Heat shock-induced enhanced susceptibility of barley to Bipolaris sorokiniana is associated with elevated ROS production and plant defence-related gene expression.

Heat stress alters plant defence responses to pathogens. Short-term heat shock promotes infections by biotrophic pathogens. However, little is known about how heat shock affects infection by hemibiotrophic pathogens like Bipolaris sorokiniana (teleomorph: Cochliobolus sativus). We assessed the effect of heat shock in B. sorokiniana-susceptible barley (Hordeum vulgare cv. Ingrid) by monitoring leaf spot symptoms, B. sorokiniana biomass, ROS and plant defence-related gene expression following pre-exposure to heat shock. For heat shock, barley plants were kept at 49 °C for 20 s. B. sorokiniana biomass was assessed by qPCR, ROS levels determined by histochemical staining, while gene expression was assayed by RT-qPCR. Heat shock suppressed defence responses of barley to B. sorokiniana, resulting in more severe necrotic symptoms and increased fungal biomass, as compared to untreated plants. Heat shock-induced increased susceptibility was accompanied by significant increases in ROS (superoxide, H2 O2 ). Transient expression of plant defence-related antioxidant genes and a barley programmed cell death inhibitor (HvBI-1) were induced in response to heat shock. However, heat shock followed by B. sorokiniana infection caused further transient increases in expression of HvSOD and HvBI-1 correlated with enhanced susceptibility. Expression of the HvPR-1b gene encoding pathogenesis-related protein-1b increased several fold 24 h after B. sorokiniana infection, however, heat shock further increased transcript levels along with enhanced susceptibility. Heat shock induces enhanced susceptibility of barley to B. sorokiniana, associated with elevated ROS levels and expression of plant defence-related genes encoding antioxidants, a cell death inhibitor, and PR-1b. Our results may contribute to elucidating the influence of heat shock on barley defence responses to hemibiotrophic pathogens.

Kinetic study of folding and misfolding of diacylglycerol kinase in model membranes.

Despite the relevance of membrane protein misfolding to a number of common diseases, our understanding of the folding and misfolding of membrane proteins lags well behind soluble proteins. Here, the overall kinetics of membrane insertion and folding of the homotrimeric integral membrane protein diacylglycerol kinase (DAGK) is addressed. DAGK was purified into lipid/detergent-free urea and guanidinium solutions and subjected to general structural characterization. In urea, the enzyme was observed to be monomeric but maintained considerable tertiary structure. In guanidinium, it was also monomeric but exhibited much less tertiary structure. Aliquots of these DAGK stock solutions were diluted 200-fold into lipid vesicles or into detergent/lipid mixed micelles, and the rates and efficiencies of folding/insertion were monitored. Reactions were also carried out in which micellar DAGK solutions were diluted into vesicular solutions. Productive insertion of DAGK from denaturant solutions into mixed micelles occurred much more rapidly than into lipid vesicles, suggesting that bilayer transversal represents the rate-limiting step for DAGK assembly in vesicles. The efficiency of productive folding/insertion into vesicles was highest in reactions initiated with micellar DAGK stock solutions (where DAGK maintains a nativelike fold and oligomeric state) and lowest in reactions starting with guanidinium stocks (where DAGK is an unfolded monomer). Moreover, the final ratio of irreversibly misfolded DAGK to reversibly misfolded enzyme was highest following reactions initiated with guanidinium stock solutions and lowest when micellar stocks were used. Finally, it was also observed that very low concentrations of detergents were able to both enhance the bilayer insertion rate and suppress misfolding.

Use of amphipathic polymers to deliver a membrane protein to lipid bilayers.

Data are presented which suggest that a class of amphiphilic polymers known as 'amphipols' may serve as a vehicle for delivering complex integral membrane proteins into membranes. The integral membrane protein diacylglycerol kinase (DAGK) was maintained in soluble form by either of two different amphipols. Small aliquots of these solutions were added to pre-formed lipid vesicles and the appearance of DAGK catalytic activity was monitored as an indicator of the progress of productive protein insertion into the bilayers. For one of the two amphipols tested, DAGK was observed to productively transfer from its amphipol complex into vesicles with moderate efficiency. Results were not completely clear for the other amphipol.

Misfolding of membrane proteins in health and disease: the lady or the tiger?

Protein misfolding is increasingly recognized as a factor in many diseases, including cystic fibrosis, Parkinson's, Alzheimer's and atherosclerosis. Many proteins involved in misfolding-based pathologies are membrane-associated, such that the bilayer may play roles in normal and aberrant folding. It can be argued that the in vivo partitioning of eukaryotic membrane proteins between folding and misfolding pathways is under kinetic control. Moreover, the balance between these pathways can be surprisingly delicate.

Mapping the oligomeric interface of diacylglycerol kinase by engineered thiol cross-linking: homologous sites in the transmembrane domain.

This work represents the first stage of thiol-based cross-linking studies to map the oligomeric interface of the homotrimeric membrane protein diacylglycerol kinase (DAGK). A total of 53 single-cysteine mutants spanning DAGK's three transmembrane segments and the first part of a cytoplasmic domain were purified and subjected to catalytic oxidation in mixed micelles. Four mutants (A52C, I53C, A74C, and I75C) were observed to undergo intratrimer disulfide bond formation between homologous sites on adjacent subunits. To establish whether the homologous sites are proximal in the ground-state conformation of DAGK or whether the disulfide bonds formed as a result of motions that brought normally distal sites into transient proximity, additional cross-linking experiments were carried out in three different milieus of varying fluidity [mixed micelles, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) vesicles, and Escherichia coli membranes]. Cross-linking experiments included disulfide bond formation under three different catalytic conditions [Cu(II)-phenanthroline oxidation, I(2) oxidation, and thionitrobenzoate-based thiol exchange] and reactions with a set of bifunctional thiol-reactive chemical cross-linkers presenting two different reactive chemistries and several spacer lengths. On the basis of these studies, residues 53 and 75 are judged to be in stable proximity within the DAGK homotrimer, while position 52 appears to be more distal and forms disulfide bonds only as a result of protein motions. Results for position 74 were ambiguous. In lipid vesicles and mixed micelles DAGK appears to execute motions that are not present in native membranes, with mobility also being higher for DAGK in mixed micelles than in POPC vesicles.

Evaluation of assays for determination of passive transfer status in neonatal llamas and alpacas.

OBJECTIVE: To evaluate several practice-adapted assays for determination of passive transfer status in crias. ANIMALS: 24 llama and 9 alpaca crias. DESIGN: Prospective study. PROCEDURE: Serum IgG concentration was measured by use of a radial immunodiffusion assay when crias were 45 to 51 hours old. Results were compared with serum gamma-glutamyltransferase (GGT) activity, serum total protein, albumin, globulin, and total solids concentrations, and results of commercially available and traditional sodium sulfite turbidity (SST) tests. RESULTS: Mean (+/- SD) serum IgG concentration was 1,762 +/- 1,153 mg/dl. On the basis of a threshold value of 1,000 mg of IgG/dl at 48 hours of age, 5 of 33 (15.15%) crias had failure of passive transfer. Serum total solids, protein, and globulin concentrations were significantly associated with serum IgG concentration, whereas serum GGT activity and serum albumin concentration were not. Serum IgG concentrations were significantly different among crias with negative, 2+, and 3+ scores on the traditional SST test. Serum IgG concentrations were not significantly different between crias with negative and 100 mg/dl scores or 100 and 300 mg/dl scores on the commercially available SST test. However, all other comparisons between crias with different scores revealed significant differences. Sensitivity and specificity ranged between 0 and 1, depending on the test and endpoint selected. CONCLUSIONS AND CLINICAL RELEVANCE: The commercially available SST test and determination of serum total protein and globulin concentrations are suitable methods for assessing passive transfer status in llama and alpaca crias.

Reconstitutive refolding of diacylglycerol kinase, an integral membrane protein.

While the formation of kinetically trapped misfolded structural states by membrane proteins is related to a number of diseases, relatively few studies of misfolded membrane proteins in their purified state have been carried out and few methods for refolding such proteins have been reported. In this paper, misfolding of the trimeric integral membrane protein diacylglycerol kinase (DAGK) is documented and a method for refolding the protein is presented; 65 single-cysteine mutants of DAGK were examined. A majority were found to have lower-than-expected activities when purified into micellar solutions, with additional losses in activity often being observed following membrane reconstitution. A variety of evidence indicates that the low activities observed for most of these mutants results from kinetically based misfolding of the protein, with misfolding often being manifested by the formation of aberrant oligomeric states. A method referred to as "reconstitutive refolding" for correcting misfolded DAGK is presented. This method is based upon reconstituting DAGK into multilamellar POPC vesicles by dialyzing the detergent dodecylphosphocholine out of mixed micellar mixtures. For 55 of the 65 mutants tested, there was a gain of DAGK activity during reconstitutive refolding. In 33 of these cases, the gain in activity was greater than 2-fold. The refolding results for cysteine replacement mutants at DAGK sites known to be highly conserved provide teleological insight into whether sites are conserved, because they are critical for catalysis, for maintenance of the proper folding pathway, or for some other reason.