Schistosoma mansoni VKRs, SmVKR1 and SmVKR2, are both implicated in reproductive activities of the parasite. In this work, we show that the SH2 domain-containing protein SmShb is a partner of the phosphorylated form of SmVKR1. Expression of these proteins in Xenopus oocytes allowed us to demonstrate that the SH2 domain of SmShb interacts with the phosphotyrosine residue (pY979) located in the juxtamembrane region of SmVKR1. This interaction leads to phosphorylation of SmShb on tyrosines and promotes SmVKR1 signaling towards the JNK pathway. SmShb transcripts are expressed in all parasite stages and they were found in ovary and testes of adult worms, suggesting a possible colocalization of SmShb and SmVKR1 proteins. Silencing of SmShb in adult S. mansoni resulted in an accumulation of mature sperm in testes, indicating a possible role of SmShb in gametogenesis.Venus kinase receptors (VKRs) are invertebrate receptor tyrosine kinases (RTKs) formed by an extracellular Venus Fly Trap (VFT) ligand binding domain associated via a transmembrane domain with an intracellular tyrosine kinase (TK) domain.  Schistosoma mansoni, S. haematobium and S. japonicum) but its massive administration in endemic areas has increased the likelihood of emergence of PZQ-tolerant parasites [Schistosomiasis is the second most important parasitic disease in the world with more than 240 million people infected in tropical and subtropical areas and is responsible for about 200 000 deaths per year . Praziquarasites . Therefoarasites .S. mansoni gametogenesis and egg production indicating that parasite kinases are interesting targets for the development of new therapeutics against schistosomes [S. mansoni [S. mansoni in 2003 [Aedes aegypti recently [S. mansoni, and they were shown to participate in the development of the ovary in female worms. SmVKR1 and SmVKR2 are two different receptors that are activated by distinct ligands [Xenopus oocytes, each receptor activated both ERK2 and Akt pathways but only SmVKR1 was able to induce JNK phosphorylation [Protein kinases, and particularly tyrosine kinases (TKs), play important roles in development and metabolism. Receptor tyrosine kinase (RTK) signaling networks are essential for many cellular activities such as growth, proliferation, differentiation or migration, and they represent major targets in anti-cancer therapy . Differestosomes –10. Part mansoni . VKRs ar mansoni ,13. Sinc in 2003 , VKRs ha in 2003 . Their frecently . Most ofrecently . Two VKR ligands . First erylation ,17.Src Homology-2 (SH2) domain-containing protein B (Shb) proteins are pleiotropic adaptor proteins that convey signals from membrane RTKs to intracellular signaling intermediates. They possess a C-terminal SH2 domain and a central PhosphoTyrosine Binding (PTB) domain both involved in interaction with phosphorylated tyrosines, as well as an N-terminal Proline-rich sequence that binds SH3 domains . Shb proS. mansoni Shb protein (SmShb) interacts with the membrane receptor SmVKR1. This interaction is dependent on the phosphorylation of the juxtamembrane Y979 residue of SmVKR1, occurs through the SH2 domain of SmShb, and allows the activation of a specific JNK signaling pathway in Xenopus oocytes. In situ hybridization shows that SmShb transcripts localize as Smvkr1 transcripts [S. mansoni.In this paper, we show that a nscripts  in the rAll experiments involving hamsters within this study have been performed in accordance with the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes  and have been approved by the committee for ethics in animal experimentation of the region Nord Pas de Calais France (authorization No. AF/2009) in the local animal house of the Pasteur Institute of Lille (Agreement No. A59-35009).S. mansoni is maintained by passage through albino Biomphalaria glabrata snails and Mesocricetus auratus golden hamsters. Adult schistosome pairs were collected by portal perfusion from infected hamsters at 42–45 days post infection.A Puerto-Rican strain of ClaI and XhoI restriction sites respectively and the obtained fragment was inserted into a pCR™4-TOPO® vector (SmShb-pCR™4-TOPO®).Total RNA of adult schistosomes was isolated using the RiboPure™ RNA Purification Kit. cDNA was prepared using the GeneRacer™ Kit with the SuperScript™ III reverse transcriptase (Invitrogen) following the manufacturer’s instructions. 5’ and 3’ ends of SmShb were determined by RACE PCR using GeneRacer™ 5’, 3’, 5’ Nested and 3’ Nested primers associated with SmShb-specific primers . Amplifihttp://blast.ncbi.nlm.nih.gov/Blast.cgi. Shb, Grb2, Grb7, Nck and Shc protein sequences were aligned using ClustalW algorithm in the BioEdit v7.2.5 software, and manually corrected. Maximum likelihood tree was built using MEGA6 [Sequences were analyzed using the LASERGENE package . BLASTp and BLASTn analyses of sequences obtained from the library screening were performed using the NCBI databank ng MEGA6  under thClaI and XhoI and the SmShb sequence was inserted in frame into the pGADT7 AD expression vector (Clontech) using the T4 DNA Ligase (Invitrogen™) (SmShb-pGADT7). The SH2 domain, amplified from SmShb-pGADT7 using SH2domainf/SmShbFLREr primers containing respectively EcoRI and XhoI sites, was inserted in frame into the pGADT7 AD expression vector. Site-directed mutagenesis of constructs was performed using the Isis DNA polymerase™ . The SmShb-pGADT7 construct was used as a template for the production of SmShbΔSH2 mutant using as primers SmShbdelSH2f/SmShbdelSH2r in which G3310 is replaced by T to generate a stop codon and to create the SmShbΔSH2 variant (AA sequence 1–1048). SmVKR1-pcDNA and SmVKR1 ICD YYRE-pGBKT7 plasmids  inserted in fusion with the Gal4-BD of the pGBKT7 plasmid (described in [Intracellular domains (ICD) of constitutively active SmVKRs (SmVKR1YYRE and SmVKR2YYRE) and of ribed in ) were usin vitro using the mMessage mMachine® T7 Kit  from pcDNA, pGBKT7 and pGADT7 plasmid constructs digested by PmeI (pcDNA) or by HindIII (pGBKT7 and pGADT7) restriction enzymes. cRNA solutions (1 mg/mL) were injected in stage VI Xenopus laevis oocytes according to the procedure previously described [++) in the incubation medium [cRNAs encoding SmVKR and SmShb variant proteins were synthesized escribed . Kinase n medium . Germinan medium . Followin medium . Immunopn medium .in vitro transcripts (corresponding to nt 616–1082 sequence of SmShb (Genbank Accession Number JN864885)) were synthesized and labeled with digoxigenin following the protocol of the manufacturer (Roche). The correct size of labeled sense and antisense transcripts was controlled by gel electrophoresis and the quality of RNA probes was checked by blotting and detection of digoxigenin using alkaline phosphatase-conjugated anti-digoxigenin antibodies, naphtol-AS-phosphatase, and Fast Red TR (Sigma). In situ hybridization was performed at 57°C for 16 h as previously described [Adult worm pairs were fixed in Bouin's solution  and embedded in paraplast . Sections (5 μm) were incubated in xylol to remove paraplast. Following rehydration, slides were treated with proteinase K (1 μg/ml) and sections dehydrated. For hybridization, escribed . Section2 atmosphere. After 7 days, 5 worm couples were picked up for Confocal Laser Scanning Microscopy (CLSM) examination and knockdown of gene expression was monitored by quantitative RT-PCR in the remaining worms.cDNA fragments of SmShb (610 bp) and Luciferase  were generated by PCR using respectively the SmShb-pGADT7 plasmid or the pGL3-basic plasmid (Promega) as templates and SmShbRNAiT7f/ SmShbRNAiT7r and LucT7f/LucT7f as primers . dsRNA wTotal RNA was extracted from worm couples using TRIzol® reagent (Invitrogen) and reverse transcription was performed using the Superscript III RT-PCR System (Invitrogen). cDNA was used as template for PCR amplification using KAPA SYBR® FAST Universal 2× qPCR Master Mix kit (Clontech) and the ABI PRISM 7300 detection system . SmShbqPCRf/SmShbqPCRr primers  were useWorms fixed in AFA  were stained for 30 min in 2.5% Hydrochloric Carmin red , and then destained in acidic 70% ethanol. Following dehydration in 70%, 90% and 100% ethanol, worms were preserved as whole-mounts in Canada balsam (Merck) on glass slides –29. CLSMS. mansoni library screening to interact with the tyrosine-phosphorylated intracellular domain of SmVKR1 in its active form (SmVKR1 ICD YYRE) [Clonorchis sinensis. Furthermore, we showed that within the Shb family, platyhelminth (trematode and cestode) and insect proteins form two individual groups linked to the common branch of invertebrate Shb proteins, which is distinct from that of mammalian Shb proteins  was shown from Y2H CD YYRE) . Using RCD YYRE)  with rolCD YYRE) . The ShbCD YYRE) ,32, and proteins .S. mansoni in order to study the specificity of the SmShb adaptor protein for the schistosome RTKs. Xenopus oocytes (The full-length protein of SmShb was used to confirm its interaction with the kinase active intracellular domain of SmVKR1 (SmVKR1 ICD YYRE) in a direct binding assay in yeast. Similar interaction assays were also performed with the kinase active ICDs of SmVKR2 and of the two insulin receptors SmIR1 and SmIR2 of  oocytes . Indeed, oocytes , confirmΔSH2) and another one which encoded only the SH2 domain (SH2SmShb). Each construct  was co-expressed with SmVKR1 ICD YYRE in Xenopus oocytes and their ability to interact with SmVKR1 was analyzed by co-immunoprecipitation. The results indicated that SmVKR1 ICD YYRE co-precipitated with SmShb as well as with its SH2 domain but not with its SH2-deleted variant (The primary function of an SH2 domain is to recognize and to interact with phosphorylated tyrosines . In orde variant  and thus variant .979ENL) fitting to this consensus and found to be located within the juxtamembrane region of the receptor. In order to analyze the importance of this motif for the binding of SmShb to SmVKR1, we produced a mutant receptor (SmVKR1 Y979F) in which Y979 was replaced by a phenylalanine as a non-phosphorylable residue. We then compared the capacity of active ICD of SmVKR1 or SmVKR1 Y979F to co-precipitate with SmShb after co-expression in Xenopus oocytes. The results showed that SmShb was not able to interact with SmVKR1 Y979F, indicating that the residue Y979 was involved and required for the interaction of SmVKR1 ICD with the SH2 domain of SmShb (SH2 domains of Shb proteins have been shown to bind phosphorylated tyrosines (pY) included in the consensus motif pYxxL . Within of SmShb .979F proteins were co-expressed with SmShb in Xenopus oocytes and membrane receptors were further activated by L-Arginine (L-Arg) binding as previously described [979 residue of SmVKR1 was required for its interaction with and for the phosphorylation of SmShb  on the SmShb binding potential of SmVKR2. Protein expression in oocytes and co-immunoprecipitation experiments were performed using the same conditions as described for SmVKR1, except that SmVKR2 receptors were activated by their specific ligands: Ca++ ions [949Y, which contains the pYxxL motif, interacted with SmShb, very likely by its SH2 domain  ) in n of JNK . To anal Results  showed tpathways ; this wapathways . These r as GVBD . The speShbΔSH2) .in situ hybridization showed that SmShb transcripts were also detected in the mature part of the ovary . Moreover, as for Smvkr1 [Previous studies have shown that Smvkr1 is expressed in various tissues of all parasite stages ,16 and phe ovary . Furtherhe ovary . A fainthe ovary . RecentlAttempts to analyse SmShb functions in schistosome biology have used its knockdown by RNA interference (RNAi) in adult worms. Seven days after electroporation with dsRNA, a decrease of about 80% SmShb transcripts was obtained, as compared to control worms electroporated with irrelevant dsRNA (dsLuc) . SmShb sAdaptor proteins are defined by their capacity to link membrane receptors to cytosolic proteins with enzymatic or transcriptional activation properties. Linking is mediated by diverse specific binding domains, like SH2 and PTB or SH3, which recognize phosphorylated tyrosines or poly-proline sequences, respectively. Shb is an adaptor protein containing an N-terminal Pro-rich domain, a central PTB domain and a C-terminal SH2 domain. The SH2 domain interacts with diverse phosphorylated RTKs preferentially at a pY-T/V/I-X-L binding site . In contShb proteins are ubiquitously expressed and generate variable complexes depending on the cell type and on the conditions of cell activation. Therefore, they have unique effects either in differentiation, proliferation, survival or motility ,20. The While much is known about the various physiological roles of Shb in mammals , the funThe protein SmShb has a structure similar to other proteins of the Shb family with the exception of the PTB domain, a sequence demonstrated to interact with the FAK focal adhesion kinase , which wS. mansoni parasite and in the insect A. aegypti [Since their first discovery in 2003 , VKRs ha aegypti  but theiS. mansoni, ie SmVKR1, SmVKR2, SmIR1 and SmIR2, expressed either in their normal or in their active kinase states, have shown that the intracellular domain of the constitutively active SmVKR1 kinase was the only one to offer binding sites for SmShb. Identical results were obtained from interaction studies in yeast and in Xenopus oocytes. We also provided evidence that the specific binding of SmShb to the phosphorylated form of SmVKR1 was mediated by its SH2 domain and through the unique motif pY979ENL located in the intracellular part of SmVKR1, just next to the transmembrane domain. Mapping of the phosphorylable tyrosines present within the SmVKR1 intracellular sequence effectively indicated that only the residue Y979 was susceptible to be part of the consensus motif pYxxL for Shb SH2 domain binding [979 was a crucial residue for the interaction between phosphorylated SmVKR1 and SmShb. Moreover, in the case of SmVKR2 which does not bind SmShb (and possesses a juxtamembrane F949EEL in place of the Y979ENL of SmVKR1), the replacement of F949 by a tyrosine residue was sufficient to allow its interaction with SmShb. These data indicated that a juxtamembrane motif pYExL in SmVKR was necessary and sufficient to allow interaction with SmShb. SmIR1 and SmIR2 insulin receptors, which do not possess similar motifs in their juxtamembrane regions, did not bind SmShb. However, it is important to mention that, in the experimental conditions used for SmVKR receptors, the activated Schistosoma EGF receptor (SER), which contains a juxtamembrane YxxL (YTNL) motif similarly to that of SmVKR1, did not bind SmShb (results not shown). This suggests that the Y+1 residue E could be required for SH2SmShb binding. Additionally, it is possible that the interaction between SmShb and SmVKRs requires the participation of specific residues surrounding the phosphorylated tyrosine, such as proline at Y-1 and cysteine at Y+4 positions. Indeed, these residues are common to SmVKR1 (PpYENLC) and mutated F949Y SmVKR2 (PpYEELC) but they are not present in SER (ApYTNLL). Further experiments are still necessary to fully define the binding site of SmShb on SmVKRs, and to check for possible interaction of this adaptor protein with several other RTKs already characterized in S. mansoni, particularly with the parasite FGF-R receptors [Anopheles gambiae was not able to interact with SmShb while inversely the SH2 domain of AgShb (XP_309808.4), which shares about 40% homology with SH2SmShb, interacted with AgVKR but not with SmVKR1 (results not shown).Indeed, analyses of the capacity of SmShb to interact with various known RTKs of  binding . Mutageneceptors . As mammeceptors , we cannXenopus oocytes and that only SmVKR1 activated the phosphorylation of JNK [Xenopus oocytes ligand-activation of SmVKR1 in the presence of SmShb resulted in JNK phosphorylation, but at the same time in a blockade of ERK and PI3K pathways and as a consequence in the inhibition of meiosis resumption in the oocyte. These results already suggested that SmShb could be involved in the initiation of a JNK cascade activation and further experiments performed with the SmVKR2 receptor mutated to be able to bind SmShb then confirmed the potential role of SmShb in the induction of JNK activation. Ligand activation of SmVKR2 F949Y in oocytes in the presence of SmShb generated a phenotype similar to that of ligand-activated SmVKR1, with a stimulation of the JNK pathway concomitant to the inhibition of ERK and PI3K pathways and GVBD. To our knowledge, the participation of Shb proteins in the activation of JNK has not been shown in mammalian cells, but their function in the regulation of ERK and Akt signaling has been described in Shb-deficient mice [In a previous study we have demonstrated that the activation of SmVKR1 and SmVKR2 induced ERK2 and PI3K/Akt/S6K pathways in n of JNK . In thisent mice . The losS. mansoni, both types of transcripts were co-localized in reproductive organs (mature oocytes and testes), suggesting that SmVKR1 and Shb proteins could interact within gametes. However, SmShb silencing in adult worm pairs did not provide solid morphological arguments for a decisive role of SmVKR1-SmShb interaction in the ovary. Knocking-down of SmVKR1 had shown an accumulation of big oocytes [SmVKR1 and SmShb were expressed in all parasite stages with a more important quantity of transcripts detected in larval stages as compared to adult parasites. However, in sexually mature  oocytes , which h oocytes . As many oocytes , are mulS. mansoni stages such as schistosomula and sporocysts.In conclusion, SmShb appears to be a component of a signal transduction platform specifically formed by activated SmVKR1. In this molecular context activated SmShb has the potential to mediate the activation of a JNK pathway and negatively modulate ERK- and Akt-dependent metabolism and proliferation pathways, supporting cell differentiation and development pathways. SmShb and SmVKR1 are expressed in all the stages of the parasite and they are particularly abundant in larval forms. It would be interesting to unravel the importance of their partnership in the development of other S1 Fig(A) Exon-intron structure of SmShb locus. Exons are shown as blue boxes and introns as green lines. Size scale is identical for introns and exons, except for I1 and I2 (larger than 30kb). (B) Schematic representation of the SmShb transcript indicating the coding sequence (in grey) with the Proline-rich domain in green and the SH2 domain in light blue. Untranslated regions (5’- and 3’-UTR) are indicated in orange.(TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.


Cyclin dependent kinases are proline-directed serine/threonine protein kinases that are traditionally activated upon association with a regulatory subunit. For most CDKs, activation by a cyclin occurs through association and phosphorylation of the CDK’s T-loop. CDK5 is unusual because it is not typically activated upon binding with a cyclin and does not require T-loop phosphorylation for activation, even though it has high amino acid sequence homology with other CDKs. While it was previously thought that CDK5 only interacted with p35 or p39 and their cleaved counterparts, Recent evidence suggests that CDK5 can interact with certain cylins, amongst other proteins, which modulate CDK5 activity levels. This review discusses recent findings of molecular interactions that regulate CDK5 activity and CDK5 associated pathways that are implicated in various diseases. Also covered herein is the growing body of evidence for CDK5 in contributing to the onset and progression of tumorigenesis. Saccharomyces cerevisia to humans. In humans there are 13 different CDKs (CDK1 - CDK13) that are highly expressed in mitotic cells [Cyclin dependent kinases are proline-directed serine/threonine protein kinases that are traditionally activated upon association with a regulatory subunit. CDKs are a part of a kinase family that has been conserved throughout evolution and can be found in species from ic cells . CDK5 waic cells , brain pic cells , and CDKic cells . An isofic cells , 9. One ic cells , while aic cells .CDK5 can be mapped to chromosome 7q36 and its expression is upregulated by the transcription factors Fos and CREB through the MEK/ERK pathway and by δFosB , 12. CDKp35 knockout mice have defective cortical lamination and adults suffered from sporadic lethality and seizures [Cdk5 knockout mice [p39-/- mice did not display any obvious abnormalities, however p35/p39 compound knockout mice displayed a phenotype identical to that of the Cdk5-/- mice [Unlike other CDKs, CDK5 is not primarily activated by cyclins. Instead it is through specific binding with the proteins p35 or p39, or their respective cleaved counterparts p25 and p29, that CDK5 becomes active , 17, 18.seizures , which iout mice . p39-/- -/- mice , suggestin vitro, under conditions of oxidative stress, p35 has been found conjugated to SUMO2 at Lys246 and Lys290, which led to increased p35/CDK5 activity [p35 has a myristolation sequence that localizes it to phospholipid membranes . Active activity .As previously mentioned, the CDK5 activator p25 is formed through cleavage of p35 by calpain. This produces both the p25 product as well as a p10 product. Cleavage of p35 occurs under stress conditions such as amyloidβ presence, excitotoxicity, or oxidative stress , 26. ThiThe functions of CDK5 activators p39 and p29 largely overlap with those of p35 and p25, respectively, however their expression throughout brain regions vary. p39 and p29 are mainly expressed in postnatal cerebral cortex and the hindbrain while p35 and p25 are largely expressed in the cerebral cortex of developing brains . The loc. This upregulation of Bcl2 and Bcl211 is observed only through cyclin I activation of CDK5, not activation via p35 [In addition to p35 and p39, cyclin I has also been shown to activate CDK5. Cyclin I-CDK5 binding targets CDK5 to the nucleus  and incr via p35 , 30. CDK via p35 , 31.While CDK5 is only activated by p35/p25, p39/p29, or cyclin I, the activity of CDK5 can be modulated by a variety of other proteins, as depicted in Figure KIP1. The formation of this complex, and consequent attenuation of Cdk5 activity was found to promote synaptic plasticity, memory formation, and dendritic growth, as cyclin E-/- mice, that had increased Cdk5 activity, were deficient in these processes [Cyclin E can directly interact with Cdk5 to reduce its activity. Cyclin E was found to sequester mouse Cdk5 away from other protein activators along with p27rocesses . While trocesses –37.Glutathione-S-transferase GSTP1) is another regulator of CDK5 activity that functions by competing with p35 for CDK5 binding. GSTP1 also reduces aberrant CDK5 activity by scavenging for molecules associated with oxidative stress and thereby decreasing the likelihood of p35/p39 cleavage to p25/p29   has been shown to upregulate e Figure . Knockdo pathway .Previously, CDK5 was thought to function in a cell cycle independent manner; however, recently the retinoblastoma protein (Rb) was discovered as a downstream target of CDK5. Expression of CDK5 leads to the phosphorylation of Rb, ultimately leading to the expression of cyclins and other cdks . The pro+2 channel (L-VDCC) at Ser783, which prevents the association of L-VDCC with syntaxin and SNAP-25, thereby preventing exocytosis of insulin from the cell [In pancreatic β cells, CDK5 activity reduces insulin secretion in response to glucose abundance Figure . This wathe cell .Within the immune system, CDK5 has been implicated in IFNγ-induced programmed death ligand 1 (PD-L1) upregulation, which allows certain cells to evade detection by the immune system. Decreased CDK5 expression led to increased expression of the PD-L1 transcriptional repressors IRF2 and IRF2BP and consequent decreased PD-L1 expression  to stimulate axon growth, it was found that hyperphosphorylation of CRMP2, as well as Tau, were implicated in the generation of neurofibrillary tangles characteristic of Alzheimer's disease . Cell stWhile CDK5 overexpression and aberrant activation are associated with neurodegenerative diseases, a loss or reduction in CDK5 activity is implicated in certain intellectual disabilities and neurodevelopmental disorders. Decreased CDK5 activity has been associated with intellectual disability in NF1 microdeletion syndrome patients  and schiElevated levels of CDK5 have been found in various mouse tumors and human malignant tumors  53, 62––62–65. Tin vitro, in which reducing p25 expression or knocking down Cdk5 expression prevented further cell proliferation. This suggests that it is the aberrant activation of Cdk5 by p25 that leads to the progression of sporadic MTC [In a transgenic mouse model of sporadic medullary thyroid carcinoma (MTC), p25 overexpression led to the development of bilateral malignant thyroid tumors, and was fatal after 30 weeks. However, arresting p25 expression at 5, 11, or 16 weeks led to 100 percent survival in all mice analyzed after 30 weeks. Similar results were discovered adic MTC .in vivo. Conversely, decreased CDK5 expression enhanced the recruitment of CD4+ T-cells to the tumor site in mice, and increased the tumor-free survival rate of the mice. CDK5 regulates the evasion of tumors from the immune system by decreasing expression of transcriptional repressors of PD-L1 expression, thus increasing the abundance of PD-L1 [CDK5 expression in medulloblastoma allows tumor cells to evade detection by T-cells of PD-L1 .in vivo by decreasing the abundance of HIF-1α. Because HCC is a highly vascularized tumor type, inhibiting CDK5 and therefore angiogenesis, could prove a promising treatment for this tumor subtype and other highly vascularized tumors [Inhibiting CDK5 activity in hepatocellular carcinoma (HCC) cells prevented angiogenesis d tumors .CDK5 associated with various cancers and neurodegenerative diseases further implicates its role in the development and progression of disease. Recently, tamoxifen (TMX), a drug currently used in breast cancer treatment, was found to decrease CDK5 activation by competitively binding with p35 and p25, and preventing their activation of CDK5. While the TMX inhibition of CDK5 activity could contribute to the anti-tumor effects of the drug, TMX treatment was also found to decrease Tau phosphorylation, suggesting a use for tamoxifen in treating Alzheimer's disease [Due to the biological and clinically relevant importance of CDK5's function in multiple cell types, CDK5 presents an attractive therapeutic target for treating a variety of conditions such as diabetes, cancer, and neurodegeneration. Additionally, the upregulation of  disease . HoweverOne of the most well studied CDK inhibitors being used in cancer clinical trials is flavopiridol, a drug developed by Tolero pharmaceuticals under the name Alvocidib. Flavopiridol was found to competivively bind to the ATP-binding pocket of CDK1, CDK2, CDK4, and CDK9, consequently inducing apoptosis in both dividing and quiescent cells. Early clinical trials with flavopiridol as a monotherapy proved ineffective in that there was a narrow window between no clinical response and severe, lethal tumor lysis. Ongoing trials involve combination therapies with other novel chemotherapy agents to overcome the limitations of flavorpiridol .50 values in the low nanomolar range  [Another relatively well studied CDK inhibitor, Dinaciclib, was found to be more efficacious than flavopiridol, with IC-350 nM) , 68. Din-350 nM) . Preclin-350 nM) .Roscovitine, marketed under the name Seliciclib, is an inhibitor of CDK5 and CDK2. Many of the clinical trials for Seliciclib were intiated determine dose-limiting toxicities of the drug alone or in combination with other chemotherapeutics. While roscovitine is used widely experimentally to inhibit CDK5 activity, it is not being intensively examined as a clinical cancer therapeutic .in vivo, which reduced neurodegeneration and improved cognitive function of transgenic mice, without affecting neurodevelopment [To potentially reduce broad undesirable off target effects of pan-CDK inhibitors, CDK5 inhibitory peptide (CIP) has been studied as a potential therapeutic for neurodegeneration. CIP specifically targets the hyperactivated state of CDK5 as mediated by p25/p29, while allowing normal activation of CDK5 by p35/p39. CDK5 inhibitory peptide (CIP) was found to inhibit the hyperactivation of CDK5 by p25 overexpression elopment . In the


GF) bread. In this study, corn flour was replaced with soy flour at different levels 5%, 10%, and 15% to produce a more nutritionally balanced GF bread. Physical and chemical properties, sensory evaluation and crust and crumb color were measured in bread samples. The results of evaluations showed that protein content of soy flour‐supplemented GF bread significantly increased from 9.8% to 12.9% as compared to control along with an increased in fat (3.3%–4.1%), fiber (0.29%– 0.38%), and ash (1.7%–2.2%) content. Moisture (27.9%–26.5%) and carbohydrate (58.3–52.3) content decreased with the incremental addition of soybean flour. The highest total score of sensory evaluation was for the bread sample containing 15% soybean flour. The evaluation of crust and crumb showed that bread samples with 15% soy flour were significantly darker than the other bread samples. In conclusion, adding higher levels of soybean flour into GF bread can improve bread quality, sensory characteristics, and nutritional properties of bread. Nutritional status in patients with celiac disease (CD) can be improved through the produce GF bread in this way.The aim of this study was to assess the effect of soy flour on nutritional, physicochemical, and sensory characteristics of gluten‐free ( An estimated up to 2% of the world population suffers from CD and a lifetime gluten‐free diet (GFD) is the only effective treatment for this disease  was supplied by Soyan Toos Company, Iran. Cassava starch , corn starch , corn flour , and rice flour  were obtained from market. Other ingredients were sodium caseinate ; fat (Sunflower oil); sugar; salt and yeast 2.1Solid ingredients corn flour, 50, 45, 40, and 35%; corn starch, 10%; rice flour,13%; cassava starch, 8.5%; soy flour, 0, 5, 10, and 15%; fat, 7%; yeast, 2.5%; salt, 1%; sugar, 3% and sodium caseinate 5% were mixed with water (460 ml) at 400 rpm for 1 min and 600 rpm for 2 min. Dough was divided into 17 parts (60 gr), then proofed at about 27°C and 80% humidity for 15–20 min. Breads were baked at 225°C for 20 min with steam. The loaves were cool down for a minimum of 2 hr at 24°C before evaluation.2.22.2.1Loaf specific volume (SLV) is considered as one of the most important criteria in evaluating bread quality since it provides quantitative measurements of baking performance  according to the method described in AOAC .2.2.3The ash was determined by burning the known weights of the samples in a muffle furnace as recommended by the AACC .2.2.4The percentage of protein was determined by Kjeldahl method as recommended by the AOAC . The con2.2.5The crude fat was determined by extracting a known weight of sample in petroleum ether  in a Soxhlet extractor AACC, .2.2.6Crude fiber was determined as recommended by the AACC .2.2.7The available carbohydrate was measured by the difference method  FAO, .2.3Bread samples prepared by different levels of soy flour were evaluated by 30 taste‐testing panel judges comprising of workers with more than 10 years of experience in baking and teachers, scientific officers and students of the School of Nutrition and Food Sciences affiliated by Shiraz University of Medical Sciences. The bread samples were served as slices including the center points, at the same time. The panelists were requested to evaluate the bread on the basis of acceptance of its color, texture, taste, and overall quality on a 5‐point hedonic scale which ranged from 1 (dislike extremely) to 5 (like extremely) for each organoleptic characteristic.2.4By a digital camera in a box with fluorescent light, the color characteristics of the sample bread loaf were determined. The photos were analyzed with the Adobe Photoshop software version 8  was used to determine the significances within treatments. Statistical analysis of the data was performed using the SPSS software .The experiments were performed in a randomized design and performed at least in triplicates. Analysis of variance (ANOVA) and Kruskal–Wallis Test were used to study the differences between samples. Duncan's multiple range test  which is in agreement with the other studies  and the lowest in control bread (1.7%). The same as other studies, the ash content increased with increasing level of soy flour in the bread samples  bread was higher (58.3%) than that of soy flour content bread samples  were measured by the panel judges and the results are presented in Table In this study, with regard to taste, texture, color, and overall acceptability, the sensory characteristics score of bread containing 15% soy flour, compared to 0, 5, and 10 percentage of soy flour, were found to be the highest.The taste is the most important factor which affects the acceptability of an edible product  was found for bread containing 15% soy flour. The score for color increased with the increase in the level of soy flour which was not significant. The color of the GF bread samples improved from creamy to brown. The darker color of GF bread samples with soy flour may be due to the presence of yellow pigment in the soybean flour and Maillard reaction during processing . It has been shown that appearance of bread is an important sensory parameter  and the bread containing 5% soy flour had the least score (3.3). The score of crust texture improved with the increase in the level of soy flour which was statistically significant  and the GF bread containing 15% soy flour had the least score (3.95). This may be due to the beany flavor of soy flour (Awasthi et al., Color together with texture and aroma, contributes to consumer preference. It is influenced by physicochemical parameter of dough (Ahmad et al., 3.3Table 4Adding soy flour can improve quality and nutritional properties of wheat bread (Islam et al., No potential conflict of interest relevant to this article was reported.


Ferroelectric domain structure has been formed under the action of electron beam scanning in congruent lithium niobate single crystal covered by surface dielectric layer. The obtained types of the domain patterns have been considered as subsequent stages of domain structure evolution. The dependence on irradiated charge density of domain density, length, and period of domain rays and stripe domain width was used for characterization of the domain structure evolution. The threshold irradiated charge density necessary for the formation of solid stripe domain has been revealed. All obtained results have been discussed in terms of kinetic approach based on the analogy between domain structure evolution and first-order phase transition. It has become achievable due to development of the domain engineering methods in nonlinear optical ferroelectric crystals . The perficients .The traditional technique of domain engineering based on application of electric field by stripe electrodes faces such problems as domain broadening and spontaneous backswitching, which hamper achieving the submicron periods , 7. Thesμm produced by e-beam for second harmonic generation with high conversion efficiency in the bulk crystals of MgO-doped LN (MgOLN) [The switching by an electron beam is very promising, because the size of the electron beam is easily scaled down to several nanometers, which allows overcoming the limitations of the traditional method. The method modification by coating of the irradiated surface by buffer dielectric layer has been successfully used to create PPLN with high homogeneity and quality –20. The  (MgOLN)  and soft (MgOLN) .It is well known that existence of the intrinsic or artificial surface dielectric layer leads to qualitative change of the mechanisms of the domain structure evolution, appearance of the nanodomain ensembles, and lack of the domain wall shape stability , 24. TheThis paper is devoted to investigation and analysis of domain structure formation and domain shapes created by e-beam irradiation in the bulk congruent lithium niobate (CLN) crystals covered by artificial dielectric layer.Z-cut plates of CLN single crystals . The irradiated Z− polar surface was covered by 2.5 μm thick AZ nLOF 2020  photoresist layer by spin-coating method. The solid copper electrode was deposited by the magnetron sputtering at the opposite polar surface and grounded during irradiation.We have studied the 0.5 mm thick Y-crystallographic axis with period of 20 μm and width of 750 nm. The stripe exposure was performed by transversal meander-scan covering of the desired area. The samples were irradiated at the fixed accelerating voltage of 14 kV and the beam current of about 1 nA. The charge density D has been used for characterization of the irradiation condition.I is electron beam current, t is exposure time, and A is irradiated area.The samples were irradiated in the vacuum chamber of the Auriga Crossbeam workstation  equipped with electron beam lithography system Elphy Multibeam . The lithography system was used for precise control of the exposure parameters and beam movement. The irradiated patterns were created using Raith Nanosuite software and represented a grating of stripes oriented along The static domain structures were revealed by selective chemical etching in pure HF for 90 s at the room temperature after removal of the dielectric layer and electrode , 27. TheD range from 1 to 56 mC/cm2. Four types of domain structures were distinguished: (1) isolated nanodomains  isoirection , (3) solirection , and (4)irection .D values. Continuous solid stripe domains appeared for D above 56 mC/cm2  have been several orders of magnitude shorter than the switching times (close to one second). Thus, the irradiation with various charge densities can be considered as an analog of the switching by field pulses of various amplitudes and durations. Therefore, the different types of domain structure have been considered as different stages of domain structure evolution. This approach allowed distinguishing five subsequent stages of domain growth: (1) discrete switching (appearance of isolated nanodomains) , (2) domD dependence of isolated domain density demonstrates three regions with different behaviors (D (below 8 mC/cm2) corresponds to intensive nucleation  keeps constant with D increase. This fact can be attributed to correlated nucleation effect  [μm) [The short and long domain rays have been separated. The short rays appeared between long ones randomly. The average period of long domain rays  . It has in wall) , 25. Thell) [μm) .D increase (The linear increase of the average ray length with increase  is due tY-crystallographic axis is caused by anisotropy of the bulk screening [The Fourier analysis of the domain patterns allows revealing angular distribution of the ray directions. The prevalent ray orientation along the creening , 25. It D increase up to 44 mC/cm2 and disappeared for larger D corresponding to formation of the flat domain wall (L = lfd/ld, where ld is the domain wall length and lfd is the length of the flat boundary (D necessary for formation of solid stripe domain.We have used charge density dependence of the average ray length and the normalized domain wall length obtained by analysis of the binary domain images for characterization of the stripe domain formation . The aveain wall . The norboundary . Both apW is the stripe domain width, W0 is the minimal width, Dth is the threshold charge density necessary for formation of stripe domain, and C is constant. The parameters of linear fitting by (We have measured the charge density dependence of domain width as a characteristic of sideways domain wall motion. The similar linear dependence  has beenW=W0+CD−DThe domain structure formation induced by electron beam irradiation has been studied in congruent lithium niobate single crystal covered by surface dielectric layer. The different types of domain structures have been revealed at the irradiated surface depending on irradiated charge density. The exposure with different charge densities has been considered as an analog of the switching. Thus, the irradiation with various charge densities can be considered as an analog of the switching by field pulses of various amplitude and duration, and the different types of domain structure have been considered as different stages of domain structure evolution.Y-crystallographic axis has been attributed to screening anisotropy. The domain merging leading to formation of stripe domain has been considered. The threshold value of irradiated charge density necessary for formation of solid stripe domain has been revealed. The linear dependence of domain width on irradiated charge density correlates with results obtained recently in other materials. All obtained results have been explained in terms of kinetic approach based on analogy of domain growth with first-order phase transition. The ray formation at the irradiated surface was attributed to ineffective screening of depolarization field due to existence of artificial dielectric layer.The processes of discrete switching and domain ray growth have been characterized by domain density, as well as length and period of domain rays as a function of charge density. The prevalent orientation of the domain rays along


The temporal relaxation of the induced PFM contrast was measured at various temperatures. The average value of the induced contrast decreases during heating for all investigated crystals. Below the freezing temperature the induced state remains stable after an initial relaxation. Above the freezing temperature the induced state is unstable and gradually decays with time. The stability of the induced state is strongly affected by the measuring conditions, so continuous scanning results in a faster decay of the poled domain. The obtained effects are attributed to a decrease of the induced polarization and backswitching of the polarized area under the action of the depolarization field.The stability of ferroelectric domain patterns at the nanoscale has been a topic of much interest for many years. We investigated the relaxation of the polarized state created by application of a local electric field using a conductive tip of a scanning probe microscope for the model uniaxial relaxor system Sr Especially light frequency conversion devices based on periodically poled ferroelectric crystals2 and high-density storage systems3 are very promising. Nanoscopic studies of the domain structure evolution under application of a local field by a conductive SPM tip provide useful insight into the polarization reversal process in ferroelectrics. The majority of these studies are concerned with thin crystals of LiNbO3 and LiTaO36 using high-voltage SPM7 due to the extremely high coercive fields of these materials.The creation of regular micron and submicron domain patterns by application of a local electric field using a conductive tip of a scanning probe microscope (SPM)xBax1−Nb2O6, SBN100x) is considered to be a prospective material for domain engineering and high-density storage systems. Prominent nonlinear-optical and electro-optical properties of SBN together with high values of piezoelectric coefficients8 open new horizons for commercialization of the domain patterned crystals. Furthermore, SBN crystals are very attractive for SPM recording due to low coercive fields8. However, it is considered that the intrinsic nanoscale compositional inhomogeneity in relaxor ferroelectrics hampers the creation of tailored domain structures in SBN9.The uniaxial relaxor ferroelectric strontium barium niobate  revealed maze-type domain structures with characteristic sizes of about a hundred nanometers11 in thermally depolarized crystals at room temperature, as well as its evolution at elevated temperatures12. The micro- and nanodomain structures formed during polarization reversal in a uniform field were also studied using PFM13.The first high-resolution studies using SPM allowed visualizing pristine static domain patterns on the polar surface of SBN crystals14. An important issue is the stability of the created domains, especially considering possible applications. It was revealed that 1D and 2D regular domain arrays recorded on polar surfaces at room temperature are essentially more stable as compared to isolated domains14. The fabrication of domain gratings with charged domain walls on the (100) nonpolar surface (X-cut) of SBN61 at room temperature and its temporal relaxation also have been demonstrated15.The field and exposure characteristics of the SPM domain recording were investigated in SBN61 crystals10. Due to the relatively low transition temperature SBN single crystals are good candidates for SPM investigation of induced domain states both in the relaxor state (above the freezing temperature) as well as in the ferroelectric state (below the freezing temperature).Most of the studies have addressed the stability of the domain patterns created by SPM at room temperature. The effect of temperature has been less investigated16. These materials have a particular polar structure characterized by appearance of so-called polar nanoregions, PNRs, at relatively high temperatures (~600 K for SBN7517). PNRs have size of a few nanometers. This relaxor state differs from the paraelectric one, but manifests an ergodic behaviour18. In particular, by application of a strong enough electrical field a macroscopically poled state can be induced, which is however unstable and reverses back to the relaxor state after the field is switched off. Strengthening of interaction between PNRs on cooling results in a transition into a non-ergodic superdipolar glass state characterized by transformation of PNRs in coarse-grained microdomains that keep nanometer scale size20. This occurs at so-called freezing temperature. When relaxor ferroelectrics are poled below the freezing temperature an irreversible transition into a ferroelectric state can be induced.SBN crystals belong to a class of relaxor ferroelectricsIn this paper, we report on a study of PFM-recorded states in SBN crystals with different transitions temperatures: SBN75 with the transition below room temperature and SBN61 (pure and Ce-doped) with the transition above room temperature. The effect of temperature on both the induced piezoresponse and the stability of the created domains was investigated. Full and a partial relaxation of the induced state was observed above and below the transition temperature, respectively. Based on obtained data we have concluded that decay of the domains is promoted by the incomplete screened depolarization field. We also found that the measuring conditions affect the stability of domains. Continuous scanning removes screening charges and accelerates domains backswitching.The experiments were done for the multi-domain state that was produced by thermal depolarization achieved by heating up the sample to 200 °C and subsequent zero-field cooling.dc voltage pulses with magnitude 100 V were applied for 1 or 10 s over a spatial grid of 3 × 3 or 5 × 5 points with 5 μm distance between the points . Whereas when the tip is elevated after finishing the first scan, the contrast is preserved even after 38 min Fig. . This faFor SBN61 at room temperature the induced polarization was essentially more stable than in SBN75. The relative piezoreponse for the polarized areas created at room temperature decreased with time to half of the initial value after 120 min Fig. . We founTf = 65 °C) for this composition. To compare the stability of the induced state below and above the freezing temperature we analyzed time dependences of the induced piezoresponse at 60 °C and 70 °C   Fig. .Figure 6We observed that in both the continuous and the discrete regime the induced domains exhibit at least a partial backswitching after removing the external field. Besides, there is a qualitative difference between experiments performed below and above the freezing temperature. For temperatures corresponding to the relaxor phase (that is above the freezing temperature) the sample comes back to the unpoled state after a certain time. The domains written below the freezing temperature are more stable. After initial gradual backswitching both the induced piezoresponse and the diameter of the created domain (in the case of discrete switching) approaches a steady value.The measured PFM signal is dominated by the electromechanical contribution due to the converse piezoelectric effect. Since the piezoelectric coefficient is proportional to the value of spontaneous polarization, the variation of the PFM signal reflects the variation of the polarization. In spite of the relatively “hard” cantilevers (the spring constant lies in range 15–50 N/m) were used, the electrostatic contribution could not be eliminated completely. It is proportional to the dielectric permittivity of the sample and the work function difference between the tip and sample surface. This electrostatic signal is unipolar and results in some asymmetry of the measured PFM signal relative to zero value  spontaneous decay of the created domain structure (backswitching), and/or 2) decrease of the magnitude of induced/spontaneous polarization.22. In PFM in absence of a macroscopic electrode, the depolarization field is screened by both surface charges  and bulk charges (intrinsic screening).The backswitching effect represents the slow partial recovery of the initial multi domain state under the action of the residual (incompletely screened) depolarization field23. The resulting domain wall will be charged to screen the arising depolarization field (intrinsic screening). It is known that such a domain wall may attain a particular zig-zag like profile to minimize the depolarizing field25. After the electric field is switched off the domain wall may start to move back towards the free sample surface, but may be also pinned by bulk screening charges. This relaxation can contribute to the observed isothermal decay of the piezoresponse, i.e. backswitching. However, separation of this contribution as well as information about structure of this domain wall cannot be obtained from the presented PFM data. This should be matter of a separate study.The studied samples with thickness from 0.2 to 0.5 mm are rather thick in PFM sense. This means that under the applied poling voltage the created domains never reach the opposite polar surface (bottom electrode)27. We assume that during scanning the tip plays the role of a movable ground electrode and removes the screening surface charges. This effectively enhances the depolarization field and leads to destabilization of the created domains and accelerated backswitching. This observation indicates that the surface screening conditions play significant role in the compensation of the depolarization field.We have observed experimentally that the depolarization effect is accelerated for continuous scanning, namely the piezoresponse contrast linearly decays with the number of full scans Figs  and 4. TWe have also observed that the induced polarization became less stable at higher temperature Figs  and 5. TOn the other hand, the decreasing piezoresponse on heating manifests the decreasing polarization on approaching the transition temperature. The temperature dependence of the relative piezoresponse Figs  and 7b cTcr = 67 °C (SBN61:Ce) is close to the value of the freezing temperature Tf measured in this crystal by analysis of the temperature dependence of the dielectric permittivity28.The extracted value of the critical temperature It should be mentioned, that the screening charge decay caused by electronic diffusion and external current occurs at any temperature below as well as above the freezing temperature. Nevertheless, we observed that isothermal depolarization is strongly accelerated above the transition temperature.Tf ≈ 65 °C12) the piezoresponse contrast induced at room temperature decays to approximately 50% of its initial value in 2 hours and remains stable hereafter  the induced domain decays relatively fast, the piezoresponse contrast disappeared after 1 hour already  scanning.2) strontium barium niobate single crystals SrxBax1−Nb2O6  with x = 0.61 and 0.75 grown by the modified Stepanov technique29 in Prokhorov General Physics Institute of Russian Academy of Sciences  and by Czochralski method30 in University of Silesia . The 0.6-mm-thick plates were cut normal to the polar axis and carefully polished.The studied samples are plates of pure and Ce-doped . PFM imaging was done using an 32 and Origin 8.6.Two atomic force microscopes were used: (1) MFP-3D  equipped with Ti/Ir-coated silicon tip-cantilever assemblies ASYELEC-02  and (2) Probe NanoLaboratory NTEGRA Aura  equipped with silicon DCP20  and DCP11  cantilevers  having diamond-like conductive coating. The experimental data were processed using software packages WSxM Supplementary Information for Temperature Effect on the Stability of the Polarized State Created by Local Electric Fields in Strontium Barium Niobate Single Crystals


It is a multi-domain protein that includes a membrane binding F-BAR domain, a homology region 1 (HR1) domain, which binds selectively to active Cdc42 and an SH3 domain. TOCA1 is involved in the regulation of actin dynamics in processes such as endocytosis, filopodia formation, neurite elongation, cell motility and invasion. Structural insight into the interaction between TOCA1 and Cdc42 will contribute to our understanding of the role of TOCA1 in actin dynamics. The  Xenopus tropicalis extracts. The importance of TOCA1 in actin dynamics has been confirmed in a range of systems  is a downstream effector of the small GTPase, Cdc42  family. It has an N-terminal membrane binding domain (F-BAR), a central Cdc42-binding domain (HR1) and a C-terminal SH3 domain. The F-BAR domain binds to and deforms PIPIn addition to its importance in understanding the pathway of actin dynamics, this resonance assignment will also be generally interesting with regards to G protein signalling. For example, HR1 domains show differential selectivity for specific G proteins. The HR1 domains from TOCA1 and from another TOCA family member, CIP4 bind to Cdc42 . The construct was expressed as a GST-fusion in E. coli BL21 cells (Invitrogen). Stationary cultures were diluted 1 in 10 and grown at 37 °C until an A600 ~ 0.8 was reached, then induced with 0.1 mM isopropyl-β-d-thiogalactopyranoside for 20 h at 20 °C. To produce isotopically labelled protein for NMR studies, cells were grown in M9 media supplemented with 15NH4Cl and 13C-glucose (Sigma-Aldrich). The proteins were purified using glutathione-agarose beads (Sigma), and eluted from the beads by cleavage of the GST-tag by HRV 3C protease prior to gel filtration on a 16/60 S75 column .The TOCA1 HR1 domain  was cloned from full-length 13C/15N-labelled HR1 domain in 20 mM sodium phosphate pH 7.5, 150 mM NaCl, 5 mM MgCl2, 5 mM DTT, 10% D2O). 15N-HSQC, 15N-separated NOESY (100 ms mixing time), 15N-separated TOCSY (48 ms mixing time), HNCA  and 13C-separated NOESY (100 ms mixing time) were recorded on an Avance AV600. NMR data were processed using AZARA (W. Boucher).All NMR experiments were run at 25 °C with 0.9 mM 15N-HSQC and 13C-HSQC experiments using 15N-separated NOESY and TOCSY experiments, 3D HNCA, HNCO, HN(CO)CA, HNCACB and HN(CO)CACB in ANALYSIS  from the random coil positions were used to generate the Chemical Shift Index (CSI), which can be used to predict the secondary structure. The short-range NOEs and the CSI for each residue are shown in Fig.


Saccharomyces cerevisiae protein kinase Rad53 is a key regulator of the DNA damage checkpoint and uses its two FHA domains to interact with multiple binding partners during the checkpoint response. One of these binding partners is the Dbf4-dependent kinase (DDK), a heterodimer composed of the Cdc7 kinase and its regulatory subunit Dbf4. Binding of Rad53 to DDK, through its N-terminal FHA (FHA1) domain, ultimately inhibits DDK kinase activity, thereby preventing firing of late origins. We have previously found that the FHA1 domain of Rad53 binds simultaneously to Dbf4 and a phosphoepitope, suggesting that this domain functions as an ‘AND’ logic gate. Here, we present the crystal structures of the FHA1 domain of Rad53 bound to Dbf4, in the presence and absence of a Cdc7 phosphorylated peptide. Our results reveal how the FHA1 uses a canonical binding interface to recognize the Cdc7 phosphopeptide and a non-canonical interface to bind Dbf4. Based on these data we propose a mechanism to explain how Rad53 enhances the specificity of FHA1-mediated transient interactions.Forkhead-associated (FHA) domains are phosphopeptide recognition modules found in many signaling proteins. The  Stress generated during DNA replication is one of the biggest hurdles proliferating cells face to preserve genome integrity. Therefore, eukaryotic cells have conserved surveillance mechanisms, known as cell cycle checkpoints, to detect and repair damage generated during DNA replication123578911Rad53 contains two forkhead-associated (FHA) domains, as well as two SQ/TQ cluster domains (SCD), flanking its kinase domain. FHA domains are commonly found in DNA damage response proteins and mediate protein-protein interactions by recognizing phosphorylated epitopes on their binding partners15161718The interaction between Rad53 and DDK is of special interest because it involves multiple interfaces of the FHA1 domain. The phosphoepitope-binding site recognizes an epitope present in DDK6-SUMO tag with a TEV protease cleavage site (pAG8586). The two protein fragments were connected directly (Dbf4(0)Rad53) or separated by a five-residue linker Rad53). Clones were confirmed by DNA sequencing . Plasmids encoding the Dbf4(0)Rad53 (pAG8801) and Dbf4(5)Rad53 (pAG8805) chimeras were transformed in BL21(DE3) cells containing a plasmid encoding rare tRNAs. Cultures were grown to A600 = 0.7, induced by addition of 1 mM isopropyl β-D-1-thiogalactopyranoside, and incubated overnight at 16 °C with orbital agitation.Dbf4-Rad53 chimeras were created by subcloning a codon-optimized fragment of Dbf4 encompassing amino acids 105–220g, and the supernatants were loaded onto a HiTrap nickel-chelating HP column  equilibrated with buffer A. The His6-SUMO-tagged chimeras were eluted with a linear gradient to 300 mM imidazole. The fractions containing the chimera were pooled and injected onto a HiPrep 26/10 desalting column  equilibrated with buffer B . The His6-SUMO tag was removed with tobacco etch virus (TEV) protease, and the tagless chimeras further purified by affinity  and size-exclusion chromatography (Superdex 75 (10/300) GL column, GE Healthcare). The purified proteins were concentrated to 9–12 mg/mL and stored in buffer B. Protein concentrations were determined using the Beer-Lambert equation with an extinction coefficient of 36,440 M−1 cm−1.Cell pellets were resuspended in buffer A  and lysed by sonication. Lysates were cleared by centrifugation at 39,000 s) distribution of sedimenting species with a resolution of 0.05 S and a maximum entropy regularization confidence level of 0.68. The solution density, solution viscosity and protein partial specific volume were calculated in SEDNTERP (ref. http://sednterp.unh.edu/), and sedimentation coefficients s were corrected to standard conditions s20,w. Weighted-average sedimentation coefficients obtained by integration of the c(s) distributions were used to create an isotherm that was analyzed in SEDPHAT 13.0a in terms of a reversible monomer-dimer equilibrium. The protein extinction coefficient at 280 nm and the interference signal increment were calculated based on the amino acid composition in SEDFIT 15.01bSedimentation velocity experiments were conducted at 50,000 rpm and 20 °C on a Beckman Coulter ProteomeLab XLI analytical ultracentrifuge following standard protocols2 and cryo-protected by addition of 10% ethylene glycol. A complete data set was collected at the X29 beamline of NSLS-I . Data was processed and scaled in HKL2000 (see 480DGESpTDEDDVVS491) was purchased from GenScript and resuspended in buffer B. The Dbf4(0)Rad53 chimera was mixed with the phosphorylated peptide at a 10-fold molar excess and incubated at 4 °C for one hour prior to crystallization trials. Crystals of the Dbf4(0)Rad53-pPEP complex were grown in 100 mM TRIS pH 8.5, and 12.5% PEG 3350 (v/v) and cryo-protected by addition of 15% glycerol. A complete data set was collected at the O8B1-1 beamline of the Canadian Light Source and processed using XDS Rad53 grew in 50 mM sodium cacodylate pH 6.5, 12% PEG 4000 (v/v), and 250 mM MgCl2000 see 30. A pho XDS see 31.Both structures were determined by molecular replacement using the FHA1 domain of Rad53 (PDB 1G6G) and the HBRCT domain of Dbf4 (PDB 3QBZ) as search models. The initial models were refined by iterative cycles of manual model building in Coot and refinement in PHENIX1H-15N] heteronuclear single quantum coherence (HSQC) spectra were acquired at 306 K using a Bruker AV-700 MHz spectrometer equipped with a 5 mm TCI cryoprobe. Samples were prepared as described by ref. 480DGESpTDEDDVVS491). Spectra were processed using NMRPipeGradient and sensitivity enhanced [lacZ reporter pSH18-34, pEG-202-Dbf4-FL-derived bait and pJG4-6-Rad53-FHA1-derived prey plasmids were transformed into DY-1 yeast strain. Cells were grown to a concentration of 2 × 107 cells/mL in Synthetic Defined (SD) medium lacking uracil, histidine, and tryptophan. Prey expression was induced for 5–6 h in 20 mL of 2% galactose-1% raffinose medium (BD Bioscience). Using a total of 5 × 106 cells, the interactions between fusion proteins were detected by the quantitative β-Galactosidase  assay. The β-Gal activity was calculated using the following equation: β-Gal activity = 1,000 × A420/(t × V × A600), where t = time of reaction (in min) and V = volume of culture used in the assay (in mL). The remaining culture was centrifuged at 2,000 g for 3 min; the cell pellets were resuspended in lysis buffer  and lysed with a bead beater . Cell lysates were clarified by centrifugation and the extracted supernatant represented the whole-cell extract. Protein concentration was quantified using a Bradford assay, and protein expression was analyzed by Western blotting. The LexA-tagged Dbf4 bait was detected using a rabbit polyclonal anti-LexA antibody (Invitrogen), and the HA-tagged FHA1 prey was detected using a mouse monoclonal anti-HA antibody (Sigma). Alexa Fluor 647 goat anti-rabbit and Alexa Fluor 488 goat anti-mouse polyclonal secondary antibodies (Molecular Probes) were used.Point mutations within FHA1 (F146A and N112A/F146A) were generated by site-directed mutagenesis using pJG4-6 FHA1 (including residues 1–165 of Rad53) as the template. Single point mutations on Dbf4 (Y198A and K200A) were generated from the pEG202-Dbf4 full-length template. All constructs were verified by DNA sequencing . Two-hybrid analysis was carried out as described previously using pEG202-Dbf4-FL as the bait and the indicated pJG4-6 FHA1 variants as the preyWe have previously shown that the HBRCT domain of Dbf4, consisting of a BRCT fold immediately preceded by a helix, is necessary and sufficient for the interaction with the FHA1 domain of Rad53s) profiles that supported a reversible monomer-dimer equilibrium Rad53 chimera at increasing loading concentrations. The sedimentation experiments demonstrated the absence of very large species and yielded cRad53 and Dbf4(5)Rad53. The chimera containing a five-residue linker readily yielded diffraction-quality crystals . The asy2) and the HBRCT  domains, a value that is below the cutoff for specific interactions as judged using the PISA serverThe FHA1 and HBRCT domains have identical architectures in the complex as in their unbound structures . HoweverThe residues of the FHA1 domain mediating the interaction with Dbf4 in the crystal structure of the Dbf4(5)Rad53 chimera are the same as those previously identified using NMRWe generated single point mutations in the FHA1 domain affecting either interface I (Phe146Ala) or interface II (Asn112Ala), as well as a double point mutation affecting both interfaces (Asn112Ala/Phe146Ala). We then measured the ability of these variants to interact with full-length Dbf4 using a yeast two-hybrid assay. As we expected from our previous work, the FHA1-Asn112Ala had a mild, yet significant, binding defect . ConversThe combination of hydrophobic and polar interactions segregated in two different contact areas could provide the means to regulate complex formation upon binding of additional partners. Since a phosphorylated binding epitope is necessary for the interaction of Rad53 with DDK, we sought to determine the structure of the Dbf4(L)Rad53 chimera bound to a phosphorylated peptide .484TDED487) that is conserved and has high phosphorylation probabilityThe fragment of Cdc7 encompassing residues 294–493 interacts reproducibly with the FHA1 domain of Rad53480DGESpTDEDDVVS491) binds to the FHA1 domain of Rad53 in a phosphorylation-dependent manner in vitro1 space group and diffracted X-rays beyond 2.3 Å resolution. We determined the structure by molecular replacement using the structures of the individual FHA1 and HBRCT domains as search models. The molecular replacement solution showed well-defined electron density for the two domains, as well as the main chain and most side chains of the phosphorylated peptide . Therefore, we used this region of the FHA1 to superimpose and compare the two complexes. As expected, the loops defining the pThr-binding groove of the FHA1 domain had larger deviations (0.34 < r.m.s.d. <0.63), caused by the binding of the phosphopeptide. Conversely, the residues in FHA1 mediating the interaction with Dbf4 were barely affected by phosphopeptide binding (0.15 < r.m.s.d. <0.35).The Dbf4:Rad53 interface is similar, but not identical, to the binary complex. Superimposition of the FHA1 domains in the binary and ternary complexes revealed that the β1 strand and the β1/β2 loop were virtually invariant Rad53 molecules defining four Dbf4:Rad53 interfaces, whereas that of the ternary complex included two Dbf4(0)Rad53 molecules defining two Dbf4:Rad53 interfaces. Superimposition of each ternary complex onto any of the binary complexes revealed that the peptide moiety could only be accommodated in half of the complexes, explaining why crystals of the ternary complex grew in different conditions. However, no other crystal contacts mediated by the FHA1 or HBRCT domains enhanced or prevented the movement in Dbf4. Therefore, phosphopeptide binding rather than crystal packing is the likely driving force of the movement.15N-labeled Rad53 upon binding Dbf4 serve as sensitive reporters to map the interface of the complexWe have previously shown that losses of cross-peak intensity in the HSQC spectrum of Yeast genetics has delineated the factors and hierarchy of interactions involved in the DNA damage response, but the molecular detail has remained elusive because most of the interactions driving the checkpoint response are transient. This problem is aggravated for ‘AND’ logic gates because they recognize two or more inputs to produce a single signal414216203644The interaction of Rad53 with the DDK complex is reminiscent of the interaction between Chk2 (the human ortholog of Rad53) and BRCA1, where the tandem BRCT repeat of BRCA1 simultaneously recognizes two distal surfaces in the FHA domain of Chk2Both Rad53 and Chk2 dimerize in solution and this is important to promote kinase activation by trans-autophosphorylation47474750In contrast to the Chk2 dimer, where the entire lateral face of the FHA domain contributes to dimer formation, Dbf4 only contacts two points on the lateral face of the FHA1 domain. Interestingly, mutations on the two surfaces are not reciprocal indicating that each partner contributes asymmetrically to the two small interfaces mediating the interaction with Dbf4, thereby suggesting a sophisticated way to gain binding-specificity without strengthening the interaction. To our knowledge, this is the first crystal structure of an FHA domain bound to a binding partner through a non-canonical interface and lays the foundation to study how FHA domains can exploit canonical and non-canonical interactions to increase binding specificity of low-affinity interactions and, in turn, extend the functional repertoire of this phosphoepitope binding module.Accession codes: Coordinates and Structure factors for the binary and ternary complexes have been deposited as Protein Data Bank entries 5T2F and 5T2S.How to cite this article: Almawi, A. W. et al. ‘AND’ logic gates at work: Crystal structure of Rad53 bound to Dbf4 and Cdc7. Sci. Rep.6, 34237; doi: 10.1038/srep34237 (2016).


The formation mechanism considers fivefold deformation twins originating from the grain boundaries in a nanocrystalline material, resulting in that fivefold deformation twins derived from a single crystal have not been reported by molecular dynamics simulations. In this study, fivefold deformation twins are observed in a single crystal of face-centered cubic (fcc) alloy. A new formation mechanism is proposed for fivefold deformation twins in a single crystal. A partial dislocation is emitted from the incoherent twin boundaries (ITBs) with high energy, generating a stacking fault along {111} plane, and resulting in the nucleating and growing of a twin by the successive emission of partials. A node is fixed at the intersecting center of the four different slip {111} planes. With increasing stress under the indentation, ITBs come into being close to the node, leading to the emission of a partial from the node. This generates a stacking fault along a {111} plane, nucleating and growing a twin by the continuous emission of the partials. This process repeats until the formation of fivefold deformation twins. Nanocrystalline (nc) metals are widely used in consumer electronics, biosensing, nanomedicine, optical systems, drug delivery, photovoltaic cells and organic solar cells, owing to their mechanical and optoelectronic properties123567910At present, the work of FTs mainly focuses on face-centered cubic (fcc) single elemental metals, such as copper (Cu)912105678101214167810121456567810121416® with an fcc structure. The formation mechanism of fivefold deformation twins is investigated in a single crystal under nanoindentation via plastic deformation.In this study, fivefold deformation twinning is performed by MD simulations in a single crystal of a Haynes alloy of the C-2000Fivefold deformation twins derived from a single crystal are not predicted by MD simulations. This is attributed to that the formation mechanism of fivefold deformation twins explains, that a simple twin is generated through partial dislocation emissions from GBs56781012141678101214567810121416® has an fcc structure with a low stacking fault energy of 1.22 mJ m−2 −2, and its CTB energy is 63 mJ m−2 −2, which is an order magnitude higher than CTB9The C-2000There is a 7.35° misfit for an ideal fivefold deformation twin, and therefore stress exists in a FT, leading to the nonuniform angles formed in a fivefold deformation twin, as shown in the insets of 9139913In this study, fivefold deformation twinning is performed by MD simulations in a single crystal of an fcc alloy. Fivefold deformation twins are observed in the single crystal under indentation. The formation mechanism of fivefold deformation twins is different from previous reports, in which the partial dislocations are emitted from the GBs. In this work, the partial dislocation is emitted from the ITBs with high energy. After the emission of the partial, a stacking fault is generated along a {111} plane, resulting in the nucleating and growing of a twin by the successive emission of the partials. A node is formed at the intersecting center of the four different slip planes. With increasing stress by the indentation, ITBs come into being close to the node, emitting a partial dislocation from the node. This makes a stacking fault generate along a {111} plane, nucleating and growing of a twin via the successive emission of the partials. The course continues, generating a fivefold deformation twin. The prediction of MD simulations sheds light on the preparation of fivefold deformation twins in single crystals and coarse-grained metals by plastic deformation.® is widely used in the corrosion-resistant engineering® is 23Cr, 16Mo, 1.6Cu, 2Co*, 3Fe*, 0.5Mg, 0.5Al*, 0.08Si, 0.01 C (wt.%), and Ni balance (* meaning the maximum). The C-2000® was selected as the fcc ternary alloy in MD simulations with chemical compositions of 61Ni, 23Cr and 16Mo (wt.%). Embedded atom method (EAM) is an effective approach to well describe the interatomic forces1920An fcc ternary Ni superalloy named C-2000rij, Fi the embedding energy, and ρi the electron density. The electron density is calculated22where fj(rij) the electron density at the site of atom i arising from atom j at a distance rij away.with For a single elemental metal, the EAM potential is composed of three functions: pair energy, electron density and embedding energy. However, for an alloy, the EAM potential consists of not only the three functions for each of the constituent elements, but also the pair energy between different elements. Thus, the EAM potential of a single elemental metal can not be directly used in an alloy. Nevertheless, by normalizing the EAM potentials of constituent elements in an alloy, a generalized EAM potential for an alloy is constructedre is the equilibrium spacing between nearest neighbors, A, B, α and β are four adjustable parameters, and k and λ are two additional parameters for the cutoff. The electron density function is addressedwhere a and b is writtenThe pair potential between different elements To smooth the embedding energy over a wide range of electron density, the embedding energy functions are divided into three parts to match values at their junctions® is constructed according to eight equations, and the parameters needed to define the EAM model are listed elsewhere212223The generalized EAM potential of the C-2000®. The single crystal was 42 nm in length, 1.5 nm in width, and 30 nm in height. The orientations of length and height were [111] and [11-2], respectively. A cylinder was used as the indenter with a tip radius of 15 nm. Displacement-controlled mode was imposed on the single crystal during indentation process. The indentation speed was 20 m/s, and the maximum indentation depth was 10 nm. Periodical boundary conditions were applied on the length and width directions, and shrink-wrapped boundary conditions were implemented on height direction. Prior to indentation on [11-2] orientation, the single crystal was relaxed by an isothermal-isobaric thermostat (NPT) at 300 K for 100 picoseconds (ps). A time step of 1 femtosecond (fs) was used during the indentation. A dwelling time was 5 ps after loading. Common neighbor analysis (CNA) was used to identify the defects induced by indentation. In CNA method, ore red line represents a TB, and multiple red lines stand for staking faults. Green color means perfect atoms, red color for hexagonal close-packed (hcp) atoms, blue color for body-centered cubic (bcc) atoms, and white color for other deformed atoms.A single crystal of MD model was constructed for the C-2000How to cite this article: Zhang, Z. et al. Formation mechanism of fivefold deformation twins in a face-centered cubic alloy. Sci. Rep.7, 45405; doi: 10.1038/srep45405 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.