BRCA2, encodes a 3418-amino acid protein that is essential for maintaining genomic integrity. Among the proteins that physically interact with BRCA2, Partner and Localizer of BRCA2 (PALB2), which binds to the N-terminal region of BRCA2, is vital for its function by facilitating its subnuclear localization. A functional redundancy has been reported between this N-terminal PALB2-binding domain and the C-terminal DNA-binding domain of BRCA2, which undermines the relevance of the interaction between these two proteins. Here, we describe a genetic approach to examine the functional significance of the interaction between BRCA2 and PALB2 by generating a knock-in mouse model of Brca2 carrying a single amino acid change  that disrupts this interaction. In addition, we have combined Brca2G25R homozygosity as well as hemizygosity with Palb2 and Trp53 heterozygosity to generate an array of genotypically and phenotypically distinct mouse models. Our findings reveal defects in body size, fertility, meiotic progression, and genome stability, as well as increased tumor susceptibility in these mice. The severity of the phenotype increased with a decrease in the interaction between BRCA2 and PALB2, highlighting the significance of this interaction. In addition, our findings also demonstrate that hypomorphic mutations such as Brca2G25R have the potential to be more detrimental than the functionally null alleles by increasing genomic instability to a level that induces tumorigenesis, rather than apoptosis.Human breast cancer susceptibility gene,  Breast Cancer2 (BRCA2) is a well-established factor associated with increased risk of hereditary breast and ovarian cancers. BRCA2 has numerous roles in maintaining the genome to prevent accumulation of mutations that can lead to cancer formation. Here, we describe the generation of a novel mouse model that has a single amino acid alteration in the BRCA2 protein that affects its interaction with another hereditary breast cancer protein, PALB2. We use these mice to examine the relevance of the interaction between BRCA2 and PALB2. We also examine the effect of disrupting one copy of Palb2 gene in the mutant mice. Our findings show that the loss of interaction between the two proteins increases cancer formation in mice. We conclude that not only are BRCA2 and PALB2 required for tumor suppression, their physical interaction is equally important.Inheritance of a deleterious mutation in  BRCA2, is a major player involved in these functions. BRCA2 recruits the DNA recombinase, RAD51, to the site of double strand breaks (DSBs), which mediates repair by homologous recombination (HR) , 1210, 1of BRCA2 . In vitrks by HR , 14–16.PALB2 are associated with increased risk of breast cancer, similar to that of BRCA2, making it an important hereditary breast cancer susceptibility gene [BRCA2, PALB2 is also classified as an FA gene (FANCN). With so many similar and overlapping functions, it is not surprising that the Palb2 null mice exhibit an early embryonic lethal phenotype similar to Brca2 null embryos. K14-Cre-mediated deletion of Palb2 loss results in mammary tumors albeit with long latency, which is significantly reduced when combined with Trp53 loss. Interestingly, the genomic profiles of Palb2;Trp53-loss associated tumors are different from those associated with Brca2;Trp53-deficient tumors in mice [Mutations in ity gene . Like BR in mice –20.The domains of BRCA2 and PALB2 interaction have been well characterized. In BRCA2, the highly conserved 10–40 residues near the N-terminus were shown to be necessary and sufficient for binding to PALB2. Within this region, three cancer-associated mutations affecting two amino acids, namely Gly25  and Trp31  were found to disrupt the interaction with PALB2. Furthermore, while W31C and W31R completely abolished the interaction with PALB2, G25R significantly reduced but did not completely abolish the interaction . A mousePALB2 suggested that the C-terminal region containing four WD40 motifs binds to BRCA2 [Functional characterization of cancer-causing mutations in to BRCA2 . It was to BRCA2 , 23. Thito BRCA2 , 24. PALto BRCA2 . PALB2 ato BRCA2 , 26.Brca2-null DT40 cells. These findings suggest that the two regions may be playing significantly overlapping roles [While the binding of PALB2 to BRCA2 is considered vital for BRCA2 function, recent studies have suggested that there may exist a functional redundancy between the PALB2-binding domain and the C-terminal DNA-binding domain of BRCA2. In chicken DT40 cells, when the PALB2-binding N-terminal region of BRCA2 was deleted, it resulted in a mild reduction in HR and a mild sensitivity to IR, camptothecin, cisplatin as well as olaparib, a PARP1 inhibitor . These cng roles . Anotherng roles . Taken tin vivo, we have generated a knock-in mouse model, with a point mutation (c.73G>A) in exon 3 of Brca2 that changes the highly conserved glycine at position 25 to arginine  cells by gene targeting  for a Brca2 null allele, to obtain Brca2G25R/Ko mice. These mice are also viable and born at expected Mendelian ratios , Brca2G25R/Ko males at 25 weeks showed the presence of some seminiferous tubules that were filled with spermatocytes and appeared similar to the tubules present in Brca2G25R/+ and Brca2G25R/G25R males . Histological analysis of  meiosis .Brca2Ko/+ and Brca2G25R/G25R chromosome spreads  along the synapsed chromosomes suggesting the presence of unrepaired DSB breaks  . As seen spreads , by thisB breaks . We also154 foci , p<0.001Brca2G25R/KO males , Brca2G25R/+, Brca2G25R/G25R, and Brca2G25R/Ko animals and monitored their tumor free survival for 2 yrs. The mean survival of the wild-type and Brca2Ko/+ mice (91 weeks) as well as Brca2G25R/+ mice (97 weeks) was not significantly different from the mean survival of Brca2G25R/G25R and Brca2G25R/Ko animals . Of these, a third also had additional tumors types , 37% had other carcinomas, 32% had multiple tumors, and only 4% developed osteosarcomas , 33% developed osteosarcomas. In addition, 17% had other carcinomas, with 22% of the animals having multiple tumors and 11% developed B-cell lymphomas  exhibited a mean survival time of 24 weeks with the majority of the animals (92%) succumbing to T-cell lymphoblastic lymphomas  analysis using an Agilent SurePrint G3 Mouse CGH Array (4X180K) chip. We examined tumors from Brca2G25R/+, Brca2G25R/G25R, Brca2G25R/Ko mice, all on Trp53Ko/+ genetic background. Based on the total region affected by copy number variation (CNV), we observed a mean genomic change of 14.99% in Brca2G25R/+;Trp53Ko/+ tumors  with an average CNV of 443 (p = 0.054)  and also had a number of regions with gains and losses  . These tumors had gains and losses similar to those seen in Brca2G25R/Ko;Trp53Ko/+ tumors and the total CNVs observed were 424(p = 0.036)  , S8 Fig.= 0.068) , S8 Fig.= 0.036) , S8 Fig.yc locus . AmplifiA2 tumors–33. TakePalb2Ko/+). The 2 lines that grew the slowest were Brca2G25R/Ko and Brca2G25R/Ko;Palb2Ko/+ , Brca2G25R/Ko (p = 0.022), Brca2G25R/G25R;Palb2Ko/+ (p = 0.016), and Brca2G25R/Ko;Palb2Ko/+ (p = 0.17) MEFs compared to the controls (Brca2G25R/Ko (p = 0.012) and Brca2G25R/Ko;Palb2Ko/+ MEFs (p = 0.009)  from early passages. We found a significant reduction in the growth of mutant MEFs of various genotypes relative to the control MEFs  . Other l= 0.009)  These reBrca2G25R mutant MEFs exhibiting delayed G2/M progression and reduced cell proliferation, we examined its effect on the genomic integrity of the cells. We examined metaphase spreads of untreated and MMC treated cells. We found very few chromosomal aberrations in untreated control MEFs (1.6–2.06 aberrations (ab)/cell). We observed a slight increase in breaks and translocations in Brca2G25R/G25R;Palb2Ko/+(2.76 ab/cell), Brca2G25R/Ko(2.74ab/cell), and Brca2G25R/Ko;Palb2Ko/+ (3.26ab/cell)  , Brca2G25R/Ko,(7.28 ab/cell) and Brca2G25R/Ko;Palb2Ko/+ (14.84 ab/cell) MEFs showed a marked increase  in aberrations  over controls MEFs (3.18–4.68 ab/cell)  (With the ctively) . After 1Brca2G25R/Ko (55% of cells) and Brca2G25R/Ko;Palb2Ko/+ (53% of cells)  along with Brca2G25R/G25R;Palb2Ko/+ , and Palb2Ko/+ (n = 5), we observed a mean decrease in protection of 19% (3 independent experiments) . The Brca2G25R/G25R cells showed a 28% loss in fork protections , which increased significantly on a Palb2Ko/+ background . Interestingly, the Brca2G25R/Ko MEFs exhibited 35% loss of protection  however, did not show any further loss when one allele of Palb2 was deleted  . Control and mutant cells were first labeled with CldU, replication was then stalled with 0.5mM HU for 1.5hrs and subsequently resumed in the presence of IdU. These cells were then analyzed for the length of the CldU and IdU labeled DNA fibers to determine whether the stalled forks had restarted or there was a new origin of replication. Overall, here was no significant difference between control and Brca2G25R/KO;Palb2Ko/+ cells suggesting that fork restart was not affected in the mutant cells  . The los, n = 4) . While tnt cells . We concPALB2 have demonstrated that it is essential for BRCA2-mediated recruitment of RAD51 as well as for protection of stalled replication forks . While   did not reveal any overt defects. We observed a small loss of fork protection in Brca2G25R/G25R and Brca2Ko/+;Palb2Ko/+ MEFs but Brca2G25R/G25R;Palb2Ko/+, Brca2G25R/Ko and Brca2G25R/Ko;Palb2Ko/+ MEFs exhibited a significant fork degradation. Our results show that although both BRCA2 and PALB2 have their own DNA binding motifs, as well as independent RAD51 binding motifs, their interaction is essential for proper recruitment of RAD51 to the sites of DNA damage and protection of stalled replication forks.Recent studies have suggested that heterozygosity of tability , 39. HowBrca2 and Palb2, a defect in RAD51 recruitment as well as a loss of protection of the replication fork leads to genomic instability. Based on the tumor predisposition of these mice, it is evident that the genomic instability contributes to tumorigenesis. Brca2G25R/Ko mice had a significantly reduced tumor free survival compared to Brca2G25R/+ as well as Brca2G25R/G25R mice, which is further enhanced on a Trp53Ko/+ genetic background. Their tumor predisposition is reminiscent of the Brca2 mutant mice that lack exon 27 (Brca2Δ27/Δ27), in terms of latency, frequency of tumor formation and enhancement on a Trp53Ko/+ genetic background [Brca2G25R/Ko animals predominately developed osteosarcomas on the Trp53Ko/+ background, unlike the Brca2Δ27/Δ27;Trp53Ko/+ mice that developed a variety of tumor types. A recent sequence analysis of the exome of osteosarcomas reported increased incidence of BRCA2 mutations as well as signatures associated with BRCA2 loss suggesting the involvement of BRCA2 in the development of osteosarcomas [Brca2G25R/Ko;Palb2Ko/+ animals that result in the least interaction between BRCA2 and PALB2 and exhibited the shortest tumor latency, predominantly developed lymphoblastic T-cell lymphomas, similar to the mice carrying a truncating mutation in exon 11 of Brca2 [As observed in the MEFs derived from mice of various genotypes with mutations in ckground , 40, 41.sarcomas –44. The of Brca2 , 37.Brca2G25R/G25R MEFs exhibited a mild defect in RAD51 recruitment and fork protection but the survival of mice was not affected except they had a mild increase in tumor formation at 24 months of age. The tumor free survival of these mice was also not altered on a Trp53Ko/+ genetic background.Overall, the tumor incidence and tumor latency in mice for the most part correspond with the degree of genomic instability observed in the MEFs. However, there were some exceptions. For example, the Brca2 is rendered functionally null by CRE-mediated recombination in the epithelial cells using cytokeratin K14 promoter (which is expressed during embryogenesis), tumor incidence is low and tumor latency is very long [Trp53 heterozygous background [Brca2G25R/Ko as well as Brca2G25R/Ko;Palb2Ko/+ mice with wild-type Trp53. Sixty-nine percent of Brca2G25R/Ko and 100% of Brca2G25R/Ko;Palb2Ko/+ mice developed tumors, with an average latency of 82 and 24 weeks, respectively. Considering these genotypes have no effect on the viability of mice, the genomic instability induced in cells must not be high enough to induce a strong apoptotic response but it must be significant enough to result in cellular transformation and tumorigenesis. Similar high tumor incidence has been reported in other mice carrying Brca2 hypomorphic alleles that result in viable mice, such as Brca2tm1Cam, Brca2Tr2014 [TRP53 or other genes that have an anti-apoptotic response.While an increase in genomic instability correlates well with an increase in tumor susceptibility, having a significantly high genomic instability may not necessarily enhance tumorigenesis . When Brery long . Althougery long . This isckground . The mosa2Tr2014 , 37. OurIn conclusion, our results provide strong evidence to support the importance of a physical interaction between BRCA2 and PALB2. In spite of their ability to independently bind to RAD51, their heterodimerization via the N-terminus of BRCA2 and the C-terminus of PALB2 is essential for their function. Disruption of this interaction not only affects the DSB repair but also the ability to protect the stalled replication forks. These unique mouse models will help distinguish the importance of HR and fork protection in BRCA2’s and PALB2’s ability to protect genomic integrity. In addition, the unique mouse models described here will be useful for preclinical studies because of their high tumor incidence and low tumor latency.Mice were treated in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals . The protocol was approved by the Animal Care and Usage Committee of NCI-Frederick (NIH) .th nucleotide of exon 3 to change codon 25  of Brca2 clone in BAC RPCI22-421-23A by recombineering in SW102 cells using the galK-based selection-counterselection method as described previously[MseI restriction enzyme recognition site. Next, a loxP-PGK-Tn5-neomycin-bp(A)-loxP cassette along with BanII and EcoRI restriction sites were inserted into intron 3 by recombineering. Finally, a 10.7 kb genomic region of Brca2 was retrieved by gap repair from the BAC into pBSK(+) plasmid containing the Thymidine Kinase (TK) gene under the control of the MC1 promoter. The resultant plasmid was the G25R knock-in targeting vector (A single base substitution (G>A) was generated in the 6reviously. The G>Ag vector .NotI and electroporated into V6.4 (129 X C57BL/6J) ES cell line as described previously [R, FIAUR ES cell clones were screened by Southern analysis of BanII and EcoRI restriction enzyme digested genomic DNA using 5’ and 3’ probes . Statistical differences in tumor types were assessed via Fisher's exact test, and survival curves were analyzed utilizing Prism Graphpad. For germ-cell visualization 3-week old testis sections were immunostained with germ cell marker (MVH) as previously described . Antibod2. Cell growth analyses were done by seeding 10cm plates with 5 X 105 cells and counting the cells every day. Metaphases were collected after a 24 hour treatment with 100nM MMC and spread as previously described [MEFs were generated from E13.5 embryos and cultured in 10% FBS at 5% Oescribed . ImmunofParaffin sections of testes were TUNEL stained using the In Situ Cell Death Detection Kit (Roche 11684817910) following the directions included with the kit.Meiotic spreads were performed as previously described. PrimaryBrca2G25R/+;Trp53Ko/+ , Brca2G25R/G25R;Trp53Ko/+,  Brca2G25R/Ko;Trp53Ko/+ (4 Osteosarcomas) and Brca2G25R/G25R;Palb2Ko/+;Trp53Ko/+ . The DNA was isolated using the Qiagen DNeasy Blood and Tissue Kit, wild-type mouse tail DNA was used as the control. The DNA was hybridized to an Agilent SurePrint G3 Mouse CGH Array 4X180K chip. The results from the array were analyzed using Nexus Copy Number from BioDiscovery.A comparative genomic hybridization array was used for 4 tumors from the following genotypes, 5 cells/ml. On pre-cleaned slides 7.5 μL of cell lysis solution  was placed near the top of the slide. Then 2.5 μL of cells were added to this drop, mixed and then left at RT for 8 minutes. The slide was then tilted to allow spreading of fibers at approximately 45° angle to allow the drop to slowly run down the slide. The slides were then air-dried. Next, the slides were fixed in methanol/acetic acid 3:1 at 4° O/N. Slides were then re-hydrated with 1X PBS, denatured in 2.5M HCL for 1 hr, washed 5X with PBS. The slides were then blocked  for 40 minutes. The slides were then labeled with mouse anti-BrdU/IdU 1:100  and rat anti-BrdU/CldU (1:500) for 2.5hrs at RT or 4° O/N. Slides were then washed 5X with 0.2% PBS-Tween 20. Secondary antibodies, Alexa-fluors anti-rat 594, and anti-mouse 488 were used at 1:300 for 1 hr in dark at RT. Slides were washed again with 0.2% PBST, air-dried and mounted with anti-fade. Fibers were then visualized and imaged at 63X. Measurements of fibers were performed utilizing ImageJ software.DNA fiber analysis was performed as previously described with minor modifications , 55. BriS1 FigA: Schematic representation of the gene targeting strategy to generate the Brca2G25R knock-in allele showing the wild type locus (Brca2WT), targeting vector, targeted allele (Brca2G25R-Neo) showing the presence of loxP-Neo-loxP cassette and the Brca2G25R knock-in allele with the point mutation and a single loxP site. The first four exons of Brca2 exons are indicated as boxes with corresponding numbers. Location of restriction sites used for Southern-based genotyping are indicated by B (BanII) and E (EcoRI). G25RGenoF and G25RGenoR designate location and direction of primers (in blue) used for PCR-based genotyping. Asterisk marks the location of the G>A mutation in the first base of codon 25 of Brca2 in exon 3. Location of probes used to detect 5′ and 3′ end targeting are shown with a solid box below the Brca2WT locus. B. Southern blot analysis showing correct gene targeting. D. Sequence read confirming G>A substitution in codon 25 of Brca2. D. Schematic representation of the Brca2 null allele (Brca2Ko/+) showing deletion of a portion of exon 11 which is replaced with human HPRT1 minigene. E. PCR-based genotyping of Brca2G25R allele utilizing the primers show in blue arrows in A.(PDF)Click here for additional data file.S2 FigA. Representative meiocytes at pachynema of  stained with SYCP3 , and RAD51  and merged (Bottom), arrows indicate the XY Body. B. Quantification of RAD51 foci along the XY body. Error bars: SD, * p<0.05. Controls are represented by either: Brca2Ko/+ or Brca2G25R/+. Abbreviations: Brca2Ko/+ = B2Ko/+, Brca2G25R/+ = B2GR/+, Brca2G25R/G25R = B2GR/GR, Brca2G25R/Ko = B2GR/Ko.(PDF)Click here for additional data file.S3 FigA.) and 7 weeks (B.). Quantification of TUNEL positive cells at 3 weeks (C.) and 7 weeks (D.). Error Bars: SD, *p<0.05.Representative images of testes with TUNEL staining (green) and counterstained with DAPI (blue) to show the cells that are undergoing apoptosis at 3 weeks ((PDF)Click here for additional data file.S4 FigA. Females and B. Males. Abbreviations: NT = No tumor observed, BCL = B-cell lymphoma, TCL = T-cell lymphoma, HS = Histiocytic sarcoma, OS = Osteosarcoma, HemS = Hemangiosarcoma, OthSa = Other sarcomas, AD = Adenomas, OthCa = Other carcinomas, MT = Multiple tumors.Distribution of tumor types found in mice by gender of indicated genotypes. (PDF)Click here for additional data file.S5 FigA. Females and B. Males. Abbreviations: NT = No tumor observed, BCL = B-cell lymphoma, TCL = T-cell lymphoma, HS = Histiocytic sarcoma, OS = Osteosarcoma, HemS = Hemangiosarcoma, OthSa = Other sarcomas, AD = Adenomas, OthCa = Other carcinomas, MT = Multiple tumors, UNK = cause of death unknown.Distribution of tumor types found in mice of indicated genotypes. (PDF)Click here for additional data file.S6 FigA. Testis cross section of Brca2Ko/+;Palb2Ko/+ mice showing normal spermatogenesis at 3 weeks and 7 weeks of age. Far left panel: Testis cross section of Brca2GR/GR;Palb2Ko/+ mouse at 25 weeks showing normal spermatogenesis. B. Kaplan-Meier tumor-free survival curves of Control, Control;Palb2Ko/+ and Brca2GR/GR;Palb2Ko/+ mutant animals (104 week study). C. Total distribution of tumors found in indicated genotypes. D. Tumor distribution broken down by gender of indicated genotypes. Abbreviations: NT = No tumor observed, BCL = B-cell lymphoma, TCL = T-cell lymphoma, HS = Histiocytic sarcoma, OS = Osteosarcoma, HemS = Hemangiosarcoma, OthSa = Other sarcomas, AD = Adenomas, OthCa = Other carcinomas, MT = Multiple tumors, UNK = cause of death unknown. Controls are represented by: Brca2+/+ and Brca2Ko/+. Abbreviations: Brca2G25R/G25R = B2GR/+, Palb2Ko/+ = P2Ko/+.(PDF)Click here for additional data file.S7 FigIndividual copy number variation (CNV) graphs for each of the heterogeneous tumors across the genome. Chromosome number is at the bottom and amount of gain of loss is indicated at the Y-axis. (+1 or -1 indicates a 2 copy gain or 2 copy loss) Abbreviations: Ly = Lymphoma, OS = Osteosarcoma, HS = Histiocytic Sarcoma, Sarc = Sarcoma.(PDF)Click here for additional data file.S8 FigA. Image of amplified region around the myc locus in Brca2G25R/G25RPalb2Ko/+ and Brca2G25R/Ko tumors on a Trp53Ko/+ genetic background. B. Percent genome change, and C. total number of copy number changes in the individual tumors. Controls are represented by: Brca2G25R/+; Trp53Ko/+. Abbreviations: Brca2G25R/+ = B2GR/+, Brca2G25R/G25R = B2GR/GR, Brca2G25R/Ko = B2GR/Ko, Palb2Ko/+ = P2Ko/+, Trp53Ko/+ = p53Ko/+.(PDF)Click here for additional data file.S9 FigA. Fold change of cell growth over the course of 5 days of MEFs of various genotypes. B. Untreated and 100nM MMC treated cell cycle profiles of MEFs of the various genotypes. C. BrdU incorporation of untreated and 100nM MMC treated MEFs of the various genotypes. Error Bars: SD, *p<0.05. Controls are represented by: Brca2+/+ . Abbreviations: Brca2+/+ = WT,Brca2Ko/+ = B2Ko/+, Brca2G25R/G25R = B2GR/GR, Brca2G25R/Ko = B2GR/Ko, Palb2Ko/+ = P2Ko/+.(PDF)Click here for additional data file.S10 FigA. RAD51 foci quantification in cells that are PCNA positive in indicated genotypes. B. Fork restart assay: The cells were pulsed with CldU for 15 minutes, then 0.5mM HU was added for 1.5 hrs to stall replication and then released into media containing IdU for 15 minutes. CldU and IdU fibers lengths were measured and plotted for the indicated genotypes. The fibers were evaluated for continuing forks (CldU followed by IdU), stalled forks , or new forks  and graphed as percentage of all fibers evaluated. Controls are represented by: Brca2+/+, Brca2Ko/+.(PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


VDR gene encoding the vitamin D receptor leading to the resistance to vitamin D. We described a female toddler with initial presentation of leg tenderness and clinical features of HVDRR including severe rickets, hypocalcemia and hypophosphatemia without alopecia. Genetic analysis revealed novel compound heterozygous mutations of p.M4I and p.H229Q in patient’s VDR gene. In cis p.M4I with FOKI-F eliminated both translation start sites of the VDR protein. The p.H229Q VDR exhibited significantly reduced VDR transactivation activity with intact dimerization with RXR. Our report expanded the mutation spectrum of HVDRR, and provided the first case of a benign variant p.M4I plus a common p.M1T polymorphism leading to a pathogenic allele.Hereditary 1, 25-dihydroxyvitamin D-resistant rickets (HVDRR), a rare recessive disease, is caused by mutation in the  VDR gene, leading to accumulation of high levels of 1,25(OH)2D3 concentration. Conversely, Vitamin D-dependent rickets type I which is caused by 1α-hydroxylase deficiency has low level of 1,25(OH)2D3. Clinical features of HVDRR include rickets, hypocalcemia, secondary hyperparathyroidism, hypophosphatemia, elevated alkaline phosphatase, with or without alopecia. Children with HVDRR are resistant to all forms of vitamin D therapy and require intravenous calcium treatment. Alopecia due to the defective VDR activity within keratinocytes, appears in approximately two-thirds of cases and is considered a marker of disease severity [Hereditary 1,25-dihydroxyvitamin D-resistant rickets , also known as vitamin D-dependent rickets type II, is a rare autosomal recessive form of vitamin D resistance due to loss of functional vitamin D receptor (VDR). With thVDR gene with T to C polymorphism converting the first translation initiation codon from ATG to ACG. In human, two VDR proteins composed of 427 amino acids  or 424 amino acids  in length can be found that are distinguished by differential initiation codons.Several polymorphisms in the VDR gene have been identified with possible pathological significance in osteoporosis, osteoarthritis, diabetes, cancer, cardiovascular disease and bone marrow density , 6. Fok VDR gene, one that alters the VDR mRNA translation initiation site and one that affects vitamin D binding activity.In this study, we described a HVDRR patient without alopecia and identified two novel mutations in the VDR gene were amplified by PCR and directly sequenced. PCR amplification primers were available upon request  nephrology cohort. Informed consent in written form was obtained from the parent of investigated individual for publication of the case details and medical images. Genomic DNA was extracted from peripheral blood leukocytes. Exons 2 to 9 and intron-exon boundaries of the  request . The refThe study protocol was approved by the Institutional Review Board of the Kaohsiung Medical University Hospital (KMUH-IRB-980010). All clinical investigation was conducted according to the principles expressed in the Declaration of Helsinki.Total RNA was collected from the peripheral blood leucocytes of the patient by standard Trizol method. The full-length H229Q VDR cDNA was obtained by reverse transcription of total RNA using the M-MLV RT-PCR kit with primers 5'-CCGGCCGGACCAGAAGCCTT-3' and 5'-CTGCTGAGTAGCCGCCAGCC-3' followed by subcloning into the pcDNA3.1/V5-His TOPO TA Expression Kit (Invitrogen). The wild-type (WT) VDR expression plasmid was generated by using the H229Q plasmid as a template with Quickchange II XL site-directed mutagenesis kit  and single primer (5'-CTCTCCATGCTGCCCCACCTGGCTGACCTGGTCAG-3') method. All plaCOS-7 and HEK293 cells were obtained from American Type Culture Collection . Expression vectors of the WT or H229Q VDR were co-transfected with the reporter plasmid D3R-Luc using FuGENE6  according to the manufacture’s protocol.2D3 followed by an additional incubation for 24 h. The cells were then lysed in 250 μl of lysis buffer and assayed for luciferase activity using the Luciferase Assay Kit  with a FLx 800 fluorescence microplate reader .COS-7 and HEK293 cells were grown in DMEM containing 10% bovine serum in 6-well tissue culture plates. Cells were transfected in triplicate with 100 ng/well WT or mutant VDR expression plasmids and 125 ng/well VDRE-luciferase plasmid using FuGene HD transfection reagent . Total protein concentration and VDR expression level were served as an internal control for transfection. Twenty-four hour after transfection, cells were incubated in DMEM containing 1% bovine growth serum with vehicle (ethanol) or 1,25(OH)p value of 0.05 was considered significant.Results are shown as means ± SE. One-way ANOVA, followed by Tukey’s honestly significant difference test, was used to calculate statistical analyses. A rd percentile, Z-score -4.5), body length of 70 cm  initially presented with bilateral leg muscle weakness, bone pain, and growth delayed. She was able to sit but unable to stand or walk without support due to bone pain. The lateral and posterior fontanels were closed, but the cranial sutures and distribution of scalp hair were normal. Her serum chemistry panel revealed several abnormalities with total calcium (Ca) 7.2 mg/dL (8.4–10.2mg/dL), phosphorus 2.1 mg/dL (2.5–4.6 mg/dL), intact parathyroid hormone 586.4 pg/ml (11–62 pg/ml) and alkaline phosphatase 2038 U/L (32–92 IU/L)(2D3 of 285 pg/ml (20–45pg/ml). The skeletal examination revealed generalized osteopenia with cupping and fraying at the metaphyseal ends of long bones of upper and lower extremities, with widening of growth plates and epiphyses (th thoracic spine (A 25-month old Han Chinese girl with body weight of 8.5 kg (<3–92 IU/L). There wpiphyses . Severe ic spine . The curic spine . There wVDR gene and the data indicated mutations at c.12G>A (p.M4I), c.687C>G (p.H229Q), and heterozygous f/F of the Fok I polymorphism (rs2228570) (in-cis and derived from her mother while f and c.687C>G (f/H229Q) were on the same allele inherited from the father , Exome Variant Server of NHLBI Exome Sequencing Project (http://evs.gs.washington.edu/EVS/), NCBI SNP database (http://www.ncbi.nlm.nih.gov/projects/SNP/), or HGMD (http://www.hgmd.cf.ac.uk/ac/index.php). Furthermore, both variants were not found in 100 healthy control Han Chinese examined by restriction fragment length polymorphism method analyzed by BsrdI or PuvII digestion (data not shown). The amino acid at position H229 is well conserved from human to fruit fly while M4 is only conserved (Neither c.12G>A (p.M4I) nor c.687C>G (p.H229Q) variants was existed in the 1000 Genomes Projects 2D3 5(OH)2D3 . Immuno-5(OH)2D3 . These r5(OH)2D3 .2D levels are unusually high which is a major clinical observation that distinguishes HVDRR from 1α-hydroxylase deficiency[2D3, was reported recently[HVDRR is a very rare form of rickets with ~100 cases reported worldwide. It is an autosomal recessive disease with males and females equally affected. Affecteeficiency. Althoug recently, 14.VDR mutations reported in the literature and Human Gene Mutation Database . Mutations that are associated with 1,25(OH)2D resistance include mutations in DNA binding domain and ligand binding domain, nonsense mutations, insertions/substitutions, insertions/duplications, deletion and splice site mutations[Currently there are more than 54 HVDRR-related mutations. Our cur2D3[The amino acid residue H229 is located in the H3 helix, which forms part of the ligand binding domain (LBD) important for the binding of 1,25(OH)2D3, 16. Sev2D3–19. H2292D3, 19. Our2D3, 20. Thi2D3.3, and 17–20ug per day of 1,25(OH)2D3 [Hypocalcemia in HVDRR patient leads to secondary hyperparathyroidism and hypophosphatemia, causing a decrease in bone mineralization and the development of rickets. Some patients improved after treated with pharmacological doses of vitamin D ranging from 5000–40000IU per day, 20–200ug per day of 25(OH)D5(OH)2D3 . When pa5(OH)2D3 . Oral ca5(OH)2D3 . Althoug5(OH)2D3 , since c5(OH)2D3 .2D3 influences adaptive immunity and inflammation through the regulation of T-cell helper 1(TH 1), TH2 production and IL-17[The influence of vitamin D deficiency on various inflammatory processes has raised concern in addition to its well-established role in systemic calcium homeostasis. 1,25(OHand IL-17. Increasand IL-17. HoweverIn conclusion, we have extended the mutation spectrum of HVDRR by studying two novel VDR mutations. Our case study demonstrates another example that severe metabolic abnormalities can present without alopecia and contributions of Fok I and a benign p.M4I variant in a HVDRR patient.


GABRB2, a schizophrenia candidate gene coding for GABAA receptor β2 subunit, is examined for possible association with heroin dependence in Han Chinese population. Four single nucleotide polymorphisms (SNPs) in GABRB2, namely rs6556547 (S1), rs1816071 (S3), rs18016072 (S5), and rs187269 (S29), previously associated with schizophrenia, were examined for their association with heroin dependence. Two additional SNPs, rs10051667 (S31) and rs967771 (S32), previously associated with alcohol dependence and bipolar disorder respectively, were also analyzed. The six SNPs were genotyped by direct sequencing of PCR amplicons of target regions for 564 heroin dependent individuals and 498 controls of Han Chinese origin. Interestingly, it was found that recombination between the haplotypes of all-derived-allele  and all-ancestral-allele  at S5-S29 junction generated two recombinants H3 (OR = 8.51) and H4 (OR = 5.58), both conferring high susceptibility to heroin dependence. Additional recombination between H2 and H3 haplotypes at S1-S3 junction resulted in a risk-conferring haplotype H5 (OR = 1.94x109). In contrast, recombination between H1 and H2 haplotypes at S3-S5 junction rescued the risk-conferring effect of recombination at S5-S29 junction, giving rise to the protective haplotype H6 (OR = 0.68). Risk-conferring effects of S1-S3 and S5-S29 crossovers and protective effects of S3-S5 crossover were seen in both pure heroin dependent and multiple substance dependence subgroups. In conclusion, significant association was found with haplotypes of the S1-S29 segment in GABRB2 for heroin dependence in Han Chinese population. Local recombination was an important determining factor for switching haplotypes between risk-conferring and protective statuses. The present study provide evidence for the schizophrenia candidate gene GABRB2 to play a role in heroin dependence, but replication of these findings is required.Substance dependence is a frequently observed comorbid disorder in schizophrenia, but little is known about genetic factors possibly shared between the two psychotic disorders.  Substance dependence is a common comorbid disorder in schizophrenia with the highest abuse rate from alcohol 43.1%-65%), followed by cannabis (50.8%), nicotine 28.5%), and cocaine dependence (23%) ; (2) 1.4 kbp region containing rs187269 (S29)  (4) 0.8 kbp region containing rs967771 (S32) [4.7 kbp upstream to 3.5 kbp upstream of 5’ end of Exon 9]. Specific primers were designed by Primer-BLAST of the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/tools/primer-blast/) with avoid-low-complexity-region-for-primer-selection parameter. Primers with less than five hits in the human genome were chosen as forward and reverse primers. The primers used to amplify these fragments were shown in Genomic DNA was extracted from peripheral blood samples by using DNA purification kit . Four intronic regions in 2), and 1 U Taq polymerase . PCR amplification consisted of an initial denaturation at 95°C for 5 minutes, 30 cycles of 30 seconds at 95°C, 30 seconds at annealing temperature optimum for each primer pair, elongation at 72°C for the length of time adjusted to the size of the desired amplicon, and followed by a final elongation step at 72°C for 7 minutes. A small amount of PCR product was electrophoresed in a 0.7–2.0% agarose gel, stained with ethidium bromide, and was visualized under ultraviolet light (UV) to confirm successful amplification.Nested polymerase chain reaction (PCR) was employed to amplify the target regions. DNA fragment was amplified in a 20 μl reaction mixture containing 10 ng of genomic DNA, 150 nM of each primer, 200 μM of each deoxyribonucleotide triphosphate (dNTP), 1.5 mmol/L magnesium chloride . Sequencing reaction consisted of an initial denaturation at 95°C for 1 minute, 30 cycles of 10 seconds at 95°C, 5 seconds at 50°C, and 4 minutes at 60°C. Products from the sequencing reaction were purified with ethanol using the same protocol. Purified products were then dissolved in 10 μl Hi-Deionized Formamide (Applied Biosystems), denatured at 95°C for 1 minute, and were sequenced using the ABI PRISM® model 3100 capillary DNA sequencer (Applied Biosystems). All SNPs  were genotyped by at least two independent researchers with the CODON CODE ALIGNER software, version 5.0.1 (http://www.codoncode.com/index.htm). Chromatogram reads with low-quality score were re-sequenced. Refer to Amplified PCR products were purified by ethanol. Absolute ethanol was added to the PCR products to a final concentration of 75% and the mixture was stored at -20°C overnight. The mixture was centrifuged at 3,000 rpm for 30 minutes and the resulting precipitate was washed in 70% ethanol for two additional rounds, which was then centrifuged at 3,000 rpm for 20 minutes. The precipitates were air-dried and dissolved in 1X Tris-Cl  and ethylenediaminetetraacetic acid  buffer. Each sequencing reaction was performed in a 15 μl reaction mixture containing ~100 ng purified PCR products, 480 nmol/L sequencing primer, and 0.75 μl BigDyeD’ and r2 [Linkage disequilibrium (LD) was calculated using the Haploview software, version 4.2 . Strengt and r2 –31. LD pHardy-Weinberg equilibrium (HWE) exact test was performed for the disease and control samples using the GENEPOP software, version 4.2 –33. EnumP-value below 0.05 was considered as a statistically significant value for an association. All P-values showing significant associations were then reviewed by the Bonferroni correction (CorrP) and global permutation test . The global permutation test was based on the null hypothesis that all odds ratios of haplotypes were equal and P-value was corrected after 10,000 permutations. Moreover, the resampling method was employed as an additional means to correct the significance of an overall association of the SNPs, as reported in Lo et al., 2007 [CorrP < 0.05) were included in the results section unless otherwise indicated.Disease association analysis was performed using the UNPHASED software, version 3.1.7 . Overalll., 2007 . This meGABRB2 previously reported for their significant association with schizophrenia and positive selection of their derived alleles [The four intronic SNPs in  alleles , 35, nam alleles , 27, res alleles . MissingPatient and control samples with missing genotype information for S1, S3, S5, or S29 or missing gender information have been excluded in this study . After eP > 0.05) and significant deviation from HWE (P > 0.05) was not observed in neither control groups. Thus, the two control groups were combined as one group (CON) in the subsequent statistical analyses.In order to equalize the sample size of the heroin dependent and control groups and exclude differences in geographical origins of subject recruitment, allelic and genotypic comparisons were made between CON1 and CON2 groups . SignifiP > 0.05) in HER and CON groups (P > 0.05). In addition, LD plot of pairwise SNPs in gender-combined combined controls (CON) showed that the four SNPs, S1, S3, S5, and S29, are moderately linked , while LD with S31 or S32 were not linked despite high D’ values   . Moreove≤ 0.122) .PCorr = 8.637x10-13, PGlobal = 0.0001). S1-S3-S5-S29, S1-S3-S29, and S1-S29 were the next strongly associated haplotypes by this order . Interestingly, only haplotypes that contain SNPs in the S1-S29 segment  but not those that contain S31 and/or S32 were significantly associated in all 1,000 resampled datasets (P < 0.05).All haplotypes showing significant association with heroin dependence after the Bonferroni correction and global permutation test contained S29 . The strP > 0.05), all-ancestral-allele haplotype (H2) showed significant association with heroin dependence  . Additional recombination between H2 and H3 haplotypes at S1-S3 junction . Interestingly, additional recombination between H1 and H4 haplotypes at S3-S5 junction created H6 haplotype, which restored the protection from heroin dependence .Given the extremely low p-values of haplotypes, containing S1, S5, S5, and S29, associated with heroin dependence, role of haplotype level recombination in susceptibility to heroin dependence was further examined. Exhaustive analysis of all possible four-SNPs haplotypes was performed to observe the combined effect of the four genetic loci in female + male and male samples. However, minimum haplotype frequency threshold was not used in this analysis to visualize active recombination of common and rare haplotypes. Homozygous derived allele haplotype (H1 haplotype) was included as a reference . While t 0.0010) . Moreove 0.0010)  resultedjunction  exacerbaTo confirm whether the flipping effect of recombination on the susceptibility to heroin dependence observed in the four-SNPs haplotypes was independent of the effect of other two markers, six-SNPs containing all markers employed in this study were analyzed. Consistent with previous findings, H2-H6 haplotypes showed the same trend in the susceptibility to heroin dependence, though only H3 and H4 haplotypes were significantly associated with heroin dependence in both female + male and male samples . In addiEffects of multiple substance dependence in susceptibility to heroin dependence were analyzed in individual 2-, 3-, and 4-SNPs haplotypes containing only S1, S3, S5, and S29. In overall heroin dependent individual (HER) group , risk-coGABRB2 in susceptibility to heroin dependence in the Han Chinese population. Although we failed to show that genetic markers in GABRB2  were associated with heroin dependence in allele and genotype levels (P > 0.05), very significant associations were detected at haplotype level, especially in haplotypes only containing SNPs in the S1-S29 segment   (GABRB2 with susceptibility to heroin dependence.We examined the role of in  0.0001) . In addi 0.0001) , indicatet al., 2004 [et al., 2007 [Results of haplotype level associations support the existence of common genetic predisposition to schizophrenia and heroin dependence. Genetic markers employed in this experiment have been replicated in studies with different populations with schizophrenia ,13. In pl., 2004 , and  as well as in both pure heroin dependence and multiple substance dependence subgroups . InteresComparison of haplotype level associations in heroin dependence suggested that genetic studies focusing on the disease associations in allele and genotype levels could result in incomplete conclusions. Protective odds ratio of the ancestral S1 and S5 alleles  was not GABRB2 have been reported to have a regulatory function of GABAA receptor activity. In particular, the derived alleles of these markers were found to increase the mRNA expression of the total β2 subunit of the GABAA receptor [A receptor consists of the β2 subunit [The four SNPs, S1, S3, S5, and S29, localized in the intronic regions of receptor . As the  subunit –20. Inte subunit , indicatGABRB2 in schizophrenia and heroin dependence. Considering the uniqueness of human recombination hotspots and positive selection of the alleles in GABRB2 [Accordingly, the present study suggested the shared genetic basis of n GABRB2 ,35, genen GABRB2 , it can S1 Dataset(XLSX)Click here for additional data file.S1 Fig(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.S8 Table(DOCX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.


S1 Fig(A) Protein purification of EBOV MS2 VLP. Proteins of various molecular weights were differentiated by gel exclusion chromatography. The target protein of EBOV VLP was obtained around 60mins. (B) The visual results of EBOV VLPs in SDS-polyacrylamide gel electrophoresis. The bands between 10KD and 15KD were EBOV VLPs. Lane M, prestained protein molecular weight marker; Lane 1, EBOV VLP with NP fragments; Lane 2, EBOV VLP with GP/L fragments. (C) Enzymatic resistance of EBOV VLPs. After treated with RNase A and DNase I at 37°C, the single stripe over 1kb of 1% agarose gel electrophoresis proved the enzymatic resistance of VLPs encapsulated with EBOV fragments. Lane M, DL2000 DNA marker; Lane 1, EBOV VLP of NP fragment without incubated with RNase A and DNase I; Lane 2, EBOV VLP of NP fragment incubated with RNase A and DNase I; Lane 3, EBOV VLP of GP/L fragment without incubated with RNase A and DNase I; Lane 4, EBOV VLP of GP/L fragment incubated with RNase A and DNase I. (D) Reverse transcription PCR (RT-PCR) of the segments extracted from EBOV VLPs. After extraction of target EBOV nucleotides fragments encapsulated in EBOV VLPs, RT-PCR and 1% agarose gel electrophoresis were done for identification. The target bands between 2kb to 2.5Kb served as the successful proofs. Lane M, DL15000 DNA marker; Lane 1, positive control for NP fragment; Lane 2, RT-PCR products of RNA extracted from EBOV VLP of NP fragment; Lane 3, positive control for GP/L fragment; Lane 4, RT-PCR products of RNA extracted from EBOV VLP of GP/L fragment; Lane 5 and 6, negative controls.(TIF)Click here for additional data file.S2 Fig(A) The results of EBOV VLP with NP fragment. (B) The results of EBOV VLP with GP/L fragment. In both (A) and (B), all of the samples in panel from 1401 to 1410 were included. In order to distinguish different two sample panels, “*” was marked to the samples from blind mail. For each sample, three duplications were conducted and the distributions were revealed in scatter plot.(TIF)Click here for additional data file.


Measurements of protein abundance changes are important for biological conclusions on protein-related processes such as activity or complex formation. Proteomic analyses in general are almost routine tasks in many laboratories, but a precise and quantitative description of (absolute) protein abundance changes require careful experimental design and precise data quality. Today, a vast choice of metabolic labeling and label-free quantitation protocols are available, but the trade-off between quantitative precision and proteome coverage of quantified proteins including missing value problems remain. Here, we provide an example of a targeted proteomic approach using artificial standard proteins consisting of concatenated peptides of interest (QconCAT) to specifically quantify abiotic stress-induced abundance changes in low abundant vacuolar transporters. An advantage of this approach is the reliable quantitation of alimited set of low-abundant target proteins throughout different conditions. We show that vacuolar ATPase AVP1 and sugar transporters of the ERDL (early responsive to dehydration-like) family and TMT2 (tonoplast monosaccharide transporter 2) showed increased abundance upon salt stress. Measurements of protein abundance changes allow conclusion about post-transcriptional regulatory processes, for example through correlation with protein activity or complex stoichiometry. Quantitative proteomic analyses are routine tasks in many experiments, and a large selection of methods using metabolic labeling or label-free approaches is available reviewed in A common workflow to address this problem is to firstly perform profiling experiments, then, secondly to define a list of relevant target proteins/peptides which will then be studied using dedicated targeted protein analysis methods . Such woThe vacuole is an important plant specific organelle with functions in storage of solutes as nutrient reservoirs, but also with important roles in adaptation to stresses, such as cold stress , salt stArabidopsis various abiotic stresses, especially cold stress, lead to accumulation of sugars, particularly glucose and fructose in the vacuole . Only in very low H/L mixing ratios, the measured ratios were lower than expected suggesting that under these conditions, the signal to noise ratio of the full scan spectra did not fully resolve the very low presence of unlabeled peptide. Different peptides showed slightly different curves reflecting variations in ionization properties.Unlabeled  and labeled  QconCATs were mixed in different ratios  to result in a total of 10 μg of protein before tryptic digest. After tryptic digestion and desalting, 5 μg of each mixture was analyzed by LC–MS/MS. The measured H/L ratio of each mixture of labeled and unlabeled peptides showed a linear relationship with the expected H/L ratio provided by the peptide mixture . Based on these results, in further experiments a mixing ratio or protein:QconCAT of 5:1 was used to ensure efficient coverage of the target peptides in the mass spectrometric analysis.To determine the limit of detection within a complex protein background, labeled QconCAT protein was spiked into different amounts of microsomal protein preparations resulting in w/w protein:QconCAT ratios of 5:1, 10:1, and 20:1. After joint tryptic digestion and desalting of these spiked protein mixtures, 5 μg of each mixture was analyzed. As expected, under conditions of the QconCAT protein being mixed with lower amounts of the microsomal protein, higher spectral counts were achieved for each of the QconCAT peptides resulting from the tryptic digestion . These results confirm that our chromatography and subsequent retention time alignment of raw data is very reproducible and that ion intensity (or ion intensity ratio) quantitation is more robust and less prone to variation than spectral counting.Next, we analyzed the reproducibility of QconCAT detection. Thus, a total of 25 μg of QconCAT protein was in-solution digested, desalted and injected five times in 5 μg injections for mass spectrometric analysis. The five injections were done either from different wells on the autosampler plate or repeatedly sampled from the same well. To determine the reproducibility between runs, detected peptides were analyzed according to variation in peak intensities, retention times and the number of recorded spectra. Across all peptides and the five injections, retention time variations from run to run was extremely low. In contrast the ion intensity variation averaged around 0.2 rsd, and the spectral counts showed largest variation (Supplementary Table S1). The peptides were chosen with high probability for good ionization properties . Using the L/H ratio, with constant amount of the heavy standard peptide (H), the ratio directly is proportional to the abundance ratio of plant-derived peptide (L) compared to its standard. Comparing two different conditions (stress treatment and control), the change in L/H ratio depicts the change in protein abundance induced by the stress condition  and the ion intensities of the spiked-in standard can be used for normalization between different samples. Thus, a change of the L/H ratio to higher (more positive) values is proportional to an increase in abundance of the peptide (and protein) while a ratio change to lower (more negative) values is proportional to a decrease of peptide and protein abundance .The QconCAT protein was spiked into vacuolar preparations of plants grown under salt stress or drought stress and respective control conditions. We were particularly interested in following the changes in protein abundance of several vacuolar sugar transporters which play an important role in adjustments of cellular solute concentrations under these stress conditions. The abundance changes in sugar transporter abundance were based on changes of proteotypic signature peptide ratios in stressed plants compared to their control conditions (operties  or basedoperties  or from operties . We thenSupplementary Table S1). However, each peptide was produced to the same molar amount: the 1 μg spiked-in QconCAT corresponded to 13 fmol of protein. With knowledge of the molar amount of standard added to the sample, the molar amount of each peptide can be calculated as follows: L/H * (mQconCAT/MWQconCAT). Such calculations revealed AVP1 as the most abundant protein with 45 ± 31 mmol/gprotein and VMA21a and VMA22 as the least abundant proteins with 0.3 ± 0.12 mmol/gprotein. Among the sugar transporters, ERDL4 and TMT2 were most abundant averaging 24 ± 3.8 mmol/gprotein and 5.8 ± 2.5 mmol/gprotein, respectively.The amount of the spiked in standard protein was 1 μg, but after tryptic digestion within the mixture with the vacuolar protein, each standard peptide was produced to different absolute amounts according to the molecular weight of each peptide, ranging from 10 to 34 ng in the final digested 5:1 mixture of protein:QconCAT . Thereby, in some cases, such as for high abundant proteins AVP1 (AT1G15690), ERDL4 (AT1G19450) and COPT5 (AT5G20650), the endogenous peptides (L) was in all cases more abundant than the spiked in standards (H) leading to large L/H ratios across conditions. In other cases, such as for low abundant VMA21a (AT2G31710), VMA22 (AT1G20770), ERDL7 (AT2G48020), and ERDL8 (AT3G05150), the endogenous peptides (L) were less abundant than the spiked-in standard (H) leading to low L/H ratios across conditions.The L/H ratios in control conditions, salt stress (400 mM NaCl) as well as control and drought conditions could be derived for several vacuolar transporters (Table 1). Among the sugar transporters, significant and large increases in abundance were observed also for ERDL7 (AT2G48020) and TMT2 (AT4G35300). ERDL4 (AT1G19450) and ENT1 (AT1G70330) were the only sugar transporter for which no significant change in abundance was measured under salt stress. Under drought stress, for most proteins a mild decrease in protein abundances was observed (Table 1). These mild changes were significant only for AVP1 (AT1G15690), ERDL7 (AT2G48020), and ENT1 (AT1G70330) which decreased from vacuolar membranes. A significant drought-induced increased abundance was observed for ERDL4 (AT1G19450) and COPT5 (AT5G20650).Under salt stress, we observed an increase of protein abundance for most of the monitored proteins. Particularly, for AVP1 (AT1G15690) abundance significantly increased almost threefold when plants were exposed to salt stress of 400 mM NaCl (Table 1). Only for COPT5 (AT5G20650) under drought stress, an increased abundance was measured based on the QconCAT quantitation while the label-free quantitation resulted in a decreased abundance.The measured abundance changes using the QconCAT-derived standard peptides were confirmed by an independent label-free quantitation using the same raw files. The very low abundant proteins VMA21a (AT2G31710), VMA22 (AT1 G20770), and ERDL8 (AT3G05150) could not be quantified in the label-free analysis. Overall, in the label-free analysis the abundance changes resulted in smaller amplitudes for some proteins, but with similar trends as in the ratio quantitation  were reproducibly detected in replicates of control and stress treatment samples allowing solid quantitation in both conditions.In the past years, several methods for absolute protein quantitation strategies were developed, such as emPAI , APEX B, the TOPr = 0.89, r2 = 0.81) with the iBAQ values calculated from a label-free analysis.The absolute quantitation of protein amounts using spiked-in standard peptides is most likely to be less accurate due to lower statistical power of only few peptides, and these peptides may not necessarily be among the most optimal peptides which otherwise are for iBAQ or TOP3 quantitation. It has been shown that quantitation using standard peptides particularly depends on good design of the peptides , and expThe observation that TMT2 protein abundance decrease during drought is surprising, given the high monosaccharide import specificity of latter carrier  and giveTable 1 and see Results) the transport characteristics have been determined  transporters. We demonstrated that the reproducibility of QconCAT detection is very high and that ion intensity ratio quantitation is more sensitive and more and robust than spectral counting.The use of these spiked-in standards clearly does exceed the range of quantitation to include more low-abundant proteins compared to a simple label-free experiment. We showed that salinity stress results in large changes in vacuolar transporter abundances, particularly for vacuolar ATPases AVP1, members of the ERDL-transporter family as well as for the monosaccharide transporter TMT2. Since salinity is known to increase the sugar content in vacuoles , we suggEscherichia coli BL21 (DE3) with the genotype B F-dcmompThsdS(rB-mB-) galλ(DE3) were purchased from Agilent Technologies. The selected QconCAT peptides (see Supplementary Table S1) were concatenated and flanked by a leader N-terminal sequence (MAGK) and a C-terminal sequence (LAAALEHHHHHH) for purification . The full QconCAT1 protein sequence is:Competent MAGKCLTAVFMLEDEKATILFDFWKHKPDASFVAEAKSLS VFGGLVSPKFSPATFSPVSSEDSNLRQSSMMGSSQVIRHGSLA NQSMILKQTTSMDKGVVALDLGRLYGTHENQSYLARPVPEQ NSSLGLREGWFDLASARDFSTDTDSEIGSPLSPQLRGTFSQLD NLSMANITKHKPDAAFLAEAKLFNLLETSHKDNDDYATDDG AGDDDDSDNDLRLLLMISSIGMTISLVIVAVAFYLKSSGEISPER EPLIKSGNAPIYYPNREATFGELFRITAAQVLEHPWIKSFTQSS PSFTQSSPSFTPTTKTGIPTALWDLMEPYAKVSAVSLSEEEIKM NGGDVTVAGSDDLKMGLTDDFETSLQVLRGATSSDDHALK VTDFGLSAFIEEGKNITDDIPGWGRLTSGVTNTDPALKLAMG PLCDLIGPRADISEEAAEIQDYIETLERSFLALASGDRGGSTMS VLSRQIQMTTTDKMALDPEQQQPISSVSRSSSGVSAPLIPKNT KPPSFSDSTIPVDSDGRQFNTIPGLMEGTAKPDYATCVKGEA SGSVFFIDGSNNQYLRPRLTSDLGASSSGGANNGKIYLHQEGF PGSRRPFIHTGSWYREIGTGNIYACKVSILLGLLKEAGEIQEYL ASLAHLPKAAVIGDTIGDPLKDPLVNLFGSLHEKHSDFEIALQ KLAAALEHHHHHH.NdeI/BamHI restriction sites of pET21a . A list of tryptic peptides and corresponding proteins they were derived from is shown in Supplementary Table S1.The plasmids encoding the proteins for QconCAT1were cloned into the E. coli using a standard transformation protocol, and frozen stocks were stored at -80°C. The QconCAT protein was expressed in E. coli cultures with a full complement of unlabeled amino acids  and [13C6]lysine (10 mg 100 ml-1). Expression was induced with IPTG , and after 5 h the cells were harvested by centrifugation at 4,000 rpm for 15 min at 4°C. Inclusion bodies containing the QconCAT were recovered by breaking the cells using BugBuster Protein Extraction Reagent  and after several washing steps centrifuged at 16,000 × g for 15 min at 4°C. For purification of the labeled and unlabeled QconCATs, the pellets of inclusion bodies were resuspended in IMAC binding buffer  and loaded onto gravity flow Ni2+-NTA sepharose columns . After several washing steps the bound QconCAT was eluted with elution buffer  in 5 ml × 1 ml fractions. Purified QconCATwas desalted by dialysis  against 100 volumes of 50 mM ammonium bicarbonate, 1 mM DTT for 3 h × 2 h at 4°C. Finally, the protein content was determined using BSA as a standard . To ensure stability, purified QconCAT was aliquoted into 10, 25, 50, and 100 μg samples and dried down in a speed vac to completely dryness and stored at -80°C. Before use, QconCAT protein was resuspended in 6 M urea, 2 M thiourea, pH 8 in Tris-HCl to result in a final concentration of 1 μg μL-1.Subsequent transformation, protein expression, and purification were performed as described in For in-solution digest the purified QconCAT protein (5 or 10 μg) was denatured using UTU buffer . After reduction in 6.5 M DTT and alkylation of cysteine residues by 27 mM iodoacetamide, proteins were digested for 3 h by LysC  at room temperature. The solution was then diluted fourfold with 10 mM Tris-HCl, pH 8.0 followed by overnight digestion with trypsin  at 37°C at 350 rpm. Finally, digested peptides were desalted over C18 STAGE- tips and dried down in a speed vacuum concentrator and stored at -80°C. For mass spectrometric analysis samples were resuspended in resuspension buffer .® RSLC C18, Thermo Scientific) on a gradient using 0.5% acetic acid as aqueous phase and 0.5% acetic acid in 80% acetonitrile as organic phase. The flow rate was set to 250 nL per minute. Peptides were eluted on a linear gradient running from 4 to 64% acetonitrile in 135 min. Spectra were using information-dependent acquisition of fragmentation spectra of multiple charged peptides within the m/z range of 300–1600. Up to 12 data-dependent MS/MS spectra were acquired for each full-scan spectrum acquired at 70,000 full-width half-maximum resolution. Fragment spectra were acquired at a resolution of 35,000. Masses of peptides of QconCAT1 were used as inclusion list to induce preferred fragmentation of the target peptides of interest.Tryptic peptide mixtures were analyzed by LC–MS/MS using nanoflow HPLC  and a hybrid quadrupole-orbitrap mass spectrometer  as a mass analyser. Peptides were eluted from a 75 μm × 15 cm C18 analytical column  using Andromeda (Protein identification and ion intensity quantitation was carried out by MaxQuant version 1.4.1.2 . Spectrandromeda  and the Arabidopsis seedlings (approximately 20 g of fresh weight) from Col-0 were smashed into small pieces and resuspended in ice-cold homogenisation buffer , protease inhibitor cocktail (Sigma-Aldrich), phosphatase inhibitor cocktail 2 (Sigma-Aldrich) and phosphatase inhibitor cocktail 3 (Sigma-Aldrich) were added from stock solutions (50 μL per 10 mL of the homogenization buffer just before use). Tissue was homogenized with a Teflon Potter-Elvehjem on ice. The homogenate was filtered through a 21 μm nylon mesh, and centrifuged at 7,500 × g for 15 min at 4°C. Finally, the supernatant was centrifuged at 48,000 × g for 80 min at 4°C. The resulting pellet was the MF and stored at -80°C.Microsomal fractions (MFs) of seedling cultures were prepared by differential centrifugation . Frozen Arabidopsis plants were cultivated for 28 days under short day conditions  at 22°C. To achieve salt stress the plants were afterward watered using tap water containing 400 mM NaCl at day 29 and at day 31. Vacuoles/tonoplasts were isolated from these plants at day 32. To generate drought stress, watering of the corresponding plants was stopped at day 28 and vacuoles/tonoplast isolation was started when dehydration symptoms were monitored . Vacuoles and tonoplast membranes were isolated following exactly the protocol described (escribed . IsolateHP-O: performed lab experiments, analyzed the data, wrote the paper. KD: performed the salt stress experiments. OT: performed drought stress experiments, sample preparation, contributed to paper writing. HN: contributed significantly to paper writing. WS: conceived the idea and wrote main parts of the paper.http://journal.frontiersin.org/article/10.3389/fpls.2016.00411The Supplementary Material for this article can be found online at: TABLE S1TABLE S1 | The peptides on QconCAT1 which was used as a spiked-in standard protein in this study.Click here for additional data file.Click here for additional data file.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Plasmodium falciparum cGMP-dependent protein kinase (PfPKG) is a key regulator across the malaria parasite life cycle. Little is known about PfPKG’s activation mechanism. Here we report that the carboxyl cyclic nucleotide binding domain functions as a “gatekeeper” for activation by providing the highest cGMP affinity and selectivity. To understand the mechanism, we have solved its crystal structures with and without cGMP at 2.0 and 1.9 Å, respectively. These structures revealed a PfPKG-specific capping triad that forms upon cGMP binding, and disrupting the triad reduces kinase activity by 90%. Furthermore, mutating these residues in the parasite prevents blood stage merozoite egress, confirming the essential nature of the triad in the parasite. We propose a mechanism of activation where cGMP binding allosterically triggers the conformational change at the αC-helix, which bridges the regulatory and catalytic domains, causing the capping triad to form and stabilize the active conformation.The  Plasmodium falciparum, which has a complex life cycle in both humans and mosquitoes. One key regulator of this process is P. falciparum cGMP-dependent protein kinase (PfPKG), the main effector of the cGMP-signaling pathway. Specifically blocking this kinase stops both replication and transmission of the parasites, suggesting that PfPKG is a promising drug target. Here we identified the carboxyl cGMP-binding domain of PfPKG serving as a gatekeeper for activation of the entire kinase by having the highest affinity and selectivity for cGMP. High-resolution crystal structures with and without cGMP allowed us to identify a novel cGMP capping triad that dynamically forms upon binding cGMP and stabilizes the activated conformation. Mutation of the capping triad forming residues not only reduces its kinase activity, but also prevents blood stage merozoite egress, demonstrating its crucial role in PfPKG activation.Malaria causes up to a million fatalities per year worldwide. Most of these deaths are caused by  Plasmodium falciparum has a complex life cycle comprising phases in both a human host and a mosquito vector [The malaria parasite o vector –3. Once Plasmodium life cycle [P. falciparum cGMP-dependent protein kinase (PfPKG) is involved in both asexual and sexual development [PfPKG blocked progression of the life cycle at multiple stages [The complex malaria parasite life cycle is a highly regulated process, but the molecular details are poorly understood. Several biochemical and genetic studies indicate that cyclic nucleotides play a critical role at a number of stages in the fe cycle ,5. Recenelopment –8 and spe stages ,7,9.P. falciparum expresses a single PKG that has similar functional domains to mammalian PKG (mPKG), but exhibits some fundamental differences in domain organization. PfPKG contains an N-terminal regulatory (R) domain including a possible auto-inhibitory (AI) sequence and a C-terminal catalytic (C) domain [PfPKG is larger than mPKG and does not contain an N-terminal dimerization domain, thus functioning as a monomer , whereas mPKG has only two (mCNB-A and B) [PfCNB-C is degenerate since cGMP binding could not be detected [Phosphate Binding Cassette (PBC), binds the sugar-ribose portion of cGMP and consists of a short helix (P-helix) followed by a loop.) domain . Both th monomer . MoreoveA and B) . A previdetected . The CNBdetected . EmbeddePfCNB-D has been shown to have the greatest effect on kinase activation [PfPKG activation was analyzed. Disabling PfCNB-D resulted in a 10-fold increase in the activation constant for cGMP (Ka:cGMP) and as much as a 50% reduction in maximal activity. In contrast, disabling PfCNB-A, B, or C showed little to no effect. Since little structural information is available for mPKG and none for PfPKG, the current model of activation is based on solution-based studies of mPKG [PfCNB-D in the presence and absence of cGMP. The data reveals not only the structural basis of cGMP selectivity, but also a PfPKG-specific capping triad that allosterically triggers kinase activation. Mutagenesis of the capping triad residues impairs kinase activation and abrogates P. falciparum blood stage merozoite egress.Cyclic GMP binding to tivation . In this of mPKG ,15. The PfPKG CNB domains have been shown to influence kinase activation to varying degrees [E. coli ∆cya TP2000, which lacks adenylyl cyclase activity [PfCNB-D domain binds cGMP with a half maximal effective concentration (EC50) of 38 nM, while the A and B domains bind cGMP with EC50 values ranging from 1 to 1.2 μM   . Additioormation .. While ifferent ,19. The  termini ,20. StruPfCNB-D:cGMP complex shows that, while the overall structure of its cGMP pocket is similar to that of the human PKG I CNB-B, its capping mechanism is completely different and the pocket is highly shielded from solvent due to the fully ordered C-helix  (Ka:cGMP) from 66 nM to 2–3 μM, whereas mutating D533 showed only a slight increase (to 330 nM) (Ka:cGMP over 28-fold (to >1.8 μM) and reduced its maximum activity by 90%  . and meas 1–853) . Singly  330 nM)  Tables. y by 90% . In summPfPKG by occupying a hydrophobic pocket adjoining the ATP-binding domain, the T618Q mutant has a bulkier side chain that prevents inhibitor binding and is no longer sensitive to compound 2 retaining kinase activity.To determine the role of the capping triad on malaria parasite development, we utilized a chemical genetic approach employing a selective PKG inhibitor  and a transgenic parasite line expressing an engineered PKG allele  that confers inhibitor resistance . While cP. falciparum transgenic lines containing an ectopic copy of the full length HA-tagged pkg gene harboring the T618Q mutation, but also containing either the R484A mutation (PKGT618Q/R484A-3xHA), the R484A/Q532A/D533A triple mutation (PKGT618Q/R484A/Q532A/D533A-3xHA) or no additional mutation (PKGT618Q-3xHA) as a control. The HA-tagged pkg genes were integrated as a single copy into a modified pseudogene locus via Bxb1 integrase mediated attB/attP recombination , in which the gatekeeper mutation was previously introduced by allelic replacement  whereas the PfPKGT618Q-3xHA control was able to grow at a similar rate to the allelic replacement control line of PfPKGT618Q . While there have been several studies reporting binding affinities of mPKG [PfPKG prior to this study were unknown. Previous mutagenesis studies of PfPKG and its orthologue from Eimeria tenella (EtPKG) showed that, while all CNB domains are required for maximal activation, disabling the CNB-D domain had the largest effect on activation [PfCNB-D having the highest affinity and selectivity for cGMP , F434 (αA-helix) and F455 (3), together with the hinge residues, L486 (αP-helix) and F513 (αB-helix) as previously defined in the CNB domain of Epac2, form a hydrophobic core that stabilizes the apo conformation [Structural comparison of the apo and cGMP bound states reveals two distinct sets of hydrophobic networks that link the PBC to the rest of the CNB domain. Careful analysis suggests that these networks transmit cGMP-induced changes at the PBC to the rest of the helical regions and allow er Figs. , 4 and 5er Figs. . The α31BC Figs.  and S4A.ormation .10-helix (I418 and F419) no longer interacts with L486 at the PBC, but slides along strand β3 and forms new interactions with I457. Finally, the αC-helix moves close to the cGMP pocket and forms the capping triad shielding the bound cGMP (10-αA helices and the αB-αC helices) helps form this Velcro-like interaction (10 and αA loop) stabilizes the flexible αB-αC loop with G519 and orients the N-terminal region of the αC-helix for the Velcro-like interaction  and hydrophobic interactions that shield the bound cGMP, although the nature of the latter interactions differs  [PfCNB- A and B have either the same or chemically similar residues that can recognize cGMP at the first three sites whereas PfCNB-C lacks most of these residues  . Sequencresidues .. These PfCNB-D is more ordered and shields the entire base of the β barrel through a Velcro-like interaction. Moreover, while a single aromatic residue at the αC-helix provides the capping interaction in human PKG Iβ, the triad formed by R484/Q532/D533 provides the capping interaction in PfPKG. Although these structures only represent a small portion of the R-domain and may suffer from artifacts associated with using truncated domains for crystallization and having different crystal packing environments, the dramatic conformational changes at the αC-helix are observed in both structures suggesting its highly dynamic nature. Since the activity of PKG is allosterically regulated by the structural rearrangement within its R-domain upon cGMP binding and the αC-helix is located between the R- and C- domains [While similar conformational changes were observed in the helical subdomains of the human PKG Iβ CNB-B, these changes at the αC-helix and the capping mechanism are very different between the two structures ,19. The  domains ,20, we hPfCNB-D and the full-length R domain, with D533A having the least reduction. The same trend is observed for kinase activation where major reductions were seen in R484A or Q532A as expected, but only a slight reduction in D533A. Mutating all three showed the highest reduction both in cGMP affinity and in activity compared to wild type. These results suggest that the interaction between R484 and Q532 plays a bigger role in cGMP binding and activation than the R484-D533 interaction. Possibly, this is because Q532 is located on the inner side of the αC-helix and is directly involved in the capping interaction with cGMP. In contrast, D533 is located on the outer surface of the αC-helix with its side chain exposed to solvent contributing much less to the capping interaction.Our binding and activation results strongly support this hypothesis. Reductions in cGMP binding affinity were seen for all individual mutants, R484A, Q532A, or D533A of PfPKG plays an essential role in egress of blood stage merozoites from erythrocytes [PfPKG prevents the release of proteins from apical organelles required for egress and reinvasion [PfPKG is the universal regulator of calcium release across the malaria parasite life cycle, which is achieved by phosphorylation of phospholipid kinases and generation of IP3 [PfCNB-D domain, abrogates PKG’s ability to promote blood-stage egress, reinvasion, and signaling through downstream effectors, further strengthening the conclusion that the capping triad formation is an integral part of the activation mechanism. Thus, we propose that the αC-helix serves as an allosteric switch that triggers activation upon cGMP binding. Additionally, the αC-helix, strategically located between the R and C domains functioning as a molecular switch for activation, is likely to be a common feature of all PKGs, though the molecular details of the capping may differ due to the highly divergent amino acid sequence in this region. Amino acid sequence comparisons with other apicomplexan parasitic PKGs strongly suggest that the capping triad is a shared feature that could be targeted for developing broad-spectrum inhibitors against this family of important parasites , 500 mM NaCl and 1 mM β-mercaptoethanol, and lysed using a cell disruptor (Constant Systems).A manually designed construct consisting of r, pQTEV . In desir, pQTEV . The domPfPKGs were eluted by linear gradient with cell suspension buffer containing 300 mM imidazole. To remove the His-tag, the sample was incubated with His-tagged tobacco etch virus (TEV) protease in a 50:1 molar ratio at 4˚C overnight and loaded onto Bio-Rad IMAC column for TEV separation. The sample without his-tag was obtained by collecting flow-through fractions. The sample was loaded onto a HiLoad 16/60 Superdex 75 gel filtration column  equilibrated with a buffer containing 25 mM Tris (pH 7.5), 150 mM NaCl, and 1 mM TCEP. For the purification of wild type and mutant full-length PfPKG, we performed an additional chromatography step using a Resource Q anion exchange column  before the final size exclusion step using a Hiload 16/60 Superdex 200 column .The lysate was cleared using ultracentrifugation and the supernatant was loaded onto an IMAC nickel column (Bio-Rad) on ÄKTA purifier . The N-terminal his-tagged PfCNB-D:cGMP complex, the protein sample was pre-incubated with 5 mM cGMP and concentrated to 50 mg/mL using a 10 kDa cutoff Amicon Ultra (Millipore). Crystals were obtained using the hanging drop method. 2 μL of protein solution was mixed with 2 μL of reservoir solution containing 0.2 M lithium sulfate, 15% ethanol, 0.1 M citrate at pH 5.5 and 10% 1,5-diaminopentane dihydrochloride. After three days incubation at 4˚C, drops produced rod-shape crystals belonging to a P3121 space group that diffracted to 2.0 Å resolution. Apo crystals were obtained at 22˚C in a solution containing 0.2 M lithium sulfate, 25% (w/v) PEG 3350 and 0.1 M Bis-Tris (pH 5.5). The apo crystal belongs to a P212121 space group and diffracted to 1.89 Å resolution. All protein concentrations were measured using Bradford assay. Crystals of the cGMP complex and the apo form were cryoprotected with 25% ethylene glycol and Paratone-N, respectively, before freezing.To obtain crystals of the Coot [PfCNB-D:cGMP structure was accomplished using the molecular replacement program Phaser-MR [PfCNB-D (residues 404–518) as a search model. Manual building of the model was also performed using Coot [Diffraction experiments were performed at the Advanced Photon Source . Diffraction data were processed and scaled using iMosflm  with satCoot , followeCoot . Phasinghaser-MR  using a ing Coot , followeing Coot . The fining Coot .D values were calculated from sigmoidal dose-response curves.The direct fluorescence polarization (FP) assay was performed following the procedure from Moll et al . Measure50 values were calculated from sigmoidal dose-response curves.For competition FP experiments, the protein concentration and the concentration of 8-Fluo-cGMP were fixed to give a polarization signal that was 50% of the maximum value obtained from direct FP measurements. The protein/8-Fluo-cGMP mixture was incubated with varying concentrations of unlabeled cGMP or cAMP . FP signals were detected as indicated above. Data were analyzed with GraphPad Prism 5.03 by plotting the polarization signal in mPol against the logarithm of the cyclic nucleotide concentration. The ECPfPKG was incubated for 2 hr at 25°C in a 384 well assay plate  in 20 μl buffer , 1 mM ATP, 10 mM MgCl2) and various concentrations of cGMP (3 nM—5 mM), respectively. Reaction mixtures without cyclic nucleotide were used as controls. For electrophoretic separation of substrate and product, a ProfilerPro LabChip  was used under the following conditions: downstream voltage-500 V, upstream voltage-1,900 V with a screening pressure of-1.3 psi. Substrate conversion was plotted against the logarithmic cyclic nucleotide concentration and activation constants (Ka) were calculated from sigmoidal dose-response curves employing GraphPad Prism 5.03.Kinase activity was determined using a microfluidic mobility-shift assay on a Caliper DeskTop Profiler . P. falciparum lines 3D7/attB [3D7/attB , 3D7/PfP3D7/attB , and 3D73D7/attB  were cul3D7/attB  in human3D7/attB ring stage parasites were co-transfected with the pPfPKG/attP expression plasmid and the bxb1 integrase plasmid (pINT) to facilitate recombination of the attP plasmid with the attB site integrated into the cg6 pseudogene locus as described . The folParasite synchronization was achieved by repeated sorbitol treatments. Highly synchronous parasites were obtained by magnet purification of mature schizonts  followed by the addition of fresh erythrocytes and a sorbitol treatment three to five hours later.PfPKGT618Q [BamHI and HpaI and replaced with a BglII-PstI-NotI-SpeI-BamHI linker  tag and the PbDT 3’UTR was amplified from pHH1-PKG-HA-3’UTRp.falc [SpeI gatcACTAGTTTACGATGTTCCTGACTATGC and 3xHA+PKG 3'UTR rev BamHI gatcGGATCCCCAACACCATTCAGAGGTTTA and cloned into the BamHI and SpeI sites of pDCattP-linkerI to make pDCattP-3xHA. The AMA1 promoter/5’UTR was amplified from pAMA1–5'-Sub2-HA3Rep20 [BamHI agtcGGATCCCAAAGAAGAAGCTCAGAGATTGCA and ama1 5’ rev NotI-PstI ttttGCGGCCGCttttCTGCAGTCGAGGGCCCTTTTGTACAAT, and cloned into pDCattP-3xHA via BamHI and NotI sites to make pDCattP-ama1 5’-3xHA. A BglII/NotI/SpeI/XhoI/PstI linker was then cloned into the BamHI and PstI site of pDCattP-ama1 5’-3xHA using annealed oligos BglII/NotI/SpeI/XhoI/PstI linker F GATCTGCGGCCGCACTAGTctcgagCTGCA and BglII/NotI/SpeI/XhoI/PstI linker R GctcgagACTAGTGCGGCCGCA to yield pDCattP-ama15’linkerII-3xHA. The resulting plasmid was then cut with PstI and SpeI in the linker and full-length PKGT618Q PCR products amplified from pTRC-PfPKGT618Q  with primers PKG-fwd-PstI agctCTGCAGATGGAAGAAGATGATAATCTAAAAA AAG and PKG-rev-NheI agctGCTAGCAAAATCTATGTCCCAGTTGTCTTC introduced. The PKGT618Q-3xHA gene in the final constructs was sequenced again.The R484A single and R484A/Q532A/D533A triple mutations were introduced into pTRC-PKGT618Q ,38. The TRp.falc  with priHA3Rep20   CCAGGATCCAAAAGAAGGAGGAGG, P2 (blasticidin deaminase 5’ coding) ATGCATGCCAAGCCTTTGTCTCAAG, P3 (hrp2 3’UTR reverse) TATGTATTTTTTTTGTAATTTCTGTG, P4 (PcDT 5’UTR forward) ATACACTTTCCTTTTTTGTCACT, P5 (M13 reverse (-48)) AGCGGATAACAATTTCACACAGGA, P6 (PF3D7_1222600 forward) GTGAATAATGCAAATCAAACTG, P7 (PF3D7_1222600 reverse) AATATTCCTGTTGTTTCCCCCTTTGTGG. All PCR amplifications were run for 30 cycles using Phusion High-Fidelity DNA polymerase (New England Biolabs) and the manufacturer’s recommended profiles.PfGAPDH 1:15000 . HRP-coupled anti-mouse, rabbit, and rat secondary antibodies (DAKO) were used at 1:6000.Schizont stage parasite cultures were harvested by centrifugation, erythrocytes lysed with 0.15% saponin in PBS and the parasite pellet washed twice in PBS. Parasites were resuspended in ice-cold lysis buffer  and incubated on ice for 20 minutes. All buffers were supplemented with complete protease inhibitor cocktail (Roche). The samples were centrifuged at 16000 g for 20 minutes at 4°C and the supernatant assayed by SDS-PAGE/western blot. Proteins were resolved on a NuPAGE Novex 4–12% Bis-Tris gel (Lifetech) and blotted on to nitrocellulose membrane. The membrane was blocked with 5% milk in PBS and probed with the following antibodies: anti-human PKG 1:2500 ; anti-HA 3F10 1:5000 ; anti P. falciparum cultures were synchronised by repeated sorbitol lysis, then either compound 2 (1μM) or DMSO was added to late trophozoites / early schizonts and cultures harvested 16h later to count newly formed ring stage parasites. Cells were fixed in 4% formaldehyde/0.1% glutaraldehyde in PBS for 30 minutes at room temperature, stained with SYBR green (Lifetech) diluted 1:10000 in PBS for 30 minutes, washed with PBS and analysed on a FACSCalibur cell analyser (Becton Dickinson). FACS data were analysed using FlowJo software. For growth assays, starting parasitaemia was 0.2% ring stages and cultured in presence or absence of compound 2 (1 μM). Samples were taken every 48h and fixed, stained, and analyzed as described above.PfCNB-D:cGMP complex and apo structures have been deposited in the Protein Data Bank (http://www.pdb.org) under accession numbers 4OFG and 4OFF.Atomic coordinates and structure factors of the S1 FigPfCNB-D are marked with arrows.Conserved residues are shaded in yellow (100% of identity), in green , and in gray (over 50% of identity). The capping motif forming residues are typed in red. The key cGMP contact residues in (EPS)Click here for additional data file.S2 FigThe αC helix from one molecule (Molecule A’) makes several contacts with the PBC region of a neighboring molecule in the crystal lattice (Molecule A). Molecule A is shown with its N3A motif colored in deep teal, the β barrel region in dark wheat, and the αB-C helices in red. Molecule A’ is shown with its PBC colored in yellow and the rest in gray. The sulfate ions are colored with carbon in yellow and oxygen in red. A zoomed in view of the crystal contact is shown on the right. The surface of Molecule A is rendered in mesh and that of Molecule A’ in transparent surface.(EPS)Click here for additional data file.S3 FigFo|-|Fc| omit map is contoured at σ = 1.0. The cGMP interacting residues and cGMP are labeled and colored by atom type  except for the carbon atoms. The carbon atoms belonging to the PBC are colored in yellow, the β4-β5 strands in dark gray, αC helix in red, and cGMP in white.The bound cGMP and cGMP pocket residues are shown in stick with electron density. A |(EPS)Click here for additional data file.S4 FigPfCNB-D:cGMP complex are aligned at the PBC. The hydrogen bond between cGMP and A484 is shown with a dotted line.(A) A hydrophobic core formed in the apo structure. Individual hydrophobic core forming residues are shown with transparent surface. The conserved hydrophobic hinge residues are marked with a dotted line. (B) Structures of the apo and (EPS)Click here for additional data file.S5 Fig10-αA and the αB-αC loops mediated through ordered water molecules. The secondary structures are shown with yellow coil. The residues that participated in the network are shown with the following color scheme: side chain carbon, black; oxygen, red; nitrogen, blue. The ordered water molecules are shown as blue spheres.An extensive hydrogen-bonding network is formed between the α3(EPS)Click here for additional data file.S6 FigThe wild type and mutant proteins elute at the same volume from a size exclusion column, suggesting that their stokes radius  are very similar.(EPS)Click here for additional data file.S7 FigT618Q, pDCattP_PKG-HAT618Q/R484A, or pDCattP_PKG-HAT618Q/Triple, respectively, were co-transfected into the 3D7/attB line. A) Schematic of the plasmids, the parental cg6/attB locus and the modified cg6/attB locus after integration of the pDCattP_PKG-HA plasmid. B) PCR analysis of transfectants to confirm integrase-mediated recombination via attB/attP. PCR templates used were plasmid prep from pDCattP_PKG-HAT618Q (lane 1), genomic DNA from parental 3D7/attB (lane 2), genomic DNA from transfectants 3D7/PKG-HAT618Q, 3D7/PKG-HAT618Q/R484A, and 3D7/PKG-HAT618Q/Triple (lanes 3 to 5). Primer binding sites and expected amplicon sizes for P1 to P5 are indicated in A). Primer pair P6/P7 amplifies the 3’ end of PF3D7_1222600) and serves as a genomic DNA control.Plasmids pINT and pDCattP_PKG-HA(EPS)Click here for additional data file.S8 FigT618Q (light grey squares), 3D7/PKG-HAT618Q/R484A (mid grey triangles), and 3D7/PKG-HAT618Q/Triple (dark grey circles). Data points represent the mean of a single experiment run in triplicates (error bars = standard deviation).Parasite growth was monitored for 7 days and parasitaemia determined every 48 hours. Parental 3D7/attB line (black diamonds), 3D7/PKG-HA(EPS)Click here for additional data file.S9 FigpfpkgT618Q in the cg6/attB locus express both pkg genes simultaneously. Active wild-type enzyme is produced from the endogenous PfPKG locus while enzyme produced from the cg6/attB locus carries the T618Q substitution conferring resistance to compound 2. Upon addition of compound 2, wild-type enzyme will be inhibited and the parasite depends on the activity of PKG-HAT618Q. This system allows us to assess whether a mutation introduced into the PKG-HAT618Q background interferes with kinase activity by asking whether it can complement endogenous PKG function in presence of compound 2.Transgenic parasites harbouring an extra gene copy of HA-tagged (EPS)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


This study was conducted to investigate whether a panel of eight genetic polymorphisms can predict the prognosis of patients with early stage non-small cell lung cancer (NSCLC) after surgical resection.We selected eight single nucleotide polymorphisms (SNPs) which have been associated with the prognosis of lung cancer patients after surgery in our previous studies. A total of 814 patients with early stage NSCLC who underwent curative surgical resection were enrolled. The association of the eight SNPs with overall survival (OS) and disease-free survival (DFS) was analyzed.CD3EAP rs967591, TNFRSF10B rs1047266, AKT1 rs3803300, C3 rs2287845, HOMER2 rs1256428, GNB2L1 rs3756585, ADAMTSL3 rs11259927, and CD3D rs3181259) were significantly associated with OS and/or DFS. Combining those eight SNPs, we designed a prognostic index to predict the prognosis of patients. According to relative risk of death, a score value was assigned to each genotype of the SNPs. A worse prognosis corresponded to a higher score value, and the sum of score values of eight SNPs defined the prognostic index of a patient. When we categorized the patients into two groups based on the prognostic index, high risk group was significantly associated with worse OS and DFS compared to low risk group .The eight SNPs (Prognostic index using eight genetic polymorphisms may be useful for the prognostication of patients with surgically resected NSCLC. Lung cancer, specifically non-small cell lung cancer (NSCLC), is the leading cause of cancer deaths worldwide, with an average 5-year survival rate of 16% . PatholoGenetic polymorphisms have been investigated for the prognostic/predictive biomarkers to guide therapeutic decisions in cancers, including lung cancer –8. For eCarcinogenesis is a multistep process characterized by the accumulation of multiple genetic and epigenetic alterations, which results in alterations in cell physiology that collectively dictate malignant growth: self-sufficiency of growth signals, insensitivity to growth-inhibitory signals, evasion of apoptosis, limitless replicative potential, sustained angiogenesis, and tissue invasion and metastasis . Thus, iWritten informed consent was obtained from all patients prior to surgery at each of the participating institutions and research protocol was approved by the institutional review boards of Kyungpook National University Hospital (KNUH), Seoul National University Hospital (SNUH), and Seoul National University Bundang Hospital (SNUBH). Eight hundred and fourteen patients with pathologic stages I, II, or IIIA (micro-invasive N2) NSCLC who underwent curative surgical resection were enrolled. Among the 814 patients, 334 cases were obtained from the KNUH, 307 cases from SNUH, and 173 cases from SNUBH. All of the patients included in this study were ethnic Koreans. None of the patients received chemotherapy or radiotherapy prior to surgery. The pathologic staging of the tumors was determined according to the International System for Staging Lung Cancer [CD3EAP] rs967591G>A; tumor necrosis factor receptor superfamily, member 10b [TNFRSF10B] rs1047266C>T; and v-akt murine thymoma viral oncogene homolog 1 [AKT1] rs3803300A>G; complement component 3 [C3] rs2287845T>C; and guanine nucleotide binding protein, beta polypeptide 2-like 1 [GNB2L1] rs3756585T>G) were selected from our published papers according to potential function and significance [HOMER2] rs1256428A>G; a disintegrin-like and metalloprotease domain with thrombospondin type 1-like 3 [ADAMTSL3] rs11259927C>T; and CD3d molecule, delta (CD3-TCR Complex) [CD3D] rs3181259T>C) were also included although not yet published. The HOMER2 rs1256428A>G and ADAMTSL3 rs11259927C>T were significantly associated with the prognosis of lung cancer after surgery, among  SNPs in 15q25 region which has been identified as a lung cancer susceptibility locus in genome wide association studies. CD3D rs3181259T>C was identified in our study investigating the SNPs in genes encoding cluster of differentiation 3 (CD3) subunits in the T-cell receptor (TCR) complex, which is vital for T-cell development and plays an important role in the recognition of antigens including tumor antigens. Among four SNPs in CD3D and CD3E promoters, only CD3D rs381259T>C was shown to be predictive of prognosis of lung cancer. In the present study, five SNPs  were tested in an increased number of patients compared with their original studies which included 811, 310, 310, 792 and 792 respectively  for OS = 3.0 x 10−5 and PL-R for DFS = 0.01) and pathologic stage (PL-R for OS = 7.0 x 10−7 and PL-R for DFS = 4.0 x 10−12) were significantly associated with OS and DFS. Sex (PL-R for OS = 0.005) and smoking status (PL-R for OS = 0.04) were associated with OS. Tumor histology was associated with DFS (PL-R for DFS = 0.04).The clinical and pathologic characteristics of the patients and the association with OS and DFS are shown in CD3EAP rs967591, TNFRSF10B rs1047266, AKT1 rs3803300, C3 rs2287845, HOMER2 rs1256428, GNB2L1 rs3756585, ADAMTSL3 rs11259927, and CD3D rs3181259) were found to be significantly associated with OS and/or DFS (P = 1.0 x 10−4 and 0.01) for CD3EAP rs967591 under recessive model, 1.77 and 1.62 (P = 0.006 and 0.004) for TNFRSF10B rs1047266 under recessive model, 1.35 and 1.17 (P = 0.03 and 0.14) for AKT1 rs3803300 under dominant model, 1.44 and 1.39 (P = 0.004 and 0.006) for C3 rs2287845 under additive model, 1.25 and 1.11 (P = 0.02 and 0.15) for HOMER2 rs1256428 under additive model, 1.33 and 1.15 (P = 0.004 and 0.08) for GNB2L1 rs3756585 under additive model, 1.52 and 1.35 (P = 0.006 and 0.02) for ADAMTSL3 rs11259927 under recessive model, and 1.45 and 1.24 (P = 0.006 and 0.05) for CD3D rs3181259 under recessive model, respectively.The 8 SNPs  and low (prognostic index <15) risk group, high risk patients were significantly associated with worse OS and DFS compared to low risk group at a much higher statistical significance compared with individual SNP analysis  were significantly associated with OS and/or DFS. Combining those eight SNPs, we designed a prognostic index to predict the prognosis of patients. After categorizing patients into high and low risk groups by the prognostic index, we found the high risk group was significantly associated with worse OS and DFS than the low risk group. Combined analysis using the prognostic index had a much better resolution in predicting the prognosis of patients compared to individual SNP analysis. The prognostic index using those eight genetic polymorphisms may be useful for identifying patients at a higher risk of disease recurrence and death after surgical resection of NSCLC, and thereby help to select patients for adjuvant chemotherapy.This study was conducted to investigate whether a panel of eight genetic polymorphisms could predict the prognosis of patients with early stage NSCLC after surgical resection. We selected eight SNPs which were associated with the prognosis of lung cancer patients after surgery from our previous studies, and evaluated those SNPs in 814 patients. The eight SNPs , the SRC family of tyrosine kinases, and β integrins, as well as PKC, and thus plays a pivotal role in a wide range of biologic responses, including cell growth, adhesion, and migration [GNB2L1 over-expression has been strongly associated with poor clinical outcomes of cancer patients [in vitro promoter assay and electrophoretic mobility shift assay (EMSA) revealed that the rs3756585 T-to-G change increased transcription factor binding and promoter activity of GNB2L1.igration –38. Studigration –41. In apatients . AccordiADAMTSL3 encodes a secreted glycoprotein with strong similarity to members of the ADAMTS  family. The ADAMTS family has been involved in various human biological processes , including connective tissue structure, cancer, coagulation, arthritis, angiogenesis and cell migration [ADAMTSL3 have been identified recently in colorectal cancer [igration . They arigration . ADAMTS-igration . Recentll cancer . T-cellsl cancer . Accumull cancer .In this study, we showed that prognostic index based on eight genetic polymorphisms was an independent predictive factor for the survival of patients with NSCLC after surgery. The prognostic index may help to predict patients’ prognoses more precisely when used in addition to pathologic stage, the single most powerful predictor of prognosis after surgical resection of NSCLC, therefore being particularly useful in selecting patients who may benefit from adjuvant chemotherapy. Because the current study partly replicated our previous studies on the association between the eight SNPs and patient survival using a relatively large number of patients, reduced chance of false positive associations is expected. However, the prognostic index needs to be further tested in future prospective studies including clinical trials. In addition, further studies are needed to understand the roles of those genes in lung cancer and to clarify the association between the SNPs and prognosis.In conclusion, this study shows that a panel of eight SNPs could be useful to identify patients with a higher risk of disease recurrence and death after surgical resection of NSCLC, and thereby help to select patients for adjuvant chemotherapy. Further studies are required to confirm the validity of these SNPs in other ethnic populations.S1 Table(DOCX)Click here for additional data file.S1 FigCD3EAP rs967591G>A, A); TNFRSF10B rs1047266 C>T, B); AKT1 rs3803300 A>G, C); C3 rs2287845T>C, D); HOMER2 rs1256428 G>A, E); GNB2L1 rs3756585 T>G, F); ADAMTSL3 rs11259927 C>T, G); and CD3D rs3181259 C>T, H). P values by Log-rank test.(PPTX)Click here for additional data file.