Molecular analysis of the Heterobasidion annosum Mitogen Activated Protein Kinase (MAPK) HaPMK

The white rot fungus Heterobasidion annosum s.l. is a basidiomycete which is considered to be the most economical important pathogen of conifer trees (Pinus, Picea and Abies) in the northern hemisphere. Presently, the knowledge on the biology and molecular aspects of the Heterobasidion pathosystem is still poor and this is the major set-back in preventing the spread of the pathogen. A deeper investigation at the molecular level of the pathogenicity factors involved during the infection process is very important to better control the disease. Intra-cellular signal-transduction pathways, and in particular the Mitogen Activated Protein Kinases (MAPKs), have been shown to play key roles in the infection cycle in many fungal pathogens, being pivotal in survival, appressorial formation, sporulation and response to various biotic and abiotic stresses. The aim of this study is to characterize a specific H. annosum MAPK, with high sequence homology to FUS3 gene (involved in mating) in S. cerevisiae and with PMK1 gene (involved in appressoria formation) in Magnaporthe grisea. In order to study the function of this MAPK in H. annosum, we performed a complementation experiment in the S. cerevisiae fus3?? mutant. Expression level profiles, proteomics and immunology studies were used to distinguish between phosphorylated/active and non-phosphorylated/inactive form of the MAPK. Some valuable insights on this kinase cascade in Heterobasidion were discovered, but further studies are required to fully understand its role in the lifecycle of this fungus

Quantification of Intrinsically Disordered Proteins: A Problem Not Fully Appreciated

Protein quantification is essential in a great variety of biochemical assays, yet the inherent systematic errors associated with the concentration determination of intrinsically disordered proteins (IDPs) using classical methods are hardly appreciated. Routinely used assays for protein quantification, such as the Bradford assay or ultraviolet absorbance at 280 nm, usually seriously misestimate the concentrations of IDPs due to their distinct and variable amino acid composition. Therefore, dependable method(s) have to be worked out/adopted for this task. By comparison to elemental analysis as the gold standard, we show through the example of four globular proteins and nine IDPs that the ninhydrin assay and the commercial Qubit(TM) Protein Assay provide reliable data on IDP quantity. However, as IDPs can show extreme variation in amino acid composition and physical features not necessarily covered by our examples, even these techniques should only be used for IDPs following standardization. The far-reaching implications of these simple observations are demonstrated through two examples: (i) circular dichroism spectrum deconvolution, and (ii) receptor-ligand affinity determination. These actual comparative examples illustrate the potential errors that can be incorporated into the biophysical parameters of IDPs, due to systematic misestimation of their concentration. This leads to inaccurate description of IDP functions

Interplay of Structural Disorder and Short Binding Elements in the Cellular Chaperone Function of Plant Dehydrin ERD14.

Details of the functional mechanisms of intrinsically disordered proteins (IDPs) in living cells is an area not frequently investigated. Here, we dissect the molecular mechanism of action of an IDP in cells by detailed structural analyses based on an in-cell nuclear magnetic resonance experiment. We show that the ID stress protein (IDSP) A. thaliana Early Response to Dehydration (ERD14) is capable of protecting E. coli cells under heat stress. The overexpression of ERD14 increases the viability of E. coli cells from 38.9% to 73.9% following heat stress (50 °C × 15 min). We also provide evidence that the protection is mainly achieved by protecting the proteome of the cells. In-cell NMR experiments performed in E. coli cells show that the protective activity is associated with a largely disordered structural state with conserved, short sequence motifs (K- and H-segments), which transiently sample helical conformations in vitro and engage in partner binding in vivo. Other regions of the protein, such as its S segment and its regions linking and flanking the binding motifs, remain unbound and disordered in the cell. Our data suggest that the cellular function of ERD14 is compatible with its residual structural disorder in vivo

Quantification of Intrinsically Disordered Proteins: A Problem Not Fully Appreciated

Protein quantification is essential in a great variety of biochemical assays, yet the inherent systematic errors associated with the concentration determination of intrinsically disordered proteins (IDPs) using classical methods are hardly appreciated. Routinely used assays for protein quantification, such as the Bradford assay or ultraviolet absorbance at 280 nm, usually seriously misestimate the concentrations of IDPs due to their distinct and variable amino acid composition. Therefore, dependable method(s) have to be worked out/adopted for this task. By comparison to elemental analysis as the gold standard, we show through the example of four globular proteins and nine IDPs that the ninhydrin assay and the commercial QubitTM Protein Assay provide reliable data on IDP quantity. However, as IDPs can show extreme variation in amino acid composition and physical features not necessarily covered by our examples, even these techniques should only be used for IDPs following standardization. The far-reaching implications of these simple observations are demonstrated through two examples: (i) circular dichroism spectrum deconvolution, and (ii) receptor-ligand affinity determination. These actual comparative examples illustrate the potential errors that can be incorporated into the biophysical parameters of IDPs, due to systematic misestimation of their concentration. This leads to inaccurate description of IDP functions

A Novel Method for Assessing the Chaperone Activity of Proteins.

Protein chaperones are molecular machines which function both during homeostasis and stress conditions in all living organisms. Depending on their specific function, molecular chaperones are involved in a plethora of cellular processes by playing key roles in nascent protein chain folding, transport and quality control. Among stress protein families-molecules expressed during adverse conditions, infection, and diseases-chaperones are highly abundant. Their molecular functions range from stabilizing stress-susceptible molecules and membranes to assisting the refolding of stress-damaged proteins, thereby acting as protective barriers against cellular damage. Here we propose a novel technique to test and measure the capability for protective activity of known and putative chaperones in a semi-high throughput manner on a plate reader. The current state of the art does not allow the in vitro measurements of chaperone activity in a highly parallel manner with high accuracy or high reproducibility, thus we believe that the method we report will be of significant benefit in this direction. The use of this method may lead to a considerable increase in the number of experimentally verified proteins with such functions, and may also allow the dissection of their molecular mechanism for a better understanding of their function

Loss of activity of each enzyme as a function of time at high temperature.

<p>Loss of activity of each enzyme as a function of time at high temperature.</p

Effect of E. coli total protein extract on the deactivation of CS by high temperature.

<p>The activity of citrate synthase was compared before and after temperature stress, between samples with and without the addition of total protein extract from cells grown at 37°C or 42°C as indicated in the grid. The addition of either type of extract significantly protects the activity of CS (shown by t-test). A slight, but not statistically significant, difference was observed between the protective effects of various cell extracts. The height of the bars represents the mean of at least 6 technical repeats, the error flags– 95% CI. The significance of the differences was assessed via an one-way ANOVA.</p

Hsp70 (A), Hsp90 (B), and GroEL (C) protect citrate synthase against temperature deactivation.

<p>The activity of the enzyme after temperature stress alone (CS AS) or in the presence of different ratios of the respective chaperone (and excess ATP where noted) was measured as described in the Materials and Methods section, and plotted on each panel. The bars represent the mean of the parallel measurements; the error bars indicate the 95% confidence intervals of each measurement. The data was normalized to the activity of the enzyme alone before it was subjected to temperature stress (CS BS). One-way ANOVA analysis was performed in each case to assess the significance of the differences of the protection effect of each chaperone compared to the CS AS. Panel D shows examples of the concentrations of chaperones used in previous experiments to assess protective activity.</p

Enzymatic activity of CS is protected by ERD10 against temperature deactivation.

<p>The activity of CS was recorded before (A) and after stress (B) of 44°C for 40min in the presence (■) and absence (•) of different concentration of ERD10 as described in the Material and Methods section. Activity is presented as percentage of the activity of the non-stressed CS without the addition of ERD10. Concentration of CS is constant at 4.5nM. The symbols represent the mean, and the error flags—the 95%CI.</p