Perinatal brain damage underlies an important share of motor and neurodevelopmental disabilities, such as cerebral palsy, cognitive impairment, visual dysfunction and epilepsy. Clinical, epidemiological, and experimental studies have revealed that factors such as inflammation, excitotoxicity and oxidative stress contribute considerably to both white and grey matter injury in the immature brain. A member of the death associated protein kinase (DAPk) family, DAPk1, has been implicated in cerebral ischemic damage, whereby DAPk1 potentiates NMDA receptor-mediated excitotoxicity through interaction with the NR2BR subunit. DAPk1 also mediate a range of activities from autophagy, membrane blebbing and DNA fragmentation ultimately leading to cell death. DAPk mRNA levels are particularly highly expressed in the developing brain and thus, we hypothesize that DAPk1 may play a role in perinatal brain injury. In addition to reviewing current knowledge, we present new aspects of the molecular structure of DAPk domains, and relate these findings to interacting partners of DAPk1, DAPk-regulation in NMDA-induced cerebral injury and novel approaches to blocking the injurious effects of DAPk1. Perinatal brain injury is a major cause of neurological deficits in newborns leading to lifelong consequences such as cerebral palsy and delayed cognitive and behavioral deficits . PerinatEmerging evidence suggests that inhibition of death-associated protein kinase 1 (DAPk1), which prevents excessive NMDA receptor (NMDAR) activation without interfering with physiological functions, provides neuroprotection in animal models of adult stroke ,8. The N2 during reperfusion, which contribute to the formation of more reactive oxygen species that damage lipids, proteins and other cell components ultimately leading to cell death [Neonatal hypoxia-ischemia or direct excitotoxic insults induce a cascade of neurotoxic events in the brain  that leall death ,13. Despll death ,15, partHowever, mounting evidence suggests that these problems might be circumvented by selectively targeting molecular components of the death signalling cascade downstream of the NR2B subunit of NMDARs. The NR2B-containing NMDARs (NR2BRs) are preferentially localized at the extrasynaptic sites, which have been linked to cell death, but not with physiological functions ,17. Alth2+/CaM) dependent serine/threonine kinases whose relatives subsume DAPk1, DAPk-1 related protein 1 , Zipper interacting protein kinase , DAPk-1 related protein 1 (DRAk-1) and DRAk-2. DAPk1, DRP-1 and ZIP kinases are usually grouped into one superfamily since they share 83% and 80% identity at the amino acid level, respectively, with DAPk1’s catalytic domain, whereas DRAk-1 and DRAk-2 share 50% identity with DAPk1. Although DRP1 and ZIPk are not reported to be directly regulated in response to cerebral ischemia, DAPk1 mRNA levels are increased in response to neonatal hypoxia-ischemia in vivo [The death-associated protein kinases include a family of calcium/calmodulin . It consist of a protein kinase domain, a calcium-calmodulin binding region of 62 amino acids, 10 ankyrin repeats each about 30 amino acids in length, two putative P loops, cytoskeletal binding region and a death domain . More thThe catalytic domain of DAPk1 is composed of 11 subdomains, which have been implicated in many cellular functions . The 3D 2+/CaM-dependent kinases that are regulated by a double-locking mechanism. DAPk1 activity is regulated through several phosphorylation sites that are located within the CaM autoregulatory domain, two of which are Ser289 and Ser308. Full activation requires both the dephosphorylation of Ser308 and CaM binding. Binding of Ca2+ recruits CaM to the autoregulatory CaM-binding segment pulling this domain out from the catalytic cleft. Dephosphorylation of Ser308 increases the affinity for CaM thereby promoting the catalytic activity at low CaM levels. It has been shown that the deletion of the CaM-binding domain from DAPk or the substitution of Ser308 to Ala, generates a constitutively active kinase thereby exhibiting greater Ca2+ independent catalytic activity and killing potential [2 in area [DAPks are Caotential ,25. The  in area .In order to further analyze the CaM binding region we retrieved, from the crystal structure of the binary DAPK-CaM complex, the DAPK catalytic domain and adjacent autoregulatory domain complexed with CaM from PDB (PDB code: 1IG1)  and critin vitro and deletion analysis in vivo determined that the death domain of DAPk1 is the major site for the interaction with tumor suppressor protein tuberin (TSC2). Thus, there is a positive association between growth factor stimulation of DAPk and mTORC1 signaling, which may ultimately affect autophagy, cell survival, or apoptosis [The death domain of DAPk1 is largely associated with protein-protein interactions, kinase activity and apoptotic functions. Deletion of the death domain is reported to abrogate the apoptotic functions of the kinase including TNF-α- and Fas-induced cell death . Bindingpoptosis . Furtherpoptosis .ab initio modeling and structural refinement [The death domain is the least studied domain of DAPk1 and no crystal structure has been reported so far. In order to identify structural features, we used bioinformatics and cheminformatics tools to build homology models of the death domain of the DAPk1 protein. I-TASSER web server was used for structure prediction, which combines the methods of threading, finement ,30. The The ankyrin repeats facilitate protein-protein communications and are implicated mostly in DAPk1 degradation. s-DAPk-1, an alternatively spliced product lacking the DAPk1 kinase domain regulates the steady-state levels of the full-length DAPk-1 and is implicated in proteasome-independent degradation pathway for DAPk1  The ankyC-terminal domains and phosphorylates it at Ser188. This phosphorylation event occurs both in vitro and in vivo in a Ca2+-dependent manner [As described above, DAPk1 is a relatively large protein with multiple domains and docking motifs that drive its regulation and function. Understanding protein interactions at the domain level gives a global view of the protein interaction network, and possibly of protein functions. However, to date only certain domains of the DAPk1 protein have been purified by crystallographic studies and the native substrates of DAPk and the molecular pathways underlying DAPk-mediated signal transduction still remain unclear to a large extent. Biochemical studies demonstrate that DAPk1 binds syntaxin-1 via their t manner . PDCD6, t manner .in vitro and in vivo. DAPk1 promotes the cytoplasmic retention of ERK1/2, thereby inhibiting ERK signaling in the nucleus ultimately promoting the apoptotic activity of DAPk1 [in vivo and in vitro [DAPk1 has been found to interact with ERK1 and ERK2 through a specific docking sequence within its death domain. Phosphorylation of DAPk1 at Ser735 by ERK2 increases the catalytic activity of DAPk1 both of DAPk1 . Furtherof DAPk1 . It was of DAPk1 . DANGER,in vitro ,40. Thusin vitro .Much of our knowledge regarding the mechanisms of hypoxic-ischemic brain damage, as well as potential therapeutic interventions, evolves from literature on experimental adult stroke models. However, there is a distinct difference to the damaging effects of hypoxia-ischemia between the adult and the neonatal brain. In some respects, the immature brain is more resistant to hypoxic-ischemic damage than its adult counterpart, but at the same time exhibiting periods of heightened sensitivity to injury, dependent on the specific developmental stage of the brain . Importain vivo and directly binds with the NMDA receptor NR2B C-terminal tail, at amino acids 1292–1304. Subsequently DAPk1 phosphorylates the NR2B subunit at Ser1303 and enhances the NR1/NR2B receptor channel conductance resulting in excessive Ca2+ influx through the receptor channels thereby triggering a cascade of intracellular events that arbitrate cell death. It has been suggested that DAPk1 potentiates NR2BR activity, thereby promoting neuronal death by activation of “calcium-activated death-signaling proteins like the NR2B–PSD95–nNOS signaling complex” [2+ influx through NMDAR channels activates calcineurin (CaN), which in turn dephosphorylates DAPk1 at pS308, leading to its activation thereby acting as an upstream activator of DAPk1 (The activity of the NMDAR-ion channel complex is tightly regulated by a number of pharmacologically distinct binding sites . Howevercomplex” . Under pof DAPk1 . It has in vitro and in vivo ischemic models [In support of a role for DAPk1 in brain injury, a selective DAPk inhibitor was shown to be neuroprotective in both c models ,8. In adc models ,48. ThusWith the evidence of DAPk1-mediated injury in cerebral ischemia and the ability of bioavailable DAPk inhibitors to rescue neuronal death, DAPk1 has emerged as an important drug-discovery target for brain disorders. Thus, by identifying the binding domains and exact interacting residues of DAPk1 we can potentially efficiently disrupt the interaction of DAPk1 with its partners, thus ameliorating brain injury. Some examples of novel, potentially neuroprotective, strategies are outlined below.A strong interest exists for ATP-competitive inhibitors, which bind to the active site within the catalytic domain that is required for the functions of DAPk complexes, resulting in down regulation of death signaling globally . HoweverCompounds that act outside the ATP-binding pocket are also of considerable interest. As discussed above, DAPk1 and DAPk2 are CaM-dependent kinases and disrupting the CaM-DAPk1 interaction by means of small molecules may be an effective mechanism to inhibit the protein. The death domain is a prime mediator of the interactions necessary for transducing a death signal. Therefore, silencers of the death domain, such as the SODD/BAG-4-related proteins, could be potential therapeutic agents . At thisC-terminal domain of DAPk1 between the ankyrin repeats and the death domain. DAPk1 forms a multi-protein survival complex with cathepsin B countering the rate of TNFR-1-dependent apoptosis and highlights the importance of developing DAPk1 inhibitors as agents to sensitize cells to stress-induced apoptosis [C-terminal tail of s-DAPk1 can regulate the ability of the protein to mimic the biological functions of DAPk1 in promoting membrane blebbing and controlling the half-life of DAPk1 protein itself. Therefore s-DAPk could be exploited to inhibit the DAPk1 functions [Cathepsin B is another protein with a novel binding domain localized in the poptosis  siRNA-bapoptosis . Similarpoptosis . The s-Dunctions ,55.Further, kinase targets are usually components of complex, interconnected signal transduction cascades comprising many protein kinases, with pathway redundancy and crosstalk between pathways . Cross tNeonatal hypoxia-ischemia triggers a cascade of biochemical and molecular events, including excitotoxicity through the NMDA receptor. Brain damage is progressive and evidence suggests a distinct difference between the immature and mature brain in the pathology and consequences of brain injury . Thus, e


The PWWP domain was first identified as a structural motif of 100–130 amino acids in the WHSC1 protein and predicted to be a protein-protein interaction domain. It belongs to the Tudor domain ‘Royal Family’, which consists of Tudor, chromodomain, MBT and PWWP domains. While Tudor, chromodomain and MBT domains have long been known to bind methylated histones, PWWP was shown to exhibit histone binding ability only until recently.The PWWP domain has been shown to be a DNA binding domain, but sequence analysis and previous structural studies show that the PWWP domain exhibits significant similarity to other ‘Royal Family’ members, implying that the PWWP domain has the potential to bind histones. In order to further explore the function of the PWWP domain, we used the protein family approach to determine the crystal structures of the PWWP domains from seven different human proteins. Our fluorescence polarization binding studies show that PWWP domains have weak histone binding ability, which is also confirmed by our NMR titration experiments. Furthermore, we determined the crystal structures of the BRPF1 PWWP domain in complex with H3K36me3, and HDGF2 PWWP domain in complex with H3K79me3 and H4K20me3.PWWP proteins constitute a new family of methyl lysine histone binders. The PWWP domain consists of three motifs: a canonical β-barrel core, an insertion motif between the second and third β-strands and a C-terminal α-helix bundle. Both the canonical β-barrel core and the insertion motif are directly involved in histone binding. The PWWP domain has been previously shown to be a DNA binding domain. Therefore, the PWWP domain exhibits dual functions: binding both DNA and methyllysine histones.enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in This article can also be viewed as an  It was predicted to be a protein-protein interaction domain The PWWP domain was later on shown to bind DNA in 2002 by Cheng's laboratory Alongside Tudor, chromodomain, MBT domains, the PWWP domain belongs to the Tudor domain ‘Royal Family’ In order to systematically study the structure and function of this domain, we purified some representative human PWWP domains and tested their binding ability to different histone peptides. The results show that PWWP domain is a weak methyllysine histone binder. Furthermore, we determined the crystal structures of the PWWP domains from seven different human proteins and three PWWP domain complex structures with histone peptides, i.e., BRPF1-H3K36me3, HDGF2-H3K79me3 and HDGF2-H4K20me3. Therefore, the PWWP domain can not only bind DNA but also histones.The PWWP domain was first identified in WHSC1 and named after the central core motif Pro-Trp-Trp-Pro of the PWWP domain in WHSC1 In order to explore the functional roles of the PWWP domains, we determined the crystal structures of the PWWP domains from 7 different human proteins, namely, BRPF1 (bromodomain and PHD finger-containing protein 1), BRPF2, BRPF3, MUM1 (melanoma associated antigen (mutated) 1), DNMT3A, DNMT3B and HDGF2 (Hepatoma-derived growth factor 2). Our structures revealed that the overall fold of the PWWP domain consists of three motifs: a canonical β-barrel core, an insertion motif between the second and third β-strands and a C-terminal α-helix bundle . The canPombe protein Pdp1 harbors a PWWP domain in its N-terminus, which was shown to bind mono-methylated histone H4K20. Because the C-terminal fragment of Pdp1 is able to bind the Pombe H4K20 methyltransferase SET9, SET9 is recruited to the H4K20me1 chromatin region through the PWWP domain of Pdp1 to increase the concentration of SET9 on chromatin and carry out the trimethylation of histone H4K20 The PWWP domain is structurally similar to other members in the Tudor domain ‘Royal family’ To better understand the histone binding ability and preference of these human PWWP domains, we used fluorescence polarization and NMR titration techniques to measure binding affinities of some representative PWWP domains to various histone peptides bearing different lysine methylation states. By fluorescence polarization assay, we found that the PWWP domains in BRPF1, BRPF2, HDGF2, MUM1 and the N-terminal PWWP domains of WHSC1 and WHSC1L1 show weak binding affinity to histones with H3K36, K3K79 or H4K20 methylation . In ordeTo shed light on the molecular mechanism of methylated histone binding by PWWP domains, we determined the crystal structures of the PWWP domain of human BRPF1 in complex with H3K36me3 and that of human HDGF2 in complex with H3K79me3 and H4K20me3.In the BRPF1-H3K36me3 complex structure, the peptide resides in a groove formed by the insertion motif, the fourth β-strand and its preceding loop from the BRPF1 PWWP domain . The triWe were also able to co-crystallize HDGF2 with both H3K79me3 and H4K20me3 peptides, which show very similar binding mode. In the HDGF2-H3K79me3 complex structure , the triDNMT3A had been shown to bind histone H3K36me3 In all these complex structures, the methyllysine binding aromatic residues are from the loop between the first and second β-strands, the N-terminus of the second β-strand and the C-terminus of the third β-strand . The hisComparison of the structural architecture of the PWWP domain to those of chromodomain, MBT and Tudor domains shows that PWWP, MBT and Tudor all have a 5-β-strand canonical core, while the chromodomain consists of three β-strands and one α-helix . OverallThe histone methyllysine binding mode exhibited by PWWP is similar to that adopted by other methyllysine binding proteins WHSC1 is located in the Wolf–Hirschhorn syndrome critical region on 4p16.3 and is disrupted by chromosomal translocation in lymphoid multiple myeloma disease de novo DNA methyltransferases and the loss-of-function mutations in human DNMT3B causes a developmental defect characterized by hypomethylation of pericentromeric repeats and are implicated in ICF  syndrome Mutations in PWWP domain-containing proteins have been implicated in different human diseases. The gene Escherichia coli BL21 (DE3)-V2R-pRARE2. The cells were grown in Terrific Broth and the protein was over-expressed by addition of 1 mM isopropyl-1-thio-D-galactopyranoside (IPTG) and incubated overnight at 15°C. Harvested cells were resuspended in 50 mM HEPES, pH 7.4, supplemented with 500 mM NaCl, 2 mM β-mercaptoethanol, 5% glycerol, 0.1% CHAPS. The cells were lysed by passing through a microfluidizer (Microfluidics Corporation) at 20,000 psi. After clarification of the crude extract by high-speed centrifugation, the lysate was loaded onto a 5 ml HiTrap chelating column , charged with Ni2+. The column was washed with 10 column volumes of 20 mM HEPES buffer, pH 7.4, containing 500 mM NaCl, 50 mM imidazole and 5% glycerol, the protein was eluted with 20 mM HEPES buffer, pH 7.4, 500 mM NaCl, 250 mM imidazole, 5% glycerol. The protein was dialyzed against buffer containing 20 mM HEPES, pH 7.4, 500 mM NaCl and 5% glyceral. TEV protease was added to combined fractions containing target proteins to remove the His-tag. All the proteins except DNMT3A were further purified to homogeneity by ion-exchange chromatography on Source 30S column (10×10) , equilibrated with 20 mM PIPES buffer, pH 6.5, and eluted with linear gradient of NaCl up to 500 mM concentration (20CV). For DNMT3A, Source 30Q column was used for ion exchange chromatography. The 15N-labeled proteins for NMR titration were purified in the same protocols as native ones except that bacteria were grew in M9 minimal medium containing 1 g/L 15(NH4)2SO4 as the sole nitrogen source. The labeled proteins were concentrated to 0.15–0.3 mM for NMR titration.DNA fragment encoding the PWWP domain of human BRPF1 (residues 1085–1213), BRPF2 (residues 925–1049), BRPF3 (residues 1056–1195), HDGF2 (residues 1–93), DNMT3A (residues 278–427), DNMT3B (residues 206–355), MUM1 (residues 406–539) WHSC1 (residues 208–368) and WHSC1L1 (residues 247–402) were amplified by PCR and sub-cloned into pET28-MHL vector (Genbank accession number: EF456735) and transformed into Purified PWWP domain proteins were crystallized using hanging drop vapor diffusion method at 20°C by mixing 1 µl of the protein solution (10 mg/mL) with 1 µl of the reservoir solution. BRPF1 (apo) and its complex with H3K36me3 peptide were crystallized in 3.5 M sodium formate, 0.1 M Tris-HCl, pH 8.5; BRPF2 in 30% PEG2K-MME, 0.20 M potassium bromide; BRPF3 in 30% PEG 4,000, 0.2 M ammonium sulfate, 0.1 M sodium cacodylate, pH 6.5; HDGF2 in 2.0 M ammonium sulfate, 0.2 M potassium/sodium tartrate, 0.1 M sodium citrate pH 5.6; HDGF2-H3K79me3 complex in 2.0 M ammonium sulfate, 5% isopropanol; MUM1 in 25% PEG 3,350, 0.1 M ammonium sulfate, 0.1 M HEPES, pH 7.5; DNMT3A in 28% PEG 3,350, 0.1 M ammonium sulfate, 0.1 M Bis-Tris, pH 6.0; DNMT3B in 30% PEG2K-MME, 0.20 M potassium bromide, 0.1 M Bis-Tris, pH 6.5. The peptides used for co-crystallization are: SAPATGGVKme3KPHRYR (H3K36me3); EIAQDFK(me)3TDLRY (H3K79me3); AKRHRKme3VLRDN (H4K20me3).Kd values.Fluorescence polarization assays were performed in 384-well plates, using the Synergy 2 microplate reader from BioTek as described in ds, 15N-1H HSQC spectra were collected with 15N-labeled samples of PWWP domains, free and with additions of increasing amounts of unlabeled H3K4me3 (1–11 aa), H3K9me3 (1–15 aa), H3K36me3 (30–41 aa), H3K79me3 (73–84 aa), p53K370me2 (365–375 aa), p53K372me2 (364–376 aa) and p53K382me2 (376–388 aa) peptides. Weighted average chemical shift variations (Δ ppm) were calculated according to the formula (Δ ppm = ([δHN]2+[δN]2)½, where δHN and δN are the changes in HN and N chemical shifts, respectively) as described in ds were estimated with the amide peaks of two selected amino acids, as shown in To map the binding site of BRPF1 and HDGF2 PWWP domain for various methylated histone peptides and estimate the corresponding KAll diffraction data were collected at 100 K and reduced with the HKL suite of programs Figure S1Electron density maps for the ligands identified in our complex structures and reported in this paper. (A) The omit density map for the H3K36me3 peptide in the BRPF1-H3K36me3 complex at 3σ contour. (B) The omit density map for the H3K79me3 peptide in HDGF2+H3K79me3 at 3σ contour. (C) The omit density map for the H4K20me3 peptide in HDGF2+H4K20me3 at 2σ contour. (D) the 2Fo-Fc density map for the bis-tris molecule in the DNMT3A-bis-tris structure. (E) the 2Fo-Fc density map for the bis-tris molecule in the DNMT3B-bis-tris structure.(EPS)Click here for additional data file.Datapack S1Standalone iSee datapack - contains the enhanced version of this article for use offline. This file can be opened using free software available for download at http://www.molsoft.com/icm_browser.html.(ICB)Click here for additional data file.Text S1Instructions for installation and use of the required web plugin (to access the online enhanced version of this article).(PDF)Click here for additional data file.


We also introduce various applications of aptamers for the diagnosis of diseases and detection of small molecules. Numerous analytical techniques, such as electrochemical, colorimetric, optical, and mass-sensitive methods, can be utilized to detect targets, due to convenient modifications and the stability of aptamers. Finally, several medical and analytical applications of aptamers are presented. In summary, aptamers are promising materials for diverse areas, not just as alternatives to antibodies, but as the core components of medical and analytical equipment.Recently, aptamers have attracted the attention of many scientists, because they not only have all of the advantages of antibodies, but also have unique merits, such as thermal stability, low cost, and unlimited applications. In this review, we present the reasons why aptamers are known as alternatives to antibodies. Furthermore, several types of  In. In63]. , and K+  64,65].,65.2+, a3.5.Numerous other aptasensors have been exploited in combination with various types of analytical equipment, such as those used for SPR, surface acoustic wave (SAW), QCM, and microchannel cantilever sensors  66–72].–72.66–72ELISA, one of the major clinical diagnostic tests available, is a versatile technique to detect almost any protein or peptide with high sensitivity. One version of ELISA, commonly referred to as sandwich ELISA, involves the simultaneous use of two antibodies or analyte-binding receptor proteins to capture the analyte or target and to report the target detection . An aptaIn addition to ELISA, another common tool for clinical diagnosis is RDT, which is a rapid and simple method for point-of-care testing (POCT). RDTs for the diagnosis of infectious diseases, such as malaria and influenza, are already in use as commercially available tests. Because of the high sensitivity of the aptamer, several aptamer-based RDT methods, for many biomarkers related to diseases, have been introduced for use in early diagnosis or convenient POCT. In particular, Liu’s group has developed a dry-reagent strip biosensor based on aptamers and functionalized AuNPs for use in thrombin analysis . In this4.2.As mentioned above, Macugen, developed by Pfizer and Eyetech, is already commercially available for AMD. This drug is a pegylated aptamer, a single strand of nucleic acid with specificity to VEGF165, which plays a critical role in angiogenesis and permeability . Additio4.3.Aptamers that bind to internalized cell surface receptors have been exploited to deliver drugs and a variety of other cargo into cells. For example, the prostate-specific membrane antigen (PSMA) is an important prostate cancer marker . The duaSmall interfering RNAs (siRNAs) have received considerable attention recently as a new class of therapeutics for a variety of diseases. The role of siRNA is to induce an RNA interference (RNAi) pathway, which regulates the expression of a specific gene. In particular, it is important to efficiently and safely deliver siRNAs into specific cells in order for the therapeutic use of the siRNAs to be effective. Levy’s group introduced an siRNA-aptamer conjugate via a modular streptavidin bridge using an anti-PSMA aptamer for prostate cancer cells (LNCaP)  89]. As. As89]. 4.4.Another application is bio-imaging, using an aptamer that is conjugated to a fluorophore, a QD, or other materials such as gadolinium, which is useful for magnetic resonance imaging (MRI). Using aptamers as imaging agents has the advantage of their being non-toxic, because oligonucleotide moieties are present in the human body. Additionally, as aptamers have high specificity for their target, accurate targeting, and rapid diffusion through the blood circulation, use of these molecules can increase the certainty of the results obtained during diagnosis or clinical analysis. Based on these advantages, aptamers have been studied as imaging agents for cell imaging as well as single-protein imaging.N-benzylcarboxyamide)-2′-deoxyuridine  on a thymidine base [Kim’s group published the results of C6 cell imaging using a Cy3-labeled AS1411 aptamer, which included a chemical modification of 5- and PSMA(−) prostate cancer cells (LNCaP and PC3), was shown by imaging to bind only to prostate cancer cells and not to normal (PNT2) or other cancer cells . Additio4.5.32P, and introducing the possibility of multiplexing detection [A Western blot analysis is an analytical technique routinely used to quantify specific proteins . The proetection .4.6.l-selectin. The recombinant human l-selectin-Ig fusion protein was successfully purified from Chinese hamster ovary (CHO) cell-conditioned medium using this aptamer affinity column [Immunoaffinity purification is a general laboratory technique used in many scientific fields, that relies on the interaction between an antigen and an antibody to purify target proteins. The use of an aptamer in chromatography has many advantages over the use of an antibody, including an equal or superior affinity and specificity to the target, a smaller size, easier modification and immobilization, better stability, and higher reproducibility. Drolet’s group developed an aptamer affinity chromatography system for human y column . Additioy column .5.in vivo applications, unmodified aptamers are highly susceptible to degradation by several nucleases, and their small size makes them liable to renal filtration, but it can be expected that the potential of aptamers for bio-applications will increase, based on their merits. Thus, further investigations into aptamers and aptasensor systems need to be carried out in order to quckly overcome existing limits.In this review article, the advantages of aptamers and their applications have been presented through descriptions of several excellent investigation projects. An aptamer is a convincing substitute for an antibody because it is very stable under hot or hazardous conditions. Since aptamers are mostly oligonucleic acids, they can easily be synthesized in quantity with high purity, and can be modified with various molecules using simple chemical reactions. Therefore, aptamers have shown great strength in a wide range of applications. Moreover, aptamers are non-immunogenic and non-toxic materials that can be exploited in medical applications, such as diagnosis and treatment of diseases. It is also noteworthy that the types of target molecules are unlimited. Diverse SELEX methods have been designed as the targets to select aptamers conveniently and quickly. Affinity chromatography and magnetic bead-based SELEX are powerful techniques for relatively large molecules such as proteins, because of their high affinity to the target and easy separation from dispensable molecules. Besides, capillary electrophoresis-based SELEX is suitable for small molecules and is a rapid method. Aptamers against biomarker related diseases can be used to diagnose these diseases. In the ALISA and RDT methods, aptamers have been used as capture-probe molecules, based on their affinity and selectivity to the targets. As a result, sensors have shown excellent performance in diagnosis of diseases. For the detection of small molecules, aptamers will be utilized pivotally, because antibody-based detection has considerable limitations, while the applications of aptamers are relatively broad. Numerous sensor techniques incorporating aptamers have been described, such as electrochemical, colorimetric, optical, and mass-sensitive methods. These aptasensors are being employed in various fields, including the medical and industrial fields. Beyond this, the use of aptamers as drugs, therapeutics, bio-imaging materials, and analytical reagents have been investigated and, as a result, the drug Macugen came into the world. In conclusion, it is certain that aptamers are a strong and versatile alternative to antibodies. Of course, aptamers still have issues that need to be resolved for purposes of biological applications. For example, if they are used as agents for


Xenopus tropicalis , we compared the toxicities and somatic mutation activities of four TALEN architectures in a side-by-side manner: a basic TALEN, a scaffold with the same truncated N- and C-terminal domains as GoldyTALEN, a scaffold with the truncated N- and C-terminal domains and an obligate heterodimeric nuclease domain, and a scaffold with the truncated N- and C-terminal domains and an obligate heterodimeric Sharkey nuclease domain. The strongest phenotype and targeted somatic gene mutation were induced by the injection of TALEN mRNAs containing the truncated N- and C-terminal domains and an obligate heterodimeric nuclease domain. The obligate heterodimeric TALENs exhibited reduced toxicity compared to the homodimeric TALENs, and the homodimeric GoldyTALEN-type scaffold showed both a high activity of somatic gene modification and high toxicity. The Sharkey mutation in the heterodimeric nuclease domain reduced the TALEN-mediated somatic mutagenesis.Transcription activator-like effector nucleases (TALENs) are facile and potent tools used to modify a gene of interest for targeted gene knockout. TALENs consist of an N-terminal domain, a DNA-binding domain, and a C-terminal domain, which are derived from a transcription activator-like effector, and the non-specific nuclease domain of FokI. Using  Gene knockout is an important method used to demonstrate the function of a specific gene. Targeted gene disruption by homologous recombination in embryonic stem (ES) cells is currently feasible only in mice  and ratsSharkey mutation into this TALEN scaffold, as Sharkey is known to enhance the DNA cleavage activity of FokI in both homodimeric and heterodimeric ZFN architectures .Multiple TALEN scaffolds have been reported in addition to the basic architecture, pTAL . A pTAL-tectures . In the tyrosinase gene of Xenopus tropicalis  using the Golden Gate assembly method   or low (80 pg) dose  . The nucS/KKR-S) . The Shatectures . For a spg) dose . The morpg) dose . In contpg) dose .A pair of left and right TALENs binds to left and right target sites, respectively . To examtyrosinase . Injection of the low dose of ΔNΔC-ELD/KKR or ΔNΔC mRNAs also led to pigment loss in 60% to 98% of eyes. Conversely, the low dose of TAL-Tyr I and -Tyr II mRNAs induced pigment loss in only 5.8% and 2% of eyes, respectively, and even the high dose of TAL-Tyr II mRNAs elicited pigment loss in only 14% of eyes. This phenotype difference may be ascribed to the distinction in architecture between TAL and ΔNΔC, namely, the truncation of the N- and C-terminal domains flanking the DNA binding domain tyrosinasg domain  Fig. 1)tyrosinastectures .supplementary material Fig. S2; Somatic mutation by four types of TALEN scaffolds was quantified by measuring the loss of restriction enzyme recognition sites between target sites in the amplicons from a genomic DNA of the injected embryos. HaeIII and PflMI recognition sites are located in the spacers of the Tyr I and Tyr II sites, respectively . When thsupplementary material Fig. S3), consistent with those using pooled genomic DNAs. These data indicate that the restriction enzyme resistance assay has a tendency to underestimate mutation rates due to the conservation of restriction sites even after DNA repair.Target site sequences were determined using a pool of genomic DNAs extracted at NF-stage 35/36 from randomly selected living embryos injected with the high dose of TALEN mRNAs , though X. tropicalis embryos injected with TALEN mRNAs synthesized using a scaffold with the truncated N- and C-terminal domains and an obligated heterodimeric nuclease domain . The frogs were maintained at 24°C. The tadpoles were staged according to Nieuwkoop and Faber . Each blastomere of two-cell-stage X. tropicalis embryos was injected with 2 nl of 50 or 10 ng/µl of each TALEN mRNA and 25 ng/µl of mCherry mRNA (TALEN mRNAs were transcribed rry mRNA . The flurry mRNA . The embEmbryos were classified according to their morphology and the loss of pigmentation in the retinal pigment epithelium at NF-stage 35/36 . Each in


PDZ domains are highly abundant protein-protein interaction modules involved in the wiring of protein networks. Emerging evidence indicates that some PDZ domains also interact with phosphoinositides (PtdInsPs), important regulators of cell polarization and signaling. Yet our knowledge on the prevalence, specificity, affinity, and molecular determinants of PDZ-PtdInsPs interactions and on their impact on PDZ-protein interactions is very limited.in vivo cell-localization studies and in vitro dot blot and Surface Plasmon Resonance (SPR) experiments using synthetic lipids and recombinant proteins. We found that PtdInsPs interactions contribute to the cellular distribution of some PDZ domains, intriguingly also in nuclear organelles, and that a significant subgroup of PDZ domains interacts with PtdInsPs with affinities in the low-to-mid micromolar range. In vitro specificity for the head group is low, but with a trend of higher affinities for more phosphorylated PtdInsPs species. Other membrane lipids can assist PtdInsPs-interactions. PtdInsPs-interacting PDZ domains have generally high pI values and contain characteristic clusters of basic residues, hallmarks that may be used to predict additional PtdInsPs interacting PDZ domains. In tripartite binding experiments we established that peptide binding can either compete or cooperate with PtdInsPs binding depending on the combination of ligands.We screened the human proteome for PtdInsPs interacting PDZ domains by a combination of Our screen substantially expands the set of PtdInsPs interacting PDZ domains, and shows that a full understanding of the biology of PDZ proteins will require a comprehensive insight into the intricate relationships between PDZ domains and their peptide and lipid ligands. PSD-95/DLG1/ZO-1 (PDZ) domains are among the most abundant modular domains in multicellular organisms. In human, there are more than 250 PDZ domains, present in over 150 different proteins that commonly control processes of cell signaling and polarity Efficient and accurate flow of cellular information relies on scaffolding proteins that coordinate the physical assemblies of signaling complexes PtdInsPs control various aspects of cell signaling, vesicular trafficking, and cytoskeleton remodeling, and emerge as important regulators of cell polarization Until now, few studies have focused on the prevalence, specificity and determinants of PDZ-PtdInsPs binding-interactions. In a first extensive study, Wu et al, baited 74 isolated purified PDZ domains and 14 PDZ tandems with liposomes prepared from bovine brain lipid extracts in vitro binding experiments. We established the affinities and specificities of these interactions and probed, for two selected cases, the interplay between peptide and PtdInsPs binding. We identify a high pI value and clusters of basic residues as common features of PtdInsPs-interacting PDZ domains and successfully predict additional PtdInsPs binding PDZ domains.In this study we aimed to clarify whether and how particular PDZ domains mediate specific PtdInsPs-interaction and whether synergistic PDZ/peptide/PtdInsPs interplay is more than anecdotal. To this end we screened the human PDZ proteome for PtdInsPs interactions combining cell-localization assays and We used cell-localization as an assay for the identification of candidate PtdInsPs interacting PDZ domains. Briefly, the screening vector encoded a fluorescent tag  linked to an ‘enhancer element’  in MCF-7, HEK293 and HeLa cells , Fig. S2in vitro. We established the identity of the subcellular compartments by co-localization experiments with known markers, and probed the PtdInsPs dependence of the localizations by treatments altering cellular PtdInsPs levels. The in vitro binding was studied by SPR, whereby recombinant PDZ domains were injected over PtdInsPs-containing liposomes. Sensorgrams were corrected for binding to reference (PtdIns-containing) liposomes, and for buffer effects (double reference subtracted), see Defined subcellular localizations of fluorescently tagged PDZ domains may be PtdInsPs and/or peptide driven. For a subset of PDZ domains we therefore investigated to what extent the cellular enrichments were PtdInsPs dependent, and correlated this with PtdInsPs binding From the twenty-four PDZ domains that localized discretely at the plasma membrane, we selected eight  and co-transfected them with PtdIns4P5-kinase (PIP5K), which increases the plasma membrane PtdInsP2 levels in vitro binding data thus suggested a potential contribution of PtdInsP3 in membrane targeting of MPP7, but we failed to demonstrate such contribution in vivo. Transient increase in plasma membrane PtdInsP3 levels by serum stimulation did not promote increased enrichment of eYFP-S1PDZ1-MPP7 , except for PtdInsP3 (25±8 µM) , suggestDZ1-MPP7  while itDZ1-MPP7 . SimilarDZ1-MPP7 . We theret al.D generally above 100 µM). CASK interacts with monophosphate PtdInsPs with low affinity (>100 µM KD) but has good affinity for polyphosphate PtdInsPs, with an apparent affinity of 25±7 µM for PtdInsP2 P2 sP2 . MPDZ_7 in vitro, with high-to-intermediate affinities P2 4′ phosphatase IpgD   . ERBP2IP) et al. suggestiin vitro approach we performed lipid blot assays using commercially available membranes pre-spotted with PtdIns, PtdIns4P, PtdInsP2 and PtdInsP3 as well as other abundant lipids  and phosphatidylserine (PS), but not with zwitter ionic lipids such as triacylglycerol (TGA), diacylglycerol (DGA), phosphatidylglycerol (PG), sphingomyelin or cholesterol. We therefore performed SPR experiments with five selected PDZ domains  and two selected lipids, PS and PE, embedded in DOPC liposomes (10% PS or 40% PE in a DOPC using 100% DOPC as reference). Although we did not observe any detectable PDZ-PE binding (data not shown), we detected low affinity interactions with PS P2 in composite liposomes: CASK, 9±2 µM; DFNB31_1 2.7±1 µM; MAGI1_6 4.5±1 µM; MAGI3_3 14±2 µM; SLC9A3R2_1 2.5±1 µM). The results confirm that a subgroup of PDZ domains interact with PtdInsPs and suggest that these interactions may be enhanced in vivo by interactions with other anionic phospholipids.As an alternative t lipids . IL16_3 D-values: DFNB31_1/Y167W for FTDTHL 46±6 µM; CASK/I157W for TKEFYA 280±20 µM) DPtdInsP2 in presence of peptide >30 µM, DPtdInsP2 in presence of 1 mM TKEFYA 6.2±2 µM, Given the overall low PtdInsPs specificities, and often modest PtdInsPs affinities, it is unlikely that PtdInsPs bindings alone target the PDZ domains to their defined subcellular compartments. Plausibly, peptide interactions are also involved in controlling the subcellular targeting. To address the interplay between PtdInsPs and peptide-binding we chose DFNB31_1 and CASK as models, taking two of the PDZ domains with well-defined cognate peptides. Cognate peptide ligands were the C-terminal peptides of usher (FTDTHL) for DFNB31_1 Thus, PDZ-peptide-PtdInsPs interactions can be competitive as well as synergistic depending on the combination of ligands, a topic that deserves systematic future investigations.The most striking common property of the PtdInsPs binding PDZ domains is a high pI value, on average higher than 9  as compaIn vivo, DFNB31_1 is enriched in nucleoli as shown by the co-localization with the nucleolar marker nucleophosmin/B23 P2 binding, while conferring a 2-fold decrease in peptide binding affinity without shifting the cellular enrichment. Taken together, the data confirm the importance of the conserved basic cluster in PtdInsPs binding and the importance of this charge cluster in defining the cellular localization of the fluorescently tagged protein.However, from the lack of complete conservation of the identified positive charge cluster it is clear that PDZ domains may interact with negatively charged PtdInsPs through alternative ways. Notably, SLC9A3R2_1, is among the highest affinity PtdInsPs binders, but has only two basic residues of the consensus positive charge cluster. Instead, this protein has a basic cluster in the loop region between beta strand 2 and 3 , which might be involved in PtdInsPs binding of this protein.To assess the functional importance of the conserved positive charge cluster, we performed a mutagenic analysis of DFNB31_1. smin/B23 . DFNB31_in vitro  and its in vitro . Similarin vitro  that hasin vitro . We estain vitro . We detein vitro , Table 2lization . Three m(in vivo . The R14in vitro PtdInsPs binding. We chose two PDZ domains with positive charge clusters similar to the main consensus (APBA3_1 and LIN7A) and two domains, INADL_7, and MPDZ_8, that share a similar basic cluster with SLC9A3R_1  and a cluster of basic residues could be a signature for PtdInsPs binders we selected four PDZ domains fulfilling the criteria that did not show-up in our screen, and tested their LC9A3R_1 . We founin vitro binding experiments we identified several new PtdInsPs binding PDZ domains and, using various lipid modifying treatments, extensively documented for some of them PtdInsPs-dependent subcellular targeting. Interestingly, we observed PtdInsPs-dependent targeting to the plasma membrane (CASK) as well as to the nucleus . Our study is the so far most comprehensive cell-based screen for PtdInsPs interacting PDZ domains. It shows that PDZ-PtdInsPs interactions commonly are in the low-to-mid micromolar range and tend to be reinforced by additional electrostatic interactions with other anionic phospholipids such as PS. Binding to cognate peptide ligands can either reinforce or compete with PDZ-lipid interactions, and some PDZ domains are likely to coincidently bind protein and lipid ligands. We established that high pI values and clusters of basic residues are common properties of PtdInsPs interacting domains, a ‘signature’ that can be used to predict additional PtdInsPs-interacting domains. It is thus now clear that a subgroup of PDZ domains interact with PtdInsPs through positively charged clusters, which should be taken into consideration when addressing the biology of PDZ containing proteins.Through a cell-based screen and complementary et alIn the current study we used isolated PDZ domains, which may have affected the outcome in several ways. Indeed, it is not always obvious how to define the borders of PDZ domains and changing the borders may alter the functional properties of the domains Of further note is that PDZ domains often make part of multi-domain proteins and thus may only provide a part of the peptide and/or lipid interactions required for targeting their host proteins to the appropriate locations. Indeed, PtdInsPs interacting PDZ domains tend to occur in tandem with other PtdInsPs binding modules, such as the PDZ domain of PICK1 being linked to a BAR domain A comparison of the cellular enrichments observed for the PDZ domains in the screening constructs with the reported localizations of the host proteins based on Gene Ontology Annotations  reveals Our screen greatly expanded the set of known PtdInsPs interacting PDZ domains and provided signatures that can be used for the identification of additional lipid binding PDZ domains. The PDZ domains we identified as PtdInsPs binders interact with lipid metabolizing enzymes such as PTEN , are parts of proteins involved in PtdInsPs dependent processes such as vesicular trafficking (APBA3), interact with nuclear-PtdInsPs or even potentially peroxisomal-PtdInsPs which paves the way for future original research on poorly investigated areas of cell biology.http://www.ebi.ac.uk/interpro/), PFAM  and SMART version 5.0 (http://smart.embl-heidelberg.de<http://smart.embl-heidelberg.de/). Ten amino acids from the original protein sequence were added on each side of the predicted PDZ domains. Predicted amino acid sequence and actual translation of cloned DNA constructs are given in 5′-GATCACATGGTCCTGCTG.Our human PDZ domain collection comprises 246 PDZ domains. The domains and their boundaries were established by cross searching Interpro . The PLCΔNES was described earlier PDZ domains were subcloned from the screening constructs into pEYFP-C2 (Invitrogen) for microscopy assays or into the pETM-11 (EMBL Heidelberg) vector for his-tagged protein expression. Coding sequences were amplified using oligonucleotides carrying restriction sites for MCF-7 cells obtained from American Type Culture Collection  were cultured in DMEM/F-12 medium (Life Technologies) supplemented with 10% fetal bovine serum (Gibco). For microscopy experiments cells were plated on eight-well chamber slides , transfected after 4 h with FuGENE (Roche Diagnostics) and fixed with 4% parafolmaldehyde the day after transfection. Fluorescence confocal micrographs were obtained with Olympus Fluoview 1000 (Olympus) and wide field micrographs with Leica AS-MDW (Leica) using appropriate filter sets. The enrichment of eYFP-S1PDZ1-PDZX, eYFP-PDZX and eYFP-PDZX-PDZX constructs in the defined subcellular regions  was scored by confocal microscopy and images of at least 30 cells from three independent experiments were analyzed for each condition. For mutagenic analysis, nucleolar enrichment of eYFP-DFNB31_1 and its mutants was scored in living cells and quantified by measuring fluorescence intensities of nucleoli and nucleoplasm and calculating the nucleolar/nucleoplasm ration after background correction. The ratio was calculated for at least 30 cells from three independent experiments and cells were classified as displaying strong nuclear enrichment if the nucleolar/nucleoplasm ration was higher than 1.2.Wortmannin (Sigma-Aldrich) or LY294002  were added to the cells for 30 min to the final concentration of 500 nM and 50 µM, respectively. Cells were treated with 800 nM of YM201636 (Chemdea) for 2 h. For ionomycin treatment cells were washed with Krebs-Ringer buffer  before addition of 10 µM ionomycin  in the same buffer. Phenylarsine oxide (PAO) (Sigma-Aldrich) was added to the cells to the final concentration of 100 µM and rapamycin (Sigma-Aldrich) to 200 nM. For serum stimulation experiments cells were serum starved for 18 h before 30 minutes stimulation with 10% fetal bovine serum (Gibco).Cells were stained as described previously E. coli ER2566 cells carrying the pETM-11 expression constructs, and purified by purified by nickel affinity chromatography as described previously N-terminally his-tagged proteins were expressed, in SPR experiments were performed in a BiacoreT100  at 25°C in 25 mM HEPES, 150 mM NaCl, pH 7.2 and 1 mM beta-mercaptoethanol with a flow rate of 30 µl/min. Liposomes were prepared and immobilized on L1 sensor chips as described previously Dot blot overlay assays were performed according the protocol of the manufacturer (Echelon Biosciences), by incubating the fat-free milk blocked membranes with 10–50 µM 6xhis-tagged proteins. Bound proteins were detected using the primary anti 6His antibody (Roche), goat-anti mouse HRP conjugated secondary antibody and ECL detection.Spectrofluorimetric measurements were performed at 25°C in 25 mM HEPES, 150 mM NaCl, pH 7.2 and 1 mM beta-mercaptoethanol in a Carry Eclipse Spectrofluorimeter (Varian). The peptides corresponded to the C-termini of syndecan-2 (TKEFYA) and usher (FTDTHL) and were of high purity , and the experiment were performed and analyzed as described previously DGN− = ΔDGNmut− − ΔDGNwt−, where ΔΔGD-N is the difference between the free energies of unfolding upon mutation.The effects of the mutations on the stability were calculated by ΔΔStructure based sequence alignments made using ClustalX and Swiss pdb viewer.Figure S1Related to  and . Control experiments showing that fluorescence intensities are comparable between constructs, fusions proteins are not cleaved or degraded, and showing the distribution of eYFP-PDZ constructs where S1PDZ1 enhancer is omitted.(PDF)Click here for additional data file.Figure S2Related to . Confocal micrographs of eYFP-S1PDZZ-PDZX constructs in MCF-7, HEK293 and HeLa cell lines.(PDF)Click here for additional data file.Figure S3Related to . Control microscopy experiments illustrating the effectiveness of lipid modifying treatments and showing additional examples of the effect or lack of effect of various treatments.(PDF)Click here for additional data file.Figure S4Related to . Sequence alignment of PtdInsPs-interacting PDZ domains.(PDF)Click here for additional data file.Table S1Related to . Oligomeric status of various recombinant PDZ domains used in this study.(PDF)Click here for additional data file.Table S2Related to . Apparent KD values for 19 recombinant PDZ domains for 5% PtdInsPs in DOPC liposomes.(PDF)Click here for additional data file.Table S3Related to discussion. Comparison of the localization of eYFP-S1PDZ1-PDZX domains and their host proteins based on Gene Ontology Annotations.(PDF)Click here for additional data file.Table S4Related to Materials and Methods. Amino-acid sequences of the PDZ domains used in this study.(XLSX)Click here for additional data file.


The direct isolation of integron gene cassettes from cultivated and environmental microbial sources allows an assessment of the impact of the integron/gene cassette system on the emergence of new phenotypes, such as drug resistance or virulence. A structural approach is being exploited to investigate the modularity and function of novel integron gene cassettes.Cass2, an integron-associated protein derived from an environmental V. cholerae. The structure defines a monomeric beta-barrel protein with a fold related to the effector-binding portion of AraC/XylS transcription activators. The closest homologs of Cass2 are multi-drug binding proteins, such as BmrR. Consistent with this, a binding pocket made up of hydrophobic residues and a single glutamate side chain is evident in Cass2, occupied in the crystal form by polyethylene glycol. Fluorescence assays demonstrate that Cass2 is capable of binding cationic drug compounds with submicromolar affinity. The Cass2 module possesses a protein interaction surface proximal to its drug-binding cavity with features homologous to those seen in multi-domain transcriptional regulators.We report the 1.8 Å crystal structure of Cass2 to be representative of a larger family of independent effector-binding proteins associated with lateral gene transfer within Vibrio and closely-related species. We propose that the Cass2 family not only has capacity to form functional transcription regulator complexes, but represents possible evolutionary precursors to multi-domain regulators associated with cationic drug compounds.Genetic analysis identifies  Vibrio genus is ubiquitous and abundant throughout the aquatic environment. It is clear that lateral gene transfer (LGT) events play a major role in the evolution and adaptation of this organism, with genetic interchange of Vibrio genes observed over a wide range of phylogenetic distances V. cholerae and V. vulnificus genomes suggests up to 20% of their content to have arisen via this route. The continued emergence of novel pathogenic clones carrying diverse combinations of phenotypic and genotypic properties significantly hampers control of the disease V. cholerae O139, one of the two strains responsible for epidemic Asiatic cholera, appears to be a result of LGT from multiple and diverse descendants of the seventh pandemic O1 El Tor strain V. choleraeV. cholerae from non-pathogenic environmental strains The Vibrio species One important mediator of LGT involves mobile gene cassettes clustered in association with integrons Vch_cass2) isolated from a strain of V. cholerae resident within a brackish coastal environment in north-eastern USA. Initial sequencing identified the gene cassette to encode a domain with some homology to the AraC superfamily of transcription activators, generally implicated in the regulation of stress response and virulence In this work, we focus on one integron gene cassette . Partial genomic sequencing has established this strain to have strong sequence identity (>90%) with known pathogenic strains of V. cholerae . The encoded protein sequence, Cass2, displays signature motifs that associate it with the superfamily AraC_E_bind  established it to be representative of an entirely distinct family of independent single-domain proteins, represented by over 1200 homologs across a range of organisms. A phylogenetic analysis of these sequence relatives (Cass2 in a distinct clade (75–79% amino acid identity) sourced from a variety of marine-dwelling bacteria. While Cass2 clearly clusters with homologs from specific Vibrio spp , a related but distinct clade displaying ∼40% amino acid identity is evident within Shewanella genomes. Representative protein sequences for members of these two clades are aligned with that of Cass2 in The gene cassette named elatives  places CVch_cass2, the genetic context for many of its homologs indicate an association with LGT. Those relatives displaying highest sequence homology are also encoded within gene cassette elements (e.g. V. cholerae MZO-2 and AM-19226), while others are found adjacent to transposon features (V. vulnificus CMCP6).Importantly, like Cass2 to be a monomer organised into a barrel-like form comprising an antiparallel β-sheet of eight strands (α atoms), and directly aligns elements β2, α2, β3 and β4 with β6, α3, β7 and β8, respectively. Despite the low sequence identity of the two subdomains (∼12%), the structural superposition coherently maps side chains Phe14, Leu34, Trp35, Tyr56 and Val69 from subdomain I to those of Phe97, Leu110, Trp111, Tyr136 and Val142 in subdomain II. These recurring side chains stabilise packing of the helices to the β-sheet and form critical elements of the ligand-binding site.Structure determination by x-ray crystallography revealed the protein  strands . FlankinCass2, a relatively flexible loop is located C-terminal to the helical portion, i.e. forming connections between α2–β3 (residues 46–50) and α3–β7 (residues 119–130) segments. These loops project from the top and bottom of the sheet, respectively  is buried deep within this cleft, flanking the pseudo two-fold axis of the protein structure. The polarity of this side chain is stabilised by hydrogen bonds to side chains of Tyr56, Tyr136, and Trp111  and α3  and the β7/β8 interstrand loop (Tyr136) are within 4 Å of this ligand. Some additional density can be distinguished in our maps belonging to a second (non-definable) ligand, extending further along this same cavity to Trp115.The central cavity enclosed between the helices of d Trp111 . BetweenCass2 relatives from Vibrio and Shewanella (Cass2 sequence family (Tyr39 being conservatively replaced in Shewanella strains)  . The domStaphylococcus aureus (PDB 3LUR), the C-terminal domain of Rob transcription factor from E. coli (PDB 1D5Y) Bacillus subtilis (PDB 3D6Z) E. coli (PDB 1JYH) E. coli Rob and GyrI domains are also members of the AraC/XylS family of transcription factors; Rob is known to control diverse regulons in prokaryotes Searches for structural homologues of Cass2 revealed several fold relatives with overlapping biological functions associated with transcription regulation. Close spatial alignment was found to putative transcription regulation protein from Cass2. A glutamate residue is preserved midway across the sheet in all the proteins, stabilised within a hydrophobic environment by surrounding Tyr side chains. The closest structural homolog from Staphylococcus aureus (PDB 3LUR), retains many of the hydrophobic side chains of the central cavity, but also possesses a cluster of polar residues  not present in Cass2 , results in the exposure of the central cavity for interaction with the cationic ligand Cass2 appears to be relatively flexible in our structure, and it is thus feasible that access to the ligand site in the gene cassette domain might occur by a similar helix-opening mechanism, perhaps coupled in this case with expulsion of the interior side chain Tyr39.The crystal structure of the Cass2 is unknown, it is clear that the domain contains a binding site suitable for hydrophobic/cationic compounds, compatible with that seen in its structural homologs. Tryptophan fluorescence was used to test for interactions of Cass2 with a set of cationic compounds known to associate with the related bacterial transcription regulators: TPP, benzalkonium chloride, chlorhexidine (E134Q)Cass2, designed to neutralise the electrostatic effects of Glu134, was additionally probed in these titrations. Cass2 contains three tryptophan residues, two of which  are observed to be in close contact to PEG from helices α2 and α3 within the binding cleft. The third side chain (Trp115 on helix α3) is somewhat more remote along the ligand cavity; it exhibits multiple rotamer forms in the crystal, possibly due to accommodation of other ligand molecules.Although the natural ligand of Cass2 in its apo form (349 nm). This is consistent with loss of solvent exposure of the Trp residues, such as might occur as the cavity closes upon ligand binding. For all three compounds at sub-micromolar concentrations, significant quenching (up to 60%) of the intrinsic fluorescence emission of Cass2 was observed in a concentration-dependent manner, as illustrated for the titration with TPP in D values were determined to be in the sub-micromolar range (D = 0.1 µM). The binding affinity determined for TPP (KD = 0.2 µM) indicates a tighter interaction with Cass2 than has been reported for the fold relative BmrR Initial fluorescence measurements in the presence of excess quantities of all three compounds detected a blue shift (5 nm) from the emission maximum of ar range . The monCass2 had little effect on its strength of binding to the monovalent compounds tested . For the divalent compound chlorhexidine, the affinity for the E134Q mutant appears to have been somewhat enhanced (KD = 0.10 µM). The electrostatic role of this glutamate thus appears to be tempered in the case of Cass2, presumably due to the large number of hydrophobic contacts within the internal binding cavity.Mutation of the central glutamate sidechain of Cass2 with loss of the glutamate charge.Our results are consistent with the earlier binding studies of BmrR to three cationic compounds  and crysCass2 successfully binds the same cationic compounds known to associate with transcriptional regulators, minimal inhibitory concentration (MIC) assays were undertaken to determine if the Vch_cass2 gene could directly confer resistance to Vibrio cells growing on media containing these compounds. Laboratory strains Vch_cass2+ and Vch_cass2 were prepared, but in the presence of all compounds, no difference in cell growth was observed for the two strains. The inability of Vch_cass2+ gene to directly confer resistance to cationic compounds points to the need for protein factors in addition to the effector domain to be present for effective regulation of their cellular metabolism.Given we can demonstrate that Cass2 sequence family not directly associated with the ligand-binding cleft stand out as strongly conserved. One encompasses the sequence motif -YESD- located from Tyr59 within the β3/β4 loop. When mapped onto the three-dimensional fold of Cass2, these side chains, in addition to residues Phe63, Thr64 and Ala66, cluster along a projected surface feature well to the “base” of the binding cleft (depicted in Cass2), is the origin of exposed Trp and Phe side chains which elongate the same surface. The entire region is relatively flexible in the crystal structure, with high B-factors observed for the loop residues.Two sequence segments of the Cass2 is overlaid with that of the two-domain Rob transcription factor Cass2 retains some of the hydrophobic features of the interface, as well as possessing protruding charged side chains, including Arg149 . In the Rob structure, the analogous side chain at this location (Arg288) participates in an electrostatic interaction across to the neighbouring DNA-binding domain. Thus, Cass2 gives every appearance of being suitably organised for interaction with a protein partner with features common to the helix-turn-helix domains utilised by its sequence relatives.A possible role for this surface becomes evident when, for instance, the structure of Cass2 and Rob differ completely from the situation found in the BmrR fold homolog, the interdomain interface of which is located on the opposite side of the effector-binding module Cass2, connecting strands β7 and β8, is relatively short (136–140) and unlikely to participate in a similar interaction. The absence of a linker helix in Cass2, oriented on the same side as the domain interface and essential for dimerisation in BmrR, further rules out this region as a putative protein-binding interface.It should be noted that the organization of both Vch_cass2 encodes a single and independent binding domain for cationic compounds. The structure (and sequence) of its protein product Cass2 readily confirms its homology to effector-binding domains associated with the AraC/XylS and MerR family of transcription regulators. These well-characterized factors are mediators of bacterial antibiotic and multi-drug resistance through their ability to both recognise effector molecules and to regulate transcription of the appropriate efflux system Our experimental evidence establishes that the gene cassette Cass2 depicts PEG in a binding site organised with features reminiscent of those of the effector modules of bacterial regulators Cass2 to be particularly well adapted for tightly binding the cationic drugs which serve as ligands to the AraC/MerR family. Hydrophobic forces appear to predominate within the binding interactions, and (unlike BmrR) the Cass2 domain is capable of binding monovalent and bivalent ligands. Within the structural framework of Cass2, a distinct loop feature extending from helix α2 edging the central sheet (residues 41–46) is proposed to undergo structural rearrangement so as to facilitate ligand entry.The crystal structure of Cass2 and genes from a group of phylogenetically-related Vibrio and Shewanella species. The crystal structure presented here therefore defines the paradigm fold for a new family of effector-binding proteins prevalent within these marine-dwelling species. Sequence variation between the two related groups of proteins is restricted to the putative hinge region (C-terminus of helix α2) as well as strand β4. Thus a slightly altered ligand accessibility may have evolved for the distinct clades outlined here.Significant sequence homologies are found between Vch_cass2 gene with mobile DNA elements, also notably evident for its group of related homologs, emphasises the mechanism by which these binding modules can be laterally transferred between species. While the presence of a DNA-binding partner appears necessary for transcription regulation, we cannot rule out the possibility that the biological function of Cass2 itself may be to provide a self-contained low-level multidrug resistance system, capable of sequestering drugs and preventing them from reaching further intracellular targets. The role of cationic drugs in treatment of cholera and inhibition of cholera toxin-internalization has been previously reported The association of the Cass2 structure consistent with a protein interaction site adjacent to the active-site cavity. We propose this to comprise a potential site for interaction of the effector-binding module with a specific DNA-binding domain, so as to mimic the organisation of the multi-domain transcription regulators. This is congruent with the more general observation that two interacting prokaryotic proteins, not necessarily encoded by neighbouring genes, may be found fused as a single chain homolog in another organism Cass2 in V. cholerae; no sequence homolog of the single-domain protein MarA (from E. coli) Cass2. However, wider sequence searches across published Vibrio genomes do reveal the existence of single-domain homologs  containing the helix-turn-helix motifs present in both MarA and Rob relatives.We have, however, noted surface features in the Cass2 protein and its relationship to other members of the AraC/XylS and MerR family reinforces the notion that gene cassettes within integron arrays generally move and rearrange independently of one other. Given that many cassettes encode single small domain proteins, loss of intervening attC site sequences may lead to permanent fusion of gene cassettes so as to instead encode a multi-domain polypeptide that confers advantage. Our recovery of an independent single domain with effector-binding capacities is significant as a possible evolutionary precursor to the multi-domain transcription regulators, of which the AraC and MerR families are examples.The overall structure of the tipA gene of S. lividans encodes single and two domain gene products. The full-length gene product (TipAL) comprises an N-terminal helix-turn-helix domain which auto-regulates the tipA gene in conjunction with a thiostrepton-binding domain. In vast molar excess, however, a shorter in-frame translational product (TipAS) comprising solely the drug-binding domain is independently transcribed Evidence for fusion events in the evolution of MerR regulators has previously been outlined V. cholerae was isolated from a brackish coastal pond  as follows. Several water samples (1 ml) were spread directly agar containing on thiosulfate/citrate/bile salts/sucrose  Vch_cass2.Strain OP4G of Cass2 was produced recombinantly in Escherichia coli strain BL21-CodonPlus (DE3)-RIPL (Stratagene) with an N-terminal affinity tag (MGSSH6SSGRENLYFQG-Cass2) using the plasmid p15TV-L. Cass2 was derivatized with selenomethionine (SeMet), as provided within the M9 SeMet media kit  supplemented with antibiotics (ampicillin (100 µg/ml), chloramphenicol (25 µg/ml)). Cells were grown at 37°C until OD600 1.2 and induced with 1 mM IPTG  prior to overnight growth at 25°C. Harvested cells (from 1 l culture) were frozen in Buffer A (50 mM HEPES buffer (pH 7.5), 500 mM sodium chloride, 5 mM imidazole, 5% glycerol) and sonicated in the presence of protease inhibitors (phenylmethylsulphonyl fluoride (0.5 mM) and benzamidine (1 mM).Cass2 was dialysed into Buffer B (10 mM HEPES buffer (pH 7.5), 500 mM sodium chloride) and concentrated to ∼20 mg/ml for crystallization. The reducing reagent tris-(2-carboxyethyl)-phosphine (0.5 mM) was added to all purification buffers.Following storage (80°C), the soluble cell fraction was loaded onto Ni-nitroloacetic affinity media (Qiagen) washed with Buffer A and eluted with Buffer A containing 250 mM imidazole. After addition of ethylenediamine tetraacetic acid , purified (E134Q)Cass2 was prepared using a commercial kit . The recombinant protein was prepared with E. coli BL21 (DE3) Rosetta cells (Merck) in Luria Bertani (LB) medium at 37°C. Following induction (0.2 mM IPTG) and growth at 20°C for 5 h, cells were recovered and the mutant protein isolated from the soluble fraction by batch affinity chromatography . Protein buffers were as above.Cass2 were grown to diffraction quality in 0.1 M citric acid (pH 3.50), 25% (w/v) PEG-3350. The crystals  were cryo-protected by immersion in paratone-N (Hampton Research) prior to flash freezing. Diffraction data was collected at 100 K using synchrotron radiation at the selenium K absorption edge .Using sitting-drop format, crystals of Diffraction data to 1.8 Å was processed using MOSFLM Cass2 reveals one chain per asymmetric unit, with electron density visible for 153 residues, including 7 residues of the affinity tag. No density was observed for residues 120–122 (Ser-Glu-His). Residues SeMet1 (strand β1), SeMet24 (helix α1) and Trp115 (helix α2) showed alternative conformations, suggestive of increased mobility within these portions of the molecule. The Ramachandran plot shows >96% of residues in most favoured regions; one outlier  occurs within an elongated loop (residues 58–66) connecting strands β3 and β4 of the central β-sheet.The structure of −10 and iterated until convergence (11 rounds) Vibrio genus. The retrieved amino acid sequences  were subjected to a phylogenetic analysis using a suite of programs within the Mobyle web interface Cass2 and to locate homologs within other domain organizations . DALI Sequence homology searches of the non-redundant database  were performed using PSI-BLAST with a set threshold E-value <10Cass2 and related mutants. Concentrated ligand solutions in Buffer B were titrated into a 400 µl sample of protein (180 nM in Buffer B) and Trp fluorescence monitored. As a control, each compound was also titrated into a 1.3 µM sample of tryptophan (99% purity) in Buffer B, a concentration selected as yielding similar fluorescence to the initial Cass2 sample prior to titration.Fluorescence assays were used to detect binding of compounds to Cass2 displayed maximum emission at 349±1 nm (apo form) and 344±1 nm (fully bound). Thus, fluorescence quenching was monitored by recording emission at 350 nm for all samples following excitation at 295 nm  with an integration time of 5 s. All readings were corrected for buffer background emission and sample dilution. Inner-filter effects were measured by titrating each compound into a 1.3 µM sample of tryptophan in Buffer B and the relative fluorescence quenching (ΔF) corrected as follows Fluorescence intensities were recorded at 22°C with a PerkinElmer LS55 fluorescence spectrophotometer using a 1 cm×0.2 cm quartz cell. When subjected to an excitation wavelength of 295 nm, F0−FC (FW0/FWC))/F0 where F0 = fluorescence intensity of protein sample, FC = fluorescence intensity of protein with added compound, FW0 = fluorescence intensity of free tryptophan solution, FWC = fluorescence of tryptophan solution with added compound.ΔF = (KD) for the interactions, ΔF was plotted against compound concentration and fitted to the following equation by non-linear regression using Kaleidagraph (Synergy software):Standard deviation was calculated for the individual ΔF values from three independent experiments. For the determination of dissociation constants (b−ΔFf) [Cass2])/(DK+[Cass2]))+ΔFf where ΔF is the relative fluorescence quenching, ΔFb is the maximum relative fluorescence quenching (ligand-saturated Cass2); ΔFf is the relative fluorescence quenching of unbound Cass2.ΔF =  assays were conducted with the cationic agents in 96-well plate format using a broth micro-dilution technique Vch_cass2+ and Vch_cass2− (control strain: Vibrio sp. DAT722+pJAK16 plasmid without Vch_cass2 gene) were grown overnight (37°C) in LB/salt medium. Subcultures  were grown at 37°C until OD600 value 0.6. Wells were inoculated with 10 µl subculture following further dilution , and growth monitored by recording OD595 after 16 h.Plasmid pJAK16+Cass2 are deposited as PDB file 3GK6.Coordinates and structure factors for


Clostridium perfringens alpha toxin/phospholipase C (CP-PLC) is one of the most potent bacterial toxins known to cause soft tissue infections like gas gangrene in humans and animals. It is the first bacterial toxin demonstrated to be an enzyme with phospholipase, sphingomyelinase and lecithinase activities. The toxin is comprised of an enzymatic N-domain and a binding C-domain interconnected by a flexible linker. The N-domain alone is non-toxic to mammalian cells, but incubation with C-domain restores the toxicity, the mechanism of which is still not elucidated. The objectives of the current study were to investigate the formation of a stable N and C-domain complex, to determine possible interactions between the two domains in silico and to characterize the in vitro and in vivo correlates of the interaction. To establish the existence of a stable N and C-domain hybrid, in vitro pull down assay and dot-Far Western blotting assays were employed, where it was clearly revealed that the two domains bound to each other to form an intermediate. Using bioinformatics tools like MetaPPISP, PatchDock and FireDock, we predicted that the two domains may interact with each other through electrostatic interactions between at least six pairs of amino acids. This N and C-domains interacted with each other in 1:1 ratio and the hybrid lysed mouse erythrocytes in a slower kinetics when compared with wild type native Cp-PLC. BALB/c mice when challenged with N and C-domain hybrid demonstrated severe myonecrosis at the site of injection while no death was observed. Our results provide further insight into better understanding the mechanism for the toxicity of Cp-PLC N and C-domain mixture. Clostridium perfringens is a Gram-positive, spore-forming, obligate anaerobe commonly found in the gastrointestinal tracts of both humans and animals, and in soil, food and sewage . But on kinetic analysis, typically, wild type Cp-PLC toxin followed a pre-steady-state kinetics, and on the other hand, N and C-domain hybrid Cp-PLC resulted in sigmoid kinetics  and lyse the host cells. The female BALB/c mice in our study, when challenged with N-domain alone did not show any observable histological abnormalities or necrosis (C. perfringens N-domain (the Bacillus phospholipase C enzyme) is non-hemolytic [As reviewed by Popoff M and Bouvet P [29], the catalytic N-domain of Cp-PLC is more likely derived from its enzyme precursors but the evolution of binding C-domain is still enigmatic, though it is found structurally identical to mammalian C2 domains belonging to Polycystin-1, Lipoxygenase, Alpha-Toxin (PLAT) superfamily. The catalytic property of necrosis . It is aemolytic ,30. The in silico, in vitro and in vivo evidence implicating a stable interaction between the N and C-domains of C. perfringens alpha toxin and also the necessity of an unhindered active cleft on N-domain after interactions as a critical factor for the toxicity. In summary, we present Figure S1Superposition of Cp-PLC (PDB ID: 1CA1) and modelled Cp-PLC N and C-domain complex with N-domain in ribbon format and C-domain in stick format. Cp-PLC N and C-domain residues are colored green and cyan, respectively and N and C-domains of the complex are colored pink and brown, respectively. The shift in orientation of C-domain is showed by arrow.(PPTX)Click here for additional data file.


Multicopper blue proteins, composed of several repetitive copper-binding domains similar to one-domain cupredoxin-like proteins, were found in almost all organisms. They are classified into the three different groups, based on their two-, three- or six-domain organization. We found orthologs of chordate six-domain copper-binding proteins in animals, plants, bacteria and archea. The phylogenetic analysis of 183 multicopper blue proteins and their copper-binding sites comparison make us think that all the modern six-domain blue proteins have originated from the common ancestral six-domain protein in the process of gene duplication and copper-binding sites loss as a result of amino acid substitutions. MCOs include such proteins as laccases E.C. 1.10.3.2), ascorbate oxidases (E.C. 1.10.3.3) and ferroxidases E.C. 1.16.3.1) .10.3.2, .3.1 .10.,,,,The most diverse group in terms of amino acid sequences, functions and prevalence in organisms among all MCBPs is formed by three-domain MCOs, such as laccases, ascorbate oxidases and yeast ferroxidases Fet3 In spite of the significant phylogenetic divergence and even different functions of MCBPs, the observed similarity of their domain organization and amino acid sequences indicate their common ancestry . However many aspects of MCBPs evolution remain unknown. In this paper we performed the phylogenetic analysis of six-domain MCBPs and supplemented the existing schemes of their origin and evolution.Amino acid sequences were obtained from the GenPept protein sequences database (http://www.ncbi.nlm.nih.gov/protein/) using the BLASTP program ,,Branchiostoma floridae sequences, XP_002593056.1 and XP_ 002593057.1, which are encoded by two genes with 55% identity. It is possible, that each of these sequences has a common ancestry with Cp and Heph, correspondingly.The phylogenetic tree for eukaryotic six-domain MCBPs (see appendix 1) is presented on Fig. 2. The majority of the sequences are categorized into two groups consisting of MCFOs and BCFs V and VIII. The MCFOs cluster consists of Cp and Heph subclusters, similarly BCFs cluster consists of factors V and VIII subclusters. Cp subcluster is clearly divided into 3 groups in concordance with the taxonomic classification: mammals, birds and fish. Since Cp coding gene has just one copy in the genomes of vertebrates ,The cluster of BCFs is also split into subclusters of factors V and VIII sequences, which are further categorized in a class-specific manner into 3 groups, corresponding to mammals, birds and fish. Amino acid sequences of oscutarin and pseutarin, two factor V - similar snake prothrombin activator components, are related to the group composed of bird’s factor V proteins sequences Сiona intestinalis. In turn MCFOs share a common ancestor with another Сiona intestinalis sequence XP_002119762.1, however the reliability of the appropriate node is small. All protein sequences mentioned above are related to chordates and form a common clade with sea urchin Strongylocentrotus purpuratus’s sequence XP_791569.2.The revealed BCFs form a common clade with sequence XP_002122537.1 from sea squirt  Chlamydomonas reinhardtii and Fox1 homologue was found in Dunaliella salina Until recently six-domain MCBPs were considered to relate to animal world only. However MCBPs Fox1 and Fox2 were found in green algaeClostridium perfringens. We also revealed MnxG sequence (ZP_02951893.1) by BLASTP analysis, but since the low degree of sequence identity between MnxG and the majority of eukaryotic MCBPs the expectation value was not significant . For this reason an additional BLASTP analysis using MnxG as a query was made in order to find its homologues. We revealed 23 amino acid sequences from 1217 to 2681amino acids long with the significant degree of the identity to MnxG  (see Appendix 2). Most sequences belong to Eubacteria with the exception of two Archaea proteins from Halorubrum lacusprofundi and Haloterrigena turkmenica. Fox1 sequence (XP_001694585.1) has been used for the multiple protein alignment construction as a phylogenetically remote six-domain MCBP with a known structure. The phylogenetic tree constructed on the basis of this alignment is presented on Fig. 3. All sequences could be divided into two clusters. The first cluster contains MCO-like protein sequences from Proteobacteria. The second one consists of MnxG-like bacterial proteins from Bacillus and Clostridium genera (Firmicutes phyla) and two sequences that belong to Halobacteria. Both clusters have a common node with ZP_01851853.1 sequence, which belongs to Planctomycetes.The obtained data is not sufficient to make any valid conclusions, however we can speculate that there could be a common parental sequence for these MCO-like proteins in some progenitor prokaryotic organisms.The existance of six-domain MCBPs in unicellular algae made us think that six-domain MCBPs could be found in bacteria. Earlier it was reported that the manganese oxidase MnxG, remotely similar to MCBPs Strongylocentrotus purpuratus, XP_002122537.1 from Сiona intestinalisand factor V of vertebrates. In factor V the amino acid residues, that form copper-binding sites, have been partially conserved. The fact, that there is a common node between XP_002122537.1 and BCFs V and VIII sequences in the phylogenetic tree, can indicate the existance of their common ancestor.Reconstruction of the possible six-domain MCBPs evolution pathways requires the comparison of MCBPs sequences from the phylogenetically remote species. However, since the low degree of the total sequence identity between MCBPs from eukaryotic and prokaryotic species we further analyzed the conservative amino acid composition of copper-binding domains, which define the structure and functions of MCBPs. For this purpose we manually matched the multiple sequence alignments of eukaryotic MCBPs and their bacterial homologues according to the copper-binding domains coordinates along Fox1 sequence (XP_001694585.1), presented in both alignments. Further we selected 19 most typical MCBPs representatives, containing all the found types of copper-binding sites composition . It can be seen, that there is at least one copper- binding site in the majority of the represented sequences. The exceptions are sequences XP_791569.2 from Branchiostoma floridae, XP_002119762.1 from Сiona intestinalis, factor VIII from Tetraodon nigroviridis and XP_001693036.1 from Chlamydomonas reinhardti) and 6 (absent in prokaryotic proteins) of six-domain MCBPs. Mononuclear copper-binding site, located in domain 4, was found only in Fox, Heph and Cp (except fish Cp) sequences.The most conservative mononuclear copper-binding sites are located in domains 2  or six-domain  MCBPs were found. Most of the prokaryotic genomes encode one- and two-domain MCBPs, however there are some exceptions. In particular, 17 of 23 prokaryotic genomes regarded here encode only six-domain MCBPs, while the remaining 6 genomes, including two archea species, encode additional two-domain MCBPs.One-domain cupredoxin is presumably the evolutional precursor of blue copper binding domains, whose multiplication and subsequent modification led to the formation of multi-domain MCBPs with typical mono- and trinuclear copper-binding sites or between domains 2 and 3, like in MCBPs of green algae, some bacteria and archea species. No MCBPs with trinuclear center located between domains 4 and 5 were described so far. However, multiple protein alignment of MCBPs copper-binding sites showed that the copper coordinating amino acid residues are partially remained for some lost mono- as well as tri-nuclear copper-binding sites in all the analyzed proteins.Despite of the significant phylogenetic divergence between MCBPs their general structure, defined by the copper-binding sites, as a whole remains unchanged. Blue copper-binding sites in the hypothetical ancestral six-domain MCBPs were expected to be localized in domains 2, 4 and 6 and inter-domain trinuclear copper binding sites – between domains 1 and 6, 2 and 3, 4 and 5. Probably further during the evolution these sites were partly or even completely lost. In the contemporary six-domain MCBPs the number of mononuclear sites varies from 0 to 3 and the number of trinuclear sites does not exceed 1. Moreover trinuclear copper binding sites were only found between domains 1 and 6, like in MCBPs of chordates, ,Tetraodon nigroviridis and Chionodraco rastrospinosus have the same structure except for the missing blue copper-binding site in domain 4.The chain of events of the modifications of the hypothetical ancestral six-domain MCBPs may be as follows. The loss of two trinuclear copper-binding sites, located between domains 1 and 6 and domains 4 and 5, has led to the occurrence of green algae MCBPs Branchiostoma floridae has two MCFO-like proteins. Finally Heph gene duplication probably took place after the sub-class of terrestrial animals had appeared. The general scheme of the modern six-domain MCBPs formation from the hypothetical six-domain ancestral protein is presented on Fig. 1.There were at least four gene duplication events  in higher eukaryotic organisms that resulted in the appearance of at least five homologous MCBPs in mammals. The first duplication of the ancestral six-domain MCBP gene probably took place not later, then on the early stages of chordate’s evolution, since two different MCBPs were found in sea squirts, while sea urchin’s MCBP gene is presented by a single copy in the genome. This duplication led to the formation of two groups of MCBPs with completely different functions: MCFOs and BCFs V and VIII. The subsequent independent duplications of these genes resulted to the appearance of BCFs V and VIII genes and Cp and Heph genes, found in vertebrates. The time of these duplications cannot be estimated on the base of the available data, although lancelet The phylogenetic analysis of six-domain MCBPs allows to assume that they have a common prokaryotic six-domain protein ancestor and were formed through the chain of gene duplications and amino acids substitutions which resulted in the loss of a part of copper-binding sites.


Angiotensin I-converting enzyme (ACE) has two functional N- and C-domain active centers that display differences in the metabolism of biologically-active peptides including the hemoregulatory tetrapeptide, Ac-SDKP, hydrolysed preferentially by the N domain active center. Elevated Ac-SDKP concentrations are associated with reduced tissue fibrosis.In silico analysis demonstrated S333W localized in the S1 pocket of the active site of the N domain with the bulky Trp adversely affecting binding of ACE substrates due to steric hindrance. Expression of mutant ACE (S333W) in CHO cells confirmed altered kinetic properties of mutant ACE and conformational changes in the N domain. Further, the S333W mutant displayed decreased ability (5-fold) to cleave the physiological substrate AcSDKP compared to wild-type ACE.We identified a patient of African descent exhibiting unusual blood ACE kinetics with reduced relative hydrolysis of two synthetic ACE substrates (ZPHL/HHL ratio) suggestive of the ACE N domain center inactivation. Inhibition of blood ACE activity by anti-catalytic mAbs and ACE inhibitors and conformational fingerprint of blood ACE suggested overall conformational changes in the ACE molecule and sequencing identified Ser333Trp substitution in the N domain of ACE. A novel Ser333Trp ACE mutation results in dramatic changes in ACE kinetic properties and lowered clearance of Ac-SDKP. Individuals with this mutation (likely with significantly increased levels of the hemoregulatory tetrapeptide in blood and tissues), may confer protection against fibrosis. ACE is constitutively expressed on the surface of endothelial cells, epithelial, neuroepithelial and cells of immune system (macrophages and dendritic cells) as a membrane-bound protein and has been designated as CD143. Two homologous domains (N and C domains) comprise the majority of the structure of somatic ACE (sACE) and each contain a functional Zn2+ binding active center reviewed in Angiotensin I-converting enzyme  is a ZnThe three-dimensional crystal structure of sACE is still unknown. However, the model of the two-domain ACE has been recently suggested Several mutations in ACE have been described: with several producing familial elevation of blood ACE due to effects on the rate of ACE shedding such as P1199L We now report a novel ACE mutation, where residue substitution (S333W) near the active site, altered kinetic characteristics of the N domain active center of somatic ACE. Mutated ACE exhibited decreased hydrolysis of physiological, N domain specific substrate Ac-SDKP, a negative regulator of the hematopoiesis The study was approved by the Institutional Review Boards of the University of Chicago and the University of Illinois at Chicago. Participants provided their written informed consent to participate in this study. The IRBs approved this consent procedure. Serum ACE levels were assessed in serum present in the University of Chicago Biobank obtained from patients with different sarcoidosis phenotypes . One patient (#27) was found to have serum ACE with an unusual kinetic characteristics –low (0.7) ZPHL/HHL ratio - normal value -1.05±0.05 ACE activity in serum/plasma or culture fluids or lysates from ACE-expressing cells was measured using a fluorimetric assay with two ACE substrates  were coated with anti-ACE mAbs th, 19th–24th) were sequenced, using primers designed by Genomic DNA was obtained from whole blood of patient # 27 and 13 exons of ACE gene (6th–12http://www.pymol.org) was used to make the S333W substitution in silico using PyMol Mutagenesis Wizard.Analysis of the N domain was based on the recently published CG codon for Ser at position 333  by mutation of the 1) TPlasmids carrying the coding sequence for wild-type ACE and above mutants were expressed in CHO cells using Plus Reagent  for transient transfection and generation of stable cell lines. Serum-free culture medium from these cells was used as a source of the secreted (soluble) ACE (wild type and mutants) for biochemical and immunological characterization. Lysate of these cells obtained with detergent Triton X-100 (0.5% in PBS) was used as a source of membrane-bound form of WT and mutant ACE.Lysates from CHO-WT-ACE cells (with ACE activity of 5 mU/ml using Hip-His-Leu) were compared to lysates from CHO-ACE–S333W cells normalized by equal protein loading by SDS-PAGE in 4–15% acrylamide Tris-HCl pre-cast SDS PAGE gels . After electrophoretic transfer of proteins to microporous PVDF-Plus membrane Western blotting was performed exactly as in N-acetyl-angiotensin I 3H),1′-phthalan]-3,3′-dione), a reagent that forms fluorescent adducts exclusively with primary amines 4) was warmed to 37°C in a 96-well plate. The assay was commenced by the addition of 10 µl  pre-warmed lysate, incubated for 30 minutes and stopped by the addition of 50 µl 1 M HCl. The solution was neutralized by the addition of 50 µl 1 M NaOH and the pH increased to 8.3 by the addition of 100 µl 500 mM K2HPO4/KH2PO4 buffer pH 8.3. 10 µl of fluorescamine  was added and the resulting mixture incubated for 3 minutes at room temperature. Fluorescence intensities were measured at λex = 390 nm and λem = 475 nm using a Cary Eclipse spectrofluorimeter (Varian Inc.). Changes in fluorescence compared to empty vector lysate were converted to reaction velocities by the use of a standard curve. The standard was constructed by complete hydrolysis of various concentrations of AcSDKP by purified N-domain using the same approach as above.An adapted plate assay was designed based on previously published approach for Screening of serum ACE activity was performed in 84 cases with pulmonary and extrathoracic sarcoidosis and unrelated patients serving as controls and resulted in the identification of a single case (#38) with ACE activity 5-fold higher than controls . FurtherThe two domains of ACE hydrolyze a range of natural and synthetic substrates, but with different efficiencies Therefore, the lower value of ZPHL/HHL ratio for patient # 27 suggested that the N domain active center could be inactivated by a mutation in the active site of the enzyme. To further investigate this, we incubated serum from patient # 27  with mAbs 3A5 and i2H5 which are anti-catalytic towards the N-domain active center 50 for enalaprilat was 2 nM for control serum and 7 nM for serum from patient # 27 (p<0.05); for teprotide IC50 values were 9.5 nM and 13.0 nM, respectively (p<0.05). Thus, the data presented in The pattern of inhibition of blood ACE activity from patient #27 was similar for the short tripeptide ACE inhibitor enalaprilat  and longIn order to characterize the conformation of blood ACE from patient #27 we performed conformational fingerprinting of blood ACE, using a panel of mAbs directed against 16 different epitopes located on the N-and C-domain of catalytically active human ACE The immunoprecipitation profile of plasma ACE from subject # 27 is quite different to that of plasma ACE from healthy volunteers with normal ZPHL/HHL ratio . The binWe demonstrated previously that binding of mAbs 1G12 and 6A12 to blood ACE dramatically (3–4-fold) increased in the presence of ACE inhibitors Therefore fine analysis of enzymatic activity, sensitivity to ACE inhibition and conformational analysis of blood ACE from patient # 27 –5 unequith exon of the N domain and 19th–23th exon of the C domain. We identified several known polymorphic variants of ACE gene in patient # 27: i) rs 12709426 , which correspond to D563G substitution - mature somatic ACE numbering th exon of ACE; ii) rs12720737 in intron 23; and iii) rs4362 .We sequenced those exons of the ACE gene from this patient which code amino acid residues comprising the N- and C-domain active sites th exon c.1119 C>G, p.S362W . Besides, the S333W substitution, an S333L substitution was found the GenBank in the same codon which should have a lesser effect on the N domain active center than the S333W substitution, due to the smaller size of the Leu side chain, compared to Trp.The mutation in the same codon of ACE coding for Ser333 of mature ACE was found previously in at least six individuals in North America  amino acid change (S333W for mature ACE protein) according to Polyphen-2 analysis  might be responsible for the changes in enzyme activity of this blood (and perhaps tissue) ACE in the affected individuals, we performed site-directed mutagenesis of human recombinant somatic ACE. These mutants of somatic ACE were expressed in CHO cells and the biochemical and immunological characteristics of these mutants were compared with WT somatic ACE.As expected, based on published crystal structures of the N domains with ACE inhibitors We also performed conformational fingerprinting of recombinant S333W ACE  in the sThe overall precipitation of ACE activity (measured with Hip-His-Leu as a substrate) from membrane-bound mutant ACE by mAbs directed to the epitopes localized on the N-domain was lower than that by mAbs to C-domain. The mean S333W/WT ratio of ACE activity precipitation for 8 mAbs to the N domain calculated from the data presented in These data suggest that the S333W substitution leads to local conformational changes in the N-domain of the mutant ACE  which then leads to changes in the hydrolysis of some substrates.We also assessed the ZPHL/HHL hydrolysis by mutant ACE bound to antibodies. The ZPHL/HHL ratio of WT ACE bound to mAbs did not change significantly except for mAb 4E3 which is strongly anti-catalytic for the C domain active center We also performed Western blotting of cell lysates from CHO-WT-ACE and CHO-S333W-ACE using mAb to denatured human ACE -1D8 which recognize sequential epitope in the C domain Our data indicates that dramatic changes in enzymatic activity of mutant S333W ACE (ZPHL/HHL ratio) could be due to two reasons: 1) the presence of a bulky Trp in the active site cleft that might affect the binding of ACE substrates due to steric hindrance; and 2) selective denaturation of the N-domain of mutant ACE, perhaps due to incorrect folding and trafficking to the cell surface.We demonstrated that this mutation (S333W) abolished substrate specificity of the N-domain at least for two artificial substrates Hip-His-Leu and Z-Phe-His-Leu. The N-terminal domain of WT human ACE cleaves Hip-His-Leu much slower than the C-domain Therefore we tested the effect of the S333W mutation on the hydrolysis of the natural substrate AcSDKP . Stable The rising burden of pulmonary fibrosis in the U.S. currently accounts for more than 40 000 deaths annually. Moreover, few effective drugs are presently available for the treatment of pulmonary fibrosis. Activation of the renin-angiotensin system (RAS) and production of Angiotensin II is associated with tissue fibrosis, therefore ACE inhibition and/or AII antagonism has been suggested as a possible anti-fibrotic therapy -see for review in vivo studies demonstrated that AcSDKP injection AcSDKP is a natural inhibitor of hematopoietic stem cell proliferation The inhibition of ACE in both mice and humans causes a 5-fold increase in AcSDKP levels In silico analysis demonstrated that this mutation was localized in the S1 pocket of the active site of the N domain and it is likely the bulky Trp (W) would affect the binding of ACE substrates due to steric hindrance. We studied the effect of this mutation on the hydrolysis of several artificial and natural substrates were determined. Mutant ACE (S333W) expressed in CHO cells decreased significantly the hydrolysis of N-domain specific substrates, including the anti-fibrotic Ac-SDKP. Besides, expression of mutant ACE in CHO cells allowed us to study immunoreactivity, conformation, rate of ACE shedding and trafficking to the cell surface for the mutant ACE. Using a novel conformational fingerprinting approach for ACE, we demonstrated that S333W mutation lead to conformational changes in whole ACE, and specifically  in the N-domain. Therefore, this study provides important insights into the mechanism by which the novel ACE mutation (S333R) results in changed kinetic characteristics of mutant ACE.In summary, we identified a patient with a novel mutation of ACE-S333W.