A model for the structure of the P domains in the subtilisin-like prohormone convertases

The proprotein convertases are a family of at least seven calcium-dependent endoproteases that process a wide variety of precursor proteins in the secretory pathway. All members of this family possess an N-terminal proregion, a subtilisin-like catalytic module, and an additional downstream well-conserved region of ≈150 amino acid residues, the P domain, which is not found in any other subtilase. The pro and catalytic domains cannot be expressed in the absence of the P domains; their thermodynamic instability may be attributable to the presence of large numbers of negatively charged Glu and Asp side chains in the substrate binding region for recognition of multibasic residue cleavage sites. Based on secondary structure predictions, we here propose that the P domains consist of 8-stranded β-barrels with well-organized inner hydrophobic cores, and therefore are independently folded components of the proprotein convertases. We hypothesize further that the P domains are integrated through strong hydrophobic interactions with the catalytic domains, conferring structural stability and regulating the properties and activity of the convertases. A molecular model of these interdomain interactions is proposed in this report.

Neonatal lead exposure impairs development of rodent barrel field cortex

Childhood exposure to low-level lead can permanently reduce intelligence, but the neurobiologic mechanism for this effect is unknown. We examined the impact of lead exposure on the development of cortical columns, using the rodent barrel field as a model. In all areas of mammalian neocortex, cortical columns constitute a fundamental structural unit subserving information processing. Barrel field cortex contains columnar processing units with distinct clusters of layer IV neurons that receive sensory input from individual whiskers. In this study, rat pups were exposed to 0, 0.2, 1, 1.5, or 2 g/liter lead acetate in their dam's drinking water from birth through postnatal day 10. This treatment, which coincides with the development of segregated columns in the barrel field, produced blood lead concentrations from 1 to 31 μg/dl. On postnatal day 10, the area of the barrel field and of individual barrels was measured. A dose-related reduction in barrel field area was observed (Pearson correlation = −0.740; P < 0.001); mean barrel field area in the highest exposure group was decreased 12% versus controls. Individual barrels in the physiologically more active caudoventral group were affected preferentially. Total cortical area measured in the same sections was not altered significantly by lead exposure. These data support the hypothesis that lead exposure may impair the development of columnar processing units in immature neocortex. We demonstrate that low levels of blood lead, in the range seen in many impoverished inner-city children, cause structural alterations in a neocortical somatosensory map.

Alpha-helical, but not beta-sheet, propensity of proline is determined by peptide environment.

Proline is established as a potent breaker of both alpha-helical and beta-sheet structures in soluble (globular) proteins. Thus, the frequent occurrence of the Pro residue in the putative transmembrane helices of integral membrane proteins, particularly transport proteins, presents a structural dilemma. We propose that this phenomenon results from the fact that the structural propensity of a given amino acid may be altered to conform to changes imposed by molecular environment. To test this hypothesis on proline, we synthesized model peptides of generic sequence H2N-(Ser-LyS)2-Ala- Leu-Z-Ala-Leu-Z-Trp-Ala-Leu-Z-(Lys-Ser)3-OH (Z = Ala and/or Pro). Peptide conformations were analyzed by circular dichroism spectroscopy in aqueous buffer, SDS, lysophosphatidylglycerol micelles, and organic solvents (methanol, trifluoroethanol, and 2-propanol). The helical propensity of Pro was found to be greatly enhanced in the membrane-mimetic environments of both lipid micelles and organic solvents. Proline was found to stabilize the alpha-helical conformation relative to Ala at elevated temperatures in 2-propanol, an observation that argues against the doctrine that Pro is the most potent alpha-helix breaker as established in aqueous media. Parallel studies in deoxycholate micelles of the temperature-induced conformational transitions of the single-spanning membrane bacteriophage IKe major coat protein, in which the Pro-containing wild type was compared with Pro30 --> Ala mutant, Pro was found to protect the helix, but disrupt the beta-sheet structure as effectively as it does to model peptides in water. The intrinsic capacity of Pro to disrupt beta-sheets was further reflected in a survey of porins where Pro was found to be selectively excluded from the core of membrane-spanning beta-sheet barrels. The overall data provide a rationale for predicting and understanding the structural consequences when Pro occurs in the context of a membrane.

Activation of single whisker barrel in rat brain localized by functional magnetic resonance imaging.

The previously established cortical representation of rat whiskers in layer IV of the cortex contains distinct cylindrical columns of cellular aggregates, which are termed barrels and correlate in a one-to-one relation to whiskers on the contralateral rat face. In the present study, functional magnetic resonance imaging (fMRI) of the rat brain was used to map whisker barrel activation during mechanical up-down movement (+/- 2.5 mm amplitude at 8 Hz) of single/multiple whisker(s). Multislice gradient echo fMRI experiments were performed at 7 T with in-plane image resolution of 220 x 220 microns, slice thickness of 1 mm, and echo time of 16 ms. Highly significant (P < 0.001) and localized contralateral regions of activation were observed upon stimulation of single/multiple whisker(s). In all experiments (n = 10), the locations of activation relative to bregma and midline were highly correlated with the neuroanatomical position of the corresponding whisker barrels, and the results were reproducible intra- and interanimal. Our results indicate that fMRI based on blood oxygenation level-dependent image contrast has the sensitivity to depict activation of a single whisker barrel in the rat brain. This noninvasive technique will supplement existing methods in the study of rat barrel cortex and should be particularly useful for the long-term investigations of central nervous system in the same animal.