Evolutionarily conserved bias of amino-acid usage refines the definition of PDZ-binding motif

<p>Abstract</p> <p>Background</p> <p>The interactions between PDZ (PSD-95, Dlg, ZO-1) domains and PDZ-binding motifs play central roles in signal transductions within cells. Proteins with PDZ domains bind to PDZ-binding motifs almost exclusively when the motifs are located at the carboxyl (C-) terminal ends of their binding partners. However, it remains little explored whether PDZ-binding motifs show any preferential location at the C-terminal ends of proteins, at genome-level.</p> <p>Results</p> <p>Here, we examined the distribution of the type-I (x-x-S/T-x-I/L/V) or type-II (x-x-V-x-I/V) PDZ-binding motifs in proteins encoded in the genomes of five different species (human, mouse, zebrafish, fruit fly and nematode). We first established that these PDZ-binding motifs are indeed preferentially present at their C-terminal ends. Moreover, we found specific amino acid (AA) bias for the 'x' positions in the motifs at the C-terminal ends. In general, hydrophilic AAs were favored. Our genomics-based findings confirm and largely extend the results of previous interaction-based studies, allowing us to propose refined consensus sequences for all of the examined PDZ-binding motifs. An ontological analysis revealed that the refined motifs are functionally relevant since a large fraction of the proteins bearing the motif appear to be involved in signal transduction. Furthermore, co-precipitation experiments confirmed two new protein interactions predicted by our genomics-based approach. Finally, we show that influenza virus pathogenicity can be correlated with PDZ-binding motif, with high-virulence viral proteins bearing a refined PDZ-binding motif.</p> <p>Conclusions</p> <p>Our refined definition of PDZ-binding motifs should provide important clues for identifying functional PDZ-binding motifs and proteins involved in signal transduction.</p

Isolation and Characterization of Glycophorin from Carp Red Blood Cell Membranes

We isolated a high-purity carp glycophorin from carp erythrocyte membranes following extraction using the lithium diiodosalicylate (LIS)-phenol method and streptomycin treatment. The main carp glycophorin was observed to locate at the position of the carp and human band-3 proteins on an SDS-polyacrylamide gel. Only the N-glycolylneuraminic acid (NeuGc) form of sialic acid was detected in the carp glycophorin. The oligosaccharide fraction was separated into two components (P-1 and P-2) using a Glyco-Pak DEAE column. We observed bacteriostatic activity against five strains of bacteria, including two known fish pathogens. Fractions from the carp erythrocyte membrane, the glycophorin oligosaccharide and the P-1 also exhibited bacteriostatic activity; whereas the glycolipid fraction and the glycophorin fraction without sialic acid did not show the activity. The carp glycophorin molecules attach to the flagellum of V. anguillarum or the cell surface of M. luteus and inhibited bacterial growth

Calpain-Mediated Degradation of Drebrin by Excitotoxicity <i>In vitro</i> and <i>In vivo</i>

<div><p>The level of drebrin, an evolutionarily conserved f-actin-binding protein that regulates synaptic structure and function, is reduced in the brains of patients with chronic neurodegenerative diseases such as Alzheimer’s disease (AD) and Down’s syndrome (DS). It was suggested that excitotoxic neuronal death caused by overactivation of NMDA-type glutamate receptors (NMDARs) occurs in AD and DS; however, the relationship between excitotoxicity and drebrin loss is unknown. Here, we show that drebrin is a novel target of calpain-mediated proteolysis under excitotoxic conditions induced by the overactivation of NMDARs. In cultured rodent neurons, degradation of drebrin was confirmed by the detection of proteolytic fragments, as well as a reduction in the amount of full-length drebrin. Notably, the NMDA-induced degradation of drebrin in mature neurons occurred concomitantly with a loss of f-actin. Furthermore, pharmacological inhibition of f-actin loss facilitated the drebrin degradation, suggesting a functional linkage between f-actin and drebrin degradation. Biochemical analyses using purified drebrin and calpain revealed that calpain degraded drebrin directly <i>in vitro</i>. Furthermore, cerebral ischemia also induced the degradation of drebrin <i>in vivo</i>. These findings suggest that calpain-mediated degradation of drebrin is a fundamental pathology of neurodegenerative diseases mediated by excitotoxicity, regardless of whether they are acute or chronic. Drebrin regulates the synaptic clustering of NMDARs; therefore, degradation of drebrin under excitotoxic conditions may modulate NMDAR-mediated signal transductions, including pro-survival signaling. Overall, the results presented here provide novel insights into the molecular basis of cellular responses to excitotoxicity <i>in vitro</i> and <i>in vivo</i>.</p></div

Ischemic brain damage induces the degradation of drebrin A.

<p>(a) Image of a mouse coronal brain slice showing the site of induction of photothrombotic ischemia (red) at the right hemisphere. The left hemisphere was used as a control. (b) Western blot analyses of drebrin A in total protein extracts of the left hemisphere (control) and the ischemic right hemisphere of the mouse brain. The DAS2 antibody was used to detect full-length drebrin A and its degradation products. The expression level of GAPDH was used as a loading control. The experiment was repeated three times with similar results.</p

Schematic representation of the signaling cascades from excitotoxic stimulation to the degradation of drebrin.

<p>NMDA induces the degradation of drebrin A in a calpain-dependent manner. Because drebrin A plays a critical role in clustering synaptic NMDARs, which transduce pro-survival signals, degradation of drebrin might modulate these signals and result in the delay or acceleration of neuronal death. The solid arrows indicate direct effects, and the dashed arrows indicate that effects have not yet been determined as direct.</p

Functional relationship between f-actin and the degradation of drebrin.

<p>(a) The effects of stabilization of f-actin using 5μM JAS and destabilization of f-actin using 2μM Lat-A on the levels of NMDA-induced proteolytic fragments of drebrin A in rat hippocampal neurons. The expression level of Hsp90 was used as a loading control. (b) Quantification of the signal intensities of full-length drebrin A shown in (a), relative to those in the control sample (DMSO/NMDA(-)). (c) Quantification of the signal intensities of the two degradation products in (a), relative to that of the upper band in the DMSO/NMDA(+) sample. The data are represented as the mean ± standard deviation of n = 4 replicates. *<i>P</i> < 0.05 and ***<i>P</i> < 0.005 by a Student’s t-test; ns, not significant.</p

Losses of f-actin and drebrin occur concomitantly after NMDA treatment.

<p>(a–h) Immunostaining of rat hippocampal neurons with antibodies against drebrin A/E (C-term), NeuN, and phalloidin (f-actin). (a–d) Control neurons. (e–h) Neurons that were treated with NMDA for 4 h. The arrows indicate NeuN-positive cells (neurons). Scale bar: 10 μm.</p

Combating herpesvirus encephalitis by potentiating a TLR3-mTORC2 axis

TLR3 is a sensor of double-stranded RNA that is indispensable for defense against infection with herpes simplex virus type 1 (HSV-1) in the brain. We found here that TLR3 was required for innate immune responses to HSV-1 in neurons and astrocytes. During infection with HSV-1, TLR3 recruited the metabolic checkpoint kinase complex mTORC2, which led to the induction of chemokines and trafficking of TLR3 to the cell periphery. Such trafficking enabled the activation of molecules (including mTORC1) required for the induction of type I interferons. Intracranial infection of mice with HSV-1 was exacerbated by impairment of TLR3 responses with an inhibitor of mTOR and was significantly 'rescued' by potentiation of TLR3 responses with an agonistic antibody to TLR3. These results suggest that the TLR3-mTORC2 axis might be a therapeutic target through which to combat herpes simplex encephalitis