Decrease of PKC precedes other cellular signs of calpain activation in area CA1 of the hippocampus after transient cerebral ischemia

One of the specific features of severe brain injury is an activation of calcium-dependent proteolysis by calpains. We have observed a significant increase of activity as early as 3 h after the insult in a well defined model of delayed ischemic neuronal death in gerbil hippocampus. At 24 h, the enzymatic activity transiently normalized, then increased again, following the place and time of selective cellular death in the CA1 region of hippocampus. The enhanced postischemic proteolysis resulted in concomitant cleavage of calpain-specific endogenous substrates like protein kinase C (PKC), fodrin and microtubule-associated protein-2 (MAP2). These effects were also time-dependent and restricted to the vulnerable, CA1 pyramidal neurons-containing the dorsal part (DP) of the hippocampus. We have also characterized the postischemic changes of six different isoforms of PKC. The vulnerable dorsal part of the hippocampus, but not its relative resistant abdominal part (AbP), exhibited a loss of PKCalpha, beta, gamma, and delta isoforms as early as 3 h after ischemic insult. However, at this time, solely in the soluble fraction of homogenate. Later (72 h), a further loss of the enzyme proteins, comprised the particulate fraction as well and resulted in an about 50% decrease of total PKCs in the vulnerable DP region. In the case of PKCalpha, the immunostaining pattern showed, in addition to the disappearance of the enzyme from the injured area, an extensive translocation into nuclei of the survived, ischemia-resistant neurones. The early decreases of PKC isoforms in the cytosol paralleled the transient calpain activation at 3h postischemia but substantially preceded the proteolysis of any other classical calpain substrates, such as fodrin and MAP2, being evidenced not earlier than 48-72 h after the insult and restricted also to the vulnerable dorsal part. In conclusion, our results of the time-dependent effects of transient global cerebral ischemia on the calpain activity, levels and localization of its several substrates suggest, that calpain-mediated proteolysis is specifically involved in the early (induction) as well as in the late (execution) phases of delayed ischemic neuronal death in the CA1 hippocampus.     

CAPRI: a Critical Assessment of PRedicted Interactions

CAPRI is a communitywide experiment to assess the capacity of protein-docking methods to predict protein-protein interactions. Nineteen groups participated in rounds 1 and 2 of CAPRI and submitted blind structure predictions for seven protein-protein complexes based on the known structure of the component proteins. The predictions were compared to the unpublished X-ray structures of the complexes. We describe here the motivations for launching CAPRI, the rules that we applied to select targets and run the experiment, and some conclusions that can already be drawn. The results stress the need for new scoring functions and for methods handling the conformation changes that were observed in some of the target systems. CAPRI has already been a powerful drive for the community of computational biologists who development docking algorithms. We hope that this issue of Proteins will also be of interest to the community of structural biologists, which we call upon to provide new targets for future rounds of CAPRI, and to all molecular biologists who view protein-protein recognition as an essential process.     

Modulation of sarcoplasmic reticulum calcium release by calsequestrin in cardiac myocytes

Calsequestrin (CASQ2) is a high capacity Ca-binding protein expressed inside the sarcoplasmic reticulum (SR). Mutations in the cardiac calsequestrin gene (CASQ2) have been linked to arrhythmias and sudden death induced by exercise and emotional stress. We have studied the function of CASQ2 and the consequences of arrhythmogenic CASQ2 mutations on intracellular Ca signalling using a combination of approaches of reverse genetics and cellular physiology in adult cardiac myocytes. We have found that CASQ2 is an essential determinant of the ability of the SR to store and release Ca2+ in cardiac muscle. CASQ2 serves as a reservoir for Ca2+ that is readily accessible for Ca(2+)-induced Ca2+ release (CICR) and also as an active Ca2+ buffer that modulates the local luminal Ca-dependent closure of the SR Ca2+ release channels. At the same time, CASQ2 stabilizes the CICR process by slowing the functional recharging of SR Ca2+ stores. Abnormal restitution of the Ca2+ release channels from a luminal Ca-dependent refractory state could account for ventricular arrhythmias associated with mutations in the CASQ2 gene.     

Repair of triple helix directed psoralen adducts in human cells.

Triple helix forming oligonucleotides can direct DNA damaging agents at specific sites in an intact double helix. In our study, triple helix formation was demonstrated in a SV40 based shuttle vector treated with psoralen linked to a 22-mer purine rich oligonucleotide. UVA irradiation caused a covalent linkage of the oligonucleotide through the psoralen to the mutational supF marker gene of the plasmid. After passage in the Jurkat human cell line the recovered vector was analysed in an indicator bacterial strain and mutants were collected. The presence of adducts in the target sequence did not reduce the yield of replicated progeny vector molecules, indicating repair of triple helix associated monoadducts and cross-links. Mutations were highly targeted to a six nucleotide long region of the target sequence. The number of target sequence mutants obtained after triple helix directed psoralen treatment was approximately 160 times higher than with free psoralen. A further investigation of the exact mechanism of the mutational process could make triple helix directed mutagenesis a more useful tool in gene therapy, antiviral therapy, and in studies on DNA repair and genome organisation.     

Distinct requirements for Ku in N nucleotide addition at V(D)J- and non-V(D)J-generated double-strand breaks

Loss or addition of nucleotides at junctions generated by V(D)J recombination significantly expands the antigen-receptor repertoire. Addition of nontemplated (N) nucleotides is carried out by terminal deoxynucleotidyl transferase (TdT), whose only known physiological role is to create diversity at V(D)J junctions during lymphocyte development. Although purified TdT can act at free DNA ends, its ability to add nucleotides (i.e. form N regions) at coding joints appears to depend on the nonhomologous end-joining factor Ku80. Because the DNA ends generated during V(D)J rearrangements remain associated with the RAG proteins after cleavage, TdT might be targeted for N region addition through interactions with RAG proteins or with Ku80 during remodeling of the post-cleavage complex. Such regulated access would help to prevent TdT from acting at other types of broken ends and degrading the fidelity of end joining. To test this hypothesis, we measured TdT’s ability to add nucleotides to endonuclease-induced chromosomal and extrachromosomal breaks. In both cases TdT added nucleotides efficiently to the cleaved DNA ends. Strikingly, the frequency of N regions at non-V(D)J-generated ends was not dependent on Ku80. Thus our results suggest that Ku80 is required to allow TdT access to RAG post-cleavage complexes, providing support for the hypothesis that Ku is involved in disassembling or remodeling the post-cleavage complex. We also found that N regions were abnormally long in the absence of Ku80, indicating that Ku80 may regulate TdT’s activity at DNA ends in vivo.     

Interactions between NEEP21, GRIP1 and GluR2 regulate sorting and recycling of the glutamate receptor subunit GluR2

Trafficking of AMPA-type glutamate receptors (AMPAR) between endosomes and the postsynaptic plasma membrane of neurons plays a central role in the control of synaptic strength associated with learning and memory. The molecular mechanisms of its regulation remain poorly understood, however. Here we show by biochemical and atomic force microscopy analyses that NEEP21, a neuronal endosomal protein necessary for receptor recycling including AMPAR, is associated with the scaffolding protein GRIP1 and the AMPAR subunit GluR2. Moreover, the interaction between NEEP21 and GRIP1 is regulated by neuronal activity. Expression of a NEEP21 fragment containing the GRIP1-binding site decreases surface GluR2 levels and delays recycling of internalized GluR2, which accumulates in early endosomes and lysosomes. Infusion of this fragment into pyramidal neurons of hippocampal slices induces inward rectification of AMPAR-mediated synaptic responses, suggesting decreased GluR2 expression at synapses. These results indicate that NEEP21-GRIP1 binding is crucial for GluR2-AMPAR sorting through endosomes and their recruitment to the plasma membrane, providing a first molecular mechanism to differentially regulate AMPAR subunit cycling in internal compartments.     

Chiral recognition of cyclic alpha-hydroxyketones by CD-sensitive zinc tetraphenylporphyrin tweezer

A combined chemical/chiroptical microscale protocol for the determination of absolute configurations of cyclic alpha-hydroxyketones is described. The hydroxyl group in cyclic alpha-hydroxyketones is converted into (3-aminopropylamino)acetate (NH2CH2CH2CH2NHCH2COOR), or more generally, according to a newly developed protocol, into (3-hydroxypropylamino)acetate group (HOCH2CH2CH2NHCH2COOR). The resultant conjugated compound forms a 1:1 host-guest complex with a dimeric zinc porphyrin tweezer, which exhibits exciton-coupled bisignate CD spectrum centered around the 420-nm porphyrin Soret band due to induced helicity between the two porphyrins in the complex. The absolute configurations of the alpha-stereogenic center is then determined by comparison of the sign of the observed CD exciton couplet of the complex with that of the preferred porphyrin twist predicted by the Merck Molecular Force Field (MMFFs) method.     

Performance of the first protein docking server ClusPro in CAPRI rounds 3-5

To evaluate the current status of the protein-protein docking field, the CAPRI experiment came to life. Researchers are given the receptor and ligand 3-dimensional (3D) coordinates before the cocrystallized complex is published. Human predictions of the complex structure are supposed to be submitted within 3 weeks, whereas the server ClusPro has only 24 h and does not make use of any biochemical information. From the 10 targets analyzed in the second evaluation meeting of CAPRI, ClusPro was able to predict meaningful models for 5 targets using only empirical free energy estimates. For two of the targets, the server predictions were assessed to be among the best in the field. Namely, for Targets 8 and 12, ClusPro predicted the model with the most accurate binding-site interface and the model with the highest percentage of nativelike contacts, among 180 and 230 submissions, respectively. After CAPRI, the server has been further developed to predict oligomeric assemblies, and new tools now allow the user to restrict the search for the complex to specific regions on the protein surface, significantly enhancing the predictive capabilities of the server. The performance of ClusPro in CAPRI Rounds 3-5 suggests that clustering the low free energy (i.e., desolvation and electrostatic energy) conformations of a homogeneous conformational sampling of the binding interface is a fast and reliable procedure to detect protein-protein interactions and eliminate false positives. Not including targets that had a significant structural rearrangement upon binding, the success rate of ClusPro was found to be around 71%.