Sample extraction techniques for enhanced proteomic analysis of plant tissues

Major improvements in proteomic techniques in recent years have led to an increase in their application in all biological fields, including plant sciences. For all proteomic approaches, protein extraction and sample preparation are of utmost importance for optimal results; however, extraction of proteins from plant tissues represents a great challenge. Plant tissues usually contain relatively low amounts of proteins and high concentrations of proteases and compounds that potentially can limit tissue disintegration and interfere with subsequent protein separation and identification. An effective protein extraction protocol must also be adaptable to the great variation in the sets of secondary metabolites and potentially contaminating compounds that occurs between tissues (e.g., leaves, roots, fruit, seeds and stems) and between species. Here we present two basic protein extraction protocols that have successfully been used with diverse plant tissues, including recalcitrant tissues. The first method is based on phenol extraction coupled with ammonium acetate precipitation, and the second is based on trichloroacetic acid (TCA) precipitation. Both extraction protocols can be completed within 2 d.     

Cathepsin D deficiency is associated with a human neurodegenerative disorder

Cathepsin D is a ubiquitously expressed lysosomal protease that is involved in proteolytic degradation, cell invasion, and apoptosis. In mice and sheep, cathepsin D deficiency is known to cause a fatal neurodegenerative disease. Here, we report a novel disorder in a child with early blindness and progressive psychomotor disability. Two missense mutations in the CTSD gene, F229I and W383C, were identified and were found to cause markedly reduced proteolytic activity and a diminished amount of cathepsin D in patient fibroblasts. Expression of cathepsin D mutants in cathepsin D(-/-) mouse fibroblasts revealed disturbed posttranslational processing and intracellular targeting for W383C and diminished maximal enzyme velocity for F229I. The structural effects of cathepsin D mutants were estimated by computer modeling, which suggested larger structural alterations for W383C than for F229I. Our studies broaden the group of human neurodegenerative disorders and add new insight into the cellular functions of human cathepsin D.     

Identification of a novel inhibitory actin-capping protein binding motif in CD2-associated protein

CD2-associated protein (CD2AP) is a scaffold molecule that plays a critical role in the maintenance of the kidney filtration barrier. Little, however, is understood about its mechanism of function. We used mass spectrometry to identify CD2AP-interacting proteins. Many of the proteins that we identified suggest a role for CD2AP in endocytosis and actin regulation. To address the role of CD2AP in regulation of the actin cytoskeleton, we focused on characterizing the interaction of CD2AP with actin-capping protein CP. We identified a novel binding motif LXHXTXXRPK(X)6P present in CD2AP that is also found in its homolog Cin85 and other capping protein-associated proteins such as CARMIL and CKIP-1. CD2AP inhibits the function of capping protein in vitro. Therefore, our results support a role of CD2AP in the regulation of the actin cytoskeleton.     

The human sef-a isoform utilizes different mechanisms to regulate receptor tyrosine kinase signaling pathways and subsequent cell fate

Negative feedback is among the key mechanisms for regulating receptor tyrosine kinase (RTK) signaling. Human Sef, a recently identified inhibitor of RTK signaling, encodes different isoforms, including a membrane spanning (hSef-a) and a cytosolic (hSef-b) isoform. Previously, we reported that hSef-b inhibited fibroblast proliferation and prevented the activation of mitogen-activated protein kinase (MAPK), without affecting protein kinase B/Akt or p38 MAPK. Conflicting results were reported concerning hSef-a inhibition of MAPK activation, and the effect of hSef-a on other RTK-induced signaling pathways is unknown. Here we show that, in fibroblasts, similar to hSef-b, ectopic expression of hSef-a inhibited fibroblast growth factor-induced cell proliferation. Unlike hSef-b, however, the growth arrest was mediated via a MAPK-independent mechanism, and was accompanied by elevated p38 MAPK phosphorylation and inhibition of protein kinase B/Akt. In addition, hSef-a, but not hSef-b, mediated apoptosis in fibroblast growth factor-stimulated cells. Chemical inhibitor of p38 MAPK abrogated the effect of hSef-a on apoptosis. In epithelial cells, ectopic expression of hSef-a inhibited the activation of MAPK, whereas down-regulation of endogenous hSef-a significantly increased MAPK activation and accelerated growth factor-dependent cell proliferation. These results indicate that hSef-a is a multifunctional negative modulator of RTK signaling and clearly demonstrate that hSef-a can inhibit the activation of MAPK, although in a cell type-specific manner. Moreover, the differences between the activities of hSef-a and hSef-b suggest that hSef isoforms can control signal specificity and subsequent cell fate by utilizing different mechanisms to modulate RTK signaling.     

The octopus: a model for a comparative analysis of the evolution of learning and memory mechanisms

Comparative analysis of brain function in invertebrates with sophisticated behaviors, such as the octopus, may advance our understanding of the evolution of the neural processes that mediate complex behaviors. Until the last few years, this approach was infeasible due to the lack of neurophysiological tools for testing the neural circuits mediating learning and memory in the brains of octopus and other cephalopods. Now, for the first time, the adaptation of modern neurophysiological methods to the study of the central nervous system of the octopus allows this avenue of research. The emerging results suggest that a convergent evolutionary process has led to the selection of vertebrate-like neural organization and activity-dependent long-term synaptic plasticity. As octopuses and vertebrates are very remote phylogenetically, this convergence suggests the importance of the shared properties for the mediation of learning and memory.     

Whorl and pollen-shed stage application of Beauveria bassiana for suppression of adult western corn rootworm

The corn rootworm complex is the most damaging insect pest of corn (Zea mays L.). This study was conducted to determine the efficacy of whorl and pollen-shed stage applications of a granular formulation of Beauveria bassiana (Balsamo) Vuillemin for control of adult western corn rootworm (Diabrotica virgifera virgifera Le Conte). The effect of application time (whorl-stage, pollen-shed) and plant surface exposed (leaves and leaf collars; silks; leaves, leaf collars, and silks) on level of beetle fungal infection were investigated. In addition, the number of colony forming units of B. bassiana in the corn leaf collar area was quantified. In the three years (1998–2000) of the study, application of B. bassiana at whorl-stage did not significantly increase beetle fungal infection. Beauveria bassiana applied to plants at pollen-shed in 1998 resulted in a significant increase in beetle infection with 51% of beetles from treated plants infected and 6.0% from control plants. Similar applications at pollen-shed in 1999 and 2000 resulted in very low infection levels. Beauveria bassiana application at pollen-shed stage significantly increased the number of colony forming units per leaf collar during all years of the study. Beetle infection with B. bassiana did not differ consistently among plant surface to which beetles were exposed for either application. Increased fungal load in leaf collars was not correlated with increased levels of adult infection. Increased rates of B. bassiana and application when beetles are present on the plants are likely needed to significantly increase infection rates.     

Death-associated protein (DAP) kinase plays a central role in ceramide-induced apoptosis in cultured hippocampal neurons.

Treatment of cultured hippocampal neurons with high concentrations of short-chain acyl ceramide derivatives, such as N-hexanoyl-D-sphingosine (C(6)-Cer), results in apoptotic cell death. We now show that death-associated protein (DAP) kinase plays an important role in mediating this effect. Upon incubation with C(6)-Cer, DAP kinase levels are elevated as early as 1 h after treatment, reaching levels 2-3-fold higher than untreated cells after 4 h. Neurons cultured from DAP kinase-deficient mice were significantly less sensitive to apoptosis induced by C(6)-Cer or by ceramide generated by high concentrations of nerve growth factor. A peptide corresponding to the 17 amino acids at the C terminus of DAP kinase protected wild type neurons from C(6)-Cer-induced death and from death induced by the addition of exogenous bacterial neutral sphingomyelinase, whereas a scrambled peptide had no protective effect, implying that the DAP kinase C-terminal tail inhibits the function of DAP kinase. Together, these data demonstrate that DAP kinase plays a central role in ceramide-induced cell death in neurons, but the pathway in which DAP kinase is involved is not the only one via which ceramide can induce apoptosis.     

Characterization of a Legionella pneumophila relA Insertion Mutant and Roles of RelA and RpoS in Virulence Gene Expression

To investigate the involvement of RelA in the regulation of Legionella pneumophila virulence, a deletion substitution was constructed in the relA gene. The relA knockout resulted in an undetectable level of ppGpp in the cells during the stationary phase, but the original level was restored when the relA gene product was supplied on a plasmid. The effect of the relA mutation was examined with two systems that are known to be expressed during the stationary phase in L. pneumophila. Pigment production was found to be dependent on the relA gene product, and only one-half as much pigment was produced by the relA mutant as by the wild-type strain. Flagellum gene expression was also found to be dependent on the relA gene product, as determined with a flaA::lacZ fusion. However, the relA gene product was found to be dispensable for intracellular growth both in HL-60-derived human macrophages and in the protozoan host Acanthamoeba castellanii. To determine the involvement of the relA gene product in expression of L. pneumophila genes required for intracellular growth (icm/dot genes), nine icm::lacZ fusions were constructed, and expression of these fusions in the wild-type strain was compared with their expression in relA mutant strains. Expression of only one of the icm::lacZ fusions was moderately reduced in the relA mutant strain. Expression of the nine icm::lacZ fusions was also examined in a strain containing an insertion in the gene that codes for the stationary-phase sigma factor RpoS, and similar results were obtained. We concluded that RelA is dispensable for intracellular growth of L. pneumophila in the two hosts examined and that both RelA and RpoS play minor roles in L. pneumophila icm/dot gene expression.     

Ubiquitylation‐dependent oligomerization regulates activity of Nedd4 ligases

Ubiquitylation controls protein function and degradation. Therefore, ubiquitin ligases need to be tightly controlled. We discovered an evolutionarily conserved allosteric restraint mechanism for Nedd4 ligases and demonstrated its function with diverse substrates: the yeast soluble proteins Rpn10 and Rvs167, and the human receptor tyrosine kinase FGFR1 and cardiac I_KS potassium channel. We found that a potential trimerization interface is structurally blocked by the HECT domain α1‐helix, which further undergoes ubiquitylation on a conserved lysine residue. Genetic, bioinformatics, biochemical and biophysical data show that attraction between this α1‐conjugated ubiquitin and the HECT ubiquitin‐binding patch pulls the α1‐helix out of the interface, thereby promoting trimerization. Strikingly, trimerization renders the ligase inactive. Arginine substitution of the ubiquitylated lysine impairs this inactivation mechanism and results in unrestrained FGFR1 ubiquitylation in cells. Similarly, electrophysiological data and TIRF microscopy show that NEDD4 unrestrained mutant constitutively downregulates the I_KS channel, thus confirming the functional importance of E3‐ligase autoinhibition.