Caspase Inhibitors of the P35 Family Are More Active When Purified from Yeast than Bacteria

Many insect viruses express caspase inhibitors of the P35 superfamily, which prevent defensive host apoptosis to enable viral propagation. The prototypical P35 family member, AcP35 from Autographa californica M nucleopolyhedrovirus, has been extensively studied. Bacterially purified AcP35 has been previously shown to inhibit caspases from insect, mammalian and nematode species. This inhibition occurs via a pseudosubstrate mechanism involving caspase-mediated cleavage of a “reactive site loop” within the P35 protein, which ultimately leaves cleaved P35 covalently bound to the caspase's active site. We observed that AcP35 purifed from Saccharomyces cerevisae inhibited caspase activity more efficiently than AcP35 purified from Escherichia coli. This differential potency was more dramatic for another P35 family member, MaviP35, which inhibited human caspase 3 almost 300-fold more potently when purified from yeast than bacteria. Biophysical assays revealed that MaviP35 proteins produced in bacteria and yeast had similar primary and secondary structures. However, bacterially produced MaviP35 possessed greater thermal stability and propensity to form higher order oligomers than its counterpart purified from yeast. Caspase 3 could process yeast-purified MaviP35, but failed to detectably cleave bacterially purified MaviP35. These data suggest that bacterially produced P35 proteins adopt subtly different conformations from their yeast-expressed counterparts, which hinder caspase access to the reactive site loop to reduce the potency of caspase inhibition, and promote aggregation. These data highlight the differential caspase inhibition by recombinant P35 proteins purified from different sources, and caution that analyses of bacterially produced P35 family members (and perhaps other types of proteins) may underestimate their activity

PENGARUH POTASSIUM PEROXYMONOPERSULFATE TERHADAP PERUBAHAN WARNA BASIS GIGI TIRUAN THERMOPLASTIC NYLON

Thermoplastic nylon denture bases are derivatives from diamine and dibasic acid monomers that has ideal features as dentures for it�s great flexibility and aesthetics, also it doesn�t cause allergies. Thermoplastic nylon has a high water absorption properties that can cause discoloration. Potassium peroxymonopersulfate is present as a component of a triple salt, free flowing, white granular and soluble in water. Potassium peroxymonopersulfate are active ingredients of the alkaline peroxide denture cleanser. Chemically, potassium peroxymonopersulfate are derivatives of hydrogen peroxide, which breaks down in water into free radicals (HO2 * and O*) that damages the pigment color. This study is aimed to determine the color stability of thermoplastic nylon denture againts the immersion time in potassium peroxymonopersulfate. Subjects of this study were 28 samples of thermoplastic nylon, rectangular shaped, with dimensions of 25 x 15 x 2.5 mm and were divided into 4 groups, containing 7 samples each. The first group, as control was immersed in aquadestilata. The second group was immersed in denture cleanser solution containg potassium peroxymonopersulfate for 12 hours. The third group was immersed in denture cleanser solution containg potassium peroxymonopersulfate for 24 hours and the fourth group was immersed in denture cleanser solution containg potassium peroxymonopersulfate for 36 hours. The absorbance changes of all samples were measured with spectrofotometer. Data obtained were then analyzed using ANOVA followed by Tukey's test. Results showed that there were significant differences in color stabilty of thermoplastic nylon on immersion of denture cleanser solution containing potassium peroxymonopersulfate (p <0.05). The conclusion of this study is there were differences in color stability of thermoplastic nylon denture base on immersion of denture cleanser solution containing potassium peroxymonopersulfate, but immersion within 12 hours are still esthetically safe

N-linked glycosylation modulates dimerization of protein disulfide isomerase family A member 2 (PDIA2)

Protein disulfide isomerase (PDI) family members are important enzymes for the correct folding and maturation of proteins that transit or reside in the endoplasmic reticulum (ER). The human PDI family comprises at least 19 members that differ in cell type expression, substrate specificity and post-translational modifications. PDI family A member 2 (PDIA2, previously known as PDIp) has a similar domain structure to prototypical PDI (also known as PDIA1), but the function and post-translational modifications of PDIA2 remain poorly understood. Unlike most PDI family members, PDIA2 contains three predicted N-linked glycosylation sites. By site-directed mutagenesis and enzymatic deglycosylation, we show here that all three Asn residues within the potential N-linked glycosylation sites of human PDIA2 (N127, N284 and N516) are glycosylated in human cells. Furthermore, mutation of N284 to glycosylation-null Gln increases formation of a highly stable disulfide-bonded PDIA2 dimer. Nevertheless, in HeLa cells, both wild-type and N127/284/516Q mutant PDIA2 proteins localize to the ER, but not the ER-Golgi intermediate compartment, suggesting that glycosylation is important for PDIA2 protein-protein interactions but not subcellular localization. Finally, we identified human major histocompatibility complex class 1 antigens (HLA-A,B,C) as potential binding partners of PDIA2, suggesting an involvement for PDIA2 in antigen presentation in addition to its previously described roles in autoimmunity and Parkinson's disease. These results further characterize this poorly defined member of the PDI family.11 page(s

C9ORF72, implicated in amytrophic lateral sclerosis and frontotemporal dementia, regulates endosomal trafficking

Intronic expansion of a hexanucleotide GGGGCC repeat in the chromosome 9 open reading frame 72 (C9ORF72) gene is the major cause of familial amyotrophic lateral sclerosis(ALS)and frontotemporal dementia. However, the cellular function of the C9ORF72 protein remains unknown. Here, we demonstrate that C9ORF72 regulates endosomal trafficking. C9ORF72 colocalized with Rab proteins implicated in autophagy and endocytic transport: Rab1, Rab5, Rab7 and Rab11 in neuronal cell lines, primary cortical neurons and human spinal cord motor neurons, consistent with previous predictions that C9ORF72 bears Rab guanine exchange factor activity. Consistent with this notion, C9ORF72 was present in the extracellular space and as cytoplasmic vesicles. Depletion of C9ORF72 using siRNA inhibited transport of Shiga toxin from the plasma membrane to Golgi apparatus, internalization of TrkB receptor and altered the ratio of autophagosome marker light chain 3(LC3) II:LC3I, indicating that C9ORF72 regulates endocytosis and autophagy. C9ORF72 also colocalized with ubiquilin-2 and LC3-positive vesicles, and co-migrated with lysosome-stained vesicles in neuronal cell lines, providing further evidence that C9ORF72 regulates autophagy. Investigation of proteins interacting with C9ORF72 using mass spectrometry identified other proteins implicated in ALS; ubiquilin-2 and heterogeneous nuclear ribonucleoproteins, hnRNPA2/B1 and hnRNPA1, and actin. Treatment of cells overexpressing C9ORF72 with proteasome inhibitors induced the formation of stress granules positive for hnRNPA1 and hnRNPA2/B1. Immunohistochemistry of C9ORF72 ALS patient motor neurons revealed increased colocalization between C9ORF72 and Rab7 and Rab11 compared with controls, suggesting possible dysregulation of trafficking in patients bearing the C9ORF72 repeat expansion. Hence, this study identifies a role for C9ORF72 in Rab-mediated cellular trafficking.17 page(s

MaviP35 and AcP35 are more active when purified from yeast than bacteria.

<p>(<b>A</b>) FLAG-tagged MaviP35 or AcP35 were purified from bacteria or yeast. The indicated concentrations of inhibitors were assayed for their ability to inhibit cleavage of 100 µM Ac-DEVD-AFC by 30 nM caspase 3. Error bars represent S.E.M. from three independent replicates. (<b>B</b>) A competitive model was used to determine the caspase 3 inhibition constants for the P35 proteins purified from yeast <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039248#pone.0039248-Brand1" target="_blank">[22]</a> and bacteria.</p

MaviP35 proteins purified from bacteria and yeast have similar secondary structures.

<p>FLAG-tagged MaviP35 proteins were purified from yeast and bacteria and then subjected to circular dichroism (CD) analyses. (<b>A</b>) Wavelength scans were performed at 20°C. The final spectra is the average result of three scans (open circles). The CONTINLL algorithm calculated the nonlinear least squares best fit (solid line) against the SP29 protein database with r.m.s.d. values≤0.073. (<b>B</b>) Table of secondary structure proportions and apparent melting temperature for MaviP35 purified from bacterial and yeast. (<b>C</b>) Ellipticity at 216 nm was measured between 20 and 90°C (open circles). The nonlinear regression analysis (dashed lines) fitted the curves to a one step transition between folded and unfolded confirmations.</p

Bacterially produced MaviP35 is more prone to aggregation <i>in vitro</i> than yeast-expressed MaviP35.

<p>FLAG-tagged MaviP35 proteins were purified from bacteria or yeast and then treated with 0 or 5 µM of the crosslinker BMH prior to analysis by SDS-PAGE and anti-FLAG immunoblotting. Arrows on the right indicate the expected positions of various oligomeric species based on the migrations of the molecular weight markers. The expected molecular weight of octameric MaviP35 (286 kDa) would be greater than that of the largest marker used, so its migration is difficult to accurately estimate. A representative immunoblot is shown from four separate experiments.</p

Caspase 3 can cleave MaviP35 purified from yeast but not bacteria.

<p>FLAG-tagged MaviP35 was purified from bacteria or yeast by affinity chromotography and gel filtration. The ∼94 kDa gel filtration fraction of the bacterial preparation (“b4” in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039248#pone-0039248-g005" target="_blank">Figure 5</a>) and the ∼72 kDa fraction of the yeast-purified sample (“y3” in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039248#pone-0039248-g005" target="_blank">Figure 5</a>) were incubated with 0, 10, 100 or 1000 ng/ml caspase 3 for 1 hour, and then subjected to SDS-PAGE and anti-FLAG immunoblotting.</p

Expression in bacteria or yeast does not substantially alter the primary structural features of MaviP35 and AcP35.

<p>FLAG-tagged MaviP35 and AcP35 were purified from bacteria or yeast. (<b>A</b>) Each sample was analyzed using Matrix Assisted Laser Desorption Ionisation-Mass Spectrometry. (<b>B</b>) Reduced and alkylated proteins were subjected to trypsin digestion, then the peptides were analyzed by LC-ESI-MS. The intensity of peaks of masses corresponding to the predicted peptide mass of unmodified or modified amino terminal tryptic peptides were measured. Based on the assumption that the peak intensities are proportional to the peptide amount, the relative amount of each peptide was calculated. The integrities of the peptides were confirmed by MS/MS analysis.</p

Analytical ultracentrifugation of MaviP35 purified from bacteria and yeast.

<p>(<b>A</b>) MaviP35-FLAG proteins were purified from bacteria (gray line) and yeast (black line) and then subjected to gel filtration. (<b>B–F</b>) Analytical ultracentrifugation of the fractions indicated in gray shading yielded the sedimentation profiles shown. The molecular weights estimated for each peak are stated. Numbers in parentheses indicate numbers of MaviP35-FLAG monomers that could comprise the major oligomeric species resolved by analytical ultracentrifugation.</p