Protein Disulfide Isomerase Regulates Endoplasmic Reticulum Stress and the Apoptotic Process during Prion Infection and PrP Mutant-Induced Cytotoxicity

<div><h3>Background</h3><p>Protein disulfide isomerase (PDI), is sorted to be enzymatic chaperone for reconstructing misfolded protein in endoplasmic reticulum lumen. Recently, PDI has been identified as a link between misfolded protein and neuron apoptosis. However, the potential for PDI to be involved in the pathogenesis of prion disease remains unknown. In this study, we propose that PDI may function as a pleiotropic regulator in the cytotoxicity induced by mutated prion proteins and in the pathogenesis of prion diseases.</p> <h3>Methodology/Principal Findings</h3><p>To elucidate potential alterations of PDI in prion diseases, the levels of PDI and relevant apoptotic executors in 263K infected hamsters brain tissues were evaluated with the use of Western blots. Abnormal upregulation of PDI, Grp78 and Grp58 was detected. Dynamic assays of PDI alteration identified that the upregulation of PDI started at the early stage and persistently increased till later stage. Obvious increases of PDI and Grp78 levels were also observed in cultured cells transiently expressing PrP mutants, PrP-KDEL or PrP-PG15, accompanied by significant cytotoxicities. Excessive expression of PDI partially eased ER stress and cell apoptosis caused by accumulation of PrP-KDEL, but had less effect on cytotoxicity induced by PrP-PG15. Knockdown of endogenous PDI significantly amended cytotoxicity of PrP-PG15, but had little influence on that of PrP-KDEL. A series of membrane potential assays found that apoptosis induced by misfolded PrP proteins could be regulated by PDI via mitochondrial dysfunction. Moreover, biotin-switch assays demonstrated active <em>S</em>-nitrosylted modifications of PDI (SNO-PDI) both in the brains of scrapie-infected rodents and in the cells with misfolded PrP proteins.</p> <h3>Conclusion/Significance</h3><p>Current data in this study highlight that PDI and its relevant executors may function as a pleiotropic regulator in the processes of different misfolded PrP proteins and at different stages during prion infection. SNO-PDI may feed as an accomplice for PDI apoptosis.</p> </div

Identification of a Lacosamide Binding Protein Using an Affinity Bait and Chemical Reporter Strategy: 14-3-3 ζ

We have advanced a useful strategy to elucidate binding partners of ligands (drugs) with modest binding affinity. Key to this strategy is attaching to the ligand an affinity bait (AB) and a chemical reporter (CR) group, where the AB irreversibly attaches the ligand to the receptor upon binding and the CR group is employed for receptor detection and isolation. We have tested this AB&CR strategy using lacosamide ((R)-1), a low-molecular-weight antiepileptic drug. We demonstrate that using a (R)-lacosamide AB&CR agent ((R)-2) 14-3-3 ζ in rodent brain soluble lysates is preferentially adducted, adduction is stereospecific with respect to the AB&CR agent, and adduction depends upon the presence of endogenous levels of the small molecule metabolite xanthine. Substitution of lacosamide AB agent ((R)- 5) for (R)-2 led to the identification of the 14-3-3 ζ adduction site (K120) by mass spectrometry. Competition experiments using increasing amounts of (R)-1 in the presence of (R)-2 demonstrated that (R)-1 binds at or near the (R)-2 modification site on 14-3-3 ζ. Structure-activity studies of xanthine derivatives provided information concerning the likely binding interaction between this metabolite and recombinant 14-3-3 ζ. Documentation of the 14-3-3 ζ-xanthine interaction was obtained with isothermal calorimetry using xanthine and the xanthine analogue 1,7-dimethylxanthine

Species-Specific Immunodetection of an Entamoeba histolytica Cyst Wall Protein.

Entamoeba histolytica causes intestinal disease in endemic settings throughout the world. Diagnosis of E. histolytica infection would be improved by the identification of biomarkers that are expressed by cysts of E. histolytica, but not by cysts of closely related commensal species of Entamoeba. Herein, we describe two novel monoclonal antibodies (1A4 and 1D3) produced against a spacer region of the E. histolytica Jacob2 lectin, an outer cyst wall protein. These reagents demonstrated no cross-reaction to E. dispar recombinant antigen and low picomolar molecular detection limits when paired in ELISA sandwich assays. In an immunofluorescence microscopy assay, the α-Jacob2 murine antibodies labeled cysts of three xenically cultured E. histolytica isolates but did not label cysts of three E. bangladeshi isolates. Monoclonal antibody 1A4 did not cross-react with xenic cultures of three E. dispar isolates, demonstrating specificity to E. histolytica, while monoclonal antibody 1D3 cross-reacted with two out of three E. dispar isolates. Both antibodies labeled cysts in formalin-fixed slides, a potential logistical advantage in some settings. The monoclonal antibody 1A4 was also used in an immunofluorescence microscopy assay with formalin-fixed stool specimens. Seven out of ten ELISA-positive stool specimens exhibited 1A4-labeled cyst-like objects, compared to one out of seven ELISA-negative specimens. These results demonstrate that antibodies generated against the flexible spacer of E. histolytica Jacob2 lectin recognize and bind to Jacob2 protein in whole cysts and are capable of differentiating Entamoeba species in fixed specimens. Thus, Jacob2 is a promising biomarker for use in diagnosing E. histolytica infection

<i>Entamoeba</i> species specificities of a-Jacob antibodies 1A4 (A) and 1D3 (B) in immunofluorescence microscopy.

<p>Smears of xenic <i>Entamoeba</i> isolates were doubly stained with 0.1% Calcofluor White M2R and an anti-Jacob primary antibody with goat anti-mouse Alexa-Fluor 488. Each data point represents the mean Alexa Fluor 488 intensity of a Calcofluor-positive, cyst-like object detected by microscopy. The data points are arranged horizontally by isolate, and black horizontal bars represent the mean Alexa Fluor 488 intensity of each isolate. The overall mean Alexa Fluor 488 intensities of the <i>Entamoeba histolytica</i> isolates (n = 3) were compared to those of <i>E</i>. <i>dispar</i> isolates (n = 3) and of <i>Entamoeba bangladeshi</i> isolates (n = 3) with the Mann-Whitney U test. <i>Eh</i> = <i>Entamoeba histolytica</i>, <i>Ed = Entamoeba dispar</i>, <i>Eb = Entamoeba bangladeshi</i>.</p

Representative photos from an anti-Jacob2 immunofluorescence microscopy assay.

<p>Isolates were doubled stained with 0.1% Calcofluor White M2R and primary anti-Jacob2 monoclonal antibody (1A4 or 1D3) with goat anti-mouse Alexa Fluor 488. The three species examined were pathogen <i>Entamoeba histolytica</i> (1^st column) and commensals <i>Entamoeba dispar</i> and <i>Entamoeba bangladeshi</i> (2^nd and 3^rd columns). Calcofluor was utilized to identify chitinaceous <i>Entamoeba</i> cysts (2^nd and 4^th rows).</p

ClustalW alignment of the Jacob2 proteins from <i>Entamoeba histolytica</i> HM-1:IMSS (EHI_044500) and <i>Entamoeba dispar</i> SAW760 (EDI_246160).

<p>The ClustalW BLOSUM matrix was used with a gap open cost of 10 and a gap extension cost of 0.1. This figure was generated in Geneious 6.0.3. Cloned residues for “EhJacob” and “EdJacob” recombinant antigens are highlighted in light gray and dark gray respectively.</p

Summary of screens for α-Jacob murine monoclonal antibodies.

<p>Summary of screens for α-Jacob murine monoclonal antibodies.</p

Immunofluorescence staining and image analysis of stool specimens stained with antibody 1A4.

<p>ELISA-positive (triangle) and ELISA-negative (square) specimens were stained with DAPI and antibody 1A4 as described in the text. Eight specimens (open symbols) exhibited no detectable cyst-like objects under the DAPI filter. The bottom panels show a typical cyst detectable by (left to right) bright field, DAPI staining, and antibody 1A4 staining microscopy conducted on a fixed stool slide.</p

Monoclonal antibodies 1A4 and 1D3 detecting <i>Entamoeba</i> recombinant Jacob2 antigens in a sandwich ELISA.

<p>Measured antigen concentration ranged from 2.4–2500 pM. Columns and error bars represent mean OD450 ± standard deviation of three replicate assays. Limit of detection (LOD) was calculated as three standard deviations above the mean OD450 of EdJacob at 2500 pM concentration.</p