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MAP2 (microtubule-associated protein 2) is a cytoskeletal phosphoprotein that regulates the dynamic assembly characteristics of microtubules and appears to provide scaffolding for organelle distribution into the dendrites and for the localization of signal transduction apparatus in dendrites, particularly near spines. MAP2 is degraded after ischemia and other metabolic insults, but the time course and initial triggers of that breakdown are not fully understood. This study determined that MAP2 resides in a complex with the NMDA receptor, suggesting that spatially localized changes may be important in the mechanism of MAP2 redistribution and breakdown after oxygen-glucose deprivation (OGD). Using OGD in the adult rat hippocampal slice as a model system, this study demonstrated that MAP2 breakdown occurs very early after OGD, with the first statistical decrease in MAP2 levels within the first 30 min after the insult. There is a dramatic redistribution of MAP2 to the somata of pyramidal neurons, particularly neurons at the CA1-subiculum border. Free radicals and nitric oxide are not involved in the damage to MAP2. NMDA-receptor activation plays a prominent role in the MAP2 breakdown. In direct response to NMDA receptor activation, calcium influx, likely through the receptor ion channel complex, as well as release of calcium from the mitochondria through activation of the 2Na(+)-Ca(2+) exchanger of mitochondria, triggers MAP2 degradation. The proteolysis of MAP2 is limited by endogenous calpain activity, likely via the spatial access of calpain to MAP2.

An intricate architecture of covalent bonds and noncovalent interactions appear to position the side chain of Lys 41 properly within the active site of bovine pancreatic ribonuclease A (RNase A). One of these interactions arises from Tyr 97, which is conserved in all 41 RNase A homologues of known sequence. Tyr 97 has a solvent-inaccessible side chain that donates a hydrogen bond to the main-chain oxygen of Lys 41. Here, the role of Tyr 97 was examined by replacing Tyr 97 with a phenylalanine, alanine, or glycine residue. All three mutant proteins have diminished catalytic activity, with the value of Kcat being perturbed more significantly than that of Km. The free energies with which Y97F, Y97A, and Y97G RNase A bind to the rate-limiting transition state during the cleavage of poly(cytidylic acid) are diminished by 0.74, 3.3, and 3.8 kcal/mol, respectively. These results show that even though Tyr 97 is remote from the active site, its side chain contributes to catalysis. The role of Tyr 97 in the thermal stability of RNase A is large. The conformational free energies of native Y97F, Y97A, and Y97G RNase A are decreased by 3.54, 12.0, and 11.7 kcal/mol, respectively. The unusually large decrease in stability caused by the Tyr-->Phe mutation could result from a decrease in the barrier to isomerization of the Lys 41-Pro 42 peptide bond.

A general approach has been developed for configurational analysis (cis or trans) of Xaa-Pro peptide bonds in proteins. This approach, which entails selective 13C labeling of Xaa and Pro residues in the protein and isotope-edited NMR, has been applied to mutants of staphylococcal nuclease with suspected altered configurations of the Lys116-Pro117 peptide bond. The technique for monitoring proline configurations is based on differences in interproton distances between the H alpha of residue Xaa and the proline H delta or H alpha protons. Short (< 2.5 A) Xaa H alpha-Pro H delta interproton distances are diagnostic for the trans configuration, whereas short (< 2.5 A) Xaa H alpha-Pro H alpha interproton distances are diagnostic for the cis configuration. Biosynthetic incorporation of [alpha-13C]Xaa and [delta-13C]proline facilitates detection of trans Xaa-Pro peptide bonds, whereas incorporation of [alpha-13C]Xaa and [alpha-13C]proline facilitates detection of cis Xaa-Pro peptide bonds. Provided that the Xaa-Pro peptide bond is unique within the protein sequence, symmetric off-diagonal NOE cross peaks in the isotope-edited NOE spectrum allow for simultaneous chemical shift assignment and determination of the prolyl peptide bond geometry. We have used this technique to determine the predominant configuration of the Lys116-Pro117 peptide bond in recombinant V8 staphylococcal nuclease A (H124L) and two of its single amino acid mutants (D77A+H124L and G79S+H124L). The results are consistent with conclusions reached on the basis of indirect arguments concerning changes in the chemical shifts of histidine 1H epsilon 1 NMR signals.(ABSTRACT TRUNCATED AT 250 WORDS)

Copines make up a family of soluble, calcium-dependent, membrane binding proteins found in a variety of eukaryotic organisms. In an earlier study, we identified six copine genes in the Dictyostelium discoideum genome and focused our studies on cpnA . Our previous localization studies of green fluorescent protein-tagged CpnA in Dictyostelium suggested that CpnA may have roles in contractile vacuole function, endolysosomal trafficking, and development. To test these hypotheses, we created a cpnA − knockout strain, and here we report the initial characterization of the mutant phenotype. The cpnA − cells exhibited normal growth rates and a slight cytokinesis defect. When placed in starvation conditions, cpnA − cells appeared to aggregate into mounds and form fingers with normal timing; however, they were delayed or arrested in the finger stage. When placed in water, cpnA − cells formed unusually large contractile vacuoles, indicating a defect in contractile vacuole function, while endocytosis and phagocytosis rates for the cpnA − cells were similar to those seen for wild-type cells. These studies indicate that CpnA plays a role in cytokinesis and contractile vacuole function and is required for normal development, specifically in the later stages prior to culmination. We also used real-time reverse transcription-PCR to determine the expression patterns of all six copine genes during development. The six copine genes were expressed in vegetative cells, with each gene exhibiting a distinct pattern of expression throughout development. All of the copine genes except cpnF showed an upregulation of mRNA expression at one or two developmental transitions, suggesting that copines may be important regulators of Dictyostelium development.