate in rd3/rd3GCAPs-/- hybrid photoreceptors, whose photoresponses remained drastically suppressed compared with the WT. However, ∼70% of the hybrid rd3/rd3GCAPs-/- photoreceptors survived past 6 months, in stark contrast to <5% in the nonhybrid rd3/rd3 retinas. GFP-tagged human RD3 inhibited GCAP-dependent activation of RetGC in vitro similarly to the untagged RD3. When transgenically expressed in rd3/rd3 mouse retinas under control of the rhodopsin promoter, the RD3GFP construct increased RetGC levels to near normal levels, restored dark-adapted photoresponses, and rescued rods from degeneration. The fluorescence of RD3GFP in rd3/rd3RD3GFP+ retinas was mostly restricted to the rod photoreceptor inner segments, whereas GCAP1 immunofluorescence was concentrated predominantly in the outer segment. However, RD3GFP became distributed to the outer segments when bred into a GCAPs-/- genetic background. These results support the hypothesis that an essential biological function of RD3 is competition with GCAPs that inhibits premature cyclase activation in the inner segment. Our findings also indicate that the fast rate of degeneration in RD3-deficient photoreceptors results from the lack of this inhibition.

An Ago2-associated capped transcriptional start site small RNA suppresses adenovirus DNA replication.

Here we show that the adenovirus major late promoter produces a 31-nucleotide transcriptional start site small RNA (MLP-TSS-sRNA) that retains the 7-methylguanosine (m7G)-cap and is incorporated onto Ago2-containing RNA-induced silencing complexes (RISC) in human adenovirus-37 infected cells. RNA polymerase II CLIP (UV-cross linking immunoprecipitation) experiments suggest that the MLP-TSS-sRNA is produced by promoter proximal stalling/termination of RNA polymerase II transcription at the site of the small RNA 3' end. The MLP-TSS-sRNA is highly stable in cells and functionally active, down-regulating complementary targets in a sequence and dose-dependent manner. The MLP-TSS-sRNA is transcribed from the opposite strand to the adenoviral DNA polymerase and preterminal protein mRNAs, two essential viral replication proteins. We show that the MLP-TSS-sRNA act in trans to reduce DNA polymerase and preterminal protein mRNA expression. As a consequence of this, the MLP-TSS-sRNA has an inhibitory effect on the efficiency of viral DNA replication. Collectively, our results suggest that this novel sRNA may serve a regulatory function controlling viral genome replication during a lytic and/or persistent adenovirus infection in its natural host.

CO2/bicarbonate modulates cone photoreceptor ROS-GC1 and restores its CORD6-linked catalytic activity.

This study with recombinant reconstituted system mimicking the cellular conditions of the native cones documents that photoreceptor ROS-GC1 is modulated by gaseous CO2. Mechanistically, CO2 is sensed by carbonic anhydrase (CAII), generates bicarbonate that, in turn, directly targets the core catalytic domain of ROS-GC1, and activates it to increased synthesis of cyclic GMP. This, then, functions as a second messenger for the cone phototransduction. The study demonstrates that, in contrast to the Ca2+-modulated phototransduction, the CO2 pathway is Ca2+-independent, yet is linked with it and synergizes it. It, through R787C mutation in the third heptad of the signal helix domain of ROS-GC1, affects cone-rod dystrophy, CORD6. CORD6 is caused firstly by lowered basal and GCAP1-dependent ROS-GC1 activity and secondly, by a shift in Ca2+ sensitivity of the ROS-GC1/GCAP1 complex that remains active in darkness. Remarkably, the first but not the second defect disappears with bicarbonate thus explaining the basis for CORD6 pathological severity. Because cones, but not rods, express CAII, the excessive synthesis of cyclic GMP would be most acute in cones.

The R838S Mutation in Retinal Guanylyl Cyclase 1 (RetGC1) Alters Calcium Sensitivity of cGMP Synthesis in the Retina and Causes Blindness in Transgenic Mice.

Substitutions of Arg838 in the dimerization domain of a human retinal membrane guanylyl cyclase 1 (RetGC1) linked to autosomal dominant cone-rod degeneration type 6 (CORD6) change RetGC1 regulation in vitro by Ca2+ In addition, we find that R838S substitution makes RetGC1 less sensitive to inhibition by retinal degeneration-3 protein (RD3). We selectively expressed human R838S RetGC1 in mouse rods and documented the decline in rod vision and rod survival. To verify that changes in rods were specifically caused by the CORD6 mutation, we used for comparison cones, which in the same mice did not express R838S RetGC1 from the transgenic construct. The R838S RetGC1 expression in rod outer segments reduced inhibition of cGMP production in the transgenic mouse retinas at the free calcium concentrations typical for dark-adapted rods. The transgenic mice demonstrated early-onset and rapidly progressed with age decline in visual responses from the targeted rods, in contrast to the longer lasting preservation of function in the non-targeted cones. The decline in rod function in the retina resulted from a progressive degeneration of rods between 1 and 6 months of age, with the severity and pace of the degeneration consistent with the extent to which the Ca2+ sensitivity of the retinal cGMP production was affected. Our study presents a new experimental model for exploring cellular mechanisms of the CORD6-related photoreceptor death. This mouse model provides the first direct biochemical and physiological in vivo evidence for the Arg838 substitutions in RetGC1 being the culprit behind the pathogenesis of the CORD6 congenital blindness.

Regulatory function of the C-terminal segment of guanylate cyclase-activating protein 2.

Neuronal responses to Ca2+-signals are provided by EF-hand-type neuronal Ca2+-sensor (NCS) proteins, which have similar core domains containing Ca2+-binding and target-recognizing sites. NCS proteins vary in functional specificity, probably depending on the structure and conformation of their non-conserved C-terminal segments. Here, we investigated the role of the C-terminal segment in guanylate cyclase activating protein-2, GCAP2, an NCS protein controlling the Ca2+-dependent regulation of photoreceptor guanylate cyclases. We obtained two chimeric proteins by exchanging C-terminal segments between GCAP2 and its photoreceptor homolog recoverin, a Ca2+-sensor controlling rhodopsin kinase (RK) activity. The exchange affected neither the structural integrity of GCAP2 and recoverin nor the Ca2+-sensitivity of GCAP2. Intrinsic fluorescence, circular dichroism, biochemical studies and hydrophobic dye probing revealed Ca2+-dependent conformational transition of the C-terminal segment of GCAP2 occurring in the molecular environment of both proteins. In Ca2+-GCAP2, the C-terminal segment was constrained and its replacement provided the protein with approximately two-fold inhibitory activity towards RK, suggesting that the segment contributes to specific target recognition by interfering with RK-binding. Upon Ca2+-release, it became less constrained and more available for phosphorylation by cyclic nucleotide-dependent protein kinase. The transition from the Ca2+-bound to the apo-state exposed hydrophobic sites in GCAP2, and was associated with its activating function without affecting its dimerization. The released C-terminal segment participated further in photoreceptor membrane binding making it sensitive to phosphorylation. Thus, the C-terminal segment in GCAP2 confers target selectivity, facilitates membrane binding and provides sensitivity of the membrane localization of the protein to phosphorylation by signaling kinases.

Guanylate Cyclase-Activating Protein-2 Undergoes Structural Changes upon Binding to Detergent Micelles and Bicelles.

GCAPs are neuronal Ca(2+)-sensors playing a central role in light adaptation. GCAPs are N-terminally myristoylated membrane-associated proteins. Although, the myristoylation of GCAPs plays an important role in light adaptation its structural and physiological roles are not yet clearly understood. The crystal-structure of GCAP-1 shows the myristoyl moiety inside the hydrophobic core of the protein, stabilizing the protein structure; but (2)H-solid-state NMR investigations on the deuterated myristoyl moiety of GCAP-2 in the presence of liposomes showed that it is inserted into the lipid bilayer. In this study, we address the question of the localization of the myristoyl group of Ca(2+)-bound GCAP-2, and the influence of CHAPS-, DPC-micelles and DMPC/DHPC-bicelles on the structure, and on the localization of the myristoyl group, of GCAP-2 by solution-state NMR. We also carried out the backbone assignment. Characteristic chemical shift differences have been observed between the myristoylated and the non-myristoylated forms of the protein. Our results support the view that in the absence of membrane forming substances the myristoyl moiety is buried inside a hydrophobic pocket of GCAP-2 similar to the crystal structure of GCAP-1. Addition of CHAPS-micelles and DMPC/DHPC-bicelles cause specific structural changes localized in and around the myristoyl binding pocket. We interpret these changes as an indication for the extrusion of the myristoyl moiety from its binding pocket and its insertion into the hydrophobic interior of the membrane mimic. On the basis of the backbone chemical shifts, we propose a structural model of myristoylated GCAP-2 in the presence of Ca(2+) and membrane mimetics.

A novel hypothesis for an alkaline phosphatase 'rescue' mechanism in the hepatic acute phase immune response.

The liver isoform of the enzyme alkaline phosphatase (AP) has been used classically as a serum biomarker for hepatic disease states such as hepatitis, steatosis, cirrhosis, drug-induced liver injury, and hepatocellular carcinoma. Recent studies have demonstrated a more general anti-inflammatory role for AP, as it is capable of dephosphorylating potentially deleterious molecules such as nucleotide phosphates, the pathogenic endotoxin lipopolysaccharide (LPS), and the contact clotting pathway activator polyphosphate (polyP), thereby reducing inflammation and coagulopathy systemically. Yet the mechanism underlying the observed increase in liver AP levels in circulation during inflammatory insults is largely unknown. This paper hypothesizes an immunological role for AP in the liver and the potential of this system for damping generalized inflammation along with a wide range of ancillary pathologies. Based on the provided framework, a mechanism is proposed in which AP undergoes transcytosis in hepatocytes from the canalicular membrane to the sinusoidal membrane during inflammation and the enzyme's expression is upregulated as a result. Through a tightly controlled, nucleotide-stimulated negative feedback process, AP is transported in this model as an immune complex with immunoglobulin G by the asialoglycoprotein receptor through the cell and secreted into the serum, likely using the receptor's State 1 pathway. The subsequent dephosphorylation of inflammatory stimuli by AP and uptake of the circulating immune complex by endothelial cells and macrophages may lead to decreased inflammation and coagulopathy while providing an early upstream signal for the induction of a number of anti-inflammatory gene products, including AP itself.

Crystal structure of rat intestinal alkaline phosphatase--role of crown domain in mammalian alkaline phosphatases.

Intestinal alkaline phosphatases (IAPs) are involved in the cleavage of phosphate prodrugs to liberate the drug for absorption in the intestine. To facilitate in vitro characterization of phosphate prodrugs, we have cloned, expressed, purified and characterized IAPs from rat and cynomolgus monkey (rIAP and cIAP respectively) which are important pre-clinical species for drug metabolism studies. The recombinant rat and monkey enzymes expressed in Sf9 insect cells (IAP-Ic) were found to be glycosylated and active. Expression of rat IAP in Escherichia coli (rIAP-Ec) led to ~200-fold loss of activity that was partially recovered by the addition of external Zn(2+) and Mg(2+) ions. Crystal structures of rIAP-Ec and rIAP-Ic were determined and they provide rationale for the discrepancy in enzyme activities. Rat IAP-Ic retains its activity in presence of both Zn(2+) and Mg(2+) whereas activity of most other alkaline phosphatases (APs) including the cIAP was strongly inhibited by excess Zn(2+). Based on our crystal structure, we hypothesized the residue Q317 in rIAP, present within 7 Å of the Mg(2+) at M3, to be important for this difference in activity. The Q317H rIAP and H317Q cIAP mutants showed reversal in effect of Zn(2+), corroborating the hypothesis. Further analysis of the two structures indicated a close linkage between glycosylation and crown domain stability. A triple mutant of rIAP, where all the three putative N-linked glycosylation sites were mutated showed thermal instability and reduced activity.