Cyclic CMP and cyclic UMP mediate bacterial immunity against phages.

The cyclic pyrimidines 3',5'-cyclic cytidine monophosphate (cCMP) and 3',5'-cyclic uridine monophosphate (cUMP) have been reported in multiple organisms and cell types. As opposed to the cyclic nucleotides 3',5'-cyclic adenosine monophosphate (cAMP) and 3',5'-cyclic guanosine monophosphate (cGMP), which are second messenger molecules with well-established regulatory roles across all domains of life, the biological role of cyclic pyrimidines has remained unclear. Here we report that cCMP and cUMP are second messengers functioning in bacterial immunity against viruses. We discovered a family of bacterial pyrimidine cyclase enzymes that specifically synthesize cCMP and cUMP following phage infection and demonstrate that these molecules activate immune effectors that execute an antiviral response. A crystal structure of a uridylate cyclase enzyme from this family explains the molecular mechanism of selectivity for pyrimidines as cyclization substrates. Defense systems encoding pyrimidine cyclases, denoted here Pycsar (pyrimidine cyclase system for antiphage resistance), are widespread in prokaryotes. Our results assign clear biological function to cCMP and cUMP as immunity signaling molecules in bacteria.

Geochemical influences on nonenzymatic oligomerization of prebiotically relevant cyclic nucleotides.

The spontaneous emergence of long RNA molecules on the early Earth, a phenomenon central to the RNA World hypothesis, continues to remain an enigma in the field of origins of life. Few studies have looked at the nonenzymatic oligomerization of cyclic mononucleotides under neutral to alkaline conditions, albeit in fully dehydrated state. In this study, we systematically investigated the oligomerization of cyclic nucleotides under prebiotically relevant conditions, wherein starting reactants were subjected to repeated dehydration-rehydration (DH-RH) regimes. DH-RH conditions, a recurring geological theme that was prevalent on prebiotic Earth, are driven by naturally occurring processes including diurnal cycles and tidal pool activity. These conditions have been shown to facilitate uphill oligomerization reactions. The polymerization of 2'-3' and 3'-5' cyclic nucleotides of a purine (adenosine) and a pyrimidine (cytidine) was investigated. Additionally, the effect of amphiphiles was also evaluated. Furthermore, to discern the effect of "realistic" conditions on this process, the reactions were also performed using a hot spring water sample from a candidate early Earth environment. Our study showed that the oligomerization of cyclic nucleotides under DH-RH conditions resulted in intact informational oligomers. Amphiphiles increased the stability of both the starting monomers and the resultant oligomers in selected reactions. In the hot spring reactions, both the oligomerization of nucleotides and the back hydrolysis of the resultant oligomers were pronounced. Altogether, this study demonstrates how nonenzymatic oligomerization of cyclic nucleotides, under both laboratory-simulated prebiotic conditions and in a candidate early Earth environment, could have resulted in RNA oligomers of a putative RNA World.

Nonenzymatic Oligomerization of 3',5'-Cyclic CMP Induced by Proton and UV Irradiation Hints at a Nonfastidious Origin of RNA.

We report that 3',5'-cyclic CMP undergoes nonenzymatic di- and trimerization at 20 °C under dry conditions upon proton or UV irradiation. The reaction involves stacking of the cyclic monomers and subsequent polymerization through serial transphosphorylations between the stacked monomers. Proton- and UV-induced oligomerization of 3',5'-cyclic CMP demonstrates that pyrimidines-similar to purines-might also have taken part in the spontaneous generation of RNA under plausible prebiotic conditions as well as in an extraterrestrial context. The observed polymerization of naturally occurring 3',5'-cyclic nucleotides supports the possibility that the extant genetic nucleic acids might have originated by way of a straight Occamian path, starting from simple reactions between plausibly preactivated monomers.

cCMP causes caspase-dependent apoptosis in mouse lymphoma cell lines.

cCMP is a cyclic pyrimidine nucleotide which binds to and activates cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG). In S49 lymphoma cells, cAMP induces apoptosis via PKA. In our present study, we examined the effect of cCMP on apoptosis in S49 mouse lymphoma cells and in PKA-deficient S49kin(-)cells. These two cell lines also lack PKG, hyperpolarization-activated cyclic nucleotide-gated channels 2 and 4 (HCN2 and HCN4) as assessed by real-time PCR. The cell-permeable analog cCMP-AM induced PKA- and PKG-independent apoptosis in S49 cells. In contrast, exchange protein activated by cAMP (Epac) activation did not induce apoptosis. cCMP induced caspase-dependent apoptosis via the intrinsic pathway, led to cytochrome c release from mitochondria and also activated the ER stress pathway. On the contrary, the extrinsic apoptotic pathway was not involved. Autophagy was not detectable after treatment with cCMP-AM in both cell lines. cAMP-AM, cGMP-AM, cUMP-AM as well as the cyclic nucleotides lacking the acetoxymethylester (AM)-group had no effect. cCMP-AM altered gene expression of the apoptotic-relevant gene Gadd45α and the immediate early response genes cFos and Nr4A1 in S49 wild-type (wt) cells. In conclusion, cCMP induces apoptosis of S49 lymphoma cells, independently of hitherto known cCMP target proteins.

PDE7A1 hydrolyzes cCMP.

The degradation and biological role of the cyclic pyrimidine nucleotide cCMP is largely elusive. We investigated nucleoside 3',5'-cyclic monophosphate (cNMP) specificity of six different recombinant phosphodiesterases (PDEs) by using a highly-sensitive HPLC-MS/MS detection method. PDE7A1 was the only enzyme that hydrolyzed significant amounts of cCMP. Enzyme kinetic studies using purified GST-tagged truncated PDE7A1 revealed a cCMP KM value of 135 ± 19 µM. The Vmax for cCMP hydrolysis reached 745 ± 27 nmol/(minmg), which is about 6-fold higher than the corresponding velocity for adenosine 3',5'-cyclic monophosphate (cAMP) degradation. In summary, PDE7A is a high-speed and low-affinity PDE for cCMP.

Binding of regulatory subunits of cyclic AMP-dependent protein kinase to cyclic CMP agarose.

The bacterial adenylyl cyclase toxins CyaA from Bordetella pertussis and edema factor from Bacillus anthracis as well as soluble guanylyl cyclase α(1)ß(1) synthesize the cyclic pyrimidine nucleotide cCMP. These data raise the question to which effector proteins cCMP binds. Recently, we reported that cCMP activates the regulatory subunits RIα and RIIα of cAMP-dependent protein kinase. In this study, we used two cCMP agarose matrices as novel tools in combination with immunoblotting and mass spectrometry to identify cCMP-binding proteins. In agreement with our functional data, RIα and RIIα were identified as cCMP-binding proteins. These data corroborate the notion that cAMP-dependent protein kinase may serve as a cCMP target.

Nucleotidyl cyclase activity of soluble guanylyl cyclase α1ß1.

Soluble guanylyl cyclase (sGC) regulates several important physiological processes by converting GTP into the second-messenger cGMP. sGC has several structural and functional properties in common with adenylyl cyclases (ACs). Recently, we reported that membranous ACs and sGC are potently inhibited by 2',3'-O-(2,4,6-trinitrophenyl)-substituted purine and pyrimidine nucleoside 5'-triphosphates. Using a highly sensitive high-performance liquid chromatography-tandem mass spectrometry method, we report that highly purified recombinant sGC of rat possesses nucleotidyl cyclase activity. As opposed to GTP, ITP, XTP and ATP, the pyrimidine nucleotides UTP and CTP were found to be sGC substrates in the presence of Mn(2+). When Mg(2+) is used, sGC generates cGMP, cAMP, cIMP, and cXMP. In conclusion, soluble "guanylyl" cyclase possesses much broader substrate specificity than previously assumed. Our data have important implications for cyclic nucleotide-mediated signal transduction.

Biosynthesis of CMP-N,N'-diacetyllegionaminic acid from UDP-N,N'-diacetylbacillosamine in Legionella pneumophila.

Legionaminic acid is a nine-carbon alpha-keto acid that is similar in structure to other members of the sialic acid family that includes neuraminic acid and pseudaminic acid. It is found as a component of the lipopolysaccharide in several bacterial species and is perhaps best known for its presence in the O-antigen of the causative agent of Legionnaires' disease, Legionella pneumophila. In this work, the enzymes responsible for the biosynthesis and activation of N, N'-diacetyllegionaminic acid are identified for the first time. A cluster of three L. pneumophila genes bearing homology to known sialic acid biosynthetic genes ( neuA,B,C) were cloned and overexpressed in Escherichia coli. The NeuC homologue was found to be a hydrolyzing UDP- N, N'-diacetylbacillosamine 2-epimerase that converts UDP- N, N'-diacetylbacillosamine into 2,4-diacetamido-2,4,6-trideoxymannose and UDP. Stereochemical and isotopic labeling studies showed that the enzyme utilizes a mechanism involving an initial anti elimination of UDP to form a glycal intermediate and a subsequent syn addition of water to generate product. This is similar to the hydrolyzing UDP- N-acetylglucosamine 2-epimerase (NeuC) of sialic acid biosynthesis, but the L. pneumophila enzyme would not accept UDP-GlcNAc as an alternate substrate. The NeuB homologue was found to be a N, N'-diacetyllegionaminic acid synthase that condenses 2,4-diacetamido-2,4,6-trideoxymannose with phosphoenolpyruvate (PEP), although the in vitro activity of the recombinant enzyme (isolated as a MalE fusion protein) was very low. The synthase activity was dependent on the presence of a divalent metal ion, and the reaction proceeded via a C-O bond cleavage process, similar to the reactions catalyzed by the sialic acid and pseudaminic acid synthases. Finally, the NeuA homologue was shown to possess the CMP- N, N'-diacetyllegionaminic acid synthetase activity that generates the activated form of legionaminic acid used in lipopolysaccharide biosynthesis. Together, the three enzymes constitute a pathway that converts a UDP-linked bacillosamine derivative into a CMP-linked legionaminic acid derivative.

A nearly isosteric photosensitive amide-backbone substitution allows enzyme activity switching in ribonuclease s.

psi[CS-NH]4-RNase S, a site specific modified version of RNase S obtained by thioxylation (O/S exchange) at the Ala4-Ala5- peptide bond, was used to evaluate the impact of protein backbone photoswitching on bioactivity. psi[CS-NH](4)-RNase S was yielded by recombination of the S-protein and the respective chemically synthesized thioxylated S-peptide derivative. Comparison with RNase S revealed similar thermodynamic stability of the complex and an unperturbed enzymatic activity toward cytidine 2',3'-cyclic monophosphate (cCMP). Reversible photoisomerization with a highly increased cis/trans isomer ratio of the thioxopeptide bond of psi[CS-NH](4)-RNase S in the photostationary state occurred under UV irradiation conditions (254 nm). The slow thermal reisomerization (t(1/2) = 180 s) permitted us to determine the enzymatic activity of cis psi[CS-NH](4)-RNase S by measurement of initial rates of cCMP hydrolysis. Despite thermodynamic stability of cis psi[CS-NH](4)-RNase S, its enzymatic activity is completely abolished but recovers after reisomerization. We conclude that the thioxopeptide bond modified polypeptide backbone represents a versatile probe for site-directed photoswitching of proteins.

Cofactor fingerprinting with STD NMR to characterize proteins of unknown function: identification of a rare cCMP cofactor preference.

Proteomics efforts have created a need for better strategies to functionally categorize newly discovered proteins. To this end, we have employed saturation transfer difference NMR with pools of closely related cofactors, to determine cofactor preferences. This approach works well for dehydrogenases and has also been applied to cyclic nucleotide-binding proteins. In the latter application, a protein (radial spoke protein-2, RSP2) that plays a central role in forming the radial spoke of Chlamydomonas reinhardtii flagella was shown to bind cCMP. cCMP-binding proteins are rare, although previous reports of their presence in sperm and flagella suggest that cCMP may have a more general role in flagellar function. 31P NMR was used to monitor the preferential hydrolysis of ATP versus GTP, suggesting that RSP2 is a kinase.