Aspergillus nidulans. We have recently shown that the LaeA ortholog of Trichoderma reesei (LAE1), a fungus that is an industrial producer of cellulase and hemicellulase enzymes, regulates the expression of cellulases and polysaccharide hydrolases. To learn more about the function of LAE1 in T. reesei, we assessed the effect of deletion and overexpression of lae1 on genome-wide gene expression. We found that in addition to positively regulating 7 of 17 polyketide or nonribosomal peptide synthases, genes encoding ankyrin-proteins, iron uptake, heterokaryon incompatibility proteins, PTH11-receptors, and oxidases/monoxygenases are major gene categories also regulated by LAE1. chromatin immunoprecipitation sequencing with antibodies against histone modifications known to be associated with transcriptionally active (H3K4me2 and -me3) or silent (H3K9me3) chromatin detected 4089 genes bearing one or more of these methylation marks, of which 75 exhibited a correlation between either H3K4me2 or H3K4me3 and regulation by LAE1. Transformation of a laeA-null mutant of A. nidulans with the T. reesei lae1 gene did not rescue sterigmatocystin formation and further impaired sexual development. LAE1 did not interact with A. nidulans VeA in yeast two-hybrid assays, whereas it interacted with the T. reesei VeA ortholog, VEL1. LAE1 was shown to be required for the expression of vel1, whereas the orthologs of velB and VosA are unaffected by lae1 deletion. Our data show that the biological roles of A. nidulans LaeA and T. reesei LAE1 are much less conserved than hitherto thought. In T. reesei, LAE1 appears predominantly to regulate genes increasing relative fitness in its environment.The putative methyltransferase LaeA is a global regulator that affects the expression of multiple secondary metabolite gene clusters in several fungi, and it can modify heterochromatin structure in  Aspergillus nidulans LaeA protein, a putative S-adenosylmethionine-dependent (SAM) methyltransferase, was originally described as a global regulator of secondary metabolism  and the phytopathogenic Fusarium fujikuroi  and Cochliobolus heterostrophus, respectively  cells, which nurse the young fruiting body during development , an early cellulase producing mutant, and the mutant strains C.P.K. 3793 (Δlae1) and C.P.K. 4087 (lae1OE) derived from it , TJW123.20 , RDIT2.1 , RJW41.A , RDIT9.32 (veA), and RCSR4.16 . Unless otherwise noted, they were grown on glucose minimal medium, or GMM  was used for plasmid construction and amplification., or GMM , with adlae1 fragment obtained from pRKA41617ptrA (containing a 2.8-kb genomic clone of the T. reesei lae1 that contained 900 bp of 5′ and 500 bp of 3′ noncoding regions) was inserted into PstI and SpeI sites of pJW53 to create pJW139.3  according to the manufacturer’s instructions. RNA blots were hybridized with 32P-labeled lae1 DNA fragments, which were generated by PCR using gene-specific primers, lae1SpeI (5′TACTAGTCTACCTCTTTCAAGGAGC) and lae1PstI (5′TCTGCAGACGAGAGATCATATATCCG).The extraction of DNA from fungi and bacteria, restriction enzyme digestion, gel electrophoresis, blotting, hybridization, and probe preparation were performed by standard methods  with 106 conidia was applied onto the bottom medium.Conidia  and ascospore  production were examined using point inoculation or overlay methods in light and/or dark conditions. In point inoculation, 1 µL of 103 spores and incubating in light and dark conditions at 37° for 5 d. Colony diameter was measured and 1-cm cores from the center were taken and homogenized with 3 mL of water. Conidia were counted using a hemocytometer under a light microscope. Conidia per milliliter were determined by standard calculation .Point inoculated colonies were cultured by inoculating on GMM plates with 106 spores in 5 mL of CHAMPS medium (0.75% agar) -coated TLC plate. Sterigmatocystin was spotted as a standard. The plates were run in chloroform/acetone (8:2) solvent and stained with 15% aluminum chloride in 95% ethanol. TLC plates were viewed under 254-nm UV light.Thin-layer chromatography (TLC) was used to assess sterigmatocystin production. One-centimeter cores were punched from the center of point inoculated plates and homogenized with 3 mL of sterile double-distilled HPfuUltra II fusion HS DNA polymerase . The bait plasmid with lae1 cDNA was constructed using two quick change primers, TlaeALexAFWD (5′CGCAACGGCGACTGGCTGGAATTCAAGCTTA TGTCTCGAAACGCTCCCAACGGGTGTG) and TlaeALexAREV2 (5′CTTGGCTGCAG GTCGACTCGAGCGGCCGTTAAGCAGAGGATTCCTCTCTTCTAGATGGC) to place lae1 into pTLex3. lae1-pTLex3 was cotransformed into the Saccharomyces cerevisiae reporter strain L40 with either pGAD424 (empty prey vector) or with A. nidulans veA cDNA in pGAD424  and TveA424REV (5′GCACAGTTGAAGTGAAC TTGCGGGGTTTTTACACCTGGTATTGGTTGAAGGTGACAACG to place vel1 into pGAD424. ve1-pGAD424 was cotransformed into the S. cerevisiae reporter strain L40 with either pTLex3 (empty bait vector), with A. nidulans laeA cDNA in pTLex3  containing 2% (w/v) glucose (SD) media. Six transformants of each combination were tested for their coloration on -UTL medium containing X-Gal.The yeast two-hybrid plasmids pTLex3 and pGAD424  using PfT. reesei were harvested after 26 hr of growth, total RNA extracted  in expression levels between different conditions were performed by Roche-NimbleGen (www.nimblegen.com). Values were normalized by quantile normalization (P < 0.05) were grouped by k-means clustering as implemented in Array Star 3.0.1 . Gene accession numbers were annotated according to version 2 of the T. reesei genome assembly (http://genome.jgi-psf.org/Trire2/Trire2.home.html), and ambiguous cases annotated manually.A high-density oligonucleotide microarray  was constructed, using 60-mer probes representing the 9143 genes of lization  were use2 tests. The microarray data and the related protocols are available at the GEO web site (www.ncbi.nlm.nih.gov/geo/) under accession number GSE22687 (platform GPL10642).Genes were classified according to their major annotation in the MIPS Functional Catalogue (FUNCAT)  and a final primer concentration of 100 nM each. All assays were carried out in 96-well plates which were covered with optical tape. Primers, amplification efficiency and R-square values are given in Supporting Information, Table S1. Measurements for tef1 were performed with both protocols for reference calculation. Determination of the PCR efficiency was performed using triplicate reactions from a dilution series of cDNA . Amplification efficiency was then calculated from the given slopes in the IQ5 Optical system Software v2.0. Expression ratios were calculated using REST Software (DNase I-treated (Fermentas) RNA (5 µg) was reverse-transcribed with the RevertAid First Strand cDNA Kit (Fermentas) according to the manufacturer’s protocol with a combination (1:1) of the provided oligo-dT and random hexamer primers. All real-time PCR experiments were performed on a Bio-Rad  iCycler IQ. For the reaction the IQ SYBR Green Supermix (Bio-Rad) was prepared for 25-µL assays with standard MgClT. reesei, we determined the transcriptional profiles of the wild-type, Δlae1, and lae1OE strains by comparisons of relative transcript levels between Δlae1 and lae1OE, respectively, vs. the wild-type strain. A total of 2743 genes were differentially expressed at least twofold level either between the parent and Δlae1 or the parent and lae1OE . Only 71 genes were down-regulated in Δlae1 and up-regulated in lae1OE .Of the 1556 genes whose transcriptional behavior was consistent with a positive action of LAE1 , 11 polyketide synthase (PKS) genes, and two NRPS/PKS fusion genes  or silent (H3K9me3) chromatin on the parental, the Δlae1 and the lae1OE strain. Under the conditions used, of the 9143 predicted genes in T. reesei, 4089 were significantly associated with at least one of the three methylation marks in at least one of each of the three strains (Table S4). Of these, only 993 of these 4089 genes (24%) showed significant regulation as measured by the perturbation of LAE1  and all encode unknown proteins. H3K9me3 constituted only a small portion of the LAE1-regulated genes , and 31 of these were found to be methylated in all three strains. As for H3K4me2 and -me3, 557 genes showed both methylation marks in all three strains  and were thus also LAE1-independent.LaeA has been proposed to counteract histone H3 lysine 9 trimethylation (H3K9me3) in the sterigmatocystin gene cluster of  of LAE1 . To testlae1OE strain but not in the Δlae1 strain. We found 148 genes that met these criteria . None of the gene families that was enriched for LAE1 regulation . Expression of T. reesei lae1 in A. nidulans partially recovered the decrease in asexual sporulation in light but not in the dark  protein, a regulator of morphogenesis and secondary metabolism in some filamentous fungi , and theT. reesei and A. nidulans LAE1/LaeA proteins, respectively, as “bait,” and the T. reesei/A. nidulans VEL1/VeA proteins as “prey.” LaeA and VeA and LAE1 and VEL1 clearly interacted with each other, as did LaeA and VEL1  in both wild-type and Δlae1 strains. In contrast to A. nidulans, we found the transcript of vel1 to be strongly down-regulated in Δlae1  and to be unaffected in lae1OE . Inspection of the normalized microarray data revealed no effect on the putative vel2 (Trire2:40551), and vos1 (Trire2:102737) orthologs of velB and vosA, respectively .In A mutant . To testA. nidulans, we also tested whether the regulation of lae1 gene expression was similar to that reported for in A. nidulans (laeA was shown to be subject to inhibition by protein kinase A (PkaA), suggesting a potential role of cyclic adenosine monophosphate (AMP). We therefore investigated the lae1 transcript abundance in T. reesei strains modulated in the catalytic subunit of protein kinase A (PKA1), adenylate cyclase, and the G-protein GNA3, using Δpka1 and Δacy1 strains , indicating similar regulation as found in A. nidulans. However, neither the adenylate cyclase mutant Δacy1 nor the constitutively activated gna3 allele showed a significant effect . We conclude that PKA1 is a negative regulator of lae1 expression in T. reesei, whereas the role of cyclic-AMP is unclear. Opposite effects of Δpka1 and Δacy1 on expression of cellulase genes in T. reesei have been noted earlier . The latter approach was useful as many filamentous fungi express several LaeA homologs , which is phylogenetically closer to T. reesei than to A. nidulans. An alignment of the T. reesei LAE1 protein with that of other published LaeA/LAE1 proteins , P. chrysogenum  to this repressive chromatin mark (T. reesei as among a total of 1021 genes responding to modulation of lae1 function, H3K9me3 was absent from most genes in the Δlae1strain, and the methylation pattern of the 13 genes that indeed exhibited H3K9 methylation was independent of LAE1. Absence of H3K9me3 from chromatin associated with bona fide genes is also the default state in N. crassa, where heterochromatic histone modification marks are almost exclusively associated with transposon relics (T. reesei. Currently, the evidence for direct histone methylation by LaeA/LAE1 is at best sparse. No true substrate has been identified yet. None of the LaeA homologs share domains found in many protein methyltransferases that specifically act on histones, e.g., SET domains found in Clr4/DIM-5/Suvar3-9, or motifs found in Dot1 (Aspergillus LaeA (A. nidulans (The precise molecular function of LaeA/LAE1 and its mechanism of action remain enigmatic. Complex formation with VeA and VeB has been demonstrated, but its precise contribution to the regulation of secondary metabolism gene clusters is not yet known, even in tin mark . Based o in Dot1 . Two equlus LaeA , or the T. reesei. This species is unique in the genus because it has escaped its evolutionary history as a mycoparasite to become a successful competitor in the use of predegraded wood (A. fumigatus (A. flavus (Georgianna et al. 2008, A. fumigatus, A. flavus, and F. verticillioides (et al. 2008; T. reesei. As all these genes function in the response of the fungus to environmental stimuli and the presence of competing organism, we interpret this such that LAE1 is involved in successful establishment of T. reesei in its environment.LaeA, in combination with the velvet family of related regulatory proteins, may be involved in supporting the development of progeny in Aspergilli, by controlling the production of chemicals to protect fruiting bodies and the production of nourishing cells for developing fruiting bodies (ded wood . Regulat


The RV144 trial showed an estimated 31% vaccine efficacy (VE) against HIV-1 infection. Two immunological correlates of risk were found in vaccine recipients: Envelope V1/V2 antibody titers and IgA binding to Envelope .+ T-cell based sieve analysis of the V1/V2 region testing for differential escape from vaccine induced epitopes as predicted using computational methods. Breakthrough peptides that differed from predicted epitopes in the vaccine insert and had reduced HLA binding affinity were considered escapes.We conducted a CD8Three of twelve epitopes in V1/V2 of the MN protein boost showed evidence of escape , with more escapes in vaccine recipients (p=0.018). We hypothesized that if escape was indicative of anamnestic responses, recipient’s HLA alleles should not modify VE. While VE was not different in A*11(+/-) subgroups (p=0.45), it was higher in A*02(+) versus A*02(-) participants . Previous analysis showed that HIV variants matching the vaccine insert at site 169 (K169)  were preferentially excluded from infections in vaccine recipients . We found significant VE against K169 in only the A*02(+) subgroup =0.01). Reanalyzing the immune correlates within A*02(+/-) subgroups, we found a direct correlation between IgA-C1 titers and infection rate in A*02(-) participants , but not in the A*02(+) participants  interaction p-value=0.05).Our exploratory analysis, driven by a T-cell based sieve effect in envelope V1/V2, revealed an association between an HLA class I allele and VE, suggesting that VE was restricted to A*02(+) participants and that IgA-C1 antibodies inhibited protective effects of other responses in A*02(-) participants. This highlights the importance of considering the effects of host genetics on VE in future HIV vaccine trials.


Trypanosoma cruzi P2β protein (TcP2β) recognizes the conserved C-terminal end of all T. cruzi ribosomal P proteins. Although this region is highly conserved among different species, surface plasmon resonance analysis showed that the scFv C5 possesses very low affinity for the corresponding mammalian epitope, despite having only one single amino-acid change. Crystallographic analysis, in silico modelization and NMR assays support the analysis, increasing our understanding on the structural basis of epitope specificity. In vitro protein synthesis experiments showed that scFv C5 was able to specifically block translation by T. cruzi and Crithidia fasciculata ribosomes, but virtually had no effect on Rattus norvegicus ribosomes. Therefore, we used the scFv C5 coding sequence to make inducible intrabodies in Trypanosoma brucei. Transgenic parasites showed a strong decrease in their growth rate after induction. These results strengthen the importance of the P protein C terminal regions for ribosomal translation activity and suggest that trypanosomatid ribosomal P proteins could be a possible target for selective therapeutic agents that could be derived from structural analysis of the scFv C5 antibody paratope.The ribosomal P proteins are located on the stalk of the ribosomal large subunit and play a critical role during the elongation step of protein synthesis. The single chain recombinant antibody C5 (scFv C5) directed against the C-terminal region of the  Trypanosoma cruzi is a protozoan parasite responsible for Chagas' disease. This is an endemic disease in Latin America that affects 18–20 million people. No vaccines are available at present and drugs used for treatment show undesirable side effects. The identification of new targets for chemotherapy is a major challenge in the control of the disease and the protein synthesis machinery has been proven to be such a target in other species. Insight into the mechanism capable of selectively blocking protein synthesis could thus lead to the discovery of new therapeutic agents.Saccharomyces cerevisiae, these families have two members, P1α/P1β and P2α/P2β Trypanosoma cruzi also possesses two different P1 and P2 proteins T. cruzi P0 protein has a C-terminal end that deviates from the eukaryotic P consensus and bears similarity to that of the L10 protein of Archaea in vitro translation in a rabbit reticulocyte lysate system T. cruzi ribosomal P proteins have been also detected T. cruzi (R13 epitope), targets the five P proteins that constitute the stalk in vitro protein synthesis by trypanosomatid ribosomes, but had virtually no effect on translation by mammalian ribosomes. We expressed for the first time an intrabody (intracellular antibody), derived from scFv C5, in trypanosomatid cells resulting in growth arrest. Therefore, we propose the ribosomal stalk as a novel potential chemotherapeutic target, and the scFv C5 paratope as a model for peptide mimetics synthesis for selective blocking of the parasite protein synthesis apparatus.The large subunit of the eukaryotic ribosome possesses a long and protruding stalk formed by the ribosomal P proteins. These proteins include P0, an approximately 34 kDa polypeptide, and two distinct, but closely related peptides of about 11 kDa, P1 and P2. All of them share a conserved, highly acidic motif at its C-terminal end. An additional P protein, named P3, has been described in plants Peptides were prepared by the solid-phase method of Merrifield as was previously described 2. From the decrease in the initial linear kinetics of the interaction in the presence of increasing amounts of inhibitor, the IC50 could be determined for each inhibitor as previously described The BIACORE 3000 system, sensor chip CM5, surfactant P20, amine coupling kit containing N-hydroxysuccinimide (NHS) and N-Ethyl-N′-dimethylaminopropyl carbodiimide (EDC), ethanolamine were from BIACORE . Biosensor assays were performed with HBS-EP buffer as running buffer  surfactant P20, pH 7.4). The low carboxylated dextran matrix (B1) was activated with 35 µl of a mixture 0.2 M N-ethyl-N-dimethylaminopropyl carbodiimide and 0.05 M N-hydroxysuccinimide at 5 µl/min. TcP2β-GST fusion protein and GST (as control) were immobilized with the standard BIACORE protocol at a density of 0.05 pmol/mm2 E. coli and purification was performed as previously described in vivo expression, the DNA encoding for scFv C5 was amplified by PCR using the following oligonucleotides carrying HindIII restriction site (C5ForHIND: 5′-GT AAGCTT GCC ATG GCC GAA GTG CAG C-3′) and BamHI restriction site (C5RevBAM: 5′-GT GGATCC CCG TTT TAT TTC CAG CTT GGT CC-3′) and inserted into pHD1700 vector as previously described T. brucei inducible cell line 5 cells/ml and counted every 24 hours. The concentration was adjusted (by diluting appropriately) every 24 hours back to 2×105 cells/ml. The cumulative number of parasites corresponds to the number of parasites per milliliter multiplied by the dilution factor for each day (day after day).Expression of scFv C5 in All procedures were performed with Discovery Studio 2.5 software from Accelrys . The scFv C5 model derives from the parental mAb 17.2 crystal structure Apo (3SGD) or in complex with R13 peptide (3SGE). The former structure was used as template to build a model antibody-H13 interaction. For this purpose, we mutated the aspartic acid 3 of R13 by serine and optimized the conformation of both the mutated residues and any surrounding residues that lay within a cut-off radius of 2 Å. Five models thus obtained were scored by the Discrete Optimized Protein Energy (DOPE). We continued analysis using the lowest energy model . Finally, the complex was subjected to a minimization step (max 500), RMS gradient 0.1 Kcal/mol by conjugated gradient.2HPO4, 1.8 mM K2HPO4, 150 mM NaCl, 2.7 mM KCl, 0.1% Tween 20, pH 7.4) supplemented with 5% (p/v) non-fat milk powder. This was followed by incubation with scFv C5 200 nM together with peroxidase-conjugated anti-His Ab (∼350 nM) , for detection of ribosomal P proteins and with peroxidase-conjugated anti-His Ab (∼350 nM) alone for intrabody detection. The Ab was diluted in the blocking solution PBS-T 1% (p/v) non-fat milk powder. Proteins on transferred membranes were revealed with tetramethyl-benzidine (TMB)  after a brief wash with dextran sulphate 1% .SDS-PAGE analysis was performed as a standard procedure using 12% acrylamide gels followed by immunoblotting. Proteins were transferred from gels onto a Hybond ECL nitrocellulose transfer membrane  using a mini trans-blot system  in transfer buffer  methanol, pH 8.3). Membranes were soaked in PBS-T  was layered onto two layers of 2 M and 1.5 M sucrose made up in Buffer A  and centrifuged 16 h at 140,000 g. The pellet corresponding to polysomes was rinsed and resuspended in a buffer containing 10 mM Tris–HCl, pH 7.5; 10 mM KCl and 1.5 mM magnesium acetate. Ribosome concentration was determined by optical density at 260 nm.Purification of ribosomes from different sources and 2; 5 mM KCl; 3 mM CaCl2; 5 mM 2-merchaptoethanol and 250 mM sucrose) and lysed with 0.2–0.4% of Nonidet P40 at 4°C. The homogenate was centrifuged 2–3 times for 20 min at 1,000 g and the final supernatant fraction containing ribosomes was layered onto a discontinuous gradient of 2 and 1.5 M of sucrose made up in the following buffer: 10 mM Tris–HCl, pH 7.5; 1 mM magnesium acetate; and 100 mM potasium acetate. The gradient was centrifuged for 16 h at 140,000 g. The supernatant was discarded and the pellet carefully rinsed and resuspended as described above.Polysomes from trypanosomatids were obtained from cultures at the exponential phase of growing. Cultures were treated with cycloheximide (50 ug/ml) for 10 min before harvesting by centrifugation at 4°C. Cells were washed twice with PBS containing 50 ug/ml cycloheximide. The cells were resuspended in Lysis Buffer ; 0.3 A260U of ribosomes and 2 uCi of [35S] methionine in a final volume of 30 ul. Reactions were performed at 30°C during 60 min and stopped by adding 150 ul of 1.5 M NaOH; 1 mM Met, 170 ug/ml BSA. After incubation for 30 min at 37°C, proteins were precipitated with 1 ml of cold TCA 25%. After 60 min on ice, the samples were filtered and washed with TCA 10% and ethanol using glass fiber filters. Radioactivity retained in the filters was measured by liquid scintillation counting. For inhibition assays, reaction mixtures were incubated on ice with the scFv C5 antibody. For inhibition reversion, the scFv C5 was preincubated with the H13/R13 peptides for 20 min. After that, reactions were initiated by incubation at 30°C.The reaction mixtures were prepared on ice and contained: 19 amino acids 50 uM each (excepting Met); 2 mM dithiothreitol; 100 mM potassium acetate; 3.5 mM magnesium acetate; 75 ug/ml wheat germ tRNA; 18 mM Hepes/KOH, pH 7.5; 1 mM ATP; 0.5 mM GTP; 7.5 mM creatine phosphate; 37.5 ug/ml creatine phosphokinase; rat liver STrypanosoma brucei was cultured in HMI-9 medium Bloodstream form of Trypanosoma cruzi, Saccharomyces cerevisiae and Homo sapiens were used to perform BLAST searches Ribosomal P protein sequences of T. brucei growth rate was analyzed by two-way ANOVA with Bonferroni's post test. All analysis were done using GraphPad Prism version 5.00 for Windows, GraphPad Software, San Diego California USA, www.graphpad.com.Translation inhibition studies were analyzed by two-way ANOVA with Bonferroni's post test. Densitometry was analyzed by one-way ANOVA with Tukey's Multiple Comparison Test. T. cruzi ribosomal P2β protein , and is able to recognize all five T. cruzi ribosomal P proteins , white letters). The mammalian counterpart epitope (H13) possesses one single, non-conservative amino acid change in the first key glutamic acid position  (Rsqr = 0.992) and P015  (Rsqr = 0.997) peptides but not with the H13 peptide. These results demonstrated the low affinity of scFv C5 for the mammal epitope. Interestingly, both P015 and H13 peptides possess one amino acid change in this motif but the replacement of glycine 8 by aspartic acid in P015 peptide seemed to be less essential than the replacement of glutamic 3 by serine in H13 peptide for scFv C5 interaction.As it was previously reported the scFv C5  recognizes the C-terminal region of In silico replacement of GLU3 by SER showed a strong reduction of the interaction energies that fall from −326.6 kcal/mol (R13 complex) to −204.9 kcal/mol (H13 complex)  T. cruzi, T. brucei, C. fasciculata and R. norvegicus by western blot. As it was previously shown T. cruzi P0, P1 and P2 proteins . The sequence of the C-terminal peptide of T. brucei shows a conservative change of Glu3 by Asp in three of the low molecular weight P proteins (Table S2), and the sequences of C. fasciculata P proteins are not known. scFv C5 clearly detected the P proteins on T. brucei and C. fasciculata extracts . Similar results were obtained analyzing equal amounts of purified ribosomes from these different organisms (not shown). In contrast, no protein bands were detected using rat extracts ( lane 4), as was expected from SPR analysis.We analyzed the ability of scFv C5 to detect the ribosomal P proteins of in vitro cell-free translation system to determine the ability of this antibody to inhibit protein synthesis. Background values of radioactivity incorporation in the absence of protein synthesis were obtained in the presence of emetine (a well-characterized elongation inhibitor). It should be mentioned that the only difference among the translation mixtures was the ribosome source, ruling out a possible non-specific effect of scFv C5 on processes upstream from translation like ATP regeneration or amino acid activation. Our ribosome preparations lack initiation activity, as we have verified using the recently characterized IF4A inhibitor hippuristanol T. cruzi ribosomes by scFv C5 was dose-dependent with an IC50 of around 62,4 nM  give no viable clones. Taking advantage from a tightly regulated, inducible expression system in T. brucei, we assessed intrabody expression in this organism. We subcloned the scFvC5 coding sequence into the pHD 1700 c-myc vector and transfected it into the T. brucei cell line 1313-514. Stable clones were obtained by serial dilution. Tetracycline induction of six different clones gives detectable protein at 24 hours post-induction and almost no detectable protein in the absence of inducer (p<0.001) at day 4, indicating a detrimental effect of the intrabody expression on the logarithmic growing phase as can be seen in Figure S1). In addition, luciferase expression or a control intrabody did not affect the growing rate of induced parasites .At present, there are no confident inducible expression systems available in Trypanosoma cruzi (Tc), Trypanosoma brucei (Tb), Trypanosoma vivax (Tv), Trypanosoma congolense (To), Leishmania infantum (Li), Leishmania braziliensis (Lb), Leishmania major (Lm), Plasmodium knowlesi (Pk), Plasmodium falciparum (Pf), Plasmodium berghei (Pb), Plasmodium chabaudi (Pc), Dictyostelium discoideum (Dd), Eimeria tenella (Et), Theileria annulata (Ta), Theileria parva (Tp), Entamoeba histolytica (Eh) and Cryptosporidium parvum (Cp). We also included the ribosomal P proteins of Homo sapiens (Hs), Arabidopsis thaliana (At) and Saccharomyces cerevisiae (Sc) as key markers (Table S1). It is important to note that two independent gene duplication events yielded P1 and P2 subtypes (α and β) in trypanosomatids and S. cerevisiae. The clusters belonging to trypanosomatids P proteins P0, P1α, P1β, P2α and P2β are all supported by high bootstrap values  of the ribosomal P proteins of all genome sequenced protozoan. We searched by homology all ribosomal P proteins (putative and previously reported ones) from the following organisms: p values . TherefoTable S2) using weblogo Leishmania P proteins posses an alanine in position 3, it rules out the possibility of an effect of the C5 antibody on this organism.Thereafter we generated a sequence alignment of the C-terminal epitope corresponding to different protozoan and mammal organisms (50 observed by SPR (R13 vs H13) together with in silico structural analysis based on the recently solved crystallographic structure of the complex Fab 17.2-R13 peptide; clarify how the scFv C5 selectively blocks protein synthesis by trypanosomatid ribosomes. Western blot and sequence data analysis showed that different trypanosomatids posses the same C-terminal epitope, all being potentially susceptible to scFv C5 activity. When T.cruzi cells were transfected for constitutive expression of scFv C5 intrabody it was not possible to complete the selection period because all cells died. Profiting from the availability of inducible vectors for T. brucei and that scFvC5 recognizes T. brucei P proteins in Western Blot, we tested the effect of scFv C5 intrabody induction on these cells. The scFv C5 intrabody could be detected at protein level using its myc tag at 24 hs post-induction. Induced parasites showed a delay in growth rate, consistent with a blockade of ribosomal translation activity. As can be seen on figure S1, it is difficult to establish a relationship between intrabody expression level and functional effect. The inhibition mechanism can be resumed as a protein (scFvC5) that needs to be translated before binding the ribosomal P proteins and thus inhibiting protein synthesis. It means that it is really difficult to obtain total inhibition because the synthesis of the intrabody will also be blocked. We think that the most probable scenario could be interpreted as an equilibrium between scFv translation, protein activity and growth inhibition. In addition, the scFv C5 was able to inhibit in vitro protein synthesis in ribosome extracts from T. brucei at a similar extent than T. cruzi ribosome extracts . Intrabodies have been successfully expressed in different cell types In the present work, we demonstrated a strong correlation between the affinity of scFv C5 for the C-terminal end of ribosomal P proteins from different species and its ability to inhibit translation in a cell-free system. The difference on ICSequence analysis showed that mammalian ribosomal P proteins possess a conserved C-terminal region slightly different from the protozoan one, being the main difference the presence of a serine in position 3 Figure 4Figure S1Growth curves of all clones assessed for intrabody expression. The growth curves and expression profile of each individual clone are shown in every row. On the left side we present the western blots of scFvC5 expression performed at 0 and 24 hours post-induction. The averaged growth curve and densitometry is shown at the bottom and corresponds to the results showed in (TIF)Click here for additional data file.Figure S2Control intrabody expression in T. brucei. Growth rate of T. brucei transfected with the control scFv S3, scFv C5 or Luciferase in presence of tetracycline using the pLew inducible system.(TIF)Click here for additional data file.Figure S3Translation inhibition. Effect of scFv C5 50 nM on in vitro protein synthesis in ribosome extracts from T. cruzi and T. brucei compared with the translation inhibitor emetine at 0.1 mg/mg. Average values for control assays were 6,000 cpm and 19,000 cpm for T. cruzi and T. brucei, respectively.(TIF)Click here for additional data file.Table S1Ribosomal P-proteins analyzed in .(DOC)Click here for additional data file.Table S2C-terminal region of the ribosomal P-proteins analyzed in . A. Different mammalian P-proteins C-terminal sequences potentially hosts of parasitic infections. B. Protozoan P1/P2 C-terminal sequences.(DOC)Click here for additional data file.


A novel immunotherapy platform is represented by the DCOne cell line, which originates from a human myeloid leukemia cell line, endogenously expresses a range of leukemia associated antigens, including PRAME and WT-1, and can be differentiated into mature dendritic cells (DC). This cell line is being developed to replace patient-derived DC vaccines. The first indication in which mature DC derived from DCOne have been tested clinically is Acute Myeloid Leukemia (AML). A Phase I/IIa study enrolled 12 AML patients (age range 58-71) who were either in CR1/CR2 (n=5) or had smouldering disease (n=7). Patients had received all available standard care, were at high risk of relapse and ineligible for available post-remission therapies. A typical 3+3 design was used, starting with 4 bi-weekly intradermal DCOne DC vaccinations of 10E6 (n=3), 25E6 (n=3) or 50E6 cells(n=6). Patients were monitored for clinical and immunological responses for 126 days. Primary endpoints were safety and feasibility, and secondary endpoints were clinical and immunological responses. Treatment was well tolerated in all patients, with only injection site reactions (< grade 2). During the study 3 patients died: 2 from infections and 1 from leukemia. Clear evidence for induction of multi-functional immune responses was obtained, including increased post vaccination delayed type hypersensitivity reactions, increases in CD4+ and CD8+ T cell proliferative responses and/or seroconversion to DCOne DC and/or AML blasts in 6 out of 9 patients. Also, 3 out of 7 patients that were evaluable by IFNγ ELIPOTs showed vaccination-induced specific T cell responses to WT-1 and/or PRAME. Patients who survived more than 6 months post-vaccination showed remarkable survival . One of these patients, with smouldering AML at entry, achieved CR2 after vaccination; one patient who was in CR2 at entry relapsed 8 months after vaccination, entered CR3 following a single low dose of 5-AZA and survived until 23 months. The two most recently enrolled patients recruited are still in remission at 12 and 9 months. We conclude that vaccination with DCOne derived DC is feasible in AML, and generates both cellular and humoral immune responses. The hypothesis that DCOne-derive DC induce immune responses against patient leukemia cells correlate with clinical benefit, will now be investigated in a multi-center randomized Phase II trial in AML patients in first remission.


The plethora of vitamin D studies over the recent years highlight the pleomorphic effects of vitamin D outside its conventional role in calcium and bone homeostasis. Vitamin D deficiency, though common and known, still faces several challenges among the medical community in terms of proper diagnosis and correction. In this review, the different levels of vitamin D and its clinical implications are highlighted. Recommendations and consensuses for the appropriate dose and duration for each vitamin D status are also emphasized. It also regulates bone metabolism through activation of the VDRs found in osteoblasts, releasing biochemical signals and leading to the formation of mature osteoclasts eoclasts .2D production D cut-off for optimum skeletal health is the level that reduces PTH to a minimum and increases calcium absorption to its maximum ,16. Seveet al. D levels (500–600 ng/mL), while 1,25[OH]D2D3 plasma concentrations were only modestly elevated (40–56 pg/mL) [versus calcitriol are presented in The clinical advantages of choosing 25[OH]D instead of calcitriol as a marker for vitamin D status has been listed by Rajasree et al. . First, et al. . Furtheret al. . Althouget al. . Hughes 2) and cholecalciferol (vitamin D3). Some studies [3 increases serum 25[OH]D more efficiently than vitamin D2. A large, single dose of vitamin D2 does not last longer than a large dose of D3. In a study conducted by Armas et al., D started dropping rapidly after 3 days among subjects given vitamin D2 whereas those on vitamin D3 sustained high levels for two weeks before dropping gradually.Multiple preparations of vitamin D and its metabolites are commercially available for supplement use. The two most common supplements are ergocalciferol  [3 levels also increased from 49 to 72 nmoL/L (19.6 to 28.9 ng/mL). It took 6 weeks for 25[OH]D levels to plateau on that regimen. In another study, Glendenning et al. compared 1000 IU of D2versus D3 in patients who had vitamin D insufficiency with subsequent hip fractures. After three months, those who were supplemented with D3 had a 31%–52% greater increase in 25[OH]D levels than those supplemented with D2. However, parathyroid hormone levels did not differ between groups D levels increased from 42.5 to 90 nmoL/L and there were no significant differences between treatment groups D status from a daily dose of 1000 IU of vitamin D2 or D3 from either calcium-fortified orange juice with vitamin D or supplement capsules for 11 weeks. The average 25[OH]D levels of all groups  went up to about 25 nmoL/L with no significant differences between groups 3D2 and the deactivation of vitamin D2 molecule. On the other hand, the vitamin D3 metabolite 1,24,25[OH]3D3 must undergo an additional side chain oxidation to be biologically deactivated 2D3 2D3 .The best indicator of vitamin D status is 25-OHD because it reflects cutaneous and dietary intake, not to mention it is the major circulating form of vitamin D . While t3, serum 25(OH)D concentrations increase by approximately 0.3 to 0.4 ng/mL (0.7 to 1.0 nmol/L) D concentrations lead to increased intracellular 1,24[OH]D concentrations. This hypothesis is not widely supported as many studies revealed that vitamin D toxicity is associated with normal or marginally elevated 1,25[OH]D D with vitamin D toxicity D . It was toxicity .(ii)Vitamin D intake raises plasma 25[OH]D levels to concentrations that exceed DBP binding capacity, and free 25[OH]D has direct effects on gene expression once it enters target cells. High dietary vitamin D intake alone increases plasma 25[OH]D. The low affinity of 1,25[OH]D for the transport protein DBP and its high affinity for VDR dominate normal physiology. This makes it the only ligand with access to the transcriptional signal transduction machinery. However, in vitamin D intoxication, overloading by various vitamin D metabolites significantly compromises the capacity of the DBP by allowing other metabolites to enter the cell nucleus. Of all the inactive metabolites, 25[OH]D has the strongest affinity for the VDR, and thus at sufficiently high concentrations, could stimulate transcription D, and these concentrations exceed the DBP binding capacity and release of “free” 1,25[OH]D which enters target cells D concentration obtained from sunshine was from a farmer in Puerto Rico with a level of 225 nmol/L . On the Vitamin D hypersensitivity syndromes are often mistaken for vitamin D toxicity. The most common is primary hyperparathyroidism. Granulomatous diseases, such as sarcoidosis, granulomatous TB and some cancers also cause vitamin D hypersensitivity, as the granuloma or the tumor may make excessive amounts of calcitriol, thus raising serum calcium levels .The present review discussed current knowledge on vitamin D physiology, its clinical relevance and evidence-based treatment options on vitamin D status correction. Caution should still be practiced by clinicians in providing vitamin D supplementation among vitamin D deficient populations, with proper monitoring using approved and certified methods. Indications for vitamin D supplementation outside the conventional calcium homeostasis should also be considered to maximize extra-skeletal benefits of vitamin D correction.


A number of neurodevelopmental syndromes are caused by mutations in genes encoding proteins that normally function in epigenetic regulation. Identification of epigenetic alterations occurring in these disorders could shed light on molecular pathways relevant to neurodevelopment.KDM5C gene, encoding a histone H3 lysine 4 demethylase, in comparison to age/sex matched controls. Loss of DNA methylation in such individuals is consistent with known interactions between DNA methylation and H3 lysine 4 methylation. Further, loss of DNA methylation at the promoters of the three top candidate genes FBXL5, SCMH1, CACYBP was not observed in more than 900 population controls. We also found that DNA methylation at these three genes in blood correlated with dosage of KDM5C and its Y-linked homologue KDM5D. In addition, parallel sex-specific DNA methylation profiles in brain samples from control males and females were observed at FBXL5 and CACYBP.Using a genome-wide approach, we identified genes with significant loss of DNA methylation in blood of males with intellectual disability and mutations in the X-linked We have, for the first time, identified epigenetic alterations in patient samples carrying a mutation in a gene involved in the regulation of histone modifications. These data support the concept that DNA methylation and H3 lysine 4 methylation are functionally interdependent. The data provide new insights into the molecular pathogenesis of intellectual disability. Further, our data suggest that some DNA methylation marks identified in blood can serve as biomarkers of epigenetic status in the brain. KDM5C, encoding histone H3 lysine 4 (H3K4) demethylase. Mutations in the KDM5C gene (MIM No: 314690) were first described as causing X-linked intellectual disability (XLID) in 2005 . DNA methylation β values are continuous variables between 0 (absent methylation) and 1 (completely methylated) representing the ratio of combined locus intensity. The β values were extracted using the Methylation Module in Illumina Bead Studio after background normalization. All samples included in the analysis passed quality control metrics including bisulfite conversion control intensity values in green channel >4000 and 99% of probes had p-values of detection of signal above background <0.01 [FBXL5 (cg02630888), SCMH1 (cg03387723), and CACYBP (cg16743289) CpG sites. Sex specific DNA methylation analysis in brain was performed using published dataset (GEO Accession No: GSE15745) [The HumanMethylation27 BeadChip  containing 27,578 individual CpG sites covering >14,000 genes was used for genome-wide DNA methylation analysis. Genomic DNA was sodium bisulfite converted with the EpiTect Bisulfite Kit according to the manufacturer’s protocol . Labeling, hybridization and scanning were performed at the Centre for Applied Genomics (TCAG) at The Hospital for Sick Children, Toronto, Canada. The methylation status of the interrogated CpG sites was measured from the intensity values of the methylated (M) and unmethylated (U) probes, as the ratio of fluorescent signals β = nd <0.01 . 6 publiSE15745) . If the U test was used for group comparisons. To adjust for multiple comparisons, a permutation-based method controlling the false discovery proportion (FDP) γ in the data, for a pre-specified confidence levels α was applied [U test-based p-values for all CpG sites were ranked from smallest to largest (from most significant to least significant). The distribution of the 1000 “best” permutation-based p-values was used to determine p-values cut offs for different confidence levels based on the percentiles of this distribution. The cut off p-values were used to determine the number of significant CpG sites at different γ and α level. The Principal Component Analysis was performed in R statistical package using median centering of the data as well as with missing-value imputation to the 10 nearest neighbors.Microarray probes with detection p-value ≥ 0.01 , 2,984 c applied . 1000 raTargeted DNA methylation analysis was performed using pyrosequencing as described by Tost and Gut . PyroseqFBXL5, SCMH1 and CACYBP we performed real-time RT-PCR expression analysis. Total RNA was purchased from Clontech for 8 tissues and 4 brain regions. Lymphoblastoild cell line established from control sample C2 RNA was extracted using RNeasy kit (Qiagen). The cDNA for quantitative real-time PCR was synthesized using VersoTM cDNA kit . Mx3005P QPCR System  with SsoFast™ EvaGreen® Supermix . Expression of each gene was determined using the comparative Ct method and normalized to the expression of the GAPDH housekeeping gene. Primers sequences are shown in Additional file To analyze tissue specific expression of KDM5C mutations, we performed genome-wide DNA methylation analyses using the Illumina Infinium HumanMethylation27 BeadChip array containing 27,578 CpG sites and covering >14,000 genes in DNA from blood samples of XLID patients and controls. Our study group was comprised of 10 XLID patients from 5 families with 5 different KDM5C mutations. Two of these mutations were frameshift mutations, resulting in premature stop codons and 3 were missense mutations, predicted to be damaging by both Polyphen and Sift algorithms [KDM5C variant was reclassified as a benign variant, as it was found in a phenotypically normal maternal grandfather in another XLID family. Clinical and demographic information for the XLID patients and controls and locations of the KDM5C mutations are summarized in Additional file To determine whether altered genome-wide DNA methylation patterns are associated with 2 = 0.95-0.99) among samples as well as high correlation of microarray data with bisulfite pyrosequencing (R2 = 0.77-0.98)  for each of 27,578 CpG sites were calculated as ratios of methylated CpG to the sum of methylated and unmethylated CpG. The reliability of Illumina Infinium data was demonstrated by high correlation  levels for pre-specified confidence levels [To identify the differentially methylated CpG sites, we compared beta-values between 10 e levels . We geneU test on all 72 possible combinations of 5 cases vs. 17 controls. We observed a large degree of consistency between the CpG sites identified in this analysis (5 unrelated cases vs. 17 unrelated controls) with the CpG sites identified in the general setting (10 cases vs. 19 controls). For example, the CpG sites found to be significantly different between cases and controls across all 72 combinations at 95% confidence level exactly coincided with the 53 CpG sites shown in Additional file To address the potential effect of relatedness on differences in DNA methylation and controls we performed additional analysis including only unrelated individuals by randomly selecting 5 mutation cases from each of the five available families and 17 unrelated controls (one randomly chosen unaffected relative and 16 unrelated controls). To identify differentially methylated CpG sites, we performed the Mann–Whitney KDM5C mutation cases from controls. Two samples, carrying the p.R1546Q variant at the C-terminal end of the KDM5C protein, consistently showed the same DNA methylation levels as control samples confirming the benign nature of this variant.We further tested if DNA methylation differences at 53 CpG sites with p-values < 2.00e-07 could differentiate cases either from controls or from the variant of unknown significance (p.R1546Q). A variety of unsupervised methods including hierarchical clustering . For these analyses, we took advantage of two published datasets that used the same Illumina HumanMethylation27 microarray platform and reported a number of methylation quantitative trait loci (mQTL) in lymphoblastoid cell lines (LCLs) [cis or trans in relation to CpG. We did not find any mQTLs at the 53 CpG sites identified in our study, suggesting that the DNA methylation alterations we observed are more likely to be associated with KDM5C mutations rather than other genetic variation that exists between cases and controls.Multiple studies have shown that at some genomic loci DNA methylation levels could be affected by sequence polymorphisms in eritable ,33-36. Ws (LCLs) . At thes-16, Fisher Exact test). All 53 top CpG candidates with the lowest FDP = 0 and the highest confidence level of 99.5%  exhibited loss of DNA methylation. The 13 CpGs with the greatest loss of DNA methylation (delta beta ≤-0.2) are shown in Table KDM5C mutation group (p = 0.035) also for CpG islands, but not for non-CpG island probes  and TSPYL5 had two CpG sites meeting the permutation p-value cut off. In order to assess the genomic extent of the DNA methylation changes for each significant gene we have evaluated DNA methylation levels, delta beta differences, and p-values in cases and controls for additional array probes for the 53 top significant genes. For a majority of the genes the second probe did not exhibit significant loss of DNA methylation. Only 8 genes had a second significant probe with p ≤ 0.05. The absolute delta beta differences for the second probes were relatively small ≤ 0.05, with the exception of the STMN1 gene having a delta beta −0.16 and −0.14 in two CpG sites , SCMH1 (delta beta = −0.45) and CACYBP (delta beta = −0.28) with the largest DNA methylation differences and two loci with smaller differences DYDC1 (delta beta = −0.1) ZMYND12 (delta beta = −0.08). Using pyrosequencing, we validated the direction of the differences for all 5 loci and observed high correlation DNA methylation levels between pyrosequencing and Illumina for overlapping CpG sites  and ZMYND2 (5 CpGs) all sites tested exhibited DNA methylation differences between cases and controls , the CpG sites more distant from the TSS exhibited overall higher DNA methylation in controls and larger differences in DNA methylation between cases and controls  with no significant differences. Interestingly, based on ChIP sequencing data for histone marks from the Broad Institute [KDM5C mutations cases frequently coincided with enhancer mark H3K4me1 in embryonic stem cell (ES) and lymphoblastoid cell lines. In contrast, the mark of active promoters H3K4me3 was shifted more towards the TSS, where DNA was hypomethylated  . This dataset analyzed genome-wide DNA methylation using Illumina Methylation450 array in DNA from whole blood, peripheral blood mononuclear cells, and granulocytes, as well as in 7 isolated cell populations  in six healthy males [KDM5C mutations cases were extracted for analysis. We did not observe differences between cell types at three CpG sites analyzed at a magnitude that could explain loss of DNA methylation in KDM5C mutations cases  and a trend towards significance for SCMH1 (p = 0.07), in the diabetes study where samples of both sexes were included [FBXL5, CACYBP and SCMH1, we observed a significant  increase of DNA methylation in females compared to males in 5 loci and a trend towards significance  in three out of 9 tested loci with the delta beta differences ranging from 0.01 to 0.06  but highly significant increase of DNA methylation in females compared to males in included   of neurologically normal individuals (GEO Accession No: GSE15745) [FBXL5 and SCMH1 with the exception of cerebellum which had intermediate methylation levels in brain (30-40%) and high methylation levels in blood (70%). CACYBP had intermediate levels of DNA methylation in both brain and blood (30-50%)  . DNA met) Figure . FurtherKDM5C is an X-linked gene that escapes X-inactivation in humans and mouse [KDM5D[Kdm5c’s escape from X –inactivation is highly variable across different tissues. The level of transcript from the inactive X allele is 20-70% of the active X allele [Kdm5c/Kdm5d are expressed in a sex-specific fashion i.e., the expression of Kdm5c is significantly higher in female brains than in male brains, and the expression of Kdm5d in males is not sufficient to compensate for the female bias in Kdm5c expression [KDM5D is reported to be expressed at lower levels than KDM5C. However, as commercially available RNA mixed from several individuals was used for this experiment the proportion of male cells present in these samples is not known [KDM5C and KDM5D, respectively. To further investigate this, we assessed FBXL5, SCMH1 and CACYBP DNA methylation levels using targeted pyrosequencing assays in blood samples with different sex chromosome constitutions and reflecting variation in KDM5C/KDM5D dosage, including 47,XXX , 47,XXY , 46,XX , 46,XY  and 45,X  in comparison to males with KDM5C mutations  and female carriers of KDM5C mutation . We found that the DNA methylation levels at these three genes generally correlated with KDM5C/KDM5D dosage for all three genes analyzed   were substantially smaller than the differences for cases missing one copy of functional KDM5C . This could be due to the fact that two copies of KDM5C or one copy each of KDM5C and KDM5D is close to saturation of H3K4 demethylase activity at target promoters. Another possible explanation of small differences between 46, XX females and 46, XY males observed in both brain and blood, is that KDM5D is expressed only at slightly lower levels than KDM5C from inactive X-chromosome in human, making 46,XX and 46,XY relatively close in their levels of H3K4 demethylase activity, in contrast to larger differences between males with KDM5C mutation vs. 45,X females, where the difference is between KDM5D and KDM5C expressed from an active X. The observation that KDM5C mutation female carriers exhibit DNA methylation levels similar to individuals with 45,X karyotype and the fact these females have highly skewed X-chromosome inactivation [KDM5C is expressed from the preferentially active X-chromosome.The differences among groups with normal or extra copies of FBXL5, SCMH1 and CACYBP promoters correlate with H3K4 demethylase activity of the proteins KDM5C/KDM5D due to an inverse relationship between H3K4 methylation and DNA methylation [Based on these data we suggest that DNA methylation levels at hylation .Advances in molecular technologies have helped to identify genetic causes in many cases of syndromic and non-syndromic forms of intellectual disability (ID) -59. HoweKDM5C mutations compared to 5 controls identified 11 upregulated genes. These transcriptional changes were not very consistent among KDM5C mutations samples, and a combination of at least 6 genes was required to distinguish cases from controls [KDM5C mutations, coincided with an enhancer mark H3K4me1 in ES and lymphoblastoid cell lines. In contrast, DNA sequences more proximal to the TSS were hypomethylated in both controls and cases and coincided with the active promoter mark H3K4me3 , are characterized by enzymatic gain of function and subsequent production of hydroxyglutarate, which inhibits several histone demethylases, including H3K9, H3K27, H3K36 and H3K4 [IDH1/IDH2 are associated with genome-wide hypermethylation in AML compared either to normal bone marrow or to AML caused by mutations in other genes. However at this point it is not clear which specific histone marks contribute directly to this DNA hypermethylation phenotype [PABPN1 and ZNF532, demonstrating loss of DNA methylation in our study  [PARK2, RFWD2, FBX040) [Interestingly, the three top candidate genes are part of ubiquitin-ligase protein degradation pathways. Synaptic network remodeling, a vital part of central nervous system function, depends on ubiquitin-mediated protein degradation at the postsynaptic membrane . Genes iUBE3A) , and aut FBX040) . Further FBX040) .GAPDH. Although it is currently not clear how loss of DNA methylation at these sites affect gene expression, we propose that abnormal expression of these genes at specific cell types/developmental stages causes disturbances in downstream pathways such as degradation of target proteins. FBXL5 has been recently discovered to be a component of an E3 ubiquitin ligase complex that targets IRP2, an iron regulatory protein 2 important in intracellular and plasma iron homeostasis [FBXL5 could result in abnormal iron accumulation, thereby contributing to seizures and/or ID phenotypes observed in males with KDM5C mutations. SCMH1 is a member of the Polycomb-group 1 complex, which not only is a transcriptional repressor, but also acts as an E3 ubiquitin ligase for the geminin protein involved in DNA replication and maintenance of undifferentiated cellular states. Specifically, SCMH1 has been shown to provide an interaction domain for geminin [SCMH1 in the regulation of human height [SCMH1 is important for short stature associated with KDM5C mutations. CACYBP (Sip) is part of the SCF-like complex, involved in ubiquitin-mediated degradation of the transcriptional activator β-catenin [We have shown that these three genes are ubiquitously expressed in human tissues at relatively low levels compared to the house-keeping gene eostasis ,76. IRP2eostasis . While Feostasis , Irp2 −/eostasis . Thus, a geminin . Recentln height , thus it-catenin ,41. β-ca-catenin -82. In a-catenin . Further-catenin , extrace-catenin .de novo DNA methylation is an important process in epigenetic reprogramming occurring at early stages of embryonic development [KDM5C mutations could at least in part result from abnormally high H3K4 di/trimethylation in the embryo, protecting DNA from de novo methylation. We expect that this state is maintained through differentiation into multiple lineages. In support of this, we found parallel sex-specific DNA methylation differences in both brain and blood at FBXL5 and CACYBP, whereas SCMH1 exhibited this difference only in blood, but not in brain. We suspect that these observed sex-specific differences are due to KDM5C/KDM5D dosage rather than the effects of sex hormones, as the DNA methylation at tested targets correlates better with sex chromosome constitution than with gonadal sex, e.g. the highest DNA methylation was observed in 47,XXX females, followed by 47,XXY males, 46,XX females, 46,XY males, 45,X females. The lowest DNA methylation is seen in males with KDM5C mutations  and temporal  maps of KDM5C targets, and to annotate how loss of FBXL5, SCMH1 and CACYBP. We have also demonstrated that loss of DNA methylation at these three genes is specifically associated with KDM5C mutations and is not observed in >900 control blood samples. In addition we have shown that DNA methylation at these three genes correlates with dosage of KDM5C and its Y-linked homologue KDM5D in blood of individuals with different sex chromosome complements. Finally we observed parallel sex-specific differences in several brain regions for FBXL5 and CACYBP, suggesting that these genes could play an important role in the ID phenotype of individuals with KDM5C mutations.We have, for the first time, identified significant multilocus loss of DNA methylation in individuals with loss of function mutations of a gene encoding histone modifying enzyme, specifically a histone H3K4 demethylase KDM5C. We have validated changes in three loci with the most prominent changes: ID: Intellectual disability; XLID: X-linked intellectual disability; H3K4: Histone H3 Lysine 4; ES: embryonic stem; AML: acute myeloid leukemia.The authors declare that they have no competing interests.Conceived and designed the experiments: DG, BHYC, DTB, RW. Performed the experiments: DG, BHYC, SJG, YL and CZ. Analyzed data BHYC, DG, AT, SJG, SC, RR, YAC. Provided materials/reagents/analysis tools/clinical data: FEA, CS, JS, CAB, JH, SW, SWS, CES. Wrote manuscript DG, BHYC, DTB, AT, SC, RW. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1755-8794/6/1/prepubTable S1. Clinical features and demographic information of the 10 male patients with X-linked intellectual disability due to KDM5C mutations, 2 male patients with a variant of unknown significance. Table S2: Primer Sequences. Table S3: Number of significant CpG sites with loss and gain of DNA methylation detected by multivariate permutation analysis for different levels of confidence (1-α) and false discovery proportion limit (γ). Table S4: The top 53 most significant CpG sites that are differentially methylated between the KDM5C mutations and normal controls with the lowest FDP = 0 and the highest confidence level of 99.5%. Table S5: The top 53 most significant CpG sites with additional CpG sites within the same genes. Table S6: GEO studies used to assess DNA methylation at at FBXL5, SCMH1 and CACYBP in blood samples of population controls. Table S7: Analysis of sex specific DNA methylation differences in blood samples from diabetes study (GSE20067). Table S8: Analysis of sex specific DNA methylation differences in brain in FBXL5, SCMH1 and CACYBP promoters.Click here for fileFigure S1. Scatter plots of DNA methylation determined by Illumina (X-axis) and pyrosequencing (Y-axis) at overlapping CpG sites. Grey diamonds are cases with KDM5C mutations, black squares are controls and grey triangles are individuals with p.R1546Q variant. Figure S2: Visual mapping of all 31 samples to the coordinate space defined by the first two principal components reveals that there is no clear separation between samples with and without KDM5C mutations. The principal component analysis (PCA) was performed using all 23,837 CpG sites. The red dots represent the 10 mutation cases, the 19 light green dots represent controls and the two dark green dots represent the benign mutation variants (p.R1546Q). Figure S3: Unsupervised hierarchical clustering of methylation data at 23, 837 CpG sites reveals that there is no clear separation between samples with and without KDM5C mutations. C1-16, are unrelated controls, UN-R1-2 are unaffected relative. R1546Q 1–2 are cases with benign variant, and the rest of the samples are KDM5C mutation cases. Figure S4: Visual mapping of all 31 samples to the coordinate space defined by the first two principal components. The principal component analysis (PCA) was performed using only the methylation levels at the 53 most significant CpG sites  as determined by pyrosequencing. The Y-axis is DNA methylation%. Two groups of samples, controls  and KDM5C mutation cases  are shown on the X-axis. Figure S6: Regional DNA methylation in SCMH1 promoter. A) Screenshot from the UCSC genome browser showing location of SCMH1 promoter exon1, intron1, CpG island, Illumina 27 K microarray probes and pyrosequencing assays and tracks for H3K4me1 and H3K4me3 in lymphoblastoid cell line (GM12878) and ES cell line (H1 h-ES) (Broad Institute Histone data). B&C) Boxplots of Illumina DNA methylation data for two microrray probes within SCMH1 gene. The Y axis shows DNA methylation levels presented as C/C + T and ranging from 0 to 1. The bottom and the top of the box are 25th and 75th percentiles respectively, the whiskers are within the 1.5 interquartile range (IQR) of the data, and the circles, are outlier data points above or below 1.5 IQR. C are controls (N = 19), K are cases with KDM5C mutations (N = 10), V are cases with p.R1546Q sequence variant. D) DNA methylation upstream of SCMH1 transcription start site as determined by pyrosequencing assay (pyro). The order of CpG sites are shown from the further upstream towards the transcription start site. Each column is an average for each of three groups of 1)10 cases with KDM5C mutations (mutation), 2)19 controls (control), and 3) two individuals with the p.R1546Q variant of unknown clinical significance (VUS). The arrows show the CpG sites from the microarray. P-values were determined by Kruskal-Wallis test between mutation cases and controls. Figure S7: Regional DNA methylation in CACYBP promoter. A) Screenshot from the UCSC genome browser showing location of CACYBP promoter, exons1&2, introns 1&2, CpG island, Illumina 27 K microarray probes and pyrosequencing assays and tracks for H3K4me1 and H3K4me3 in lymphoblastoid cell line (GM12878) and ES cell line (H1 h-ES) (Broad Institute Histone data). B&C) Boxplots of Illumina DNA methylation data for two microrray probes within CACYBP gene. The Y axis shows DNA methylation levels presented as C/C + T and ranging from 0 to 1. The bottom and the top of the box are 25th and 75th percentiles respectively, the whiskers are within the 1.5 interquartile range (IQR) of the data, and the circles, are outlier data points above or below 1.5 IQR. C are controls (N = 19), K are cases with KDM5C mutations (N = 10), V are cases with p.R1546Q sequence variant. D) DNA methylation upstream of CACYBP transcription start site as determined by pyrosequencing assay (pyro). The order of CpG sites are shown from the further upstream towards the transcription start site. Each column is an average for each of three groups of 1)10 cases with KDM5C mutations (mutation), 2)19 controls (control), and 3) two individuals with the p.R1546Q variant of unknown significance (VUS). P-values were determined by Kruskal-Wallis test between mutation cases and controls. **** is p <0.0001, * is p <0.05. Figure S8: Regional DNA methylation in ZMYMD12/PPCS promoter. A) Screenshot from the UCSC genome browser showing location of ZMYND12 and PPCS promoter, CpG island, Illumina 27 K microarray probes and pyrosequencing assays and tracks for H3K4me1 and H3K4me3 in lymphoblastoid cell line (GM12878) and ES cell line (H1 h-ES) (Broad Institute Histone data). B&C) Boxplots of Illumina DNA methylation data for two microrray probes within ZMYND12 gene. The Y axis shows DNA methylation levels presented as C/C + T and ranging from 0 to 1. The bottom and the top of the box are 25th and 75th percentiles respectively, the whiskers are within the 1.5 interquartile range (IQR) of the data, and the circles, are outlier data points above or below 1.5 IQR. C are controls (N = 19), K are cases with KDM5C mutations (N = 10), V are cases with p.R1546Q sequence variant. D) DNA methylation upstream of PPCS transcription start site and within exon1/intrion1 of ZMYND12 as determined by pyrosequencing assay (pyro). The order of CpG sites are shown from the further upstream towards the PPCS transcription start site. Each column is an average for each of three groups of 1)10 cases with KDM5C mutations (mutation), 2)19 controls (control), and 3) two individuals with the p.R1546Q variant of unknown significance (VUS). P-values were determined by Kruskal-Wallis test between mutation cases and controls. **** is p <0.0001. Figure S9: Regional DNA methylation in DYDC1/DYDC2 promoter. A) Screenshot from the UCSC genome browser showing location of DYDC1/DYDC2 promoter, CpG island, Illumina 27 K microarray probes and pyrosequencing assays and tracks for H3K4me1 and H3K4me3 in lymphoblastoid cell line (GM12878) and ES cell line (H1 h-ES) (Broad Institute Histone data). B&C) Boxplots of Illumina DNA methylation data for two microrray probes within DYDC1 gene. The Y axis shows DNA methylation levels presented as C/C + T and ranging from 0 to 1. The bottom and the top of the box are 25th and 75th percentiles respectively, the whiskers are within the 1.5 interquartile range (IQR) of the data, and the circles, are outlier data points above or below 1.5 IQR. C are controls (N = 19), K are cases with KDM5C mutations (N = 10), V are cases with p.R1546Q sequence variant. D) DNA methylation upstream of DYDC1 transcription start site as determined by pyrosequencing assay (pyro) overlapping Illumina CpG site cg17703212. Column is an average for each of three groups of 1)10 cases with KDM5C mutations (mutation), 2)19 controls (control), and 3) two individuals with the p.R1546Q variant of uknown significance (VUS). P-values were determined by Kruskal-Wallis test between mutation cases and controls. **** is p <0.0001. Figure S10: Expression of FBXL5, SCMH1 and CACYBP in a panel of human tissues: BT -brain total, FB- fetal brain, CC-cerebral cortex, CB-cerebellum, TL- temporal lobe, FL –frontal lobe, SC –spinal cord, K-kidney, H-heart, L-liver, SM-smooth muscle, SK-skeletal muscle, LCL-lymphoblastoid cell line. Error bars are standard deviation of duplicated q-PCR experiments. Figure S11: DNA methylation levels of three CpG sites cg02630888 (FBXL5), cg03387723 (SCMH1), cg16743289 (CACYBP) in different blood cell types (GSE35069). The cell types are whole blood, peripheral blood mononuclear cells (PBMC), granulocytes and seven isolated cell populations , and eosinophils (Eos)) which are each shown by different colour. Y-axis is beta methylation value determined by Illumina methylation450 array (GSE35069). Each column represents mean methylation from 6 samples from healthy males, error bars are standard deviation from the mean.Click here for file


Drosophila melanogaster crammer is a novel cathepsin inhibitor involved in long-term memory formation. A molten globule-to-ordered structure transition is required for cathepsin inhibition. This study reports the use of alanine scanning to probe the critical residues in the two hydrophobic cores and the salt bridges of crammer in the context of disorder-to-order transition and cathepsin inhibition. Alanine substitution of the aromatic residues W9, Y12, F16, Y20, Y32, and W53 within the hydrophobic cores, and charged residues E8, R28, R29, and E67 in the salt bridges considerably decrease the ability of crammer to inhibit Drosophila cathepsin B (CTSB). Far-UV circular dichroism (CD), intrinsic fluorescence, and nuclear magnetic resonance (NMR) spectroscopies show that removing most of the aromatic and charged side-chains substantially reduces thermostability, alters pH-dependent helix formation, and disrupts the molten globule-to-ordered structure transition. Molecular modeling indicates that W53 in the hydrophobic Core 2 is essential for the interaction between crammer and the prosegment binding loop (PBL) of CTSB; the salt bridge between R28 and E67 is critical for the appropriate alignment of the α-helix 4 toward the CTSB active cleft. The results of this study show detailed residue-specific dissection of folding transition and functional contributions of the hydrophobic cores and salt bridges in crammer, which have hitherto not been characterized for cathepsin inhibition by propeptide-like cysteine protease inhibitors. Because of the involvements of cathepsin inhibitors in neurodegenerative diseases, these structural insights can serve as a template for further development of therapeutic inhibitors against human cathepsins. Drosophila melanogasterCysteine proteases, which are responsible for the processes of intra- and extra-cellular protein degradation and turn-over D. melanogaster, also known as Drosophila cytotoxic T lymphocyte antigen-2 (CTLA-2)-like protein, (D/CTLA-2), is of particular interest D. melanogasterResearch over the past two decades has reported several propeptide-like cysteine protease inhibitors Multiple sequence alignment of crammer with its homologs indicates that several aromatic residues in the hydrophobic cores and charged residues of the salt bridges in crammer are highly conserved among cathepsin propeptides and cytotoxic T lymphocyte antigen-2α and β (CTLA-2α and 2β). Using site-directed mutagenesis, Wiederanders et al. showed that W28, W31, and W52 of human cathepsin S propeptide are essential for the inhibition of mature cathepsin S, and that the aromatic stacking of these residues stabilizes the core structure of the propeptide domain Based on structural information obtained previously L-trans-epoxysuccinyl-leucylamido (4-guanidino) butane (E-64) and Z-Phe-Arg 7-amido-4-methylcoumarin hydrochloride (ZFR-AMC) were obtained from Sigma-Aldrich . β-Mercaptoethanol and chloramphenicol were supplied by Merck Research Laboratories . Ampicillin, isopropyl β-D-1-thiogalactopyranoside (IPTG), and dithiothreitol (DTT) were purchased from MDBio Inc. . Ethylenediaminetetraacetic acid (EDTA) was purchased from USB Corporation . Glacial acetic acid was supplied by Echo Chemical Company, Ltd. . All chemicals used were of analytical grade quality.E. coli Rosetta (DE3) strain . The cells were incubated at 37°C in lysogeny broth (LB) 600) of the cell suspension reached 0.7, IPTG was added to the cell culture at a final concentration of 1 mM to induce recombinant protein overexpression. 15N-labeled recombinant proteins were obtained from E. coli cultures in M9 minimal medium 15NH4Cl  E. coli cells were harvested by centrifugation at 6,000× g for 20 min, and the resulting cell pellets were lysed with glacial acetic acid. The lysate was subsequently subjected to centrifugation at 30,700× g for 20 min, and the supernatant was collected and dialyzed against Milli-Q water at 4°C overnight. A second centrifugation at 30,700× g for 20 min was then used to remove any precipitants. All recombinant proteins were purified using a C18 semi-preparative column  coupled to a 1100 Series reverse-phase high performance liquid chromatography (RP-HPLC) system . A linear water/acetonitrile gradient (from 29% to 55% acetonitrile over 40min) was used for protein separation at a flow rate of 1 ml/min. The purified protein fractions were characterized by an Autoflex III MALDI-TOF Mass Spectrometer . Protein concentrations were determined using the Bio-Rad Protein Assay  with bovine serum albumin as the standard.WT was cloned into a pAED4 vector Drosophila cathepsin B (CTSB) were performed as described previously E. coli BL21-Gold (DE3) cells  and then cultured in LB containing ampicillin (50 mg/ml) at 37°C. When the OD600 of the culture reached 0.7, the protein was induced by IPTG  for 3.5 h. Cells were harvested by centrifugation at 4,000× g for 20 min and lysed by sonication. The lysates were further centrifuged at 16,000× g for 30 min at 4°C. The overexpressed CTSB existed mainly in the inclusion body and therefore in vitro refolding was required to obtain purified CTSB. The supernatant and cell lysate were discarded, and the pellets were dissolved in 40 ml of 6 M guanidine hydrochloride buffer (50 mM Tris/HCl (pH 8.0) 150 mM NaCl, 5 mM EDTA, and 10 mM DTT, 6 M guanidine-HCl). For in vitro refolding, the denatured protein solution was diluted into 1 liter of 50 mM Tris/HCl (pH 8.5), 150 mM NaCl, 5 mM EDTA, 10 mM reduced glutathione, 1 mM oxidized glutathione, and 0.5 M arginine and incubated overnight at 4°C with stirring to facilitate refolding. The protein solution was subsequently concentrated and dialyzed against 25 mM NaH2PO4, pH 7.0, 0.5 M NaCl at 4°C, to yield approximately 50 ml of protein solution.The expression and purification of Procathepsin B must be autoprocessed . The autoprocessing procedure has been described elsewhere previously ex = 380 nm) The concentration of active CTSB was determined using the cysteine protease inhibitor, E-64 http://agadir.crg.es) and CD spectroscopy was used to determine the protein stability of the mutants at pH 4.0 and 6.0. Thermal denaturation experiments were conducted by monitoring changes in the CD signal at 208 nm between 4°C to 96°C in 2°C increments. The thermodynamic properties of crammer variants were calculated by fitting the unfolding curves with the following equation Yobs is the observed CD signal at a given temperature, T in Kelvin (K). Yn and Mn represent the intercept and the slope of the pre-transition straight line, respectively. Yd and Md represent the intercept and slope of the post-transition straight line, respectively. After curve fitting, enthalpy (ΔH) and entropy (ΔS) of mutants were determined (u) was also deduced on the basis of the Gibbs free energy equation. Moreover, the intrinsic fluorescence of crammer variants was monitored at pH 4.0 and 6.0 using a fluorescence spectrophotometer . The protein concentrations were 30 µM. The excitation wavelength used was 280 nm and fluorescence emission was monitored between 290 nm and 400 nm.Far-UV CD and fluorescence spectroscopies were performed using previously described procedures termined . The unf15N-labeled double mutants were buffered in 10 mM citric acid-sodium phosphate buffer with 10% D2O (v/v) for heteronuclear NMR studies at pH 4.0 and 6.0. 1H-15N heteronuclear single quantum correlation (HSQC) spectra were recorded at 25°C using a 600 MHz NMR spectrometer  equipped with a 1H/13C/15N triple resonance room temperature probe. All NMR data were processed using TopSpin (Bruker) and NMRPipe www.cgl.ucsf.edu/home/sparky).Uniformly Crammer contains seven aromatic residues in the hydrophobic Cores 1 and 2. These residues are located in the α-helix 1 , loop 1 (Y20), and the α-helix 2 (Y32) for hydrophobic Core 1, and the α-helix 2 (F46) and loop 2 (W53) for the hydrophobic Core 2 . Four sain vitro through the formation of an intermolecular disulfide bond through the cysteine residue at position 72, Cys72. The replacement of Cys72 with Ser  completely abolishes the covalent dimer formation The WT crammer dimerizes u) and the population of denatured state at 20°C  are 1.70 (D6-R29) and 2.77 (R28-E67) kcal/mol, respectively  also ext pH 6.0 , and theectively . Hence, ectively . The dismax = 326.5 nm), indicating that these residues become buried within the hydrophobic core of a folded state . For C72S/W53A, a blue-shift of its λmax value is from 336 nm to 326 nm when the pH increases from 4.0 to 6.0. The solution structure of C72S shows that W9 is buried in the hydrophobic Core 1 , whereas W53 is more accessible to solvent . The replacement of W9 with alanine causes greater disruption of the structural elements and stability which are associated with the pH-dependent disorder-to-order transition.This study uses intrinsic fluorescence to assess the effects of the mutations on the folding transition of crammer. At pH 4.0, C72S exhibits an emission maximum at 336.5 nm , indicated state . This bled state . CrammerOther mutants in the hydrophobic Core 1  have lower MEWD values (5–8 nm) . These d1H-15N HSQC spectrum (most of which can be superimposed on those observed for C72S at pH 6.0) 1H-15N HSQC spectra at pH 4.0 and at pH 6.0. 1H-15N HSQC spectra at pH 6.0 X3(F/Y)X3YX12(F/Y/W) (Crammer has a high sequence homology (>36%) with the propeptides of cysteine proteases , and its2(F/Y/W)  that is 2(F/Y/W) . In cont2(F/Y/W) . TherefoDrosophila procathepsin B to clarify the role of W53 in crammer. This model was superimposed to the structures of procathepsins S (CTSS), K (CTSK), and crammer , but these two residues have different effects on the Trp fluorescence emission. C72S/W53A apparently exhibits a larger blue-shift than C72S/W9A . This su crammer . ResultsThis study also investigates the importance of the salt bridges in crammer. The proposed double mutant cycle analysis shows that D6-R29 and R28-E67 are critical for crammer stability . The repDrosophila crammer in terms of CTSB inhibition, structure, stability, and disorder-to-order transition. The results of this study show that the conserved aromatic residues W9, Y12, Y20, and Y32 at the hydrophobic Core 1 are critical for folding transition and CTSB inhibition. These residues form π-π interactions and van der Waals contacts to maintain the integrity of the hydrophobic core. This in turn promotes the pH-dependent molten globule-to-ordered structure transition in crammer. The propeptides of human cathepsins adopt different steric arrangements at these corresponding residues, but they still adopt similar π-π stacking interactions to stabilize the hydrophobic core and the overall folding. Hence, this π-π stacking is required for the propeptides and crammer to trigger efficient inhibition activity against cysteine protease. The solvent-exposed W53 at the hydrophobic Core 2 of crammer probably directly interacts with PBL of Drosophila CTSB to express its CTSB inhibition. Finally, the conserved charged residues in crammer provide an interaction network to promote the helix formation and maintain thermostability. Specifically, the disruption of the R28-E67 salt bridge in crammer apparently disturbs the structure transition, thus reducing CTSB inhibition. These results provide an insight into the structural and functional roles of key amino acids in crammer, which is useful for the development of cysteine protease inhibitors.This study uses alanine scanning to identify the critical amino acids of Figure S1Comparison of protein structure and stability of C72S and all double mutants at pH 6.0. To clearly realize the relationship of protein structure and stability upon the mutation, we made a figure to compare the ellipticity (A), the MEWD value (B), the unfolding free energy (ΔGu) (C), and the fraction unfold (D) at 20°C. The CD signal of each mutant was recorded at 208 nm and presented in millidegree. The MEWD values were obtained from the results of the intrinsic fluorescence measurement. The fraction unfold and the unfolding free energy were determined from the thermal denatureation curves u as compared with those of C72S ( of C72S  and S1. (TIF)Click here for additional data file.Figure S2Thermal denaturation of C72S and salt bridge mutants. Thermal unfolding curves of C72S, double mutants  and triple mutants (C72S/D6A/R29A and C72S/R28A/E67A) were monitored at 208 nm from 4°C to 96°C at pH 6.0.(TIF)Click here for additional data file.Figure S3int) at pH6.0. (A)Coupling energy of salt bridge  The scheme shows that the pair-wise interaction energy (ΔΔGint) is calculated from the unfolding free energy (ΔGu) of wild-type (WT) protein, single-mutants (M+ve and M−ve), and double-mutant (DM). The substitutions are indicated inside the boxes and the ΔΔGu values for processes A–D are shown along the arrows. The ΔΔGu value is the difference of the unfolding free energies due to mutation, The ΔΔGint value is then calculated using an equation that is showed in the figure. The circles, labeled with “−”, “+”, and blank signs mean a negative charged residue, a positive charged residue and an alanine substitution, respectively. (B) The coupling energy (ΔΔGint) for salt bridge, D6-R29, is 2.77 kcal mol−1. (C) ΔΔGint for salt bridge, R28-E67, is 1.70 kcal mol−1. The positive ΔΔGint indicates that these two salt bridges have significantly contribution to the stability of crammer.(TIF)Click here for additional data file.Figure S41H-15N-HSQC spectra of the hydrophobic core 1 double mutants of crammer at pH 4.0.(TIF)Click here for additional data file.Figure S5Digestion of crammer single mutants by CTSB. In order to evaluate the proteolysis resistance, 3 µM of each single mutant  and wild-type crammer (Cer) were incubated with Drosophila cathepsin B  in 100 mM sodium acetate (pH 5.0), 1 mM EDTA and 2 mM DTT at 25°C for 1 and 2 hours. The digested protein solutions were further analyzed by 13% (w/v) Tricine-SDS/PAGE. The molecular weight of single mutants is ∼9.5 kD. Cer and C72S are resistant to CTSB digestion, but, however, W9A, F16A, and R28A exhibited onset of digestion after incubated with CTSB for 1 and 2 hours.(TIF)Click here for additional data file.Figure S6Structural alignment of atoms of crammer with the propeptides of three human cathepsins. Superimposition of the Cα atoms of crammer  with those of the human cathepsin propeptides L  Orientation of the aromatic residues in the hydrophobic core 1 of crammer. (B) Superposition of the conserved aromatic residues of the propeptides of human cathepsin L, K and S with those of crammer. (C) Orientations of the aromatic residues in the hydrophobic cores of the propeptides of human cathepsins L, K, and S. The picture was prepared with PyMOL (TIF)Click here for additional data file.Figure S7Structural alignment of crammer, the human procathepsins K and S, and the modeled structure of Drosophila procathepsin B. 3D coordinates for crammer (yellow), and human procathepsins K (blue) and S (red), are taken from the PDB (entries 2KTW Drosophila procathepsin B (light grey) structure is modeled using Modeller Drosophila procathepsin B and crammer is 10.2 Å. Insert: Expanded view of the interactions between the conserved aromatic residues of the propeptides and the prosegment binding loop (PBL) of mature cathepsin: W53 of crammer and W27 of the propeptide of Drosophila procathepsin B interact with W261 of PBL of mature Drosophila CTSB. Additionally, Y58 of human procathepsin K, and Y56 of procathepsin S make contacts with the aromatic residues of the PBL of mature human cathepsin K at Y150, andcathepsin S at Y153. The picture was prepared with PyMOL (TIF)Click here for additional data file.Figure S8Expression profiles of double mutants cycles . The protein samples were analyzed by 13% (w/v) Tricine-SDS/PAGE. An arrow indicates the expected position of the proteins. M, protein molecular weight standard; C, uninduced control without IPTG induction; I, expression of the double mutant cycles after IPTG induction.(TIF)Click here for additional data file.Table S1Thermal dynamic parameters of mutant proteins at pH 6.0. The entropy (ΔS) and the enthalpy (ΔH) of mutants are determined from the thermal denaturation curves u) at 277K and 293 K can be deduced according to Gibbs free energy equation.(DOCX)Click here for additional data file.Table S2Analysis of the π-π interactions of crammer and the propeptides of human cathepsins L (HCTSL), K (HCTSK) and S (HCTSS).(DOCX)Click here for additional data file.Table S3Hydrophobic contact analysis. The software, Ligplot, (DOCX)Click here for additional data file.Table S4The predicted helical propensities of double mutants in salt bridges by the agadir program (http://agadir.crg.es).(DOCX)Click here for additional data file.